Your Practical Guide To Basic Laboratory Techniques: A Sponsored Supplement To Science

A Sponsored Supplement to Science
Your Practical Guide to

Basic Laboratory Techniques
Produced by the
Science/AAAS Custom
Sponsored by Publishing Office
B I O LO GY ¡ E A RT H ¡ E N V I R O N M E N T ¡ L I F E ¡ I N T E R D I S C I P L I N A RY ¡ P H YS I CA L ¡ M AT E R I A L ¡ S O C I A L S C I E N C E S
O P E N A C C E S S , D I G I TA L , A N D F R E E T O A L L R E A D E R S
Pushing the Boundaries of Knowledge

As AAAS’s first multidisciplinary, open access journal, Science Advances publishes
research that reflects the selectivity of high impact, innovative research you expect
from the Science family of journals, published in an open access format to serve a
vast and growing global audience. Check out the latest findings or learn how to
submit your research: ScienceAdvances.org
TABLE O F C ON T E N T S
Introductions
2 Ready . . . Set . . . Pipet!
Sean Sanders, Ph.D.
Your
Science/AAAS
Practical 3 Taking the First Steps

Ferencz Paldy, Ph.D.
Guide to
Head of Segment Marketing Academia, Sartorius

Fiona Coats, Ph.D.
Basic
Head of Life Science Research Marketing, Sartorius
Laboratory Feature Articles

Techniques

4 Building Skills in Basic Lab Techniques:
Useful Tips from the Experts
Mike May, Ph.D.
7 The Paperless Lab

Chris Tachibana
Technical Notes
10 How to Avoid Contamination in Pipetting
13 Concentration to a Defined Final Volume with

Vivaspin® Turbo 15, Vivaspin® Turbo 4 and
Vivaspin® 500
15

Cell Culture Expansion in Fully Closed Erlenmeyer
Shake Flasks Outside the Biosafety Cabinet with
MYCAP™ CCX
20 How to Achieve Optimal Weighing Performance
Cover Image: © abscent/shutterstock.com

24 Vivaflow® and Vivaspin® Workflow in Protein
Research Laboratories
Original content (pages 4–9) has not

been peer-reviewed or assessed
by Science.
29 Minimizing Syringe Filter Consumption for Monoclonal
This booklet was produced by the Science/ Antibody Harvest from CHO Cell Culture Supernatants
AAAS Custom Publishing Office and
34
sponsored by Sartorius.
Correlation Between Colony Forming Units and Genome
Editor: Sean Sanders, Ph.D.
Proofreader/Copyeditor: Bob French
Copies of 9 Different Mycoplasma Species Using Quantified
Designer: Amy Hardcastle CFU and GC Standards for Validation
ROGER GONCALVES,
ASSOCIATE SALES DIRECTOR
Custom Publishing
Europe, Middle East, and India

38 Scouting Protein Purification Conditions Using
Vivapure Centrifugal Ion Exchange Membrane Absorbers
rgoncalves@science-int.co.uk
+41-43-243-1358
© 2018 by The American Association
42
for the Advancement of Science.
All rights reserved. 26 October 2018 Additional Resources
1
SCIENCE sciencemag.org
Y O U R PR A C T I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
“
Y
ou need to learn to walk before you can run” is a
saying many of us probably heard when we were
children. The clear message here is that there are
some basic skills we need to master before we can
move on to the next level. And there are plenty of
good reasons that following this mantra will set one up for success,
not the least of which—to continue the metaphor—is to avoid tripping
and falling on your face.
In a scientific laboratory, there are also fundamental skills that

require mastering before more complex tasks can be undertaken.
Building a solid foundation of core lab skills is critical not only to
producing accurate, reproducible experimental results, but also
to prevent damage to expensive equipment and maintain a safe
environment for ourselves and our fellow labmates.
Gaining competence in accurately weighing dry reagents is a

Ready . . . Set critical skill, particularly when making stock solutions that might be
used across multiple experiments and by multiple researchers in the
. . . Pipet! lab. When an experiment doesn’t work, we often don’t know why—
but we certainly don’t want its failure to be the result of incorrectly
It behooves all prepared solutions due to poor weighing proficiency.
researchers to ensure Filtration is a foundational technique used ubiquitously in the

biological sciences and is an essential step in many protocols. One
that their core lab of its common applications is the generation of clean water needed
skills are solid and in many aspects of lab work, but probably most importantly for
making up and diluting reagents. Impurities in improperly filtered
up to date. water, even at low levels, can negatively impact biological processes
or, even worse, generate spurious results. Filtration is also essential
for purification and/or concentration of solutions as well as the
sterilization of biological reagents for which autoclaving is not an
option due to heat sensitivity.
Most, if not all, life science laboratories have at least one set of
micropipettes. If they’re lucky, some might even have a set for each
researcher. Correct pipetting technique for small volumes of reagents
is an essential skill for researchers performing almost any type of
molecular biology experiment. Knowing how to accurately pipet
a range of fluids—from viscous glycerol to highly volatile phenol—
can make the difference between a successful experiment and yet
another confusing result. And anyone who remembers learning to
pipet will recall that it’s nowhere near as easy as it looks.
With increasing focus in the scientific community on reproducibility

of results, it behooves all researchers to ensure that their core lab
skills are solid and up to date. The latest advances in lab techniques
need to be studied and absorbed, and basic skills revisited and
refreshed. In other words, keep practicing your walking skills so that
you’re able to sprint when it’s really needed!
Sean Sanders, Ph.D.

Senior Editor, Custom Publishing
Science/AAAS
2 sciencemag.org SCIENCE
INTRO D U C T I ON S
I
n a scientific world that is more competitive than ever before,
it is imperative to gain a deep understanding of biological no-
velties and phenomena at both a macro and micro scale, and to
do this as quickly and accurately as possible. This knowledge
will potentially enable scientists to formulate novel hypotheses,
Taking the make new discoveries, and share their findings with the world. The
creation and dissemination of scientific information is the corner-
First Steps stone of scientific and societal advancement.
“A Journey of a With such a strong focus on exciting discoveries—like the next

generation of cancer therapies—it is easy to forget that it all starts
Thousand Miles with the basics. As the Chinese philosopher Laozi once said, “The
Begins with a Single journey of a thousand miles begins with a single step.“ Every cell
culture medium, and every sample of DNA, RNA, or purified protein,
Step.”—Laozi needs at some point during the experimentation process to undergo
a variety of different treatments. These materials may need to be
dissolved or diluted in purified water, weighed, filtered, pipetted,
or generally experience aseptic handling or transfer. All these small,
seemingly insignificant steps and minor details tend to be forgotten
as a user gains experience and confidence in the daily routines of their
laboratory, or even disregarded when it comes to complete beginners.
Since nothing that stands the test of time can have a weak foun-
dation, it is extremely important for today’s young scientists entering
the lab world for the first time to be able to build a robust foundation
in basic lab techniques, starting on day one. This underpinning is
crucial to their future success. It is equally important that experienced
scientists revisit these basic topics in order to remedy potential
misconceptions, and to fill in the gaps in their knowledge that have
developed over time.
Sartorius, a global laboratory products and services supplier for the

academic and (bio)pharma markets, has been dedicated to providing
solutions that strengthen scientific experimentation for more than
140 years. Sartorius engages with its customers over the full spectrum
of their work, catering not only to their basic laboratory needs (such
as weighing, pipetting, and filtering), but also by offering high-end
and high-throughput (live) cell-analysis instrumentation. By offering
this booklet in partnership with Science/AAAS, we hope that we can
contribute to building a secure and prosperous scientific future for
the benefit of all stakeholders involved.
Ferencz Paldy, Ph.D.

Head of Segment Marketing Academia, Sartorius
Fiona Coats, Ph.D.

Head of Life Science Research Marketing, Sartorius
SCIENCE sciencemag.org 3
Building Skills in Basic Lab Techniques:

Useful Tips from the Experts
Safety and competency in a science laboratory depend on a set of basic skills. As science advances, so do some of the
capabilities required for it. Nonetheless, some skills are almost as old as science itself, and these remain vital—even
though the way of doing these tasks has evolved. With both old and new techniques, beginner and experienced scientists
alike need to maintain their competency in the use of numerous standard methods. Even after learning and mastering a
technique, a refresher never hurts, and keeping current on changing methods maintains the foundation of a lab and the
integrity of its findings. By Mike May, Ph.D.
H
ere, we’ll explore familiar, everyday methods along precise weighing on a top-loading balance,” says Kevin Olsen,
with some newer ones—all aimed at helping scientists instrumentation specialist in the chemistry and biochemistry
build and maintain a skillset. Many of these skills will department at New Jersey’s Montclair State University. “It is
apply to various applications. For example, Donald important to understand the limitations of whatever kind of
Spratt, assistant professor of chemistry and biochemistry at balance you are using.”
Clark University (Worcester, Massachusetts), says, “Protein For instance, any balance produces a more accurate weight
scientists need, for example, to have excellent planning and for larger over smaller samples. “This is why we typically weigh
organizational skills so they can design and successfully execute out an analytical standard in the grams range and dilute it rather
their experiments.” He adds, “These skills are translatable to many than weighing the same material in the milligram range,” Olsen
different scientific disciplines.” In fact, most lab skills build on explains. “Different balance models have different features and if
others and help scientists learn new ones. they are used incorrectly, the weighing may not be accurate.”
For every balance, keeping it clean and calibrated impacts all
Weigh it right weight measurements. So, a little care goes a long way.
Weighing samples is one of the oldest procedures in all of
science. It’s one of the first things that scientists learn how to do, Proper pipetting
and a skill that most of them need throughout their careers. The After weighing samples, the next most common technique,
ubiquity of weighing makes it a top-priority skill for scientists at at least in the biological sciences, might be pipetting. For
all levels. The first step to weighing involves picking the right some scientists, pipetting could even be the most important
balance. “People do not need a four-place analytical balance for skill to master. To get it right, scientists need to pay attention,
routine powder dispensing, and conversely they cannot achieve and not just to the proper technique. In fact, becoming
distracted is a common mistake in pipetting, according to
Tamara Mandell, associate director of education and training
at the University of Florida’s Biotility, a center that prepares
people for the biotech industry.
4
FEATURE A RT I C L E S
Others agree on the value of the right attitude with this

process. “The most important thing to keep in mind while
pipetting is slowing down and taking my time,” says VJ Tocco, One critical role
lecturer in the department of chemical engineering at the
University of Florida, Gainesville. “Sometimes, I get tempted to
for filtration in
rush, which can lead to mistakes.” these industries
Tocco suggests other things to remember as well, including
picking the right pipette. “You should use the pipette that dis- is sterilization,
penses the smallest volume,” he says. “For example, to pipet
18 microliters of fluid, use the 20-microliter pipette, not the
since the use of
100-microliter pipette.” And the pipette tip should be wet before heat to sterilize
using it. As Tocco says, “It’s best to aspirate liquid and dispense it
at least once before actually pipetting your liquid.” Lastly, Tocco would cause
reminds scientists to take their time and not to “aspirate so quick-
ly that bubbles form in the solution.” Those bubbles cause errors undesirable
in volume measurement. product
Purifying the processes degradation.
Many protocols in a lab require a variety of solutions,
including culture media, buffers, and more. And these solutions
usually require water. In most cases, not just any water will do.
Instead, water for lab processes must be filtered and purified,
and the application determines the level of purity required. Filtering fluids
IMAGES: (FROM TOP) © M.LORAINE/SHUTTERSTOCK.COM; MAKYZZ/SHUTTERSTOCK.COM
According to ASTM International, water can be categorized To remove unwanted solids from a sample and increase
as Type I–IV, with Type I being the purest. One metric that distin- purity, scientists often use various forms of filtration, which
guishes these categories is resistivity (Ω-cm); water with fewer extend from a simple piece of filter paper in a funnel to
impurities shows higher resistivity. For example, the resistivity advanced membrane-based devices. Many molecular methods
of Type I and IV water is 18 and 0.2 megaΩ-cm, respectively. include filtration to concentrate a sample. Filtration is used
The less-pure Type IV water can be used as a source for a lab extensively to concentrate and purify proteins or DNA,
distiller, for example, and ultrapure Type I water is used for cell for example, for crystallography studies or for use in the
culture, gas chromatography, high-performance liquid chroma- polymerase chain reaction (PCR).
tography (HPLC), and other applications that are very sensitive Filtration processes can also be distinguished by general
to impurities. application. One of the most common applications for
The secret is matching the right water to an application, and analytical filtration is sample preparation for HPLC. Filtering out
not overspending to make water that is more purified than particles is essential to prevent blocking of the column, which
necessary. The volume of water necessary will also determine can lead to failure of the analysis; it also reduces background in
how to make it. In some situations, a water system for a lab the chromatogram and improves sensitivity and accuracy.
is enough, while other applications require building-wide The biotechnology and pharmaceutical industries also re-
purification systems. In the latter case, a building-wide system quire filtration in many processes, using a variety of membranes
might make reasonably pure water, for example, Type III; and and devices that often have the added requirement of meeting
then lab systems can further treat that water as needed. specific criteria, such as ASTM International standards or good
manufacturing practices (GMP) regulations. One critical role for
filtration in these industries is sterilization, since the use of heat
to sterilize would cause undesirable product degradation.
Keeping cultures healthy

From basic science to biotechnology and pharmaceutical
sciences, many labs include cell or tissue culture as a standard
method. The basic idea of keeping cells alive in culture is over
130 years old—starting in 1885 with work by German zoologist
Wilhelm Roux, who cultured chicken embryonic cells in a
saline solution. Today, scientists culture cells in two and three
dimensions, even mimicking complete organs in some cases.
Despite its increasing complexity, some of the steps for cell
culture are easier than ever.
5
Processing proteins
Many protocols in life science and clinical labs involve pro-
teins. When asked about the top skill required for working with
these molecules, Daniel J. Kosman, SUNY Distinguished Profes-
sor in biochemistry at the University of Buffalo’s Jacobs School of
Medicine and Biomedical Sciences, picks the ability to use fast
protein liquid chromatography (FPLC), which can isolate proteins
in a mixture. He also notes that protein scientists must be able to
perform heterologous expression, in which DNA or RNA from one
species is expressed in another to create a specific protein. With
this technique, though, Kosman notes that the key challenges are
ensuring the “correct folding and posttranslational modification of
heterologously expressed proteins.”
Spratt also points out the need for protein-expression capabili-
ties. When asked about the most common technique for obtain-
ing proteins for further research, he selects bacterial expression
in Escherichia coli using recombinant DNA technology, calling it
Scientists who have been thinking about “the most common and cheapest way to make a protein.” With this
writing all along can get a head start by using technique, the overexpressed protein “can then be purified using
chromatography, based on its unique physicochemical properties,
an electronic lab notebook to keep track of
such as size, charge, affinity, solubility, and/or oligomeric state,”
protocols and results. Spratt explains. “Once the protein is pure, it needs to be quanti-
fied prior to further biochemical examination.”
In the early 1980s, for example, I worked in a cell-culture lab, In fact, getting adequately pure protein for downstream tech-
and we made most of what we needed, including materials like niques can be challenging. “Many protein biochemists have
rat-tail collagen to coat the coverslips on which the cells grew. to contend with frustrating obstacles, including protein yield,
Today, scientists can purchase a wide variety of media and solubility, and degradation issues,” Spratt says. “Speaking from
reagents as well as labware designed for specific culture tech- personal experience, it can take many attempts to overcome
niques, such as 3D culture. these challenges.”
Still, some of the key skills remain the same. “The most impor- That brings up perhaps the most crucial lab skills of all: patience
tant aspect of tissue culture is good sterile technique,” says Katy and persistence.
Phelan, director of the cytogenetics laboratory at Florida Can-
cer Specialists & Research Institute (Fort Myers, Florida). “This Writing up the results
applies to initial setup of cultures as well as feeding, subcultur- Once those skills pay off, it’s time to write. Scientists who have
ing, and cryopreservation.” This means that everything—culture been thinking about writing all along can get a head start by using
media and additives, pipettes, culture vessels, and other equip- an electronic lab notebook to keep track of protocols and results.
ment—must be kept sterile and tested to confirm sterility. “Prac- At the very least, they can cut and paste methods and results to
ticing good sterile technique will reduce the chance that cul- get started on an article.
tures will become contaminated,” Phelan explains. “Valuable cell Beyond collecting all the information, more challenges arise in
lines can be lost or compromised due to failure to practice good knowing how to describe the work. For even seasoned writers,
sterile technique.” In fact, keeping cultures contamination-free it’s worth reading “The Science of Scientific Writing” by writing
is one of the biggest challenges of this general method. consultant George Gopen and Judith Swan, associate director for
Plus, it’s crucial to ensure that a culture includes only what is writing in science and engineering at Princeton University (Ameri-
intended. “A common mistake in cell culture is sample mix-up can Scientist, November–December 1990). As they concluded, “In
or cross-contamination of samples,” Phelan explains. “Various real and important ways, the structure of the prose becomes the
techniques can be employed in an attempt to prevent this error, structure of the scientific argument.”
such as working with only one sample at a time in the tissue cul- To build the best structure, make an outline or develop
ture hood, avoiding the use of prelabeled flasks or petri dishes, some organization before writing begins. It doesn’t need to be
and double-checking two unique identifiers on all paperwork a formal system of Roman numerals or capital letters, but just
and culture vessels.” something that works for the writer. A research article comes
with an overall organization, including introduction, methods,
PHOTO: © ABSCENT/SHUTTERSTOCK.COM
Increasingly, scientists must ensure the integrity of cultures.

“In a research lab, a sample mix-up can lead to false and discussion, and conclusion. So it’s worth making time to organize
unreliable results,” Phelan notes. In a diagnostics lab, however, topics within each section. In short, know what you want to write
such an error could be deadly for a patient. Many journals before you write it.
require that researchers authenticate cell lines used, and this Writing and the other techniques described here take time and
can be done using DNA fingerprinting. The American Type practice. Also, these scientific skills should be refreshed as need-
Culture Collection (ATCC), says Phelan, “actually provides a ed. Only then can scientists produce their best work.
service for human cell authentication and has an online course
called Cell Line Authentication Training.” Mike May is a publishing consultant for science and technology.
6
LIFE SCIENCE TECHNOLOGIES
DIGITAL LAB MANAGEMENT
maintain neutrality throughout,” says Shawn Douglas, LIMSwiki
DIGITAL LAB MANAGEMENT
curator, “avoiding marketing and self-promotion. The wiki is an
evolving tool, and we’re always looking for quality contributors.”
LIMSwiki
maintain provides
neutrality definitions
throughout,” for Shawn
says terms such as ELN
Douglas, (elec-
LIMSwiki
curator, “avoiding marketing and self-promotion. The wiki experi-
tronic laboratory notebook, generally used to document is an
ments) and
evolving tool,LIMS
and we’re (laboratory
alwaysinformation
looking formanagement
quality contributors.” systems,
traditionally used for definitions
LIMSwiki provides tracking standardized
for terms such processes
as ELN such (elec-as
production). But the distinction between
tronic laboratory notebook, generally used to document experi- informatics products
is blurring,
ments) and LIMSsays Markus
(laboratory Dathe, good manufacturing
information managementpractice systems,
and computer
traditionally usedsystem validation
for tracking coordinator
standardized at Roche,
processes such because
as
“convergence is happening.” ELNs,
production). But the distinction between informatics products LIMS, and equipment soft-
ware are expanding functions, interconnecting,
is blurring, says Markus Dathe, good manufacturing practice and overlapping.
Informatics
and computerpackages increasingly
system validation aim to cover
coordinator the entire
at Roche, life-
because
cycle of an R&D project including
“convergence is happening.” ELNs, LIMS, and equipment soft- reagent inventories, regulatory
forms,
ware areand work requests
expanding functions, in addition to experimental
interconnecting, details.
and overlapping.
Most researchers
Informatics packages start small, though,
increasingly aim to with a homegrown
cover the entire life- ELN
with of
cycle protocols
an R&D in text documents
project including reagent and electronic
inventories, data regulatory
files.
forms,“Everyone
and worksees the value
requests of ELNs,
in addition to from scientistsdetails.
experimental to principal
investigators to lab managers,” says
Most researchers start small, though, with a homegrown ELN Erik Alsmyr, senior director
The Paperless Lab of software development for the Accelrys

with protocols in text documents and electronic data files.
Contur’s
“Everyone iLabber)
sees for the small-to-medium-sized
value of ELNs, from scientists
Notebook
research
(previously
to groups.
principal
Alsmyr says most labs start with all-purpose
investigators to lab managers,” says Erik Alsmyr, senior director organizing and
The Paperless Lab

Some scientists keep experimental records on sticky notes.
Some groups maintain ordering information in the head of a
single technician. But for researchers looking for more sta-
ofsharing
software
Contur’s
(IP)
software
ize theyiLabber)
development
need more
protection.
such asfor
for storage
Electronic
Evernote
the Accelrys or SharePoint,
Notebookthen
capacity or intellectual
small-to-medium-sized
systems provide
research
24/7
real-
(previously
property
global
groups.
access
Alsmyr says most labs start with all-purpose organizing and
ble, searchable, and sharable records,
Some scientists keep experimental records on sticky notes. digital options such as sharing software such as Evernote or SharePoint, then real-are
to your records, says Alsmyr, and most commercial ELNs
electronic laboratory notebooks (ELNs)
Some groups maintain ordering information in the head of a and laboratory infor- compliant
ize they need with
more regulatory
storagerequirements for electronic
capacity or intellectual records,
property
mation management systems (LIMS) are
single technician. But for researchers looking for more sta- readily available. for example Part 11 of the Code of
(IP) protection. Electronic systems provide 24/7 global accessFederal Regulations Title 21,
Scientists can start with a simple online notebook
ble, searchable, and sharable records, digital options such as or choose which covers the U.S. Food and
to your records, says Alsmyr, and most commercial ELNs areDrug Administration, and Euro-
a complete lab management package to
electronic laboratory notebooks (ELNs) and laboratory infor- track the entire life- pean Union Annex 11 for the European
compliant with regulatory requirements for electronic records, market.
cycle of
mation their projects.
management By Chris
systems (LIMS)Tachibana
are readily available. Researchers
for example Part 11 areofstilltheslowCode adopters,
of Federal though, particularly
Regulations Title at
21,
A
Scientists can start with a simple online notebook or choose universities. That’s why LabArchives
which covers the U.S. Food and Drug Administration, and offers a free ELN in Euro-
ad-
a complete paperlab notebook
management seems like it should
package to track last
theforever.
entire After
life- dition
pean to a subscription-based
Union Annex 11 for the European version market.
with more storage and
all, Gutenberg Bibles
cycle of their projects. By Chris Tachibana have survived since the 1400s. features. “Our research says that in
Researchers are still slow adopters, though, particularly academia, about 95%atof
Still, paper is not perfect. Consider these true stories: universities. That’s why LabArchives offers a freeBeutler,
scientists still use a paper notebook,” says Earl ELN in LabAr-
A
ad-
At an Australian university, 30 years of notebooks
paper notebook seems like it should last forever. After became a chives’ chief executive officer. Beutler,
dition to a subscription-based version with more storage whose entire familyandare
pile of loose pages after
all, Gutenberg the have
Bibles bindings crumbled
survived sinceduring relo-
the 1400s. scientists
features. (including
“Our research a Nobel
says that Prize in winner),
academia, thinks
about it’s95%
time of for
cation. Still,
In the United
paper States,
is not a postdoc
perfect. Consider spent
these days
truecombing
stories: labs to go
scientists digital.
still use a“I’ve paper worked
notebook,”around smart,
says Earltechnologically
Beutler, LabAr-
Atthrough three-ring
an Australian binders30
university, foryears
experimental
of notebooks details requested
became a proficient
chives’ chief scientists
executive my entireBeutler,
officer. life,” hewhose
says, “and entireI’m amazed
family are
by reviewers. In a positive example of going
pile of loose pages after the bindings crumbled during relo- paperless, a Swiss scientists (including a Nobel Prize winner), thinks it’s timeprinting
that their state-of-the-art is still taking a photo of a gel, for
contract
cation. manufacturing
In the United States, organization
a postdocwowed spent daysclients with real-
combing it out,
labs anddigital.
to go gluing“I’ve it into a paper
worked notebook.”
around smart, technologically
time, online chromatography runs of their
through three-ring binders for experimental details requested samples. Electronic Realizing that adhesives
proficient scientists my entire life,” he says, disintegrate and “andnotesI’m on laptops
amazed
laboratory tools have definite advantages,
by reviewers. In a positive example of going paperless, a Swissbut scientists have don’t have the strongest IP protection, universities
that their state-of-the-art is still taking a photo of a gel, printing are buying
been reluctant adopters. The major barriers
contract manufacturing organization wowed clients with real- for going digital are informatics site licenses that
it out, and gluing it into a paper notebook.” cover entire departments, says
cost,online
time, the activation energy required
chromatography to change
runs of their samples. work habits, and
Electronic Beutler.
RealizingThisthatremoves
adhesives the cost barrier for
disintegrate and scientists
notes onand ensures
laptops
the daunting
laboratory tools number of options.
have definite advantages, but scientists have proper
don’t have archiving
the strongestof potentially patentable
IP protection, results. LabArchives
universities are buying
PHOTO: PAVEL IGNATOV/SHUTTERSTOCK.COM
been reluctant adopters. The major barriers for going digital are also targets
informatics anlicenses
site audience thatthat doesn’t
cover entirehave paper nostalgia:
departments, says
Where to Start
cost, the activation energy required to change work habits, and students. “Many of our users
Beutler. This removes the cost barrier for scientistsare academic researchers who
and ensures
LIMSwiki is an excellent
the daunting number of options. starting point for laboratory infor- teach, so we created our classroom
proper archiving of potentially patentable results. LabArchives ELN at their request,” says
matics newbies. The online resource is a community service also targets an audience that doesn’t have paper nostalgia: and
Beutler. “It lets instructors provide background information
PHOTO: PAVEL IGNATOV/SHUTTERSTOCK.COM
from the
Where to StartLaboratory Informatics Institute, a trade organization give and “Many
students. grade assignments
of our users are electronically. The largest who
academic researchers class it’s
founded
LIMSwiki in 2006
is an by LabLynx,
excellent a vendor
starting pointofforbrowser-based
laboratory infor- re- been so
teach, used weincreated
was more our than 2,000 ELN
classroom students.”
at their request,” says
searchnewbies.
matics management software.
The online LabLynx
resource emphasizesservice
is a community transpar- Tammy
Beutler. Morrish
“It lets is an academic
instructors researcher information
provide background who went digital and
ency, for example in pricing, and LIMSwiki
from the Laboratory Informatics Institute, a trade organizationprovides prices when from day one, setting up her laboratory
give and grade assignments electronically. The largest class with Labguru, a web-it’s
possibleinin2006
founded its up-to-date
by LabLynx, vendor descriptions.
a vendor “We’ve tried
of browser-based re- to based
been usedresearch
in wasmanagement
more than 2,000 system. As a postdoc, Morrish
students.”
search management software. LabLynx emphasizes transpar- Tammy Morrish is an academic researcher who went digital
ency, for example in pricing, and LIMSwiki provides prices when from day one, setting up her laboratory with Labguru, a web-
possible in its up-to-date vendor descriptions. “We’ve Originallytried to
published based
25 research
July 2014 management system. As a postdoc, Morrish
in SCIENCE
468 sciencemag.org/products SCIENCE

7
Y O U R PR
DIGITAL A C TMANAGEMENT
LAB I C A L G U IDE TO BA SIC LA BORATORY TECHNIQUES
kept a homemade database of ministration encourage electronic documentation, Dathe says,

project resources but wanted “The pharmaceutical industry is generally conservative, and it’s
an advanced, sharable system often easier and cheaper to stay with a paper system that is
when she started as an assis- known to be accepted by regulatory agencies.”
tant professor at the University At LEO Pharma in Denmark, head of discovery informatics
of Toledo Biochemistry and and data management Ulrik Nicolai de Lichtenberg developed
Cancer Biology Department. a model for committing to a commercial informatics system.
That’s a great time to set up Start with in-depth stakeholder analyses, he says. Define your
a new system, she says, be- needs and goals and “how much pain you can put up with,”
cause you know all the mice, meaning the money, time, and effort available for implementing
cell lines, and plasmids you a new system. Realize that your ELN or LIMS is just a part of an
have available for projects. information ecosystem. LEO Pharma chose the Accelrys ELN
In this [nearly sci-fi] vision Morrish praises Labguru’s for its Medicinal Chemistry R&D Department, but the ELN is
of the future laboratory, customer service and says just one element in a comprehensive infrastructure designed by
the system is a huge time- de Lichtenberg’s team. Their system will capture, validate, and
scientists simply saver. It streamlines ordering permanently store records so they are accessible, searchable,
do their work while an by putting product numbers, and legally defensible in case of IP disputes. It’s a complex
vendors, and current orders in project and de Lichtenberg recommends seeking advice from
automated tracking one place, she says. Labguru independent consultants who understand the ever-changing
system simultaneously holds her laboratory’s mouse informatics market.
records with full genotypes,
keeps records. and plasmid information in- Looking to the Cloud And Beyond
cluding maps. Morrish says Michael Elliott, chief executive officer of Atrium Research
the system is particularly helpful for locating items. “Think how & Consulting, advised de Lichtenberg and endorses his ap-
much time we waste looking for things,” she says. “Now when proach. “Don’t get enamored with a demo,” he says. “Look
I need something, even if other people aren’t around to ask, I under the hood and check out the capabilities of an informatics
can type it into the database and find it. Of course,” she adds, system.” Clients dream of a single system that streamlines pro-
“people have to put things back where they found them.” Her cess management and securely and permanently stores data
lab has a technician who checks inventories against the data- while rapidly retrieving needed information. An ideal system
base weekly. would even find “dark data”—previous work that could answer
At a higher level, the system facilitates group interactions, current research questions but is buried in disorganized files.
for example by making data sharing easy. It also teaches best Clients want scalability, a user-friendly interface, and outstand-
practices. “It helps students learn that with any database,” says ing global support. However, products vary in these capabilities,
Morrish, “you have to enter information correctly and consis- says Elliott. “Don’t choose based on a presentation or brand
tently or you won’t be able to find it.” name. Think carefully about your needs now and in the future.”
If expandability and ease of use are priorities, a cloud-
Going Digital But Maintaining Control based system, for example from Core Informatics, might
Science-based businesses also appreciate the efficiency of be the answer. In principle, the cloud can house unlimited
digital research management, but long-term stability is a high amounts of data and has a familiar interface since accessed is
priority, too. “The challenge is assuring the accessibility and through a web browser. Brower-based systems don’t require
usability of data 20 years from now,” says Dathe. Choosing specialized software, so they’re easy to upgrade. Informatics
a major informatics supplier such as IDBS, PerkinElmer, or vendors are also creating user-friendly modular packages.
Accelrys might give some assurance of permanence, but Similar to choosing mobile phone apps, users select only the
the market is so dynamic that any vendor will likely undergo components they need.
changes. In the past decades, Thermo Fisher Scientific Also on the horizon is greater mobility and compatibility.
acquired InnaPhase; PerkinElmer purchased Labtronics, Researchers are taking smartphones and tablets into the
CambridgeSoft and ArtusLabs; Accelrys, which has its laboratory so informatics developers are making products
PHOTO: BRUCE ROLFF/SHUTTERSTOCK.COM
own lengthy merger and acquisition history, was recently compatible with handheld devices. Increasingly, data needs
acquired by the French software company Dassault. Still, after to be compiled across different instruments and informatics
consolidating, companies strive to retain users. “We still carry platforms, so Pedersen says he is personally pushing
software developed in the 1990s and we’ve always shown for increased standardization to facilitate information
customers a path forward,” says Leif Pedersen, senior vice sharing. Ever the realist, though, Elliott says progress
president at Accelrys. in standardization is slow because even within a single
Nonetheless, industries are not uniformly adopting laboratory department, users might employ different terminology and
informatics. Although agencies such as the Food and Drug Ad- definitions. The force that could drive both standardization
Originally published 25 July 2014 in SCIENCE
8
SCIENCE sciencemag.org/products 469
of scientific informatics Featured Participants Santa Cruz has traveled

and better data integra- Accelrys University of Toledo the entire DIY lab notebook
tion, says Elliott, “is www.accelrys.com www.utoledo.edu journey. Boettiger started
the move toward more Atrium Research & Consulting keeping publicly accessible
collaborative work.” www.atriumresearch.com lab records in the Open-
Additional Resources
To the wish list of LabArchives WetWare platform. “It’s a
Core Informatics
informatics improvements, www.labarchives.com bit radical,” says Boettiger.
www.corelims.com
Dathe adds features that Labguru “Anyone can go in and
Evernote
give data context: when www.labguru.com edit other peoples’ notes,
www.evernote.com
and where they were LabKey Software although that rarely hap-
GitHub
collected and for what www.labkey.com pens.” After OpenWetWare,
www.github.com
project. Data should be LabLynx Boettiger moved to plat-
IDBS
linked to relevant molecular www.lablynx.com forms that give him increas-
www.idbs.com
and clinical information and LIMSwiki ing control over his research
Jekyll
the entire data-generating www.limswiki.org records, starting with Word-
www.jekyllrb.com
process, including the type LEO Pharma Press, which is usually used
OpenWetWare
and status of equipment www.leo-pharma.com for blogging. Boettiger now
www.openwetware.org
used. “Without context,” Roche uses the online software
PerkinElmer
says Dathe, “the mountain www.roche.com development site GitHub as
www.perkinelmer.com
of data we can collect is University of California, his note-book and Jekyll
Thermo Fisher Scientific
meaningless.” Santa Cruz website-generating soft-
www.thermofisher.com
www.ucsc.edu
ware to publish his note-
Being Open-Minded book online.
Scaling the data mountain is Britt Piehler’s job. Piehler is A blog-type ELN creates a robust, cached history of your
president of LabKey Software, which develops tools for data research, says Boettiger. It discourages fraud because any
management and integration. The trend toward globalization changes leave records. You choose what is public, private,
and multisite collaboration, he says, means project managers and password protected. And think of the advantages when
must coordinate data collected at far-flung sites under diverse talking to people at conferences or answering reviewer
conditions with a variety of instruments. “That’s where requests, he says. You can just pull up records on a handheld
LabKey comes in,” says Piehler. “We build tools for specific device to see what you tried and when, and how it worked out.
tasks, usually data integration for multisite collaborative
projects that need to standardize heterogeneous data.” An What’s Next
unusual feature of LabKey Software is that its product is open “The trends in laboratory records,” says Boettiger, “are
source. toward more open and collaborative, more secure, and
“We grew out of the academic community,” says LabKey’s more automated.” Although Boettiger and Dathe should
Science Outreach Director Elizabeth Nelson, “so we believe have different perspectives as an ecology researcher in
it’s an advantage for the software platform to be freely Santa Cruz and a pharma development and information
available.” Open source code allows researchers to tailor their technology specialist in Basel, respectively, they share a nearly
systems, says Piehler, and building and sharing LabKey tools sci-fi vision of the future laboratory. In this vision, scientists
creates a community. simply do their work while an automated tracking system
If the code is free, what does LabKey offer? simultaneously keeps records. Barcoding will note reagents,
“Customization,” says Piehler. LabKey Software experts samples, and instruments used, providing context to the data
can create tools that directly address Dathe’s call for for subsequent analysis. The entire process will be recorded,
giving context to data, for example by adding demographic showing the provenance of every byte and definitively
information. And in August 2013, open source and open establishing IP claims. “It will give a much more extensive
access came together via LabKey to promote scientific record that can be transparent or shared if you want,” says
transparency and reproducibility. For a clinical trial of a Boettiger. A fully automated system would simplify research
vasculitis therapy published in the New England Journal of by capturing experimental details with no manual data entry.
Medicine, the LabKey open source platform was used to Then, all we’d need is a robot to return reagents to the right
create a web portal with free public access to participant-level shelves.
data, stripped of identifying information.
Researchers who are committed to transparency and are
also do-it-yourselfers have a choice of open source workflow Chris Tachibana is a science writer based in Seattle, USA, and
management tools. Carl Boettiger, an ecology and evolu- Copenhagen, Denmark.
tion postdoctoral researcher at the University of California, DOI: 10.1126/science.opms.p1400087
Originally published 25 July 2014 in SCIENCE
470 9
sciencemag.org/products SCIENCE
How to
HowAvoid
to Avoid Contamination in Pipetting
Contamination in Pipetting
#05
Application #01
Note
Practical methods #02
for avoiding
contamination in
pipetting. #03
#04
10
APPLICATION N OT E S
Introduction tips manufactured from 100% virgin polypropylene in

Preventing contamination in pipetting is paramount to a high-quality manufacturing facility do not contain
achieving reliable results. It requires identification of the leachables. It is recommended that you confirm this with
potential contamination mechanisms in order that they the tip manufacturer.
can all be addressed.
In daily laboratory work, pipette-to-sample contam-
Aerosols, suspensions of solid or liquid particles in a ination can be avoided by following these simple
gas, are formed in many laboratory activities such as guidelines:
pipetting with air-displacement pipettes, and aerosols – Select a tip with the relevant purity class for your
are the major contamination source in pipetting. They application.
may transfer into the pipette body when non-filter – Use (sterilized) filter tips or positive displacement
pipette tips are used and consequently contaminate tips. Alternatively, you may be able to use tip-cone
subsequent samples. A slow and careful pipetting filters with some manufacturers’ pipettes. The filters
rhythm helps minimize aerosol formation. prevent aerosols from reaching the pipette body and
potentially contaminating subsequent samples.
This paper addresses the three contamination types – Always change the pipette tip after each sample.
that originate from pipetting: pipette-to-sample – Regularly autoclave, or disinfect, the pipette or
contamination, sample-to-pipette contamination, and the components that may come into contact with
sample-to-sample contamination. the sample.
Pipette-to-Sample Contamination Sample-to-Pipette Contamination

This type of contamination occurs when a contaminated This type of contamination takes place when the
pipette or pipette tip contaminates the sample. pipetted liquid or aerosol particles from it enter the
pipette body. To minimize the risk of sample-to-
Pipette tips are available in multiple purity grades from pipette contamination, the following precautions are
most manufacturers. Purity grades can be divided into recommended:
three categories: – A lways release the pipette’s push button slowly to
– no purity certification prevent aerosol formation and uncontrolled liquid
– certified free of contaminants like DNase, RNase, splashing within the pipette tip.
and endotoxins – Hold the pipette in a vertical position during pipetting
– sterilized to be free of microbial life and store the pipette in an upright position. This
prevents liquids from running into the pipette body.
Contaminants such as DNase, RNase, and endotoxins
are difficult to remove by any sterilization method, so Sample-to-Sample Contamination
it is very important to prevent contamination during Sample-to-sample contamination (or carryover
manufacturing. The absence of these contaminants is contamination) occurs when aerosol or liquid residue
separately tested, usually by a third-party laboratory. from one sample is carried over to the next sample.
Sterilization after manufacturing ensures that the tips This may take place, for example, when the same
do not contain any microbial life (bacteria, viruses, etc.) pipette tips are used multiple times. To avoid carryover
when delivered to customers. contamination:
– Use filter tips or positive displacement tips to prevent
Pipette tips can also be a potential source of leachables aerosol transfer from the sample into the pipette
– trace amounts of chemicals originating from materials body, and again to the next sample. Alternatively,
or process equipment that can contaminate the filters can be used on pipette tip cones.
samples. Examples of potential leachables are – Always change the pipette tip after each sample.
heavy metals, UV stabilizers, antioxidants, pigments, – If you suspect pipette contamination, autoclave or
release agents, biocides, and surfactants. High-quality disinfect the pipette according to the manufacturer’s
instructions.
11
Definitions:
Decontamination Any activity that reduces microbial load to Antisepsis The application
Definitions: prevent contamination. Includes methods chemical to liv
for sterilization, disinfection, and antisepsis. microorganism
Decontamination that reduces microbial The
Any activity Sterilization loaddestruction
to of all microbialThe
Antisepsis life,application ofDNase
an antimicrobial Powerful enzym
including bacterial endospores.chemical
prevent contamination. Includes methods Can be to living tissue to destroy DNA by hydrol
for sterilization, disinfection, and antisepsis.
accomplished, e.g., using steam,microorganisms.
heating, Even trace amo
Sterilization The destruction of all microbial life,chemicals, or radiation.
DNase Powerful enzymes (nucleases) that degrade low or no yield
including bacterial endospores. Can Autoclaving
be DNA by hydrolyzing it into short fragments. PCR, or to degr
Autoclaving (moist heat) is an efficient
accomplished, e.g., using steam, heating, Even trace amounts of DNases can lead purification. Co
to
sterilization method for laboratories. A hot,
chemicals, or radiation. low or no yields in DNA techniques such contact,
as saliva,
pressurized, and saturated steam is applied
PCR, or to degradation during DNA
Autoclaving to destroy microorganisms and
Autoclaving (moist heat) is an efficient RNase Powerful enzym
purification. Contamination sources: human
sterilization method for laboratories.decontaminate,
A hot, e.g., laboratorycontact,
plastic and the degradatio
saliva, bacteria.
pressurized, and saturated steam is applied
glassware. Exposure time and temperature fragments. Ver
to destroy microorganisms and are critical.RNase
Moreover, the steam Powerful
needs toenzymes (nucleases) that catalyzedifficult to rem
decontaminate, e.g., laboratory plastic and
penetrate the to
through the entire load degradation
be of RNA into short oils from skin,
glassware. Exposure time and temperature
efficient. fragments. Very stable enzymes that are bacteria.
are critical. Moreover, the steam needs to difficult to remove. Contamination sources:
Disinfection
penetrate through the entire load toThe
be elimination of virtually all pathogenic Endotoxins
oils from skin, as well as hair, tears, Lipopolysaccha
efficient. microorganisms (excluding bacterial
bacteria. are part of the
Disinfection endospores)Endotoxins
The elimination of virtually all pathogenic and reduction of the microbial
Lipopolysaccharides, large molecules thatGram-negative
microorganisms (excluding bacterial contamination to an acceptable level.
are part of the outer membrane of Salmonella, Sh
A practical
endospores) and reduction of the microbial method for surface Gram-negative bacteria such as E. coli, Haemophilus. C
contamination to an acceptable level.decontamination. The disinfectant (e.g., , Shigella, Pseudomonas, and impair the grow
Salmonella
A practical method for surface halogens), . Cause fever in humans andreleased into th
alcohols, phenolic compounds, Haemophilus
concentration, and exposure time
decontamination. The disinfectant (e.g., should
impair be
the growth of cell cultures. Are bacteria die an
alcohols, phenolic compounds, halogens), released into the environment when
selected according to the assumed Contamination
concentration, and exposure time should be
contamination type. bacteria die and the cell wall is destroyed.present wherev
selected according to the assumed Contamination sources: Endotoxins are i.e., air, water,
contamination type. present wherever bacteria are able to grow, non-sterile env
i.e., air, water, soil, skin, raw materials, any
non-sterile environment.
Specifications subject to change without notice. Printed and copyrighted by Sartorius Biohit Liquid Handling Oy.
Publication No.: SUL1002-e 150601 ∙ Order No.: 85037-550-68∙ Ver. 06| 2015
Sartorius Lab Instruments GmbH

Sartorius Lab Instruments GmbH & Co. KG
Weender Landstrasse 94-108 Weender Landstrasse 94-108
37075 Goettingen, Germany 37075 Goettingen, Germany
Phone +49.551.3080 Phone +49.551.3080
Fax +49.551.308.3289 Fax +49.551.308.3289
Sartorius Biohit Liquid Handling Oy Sartorius Biohit Liquid Handling

Laippatie 1 Laippatie 1
00880 Helsinki, Finland 00880 Helsinki, Finland
Phone +358.9.755.951 Phone +358.9.755.951
Fax +358.9.755.95.220 Fax +358.9.755.95.220
www.sartorius.com www.sartorius.com
12
How Concentration toinaPipetting

to Avoid Contamination Defined Final Volume
with Vivaspin® Turbo 15, Vivaspin® Turbo 4
#05
and Vivaspin® 500
Application #01
Rik McRae1, Hannes Landmann2,* Note
1. Sartorius Stedim Lab Ltd, Sperryway, Stonehouse, Gloucestershire, GL10 methods
Practical 3UT, UK #02
for20,
2. Sartorius Lab Instruments GmbH & Co. KG, Otto-Brenner-Straße avoiding
37079 Göttingen, Germany
*
Correspondence contamination in
E-Mail: hannes.landmann@sartorius.com pipetting. #03
#04
Abstract
This short Application Note describes how you can use Vivaspin® Turbo 15, Vivaspin®
Turbo 4 and Vivaspin® 500 concentrators to concentrate to defined final volumes. By
adding a particular volume to the filtrate vessel prior to the concentration, the final
volume of the concentrate can be adjusted accurately.
13
Introduction
It is sometimes desirable to be able to preselect a defined final volume for a concentration step, especially when
parallel concentrations are being performed. Vivaspin ® centrifugal concentrators have a built-in deadstop feature,
which prevents overconcentration to dryness. Due to the fast concentration rates possible with the
patented vertical membrane design in the Vivaspin ®, the drying out of the sample would otherwise be a possibility.
Introduction – Sartorius Precision Lab Balance
This note describes a method for achieving reproducible defined® D-16C
– Centrisart final volumes using
Centrifuge with Vivaspin
swing out rotor Turbo 15, Vivaspin ®
® for 50 ml
It is sometimes desirable to ®be able to preselect a defined final and 15 ml falcon tubes
Turbo 4 and Vivaspin 500 centrifugal concentrators. The method does not rely on the deadstop pocket but is
volume for a concentration step, especially when parallel concen- – Centrisart A-14C Centrifuge with fixed angle rotor
increasing the performed.
trations are being retained Vivaspin
volume ® by adding liquid to the filtrate vessel prior to centrifugation.
centrifugal concentrators for 24 1.5 | 2.2 ml tubes
have a built-in deadstop feature, which prevents overconcentration
Equipment
to dryness. Due to the fast concentration rates possible with the Reagents
Reagents
–patented
Vivaspinvertical
® membrane
Turbo 15 10 design the Vivaspin®, the drying
kDainMWCO 1 mg/ml Bovine Serum Albumin labelled with Bromophenol blue
1 mg/mL Bovine Serum Albumin labeled with
out of the sample would otherwise be a possibility.
– Vivaspin Turbo 4 10 kDa MWCO
®
Bromophenol blue
–This
Vivaspin 500a 10
note describes
®
kDafor
method MWCO
achieving reproducible defined Methods
final volumes using Vivaspin® Turbo 15, Vivaspin® Turbo 4 and
– Tacta® 5 mL mechanical pipette and Optifit pipette tips Methods
Vivaspin 500 centrifugal concentrators. The method does not 1. Add defined amount of water to the filtrate tube (see table
–rely
Tacta
on the1000 μL mechanical
deadstop pipette the
pocket but is increasing andretained
Optifitvolume
pipette tips 1. Add defined amount of water to the filtrate tube (see
below).
by adding liquid to the filtrate vessel prior to centrifugation.
– Tacta 200 μL mechanical pipette and Optifit pipette tips table
2. Put the below).
concentrator insert into the filtrate tube
and 2. Put thesolution.
add sample concentrator insert into the filtrate tube and
-Equipment
arium pro ultrapure water system
®
3. Close the concentrator cap (for Vivaspin® Turbo 15 or
® add sample screw
solution.
– Vivaspin
– Sartorius Turbo 15 10kDa MWCO
Precision Lab Balance Vivaspin Turbo 4) or close the cap (Vivaspin® 500) and place
®
– Vivaspin® Turbo 4 10kDa MWCO in the3.centrifuge.

Close the concentrator screw cap (for Vivaspin ® Turbo
–– Vivaspin
Centrisart ®
D-16C Centrifuge with swing-out rotor for 15 or theVivaspin
sample. ® Turbo 4) or close the cap (Vivaspin ®
®
500 10kDa MWCO 4. Concentrate
– Tacta
50 mL5 mland 15 mL pipette
mechanical falconandtubes
Optifit pipette tips 5. Remove the concentrator
500) and placeinsert andcentrifuge.
in the recover the concentrate
– Tacta 1000 µl mechanical pipette and Optifit pipette tips with a pipette.
–– Tacta
Centrisart A-14C Centrifuge with fixed-angle
200 µl mechanical pipette and Optifit pipette tips
rotor 4. Concentrate the sample.
- arium
for 24
®
1.5ultrapure
pro | 2.2 mL tubes
water system 5. Remove the concentrator insert and recover the
concentrate with a pipette.
Results
Results
Results for Vivaspin® Turbo 15
Volume of water added Volume of sample solution added Spin conditions Final concentrate volume
to the filtrate tube to the concentrator insert (average of 8 devices)
11.5 mL 15 mL 20 min @ 4,000 x g 1.50 ± 0.02 mL
9.5 mL 15 mL 20 min @ 4,000 x g 0.96 ± 0.01 mL
7.5 mL 15 mL 20 min @ 4,000 x g 0.53 ± 0.02 mL
Results for Vivaspin® Turbo 4

2.0 mL 4 mL 20 min @ 4,000 x g 0.34 ± 0.03 mL
1.5 mL 4 mL 20 min @ 4,000 x g 0.15 ± 0.02 mL
1.2 mL 4 mL 20 min @ 4,000 x g 80 ± 10 µL
Results for Vivaspin® 500 in 40° fixed-angle rotor

500 µL 500 µL 15 min @ 15,000 x g 103 µL ± 13 µL
Sartorius Lab Instruments
380 µL 500 µL 15 min @ 15,000 x g 51 µL
GmbH± 11 µLKG
& Co.
Otto-Brenner-Strasse 20
250 µL 500 µL 15 min @ 15,000 x g 30 µL ± 5 µL
37079 Goettingen, Germany
200 µL 500 µL 15 min @ 15,000 x g 23 µL ± +49.551.308.0
Phone 7 µL
email: LF-info@sartorius.com
www.sartorius.com
Conclusion
Conclusion Sartorius Lab Instruments
Reproducible defined final concentrate volumes can beGmbH
quickly
USA Toll-free +1.800.635.2906
and easily achieved
& Co. KG with Vivaspin ® Turbo 15,
UK +44.1372.737159
Otto-Brenner-Strasse 20 France +33.1.70.62.50.00
Vivaspin ®
ReproducibleTurbo
defined4, and
final Vivaspinvolumes
concentrate ®
500.can be quickly 37079 Goettingen, Germany Italy +39.0362.5557.11
® ®
and easily achieved with Vivaspin Turbo 15, Vivaspin Turbo 4, Phone +49.551.308.0 Spain +34.913.586.095
®
14 Vivaspin 500.
and email: LF-info@sartorius.com Russian Federation +7.812.327.53.27
www.sartorius.com Japan +81.3.3740.5408
USA Toll-free +1.800.635.2906 Specifications subject to change without notice.

Cell Culture Expansion in Fully Closed

How Erlenmeyer Shake
to Avoid Contamination Flasks Outside the
in Pipetting
Biosafety Cabinet with MYCAP™ CCX
#05
Charles Meadows1* (Senior Product Manager), Gregory Bremer1 (Upstream Engineer,
Research & Development), Dr. Michael Zumbrum1 Application
(Director of Research & Development,
New Oxford)
#01
Note
1. Sartorius Stedim Biotech GmbH, August-Spindler-Strasse 11, 37079 Goettingen
*
Correspondence
E-Mail: charles.meadows@sartorius-stedim.com
for avoiding
contamination in
pipetting. #03
Abstract #04
Expansion of suspension cell culture from cell banks to seed bioreactor is performed
through passages of successively larger Erlenmeyer shake flasks. The traditional cap of an
Erlenmeyer flask is unscrewed for each fluid transfer. Risk of contamination is mitigated by
performing these fluid transfers in a biosafety cabinet (BSC) or laminar flow hood.
Work in a BSC is not preferred because of high maintenance and operating costs, intensive
cleaning and decontamination procedures, and the risk and inconvenience of performing
operations in the BSC.
Despite working in a BSC, expansion processes include passages with backup flasks to be
used in the case of contamination. Backup flasks are a material waste and multiply labor-
intensive BSC work.
Sartorius’ MYCAP™ CCX includes integral tubing and a specially designed gas exchange
cartridge. Integral tubing supports good aseptic technique to prevent contamination.
All fluid transfers are done outside the BSC. The gas exchange cartridge has a high filter
surface area to support passive gas exchange and vibrant cell growth in the incubator.
15
Introduction
Bottle closures with integral tubing are widely
used in bioprocessing because they reduce or
eliminate the risk of contamination from poor
aseptic technique. Good aseptic technique
is especially important upstream where
preserving axenic, or monoculture conditions is
compulsory.
Tubing materials of the cap closure are

commonly thermoplastic elastomer (e.g.,
C-Flex®), which can be aseptically welded to
another tube of the same size. Alternatively,
aseptic connecting devices (Sartorius Opta®,
Colder Aseptiquik®, Pall Kleenpak®, etc.) may
be installed at the tube ends. In either case,
the bottle can be aseptically connected to receive or Traditional flasks have a filter membrane embedded in
dispatch fluids in non-classified spaces without the risk the cap. The arrangement allows for unrestricted air flow
of introducing a contaminant. across the entire filter surface. However, the filter mem-
brane occupies the entire cap surface, leaving no room for
Key customers approached Sartorius to improve aseptic integral tubing for aseptic fluid transfers.
technique in cell expansion with the following objectives:
– Eliminate contamination risk Sartorius’ Solution
– Enable fluid transfers in non-classified spaces The manufacturing process of the patented MYCAP™
– Reduce waste from requisite backup passages bottle closure is an enabling technology. Components,
– Achieve comparable culture growth rates & usually tube assemblies, are inserted into pre-formed
doubling times to incumbent expansion methods holes. Silicone elastomer is dispensed into the cap to
hermetically seal the installed components in
Cellular respiration consumes O2 and produces CO2 as a place and to create the highly compliant, plasticizer-free
byproduct. Cell cultures starved of O2 will not propagate. bottle closure.
Cultures with an overabundance of CO2 become acidic
and impair cell viability. The exchange of O2 and CO2 Inserted components are not restricted to tube
across the filter membrane is critical to cell growth. assemblies. Sartorius developed the MYCAP™ CCX
gas exchange cartridge with the following objectives:
It is customary to attach a disc filter to integrated tubing – Provide adequately large filter surface area
in a cap for air venting during fluid transfer. Early testing – Allow unrestricted air flow across filter membrane
of closures on Erlenmeyer flasks with a 50 mm disc – Reduce the filter footprint allowing space for
filter showed slowed or fully halted cell growth. Despite integral tubing
the large filter surface area (3 in2 | 20 cm2) of the 50 mm
disc filter, the gas exchange across the membrane The MYCAP™ CCX gas exchange cartridge is a three-
was inadequate for cell growth. Air flow through the dimensional, stadium-shaped part. Two generous 0.2 μm,
membrane is restricted at the 8 in. (3.2 mm) orifice of hydrophobic filter membranes extend into the neck of the
the hose barb on the filter housing. Cell growth resumed flask. The orientation of the filter membranes protects and
once the culture was moved to a flask with the traditional places them in position for unrestricted gas exchange
flask cap. between the culture and the incubator environment. The
stadium shape conserves space on the cap for integral
tubing for media addition, inoculum addition, sampling
and transfer.
16
e filter membrane.
The pH change of the solution on flasks with the MYCAP® CCX cap
The pH
and change
flasks with of
thethe solution vented
traditional on flasks
capwith
arethe MYCAP
virtually CCX cap
identical.
®
p.
p. and flasks with the traditional vented cap are virtually identical.
ntire
ntire
re 1L Flask
Gas Exchange Study
re Sartorius performed an evaluation to
id
14,00
7,50 1L Flask
uid 7,40
7,50
12,00
14,00 compare gas exchange across the MYCAP™
7,30
10,00
12,00
CCX cap closure and the traditional vented
CO2 Concentration
7,40
pH of Solution
cap closure.
8,00
10,00
7,20
7,30 pH MYCAP™® CCX 1L
CO2 Concentration
pH of Solution
6,00
8,00 pH Traditional 1L
7,10
7,20
pH MYCAP
CO
®
CCX 1L
Concentration
2
7,00 4,00
1L and 3L flasks were modified to accept a
7,10
ure 6,90
7,00
2,00
4,00
CO2 Concentration
pH probe in the side wall so that the probe

ure
es, 6,80
6,90
2 3 4 5 6 7 8 9 10
0,00
2,00
Time (hours) would be in direct contact with solution to

es,
sed 6,80
2 3 4 5 6 7 8 9 10
0,00
Time (hours)
read pH changes. Flasks were filled with
sed
phosphate buffered saline (PBS) solution
le 3L Flask
containing sodium bicarbonate buffer. Test
le
14,00
7,50 3L Flask
7,40
7,50
12,00
14,00 articles were placed in an incubator and CO2
7,30
10,00
12,00
concentrations changed every two hours.
CO2 Concentration
7,40
pH of Solution
8,00
10,00
7,20
7,30 pH MYCAP® CCX 3L
CO2 Concentration
pH of Solution
Change in pH of the solution indicates gas

6,00
7,10
7,20 ™
pH MYCAP
CO
®
CCX 3L
Concentration
2
4,00
6,00
exchange across the filter membrane.
7,00
CO2 Concentration
6,90 2,00
4,00
7,00
6,80
6,90 0,00
2,00
The pH change of the solution on flasks with

2 3 4 5 6 7 8 9 10
g
Time (hours)
Cell Growth Study Conclusion
6,80 0,00
2 3 4 5 6 7 8 9 10
the MYCAP™ CCX cap and flasks with the
g
Time (hours)
Sartorius performed a study comparing cell growth in flasks with traditional vented cap are virtually identical.
the MYCAP
Cell
®
CCX cap to flasks with the traditional vented cap.
Growth Study Expansion of suspension cell cultures using Erlenmeyer
Conclusion
Sartorius performed a study comparing cell growth in flasks with a BSC is a labor-intensive process. The flask’s cap is remo
the MYCAP CCX cap to flasks with the traditional vented cap.
® at eachofpassage
Expansion suspension andcell
fluid transfers
cultures including
using Erlenmeyermedia addi
flasks
Thaw Cell Growth Study
500 mL inoculation
a BSC and sampling
is a labor-intensive areThe
process. done, typically
flask’s cap is by hand-pi
removed
Sartorius performed a study comparing cell
These
at each operations
passage are performed
and fluid under
transfers including laminar
media flow in th
addition,
Thaw growth in flasks with the MYCAP™ CCX cap to
500 mL inoculation and sampling are done, typically by
BSC to prevent contamination. Yet, contamination risk p hand-pipetting
flasks with the traditional vented cap.
Passage 1 These
sooperations
back-up flasks are performed under laminar
are maintained for use in flow
theinevent
the of
500 mL 500 mL
BSCcontamination.
to prevent contamination.
In a GMP seed Yet, contamination
expansion process, risk persists
a typi
Passage 1 CHO DG44
so back-up flasks cells
are were directly
maintained for thawed
use in into
the a of a
event
passage requires three to four operators; the hood techn
500 mL 500 mL traditional In
contamination. flask and seed
aand
GMP thenexpansion
split into two trains:
process, a typical
Passage 2 hood assistant data/batch record recorder(s).
1000 mL 500 mL 500 mL 1000 mL
Train
passage 1 utilized
requires MYCAP
three to
™
four CCX flasks;
operators; and
the hood technician,
Passage 2 hood Train 2
assistant utilized
andhas traditional
data/batch flasks. Cells
record allowing were
recorder(s).
MYCAP ®
CCX integral tubing for aseptic fluid
1000 mL 500 mL 500 mL 1000 mL sub-cultured consecutively for three additional
in the open space of a workbench. The number of opera
Passage 3 MYCAP passages
®
CCX hasinintegral
varioustubing
size flasks
allowingup to
for3000 mL.
aseptic fluid trans
3000 mL 500 mL 500 mL 3000 mL in half, contamination risk is eliminated and wasteful ba
Passage 3
in the open space of a workbench. The number of operators is
flasks are not necessary.
3000 mL 500 mL 500 mL 3000 mL Two-tailed
in half, T-Tests
contamination were
risk performed
is eliminated and wasteful back-up
Passage 4 flaskscomparing
are not necessary.
the doubling times between
Carefully™controlled conditions for cell growth in a shak
3000 mL 500 mL 500 mL 3000 mL MYCAP CCX and traditional flasks of
Passage 4 in an controlled
Carefully incubator are required. In particular,
growth inthe unrestric
3000 mL 500 mL 500 mL 3000 mL the same size. conditions
There wasfor nocell
statistically a shake flask
Figure
Fig. 1: Cell
1: Cell Growth
Growth Study Study
Process Process
Diagram Diagram exchange
in ansignificant of
incubator are CO and
required. O between
In the
particular, cell
the culture and th
unrestricted
difference in growth rates between
2 2
Fig. 1: Cell Growth Study Process Diagram incubator

exchange of COenvironment
and O2with is critical
between to culture
achieving
theconfidence
cell andtargeted
the c
the two systems,
2 a 95%
density
incubator and viability.
Table 1: Process Parameters level.environment is critical to achieving targeted cell
Incubator Parameter Description | Set Point density and viability.
Incubator
TemperatureParameter Description
36.8°C | Set Point Gas exchange, as measured by a change in pH of solutio
Average culture doubling times for each flask
Temperature 36.8°C Gas response
exchange,toasa measured
change inby concentration,
COa2 change in pH ofbetween
solution in MYC
Carbon Dioxide % 7.5% size were graphed. The graph (next page)
Carbon Dioxide % 7.5%
and traditional
response to a changeflasksin COwas compared
concentration, and found
between to be
MYCAP sub
®
CC
Agitation 500 mL, 1000 mL 120 rpm illustrates the comparability2
of doubling times
and equivalent.
traditional flasks was compared and found to be substantia
Agitation 500 mL,mL
3000 1000 mL 12080rpm
rpm for MYCAP™ CCX flasks and traditional flasks.
equivalent.
3000 mL 80 rpm
Table 1: Process Parameters Successful passages in an expansion process are benchm
Table 1: Process Parameters Successful passages
cell growth ratesinandan expansion
cell culture process are benchmarked
doublings. A comparis
cell culture
growth rates and cell culture doublings.
doublings between MYCAP CCX and ® A comparison of c
traditional
CHO DG44 cells were directly thawed into a traditional flask and 17
CHO culture doublings betweenwereMYCAP
®
CCXbeand traditional flasks
thenDG44 cells two
split into weretrains.
directly thawed
Train into a MYCAP
1 utilized traditional
® flask
CCX and
flasks; ®
across 4 passages found to
across 4 passages were found to be equivalent.
equivalent.
The graph illustrates the comparability of doubling times for
YMYCAP
®
CCX
O U R PR A C T I C A flasks
L G U IDE and
TO BA SIC traditional
LA BORATORY flasks.
TECHNIQUES
st
Th
Culture Doubling Times between Traditional Flasks and MYCAP® CCX
30
th
25
M
20 to
M
Doubling Time
Culture
(hr)
15
te
10
0
500 mL Flask 1000 mL Flask 3000 mL Flask
Traditional Flask MYCAP CCX Flask

®
Conclusion Carefully controlled conditions for cell growth in a

Expansion of suspension cell cultures using Erlenmeyer shake flask in an incubator are required. In particular,
flasks in a BSC is a labor-intensive process. The flask’s the unrestricted exchange of CO2 and O2 between the
cap is removed at each passage and fluid transfers cell culture and the incubator environment is critical to
including media addition, inoculation and sampling are achieving targeted cell density and viability.
done, typically by hand-pipetting. These operations
are performed under laminar flow in the BSC to prevent Gas exchange, as measured by a change in pH of
contamination. Yet, contamination risk persists, so solution in response to a change in CO2 concentration,
backup flasks are maintained for use in the event of a was compared between MYCAP™ CCX and traditional
contamination. In a GMP seed expansion process, a flasks and found to be substantially equivalent.
typical passage requires three to four operators; the
hood technician, hood assistant and data/batch record Successful passages in an expansion process are
recorder(s). benchmarked by cell growth rates and cell culture
doublings. A comparison of cell culture doublings was
MYCAP™ CCX has integral tubing allowing for aseptic compared between MYCAP™ CCX and traditional
fluid transfers in the open space of a workbench. flasks across 4 passages were found to be equivalent.
The number of operators is cut in half, contamination
risk is eliminated and wasteful backup flasks are MYCAP™ CCX should be considered a suitable
not necessary. replacement for traditional Erlenmeyer flasks to reduce
waste, eliminate contaminations and streamline cell
expansion operations.
18
Recommendations MYCAP™ CCX Validation Template Tool:

A validation study comparing rates of growth in – Supports up to 6 Passages
MYCAP™ CCX flasks with traditional flasks should – Complete MYCAP™ CCX materials list including “Where
performed before implementing. Sartorius offers the Used Guide”
MYCAP™ CCX Validation Template Tool to streamline – Record and maintain experimental conditions; flask
experimental design,Template
MYCAP® CCX Validation data collection
Tool: and data analysis. size, culture volume, shaker speed, incubator
– Supports up to 6 Passages temperature, CO2 concentration
– Complete
The MYCAP® CCX
Tool generates materials
charts list including
to visualize ‘Whererates
growth Used and – Compare against required performance criteria; growth
Guide’
performs the Student’s T-Test to compare the datasets. rate, cell count targets, cell viability
– Record and maintain experimental conditions; flask size, culture
– Visual and Statistical Analysis including:
volume,™shaker speed, incubator temperature, CO2 concentration
MYCAP
– Compare CCX
againstValidation Template Tool
required performance makes
criteria; it quick
growth rate, – Doubling Time and Growth Rate Graphs at
and easy to make a scientifically
cell count targets, cell viability sound and informed each Passage
decision if Statistical
– Visual and MYCAP™Analysis
CCX isincluding:
an acceptable replacement – Overall Doubling Time and Growth Rate Graphs
of–incumbent
Doubling Time and Growthfor
technology Rate
useGraphs at each Passage
in a production – Student’s T-Test
– Overall Doubling Time and Growth Rate Graphs
process.
– Student’s T-Test
Growth Rate at each Passage Overall Growth Rate

0,0350 0,0350
0,0300 0,0300
0,0250
0,0250
Growth Rate
0,0200
Growth Rate
MYCAP™ CCX 0,0200

MYCAP® CCX
0,0150 Traditional Flask
Traditional Flask
Acceptable Growth 0,0150
0,0100
0,0100
0,0050
0,0000 0,0050
1000 mL 1000 mL 3000 mL 3000 mL 3000 mL Seed Reactor
Vessel Size 0,0000
Overall Doubling Time

Doubling Time at each Passage
35,00
30,00
ithout notice. Copyright Sartorius Lab Instruments GmbH & Co. KG. Printed in the EU on paper bleached without chlorine.
30,00
25,00
25,00
Doubling Time (hrs)
20,00
Growth Rate
15,00 20,00
MYCAP™ CCX MYCAP® CCX
Traditional Flask Traditional Flask
10,00 15,00
Acceptable Doubling Time
5,00 10,00
0,00 5,00
1000 mL 1000 mL 3000 mL 3000 mL 3000 mL Seed Reactor
Vessel Size
0,00
MYCAP™ CCX and Opta ® are registered trademarks of Sartorius-Stedim Biotech

AseptiQuik ® is a registered trademark of Colder Products Company
· Order No.: 85037-561-00 · Ver. 01 | 2018
Kleenpak ® is a registered trademark of Pall Corporation

C-Flex ® is a registered trademark of St. Gobain Performance Plastics
MYCAP® CCX and Opta® are registered trademarks of Sartorius-Stedim Biotech Sartorius Lab Instruments
AseptiQuik® is a registered trademark of Colder Products Company GmbH & Co. KG
Kleenpak® is a registered trademark of Pall Corporation Otto-Brenner-Strasse 20
37079 Goettingen, Germany 19
C-Flex® is a registered trademark of St. Gobain Performance Plastics
Phone +49.551.308.0
How to Achieve
Optimal Weighing Performance
How to Avoid Contamination in Pipetting
#05
Application #01
Note
for avoiding
contamination in
pipetting. #03
#04
Introduction
With full-resolution 1 μg readability up to 61 g, the new Sartorius high-capacity microbalances are pushing
back the limits of what is possible in weighing technology: They set a new record in accuracy with 60 million
divisions. Their exceptional weighing performance and the impressive quality of their weighing results are
clearly revealed when they are checked with certified weights.
But perfect measurement of weights is not the application this balance was designed for. Sartorius high-
capacity microbalances enable optimal minimum weights within the USP 41 operating range to be measured
in heavy glass vessels, such as long-necked, volumetric flasks.
20
Choose a
1
Direct weighing of even the smallest quantities of a
substance in large glass flasks enables straightforward, Quiet Pla
accurate and efficient preparation of stock solutions
and reference standards, e.g., for HPLC analysis. This 1. The table should be
eliminates the need for transferring a microsample from a whenever possible,
synthetic stone.
weighing boat into a volumetric flask, which can result in
1. 2. 3. 2. Avoid causing the
errors. Weighing directly in a large container reduces both deflect even slight
sample loss and contamination. use it to prop up y
3. Set up the balance
This application requirement that a balance needs to 4. 5. 6. location. Ensure th
meet poses an even greater challenge to its weighing or engines that gen
electromagnetic fi
technology. The reason is that the smaller the sample
Magnetism must b
quantities used, the greater the relative measuring errors may not be made o
7.
become; and the larger the tare container size employed, 4. Do not position th
the higher the influence of environmental conditions will of the room, but n
be on weighing accuracy. To ensure high accuracy during better, in the corne
is where the vibrat
weight measurements and excellent repeatability of the
generally at their l
results, you need to observe certain basic rules and
requirements.
External environmental influences or improper handling

can lead to inaccurate results or poor weighing
performance, which are not caused by the balance.
1
Choose a Stable Weighing 4. Do not position the table in the middle of the
Table in a Quiet Place to Set room, but near a wall or, even better, in the
Up Your Balance corner of a room, as this is where the vibration
amplitudes are generally at their lowest.
1. The table should be solid-built and, whenever
possible, be made of stone or synthetic stone. 5. Avoid exposing your balance to sunlight and
infrared radiation emitted by lamps or heaters.
2. Avoid causing the tabletop to sag or deflect
even slightly; for example, never use it to prop 6. The location may only be slightly ventilated.
up your arm. Exposure to drafts needs to be avoided, and the
air flow rate should be below 0.2 m/s.
3. Set up the balance in a vibration-free location.
Ensure that there are no machines or engines 7. Cold air currents from air conditioners may not
that generate vibrations or electromagnetic pass directly across or over the draft shield,
fields near the balance. Magnetism must be as this can result in an inversion layer of air
ruled out (e.g., tables may not be made of inside the draft shield. This, in turn, can cause
stainless steel). unstable weight readouts.
21
Work in the Lab under Consistently
2
2
Constant Climate Conditions.
Work in the Lab under
Consistently
1. Avoid significant Constant
temperature changes 4. Use the Sartorius ionizer option to
or spikes. nate electrostatic influences. Elec
2. Keep theClimate Conditions
relative humidity as constant charges on glass vessels dissipate
1. >40% 2. 3. as possible. Prevent the relative humidity slowly, particularly when these ve
1. Avoid significant temperature changes
from dropping below 40%, as this will or surfaces,
very clean spikes. especially whe
significantly increase interference by static are used freshly from a laboratory
electricity. ware washer. Electrostatic influen
2. Keep the relative humidity as constant as
4. 3. Use the Sartorius climate sensor option easy to detect by the continuous
possible.
(temperature, Prevent
barometric the and
pressure relative humidity from
weight readouts. Increase the air
dropping below 40%,
relative humidity) to monitor climate as this will significantly
to levels up to 60%, and use an io
conditions.increase interference by static electricity.
reduce these effects on the result
weight readings.
3. Use the Sartorius climate sensor option
(temperature, barometric pressure and relative
humidity) to monitor climate conditions.
4. Use the Sartorius ionizer option to eliminate

electrostatic influences. Electrostatic charges
on glass vessels dissipate only very slowly,
particularly when these vessels have very
clean surfaces, especially when they are used
freshly from a laboratory glassware washer.
Ensure That the Balance Is
3
Electrostatic influences are easy to detect by
the continuous drift of weight readouts. Increase
Leveled and Calibrated.
the air humidity to levels up to 60%, and use an
ionizer to reduce these effects on the resulting
1. Sartorius high-capacity micro
weight balances will
readings.
isoCAL support you in using the calibration | ad-
justment function isoCAL, and the Q-Level
3
1. 2. function implemented in the balance
for leveling continuously maintains the
accuracy of the weighing results within
a narrow toleranceEnsure
range. That the Balance is
2. Moreover, routinelyLeveled and Calibrated
check the balance
using an external, certified weight.
1. Sartorius high-capacity microbalances will
support you in using the calibration | adjustment
function isoCAL; and the Q-Level function
implemented in the balance for leveling
continuously maintains the accuracy of the
weighing results within a narrow tolerance range.
2. Moreover, routinely check the balance using an

external, certified weight.
22
During the Measuring Sequence,
4
4
Ensure That …
1. … the vessels usedDuring the next
are acclimatized Measuring
4. Avoid touching a vessel with your bare
to your balance; Sequence,
i.e., have adapted toEnsure
the That
fingers ...times, as a single fingerprint
at all
temperature conditions in the same room. can weigh up to 50 μg and therefore have
1. 2. 3. 2. … you do not 1. ... the
touch vessels used
the container with are acclimatized
a major impact nexton to
theyour
accuracy of your
your hands whenbalance,
positioningi.e.,
it on the weight measurement
have adapted to the temperature result.
weighing pan or in a sample holder. 5. When weighing, ensure that no powder
conditions in the same room.
Touching the sample vessel with your falls onto the weighing pan next to the vess
••• ••••
4.
••••••• •
•
5. 6. hand usually as this will mean that the displayed sample
increases the 2. ... you doofnot
temperature the touch
vessel. the container
weight is with youris actually in the vessel.
not what
Buoyancy and airhands
currentwhen
effectspositioning
influence 6.it Avoid
on thetheweighing
complete pan
interchange of air whe
weighing results. or in a sample
Remember that itholder.
takes Touching
openingthethesample
draft shield by opening only o
7. 8. ten minutes for these
vessel effects
withtoyour
subside. door,increases
hand usually where possible.
the Opt for using the dra
Use a pair of tweezers or forceps to posi- shield learning capability to open the door
temperature of the vessel. Buoyancy and air
tion the vessel. only as far as actually necessary.
3. Avoid placing yourcurrent effects
hand inside the influence
draft 7.weighing results.
Carefully place the tare container on
Remember
shield to ensure that that it takes ten
no unnecessary theminutes
weighingfor these
pan or in the sample holder.
interchange of aireffects
outsideto subside.
and inside theUse a pair
Avoidofapplying
tweezers anyorexcessive force.
draft shield takesforceps
place andtothat no heat 8.
position the vessel.Do not lean on or against the weighing
is transferred into the draft shield. table or rest your arm arm on it during
the weighing procedure.
3. Avoid placing your hand inside the draft shield
to ensure that no unnecessary interchange of air
outside and inside the draft shield takes place
and that no heat is transferred into the draft
shield.
4. Avoid touching a vessel with your bare fingers at

all times, as a single fingerprint can weigh up to
50 μg and therefore have a major impact on the
accuracy of your weight measurement result.
5. When weighing, ensure that no powder falls

onto the weighing pan next to the vessel, as this
will mean that the displayed sample weight is
not what is actually in the vessel.
6. Avoid the complete interchange of air when

opening the draft shield by opening only one
door, where possible. Opt for using the draft
shield learning capability to open the door only
as far as actually necessary.
7. Carefully place the tare container on the

weighing pan or in the sample holder. Avoid
applying any excessive force.
8. Do not lean on or against the weighing table

or rest your arm on it during the weighing
procedure.
23
® ®
Vivaflow and Vivaspin Workflow
Vivaflow ®
and Vivaspin ®
Workflow
in
HowProtein Research
to Avoid Contamination
in Protein Research Laboratories Laboratories
in Pipetting
#05
Application #01
Note
Practical methods
for avoiding
#02 Appl
contamination in
pipetting. #03 Note
#04
24
to final concentration | desalting of the to 2 l using dd-H2O. After every prepara-
purified protein. This protocol shows in tion, concentration and purification step,
detail the recoveries after each step along 1 ml sample was set aside for SDS gel
with the time needed for every purification analysis at the end of the preparation.
and concentration step.
Ion Exchange chromatography was cho-
Concentration and Purification Efficiency
of and efficacyPart 1 – cycle
of a multiple Creating sen as and Concentrating
the method of choice for purifying
Proteins in Cell Culture Supernatant
experimental procedurethe Culture Medium the cell culture supernatant,
was performed lysozyme from
Using Sartorius Vivaflow ®, Vivaspin
®
using Vivaflow
® tangential flow cassettes especially from the “contaminant” BSA. For
for initial concentration and diafiltration this, the 2 l cell culture supernatant needed
and Vivapure Products
®
2 bottles
of a cell culture supernatant, (4 g)byof RPMI-1640
followed were dissolved
to be concentrated into 1.8
and then diafiltered to L
Vivapure® Ion Exchangedd-H O, andfor4 g ofadjust
spin columns
2
sodium acetate
the sample was
to the added.
starting conditions
This protocol demonstrates how the Vivaflow cassettes,
the®protein purification step and finally needed for the ion exchange chromatogra-
Vivaspin® 20 ultrafiltration devices for the phy binding step.
Vivapure ® Ion Exchange spin columns and Vivaspin ® The pH was adjusted to 7.2 using 1M HCl. 2 g of BSA
final sample concentration and desalting.
devices can be used in order to perform a An complete
artificial mixture ofand 1 g inofa lysozyme
proteins RPMI- Forwere added as
concentration andprotein samples,
diafiltration, the
protein purification workflow, from concentration andmedium was
1640 culture meant to to
created bemimic
separated by ®chromatography.
Vivaflow 200 was used with a 5The volume
kDa PES
®
the type of
diafiltration of the original protein source, a cell cultureproduct that many researchers membrane. Vivaflow 200
of the cell culture supernatant sample was brought up to is a ready-to-
culture using e.g. the UniVessel device. use laboratory crossflow cassette in an
supernatant, to final concentration | desalting of the 2 L using dd-H O. After every preparation, concentration,
This procedure further reflects a method 2 acrylic housing, which allows caustic clean-
purified protein. This protocol shows in detail
that the
can be adapted toand purification
a large number of step, ing 1
andmL4-5sample wascassettes
re-uses. Two set asidecan for
be SDS
recoveries after each step along with the time needed
protein gel analysis
purification protocols, adapting at the end
run inofparallel
the preparation.
for the concentration of up
for every purification and concentration step.MWCOs and device sizes where necessary. to 5 l sample volumes. For the 2 l sample
to be concentrated in this experiment, one
cassetteIon
wasexchange
sufficient. Achromatography
Masterflex pump
with anwas
Easychosen
Load, sizeas16 the
pumpmethod
head was
used toofrunchoice for purifying
the Vivaflow ®
200 cassette.
®
Figure lysozyme
1a. and 1 b. show
fromthe theVivaflow 200
cell culture
set up before and during the concentration
process.supernatant, especially from the
“contaminant” BSA. For this,
®
the 2 L200
The Vivaflow system
cell was set
culture up and
supernatant
run at 3 bar. Once 1.8 l of filtrate had been
needed to be concentrated and
collected, the pump was stopped, the tubes
removedthen
fromdiafiltered to adjust
the cell culture mediumthe
sample
concentrate and to the and
filtrate starting conditions
the Vivaflow ®
systemneeded
was purged with dd-H O. This
for the ion2 exchange solu-
tion now contained a 10 fold concentration
chromatography binding step.
of the constituent proteins from the origi-
nal culture-medium.
For concentration and
diafiltration, the Vivaflow® 200
was used with a 5 kDa PES
membrane. Vivaflow® 200
is a ready-to-use laboratory
crossflow cassette in an acrylic
Efficiency and efficacy of a multiple cycle experimental housing, which allows caustic cleaning and 4-5 reuses.
procedure was performed using Vivaflow® tangential Two cassettes can be run in parallel for the concentration
flow cassettes for initial concentration and diafiltration of up to 5 L sample volumes. For the 2 L sample to
of a cell culture supernatant, followed by Vivapure ® Ion be concentrated in this experiment, one cassette was
Exchange spin columns for the protein purification sufficient. A Masterflex pump with an Easy Load, size 16
step and finally Vivaspin ® 20 ultrafiltration devices pump head was used to run the Vivaflow® 200 cassette.
for the final sample concentration and desalting. Figure 1a and 1b show the Vivaflow® 200 setup before and
An artificial mixture of proteins in a RPMI-1640 during the concentration process.
culture medium was created to mimic the type of
product that many researchers culture using, e.g., the The Vivaflow® 200 system was set up and run at 3 bar.
UniVessel device. This procedure further reflects a Once 1.8 L of filtrate had been collected, the pump was
method that can be adapted to a large number of stopped, the tubes removed from the cell culture medium
protein purification protocols, adapting MWCOs concentrate and filtrate, and the Vivaflow® system was
and device sizes where necessary. purged with dd-H2O. This solution now contained a
10-fold concentration of the constituent proteins from
the original culture medium.
25
A BCA protein detection test conveyed a 100% recovery Part 2 – Buffer Exchange of Culture
AofBCA protein
protein detection
after test concentration
this first conveyed a 100%step.recovery of 1
Table Medium
Part 2 – BufferConcentrate exchange of culture medium concentrate
protein after this first concentration step. Table 1 indicates the The Vivaflow® 200 system was used for fast and easy diafiltration.
indicates the time needed for the sample concentration.
time needed for the sample concentration. To this end, the diafiltration cup, a Vivaflow® accessory, was filled
A BCA protein detection test conveyed a 100% recovery of Thethe
Part
with 2Vivaflow
– 200
Buffer
®
200 System
exchange
ml concentrated was Figure
of sample.
culture used 2for
medium fastthe
concentrate
shows and diafiltra-
protein after
A BCA this first
protein concentration
detection step. aTable
test conveyed 100%1 recovery
indicatesofthe easy
The set diafiltration.
Vivaflow
up. The®
200 system
Vivaflow To ® this
was end,
used
Part 2 – Buffer exchange of culture medium concentratehowev-
tion 200 system forthefast
was diafiltration
setand up easy
as cup,
diafiltration.
before,
®
time protein
neededafter
for the
thissample concentration.
first concentration step. Table 1 indicates the To
er this
aThe
Vivaflowend, the
attaching
Vivaflow ®
an diafiltration
® accessory,
additional
200 system was cup,
was
tubeusedtoathe
Vivaflow
filled andaccessory,
fastwith
for diafiltration the lid
easy 200 was
and mL filled
placing
diafiltration.
time needed for the sample concentration. with
this the
new 200
inlet
concentrated sample.
To this end, ml
thetube concentrated
into
diafiltration a 25 mMsample.
cup, Sodium
a Figure
Vivaflow Acetate 2 shows
Figure 2 shows the diafiltration
® (pH
accessory, the
5.5)
was diafiltra-
buffer
filled
tion
withset
(needed
setup. theup.
to 200
The
ThemlVivaflow
re-adjust the ®®sample
concentrated
Vivaflow
200 sample.
200
system
system
was
concentrate
Figureset2for
was
up as before,
the
shows
set upionic
theas howev-
starting
diafiltra-
before,
er attaching
conditions
tion set up.ofThe an additional
theVivaflow
ion exchange ® tube to the diafiltration lid and placing
chromatography
200 system was set up asstep which
before, was to
howev-
however
this newThis inletattaching
tube an25 additional tube to the diafiltration
er attaching
follow). an
leads tointo
theaconcentration
additional mMtoSodium
tube ofAcetate
the diafiltration
the sample (pHand
lid 5.5)
in buffer
placing
the reser-
lid
(neededand
newtoplacing
thisand
voir re-adjust
inlet
the tube extent this
the
into inanew
sample
25 mM
which inlet
the tubeAcetate
concentrate
Sodium
original into forathe
buffer (pH25 mM
is ionic
5.5)
removed Sodium
starting
buffer and
Acetate
(needed to
conditions
collected as(pH the5.5)
ofre-adjust
waste ion buffer
the sample
exchange
(filtrate), (needed
new concentrate to readjust
chromatography
buffer (25 mMfor thestep
Sodium the
ionic sample
starting
which was to
Acetate)
is conditions
follow).
suckedThis
concentrate into of the
leads
the for ion
tothe
closed exchange
the ionic
system, chromatography
concentration
starting
gradually ofconditions
the
leading steptowhich
sample ainofthe was
the
buffer to
reser-
ion
follow).
voir
exchangeand This
to the
while leads
extentto the
keeping in concentration
which
the samplethe of thebuffer
original
volume sample
constant is in the
removed
at 200 reser-
and
ml.
exchange chromatography step which was to follow).
voirsystem
collected
The and as towasthe
waste extent 3inbar.
(filtrate), whichnew the 1original
buffer (25 buffer
mMhad is been
removed
Sodium and
Acetate)
This
collectedleads as torun
waste the atconcentration
(filtrate),
Once
new buffer
l ofof buffer
(25 the
mM sample
Sodium in the
Acetate)
Figure
Fig. 1a 1and
1a. and 1b: Vivaflow
b: Vivaflow ® ®
200 set up 200
beforesetup before
(1a) and (1a)theand
during (1b) sample is sucked into the closed
exchanged, the filtration was stopped. system, gradually leading to a buffer
during (1b) the sample concentration process. reservoir
is sucked while
exchange and
into the to the system,
closed
keeping extent in volume
which
gradually the original
leading to a bufferbuffer
concentration process. The 200 ml solution now the sample
contained the correct constant
bufferatto200 ml.
maintain
exchange
is
The removed
system while
was andkeeping
run at 3 the
collected
bar. sample
Once as 1 volume
waste
l of constant
(filtrate),
buffer had at new
been 200 ml. buffer
the stability of the proteins of interest for the next part of the
The system was run at 3 bar. Once 1 l of buffer had been
Vivaflow ® 1 b: Vivaflow® 200 set up before (1a) and during (1b) the sample
200 (5 kDa MWCO)
Fig. 1a. and (25 mMand
exchanged,
protocol sodium
the theacetate)
hadfiltration correct waspH isandsucked
stopped. into the closed
salt concentration for the ion
Table 1:1a.
Fig. Vivaflow
concentration and
process. 200, ®PES,
1 b: ®Vivaflow 200 set5upkDa MWCO
before (1a) and during (1b) the sample exchanged, the filtration was stopped.
The 200
system, ml solution now contained the correct buffer to maintain
Filtrate Volume (m
concentration
concentration L)
speed.
process. Time taken (h:min:s) The 200 mlgradually
exchange binding
solution nowstep. leading
BCA protein
contained to the
a buffer
correctexchange
quantification again
buffer to maintain while
showed
athe stability
100%
keeping protein
the stability theof the proteins
recovery.
of sample
the proteins
of
volume interest
of interest
for
constant the
for the next
next
at 200 part
part mL.
of the
of theThe
0Vivaflow® 200® (5 kDa MWCO) 0:00:00 protocol and had the correct pH and salt concentration for theionion
Vivaflow 200 (5 kDa MWCO) protocol and
system washad run theatcorrect
3 bar.pHOnce and salt 1 concentration
L of buffer had for the been
100
Filtrate Volume (mL(m) L) 0:03:16
Time taken (h:min:s) exchange
Table 2 showsbinding the step.
time BCA
needed protein
for quantification
diafiltration
exchange binding step. BCA protein quantification again showed of again
200 ml showed
sample
Filtrate Volume
Part
200 2 – Buffer exchange of culture Time taken
medium
0:06:50
(h:min:s)
concentrate aexchanged,
a100%
against
100%1000protein
protein mlthe filtration
recovery.
exchange
recovery. buffer,was stopped.
again using Vivaflow® 200 with
0 0 0:00:00
0:00:00
he The Vivaflow® 200 system was used for fast and easy diafiltration. a 5 kDa PES membrane.
300
100 0:10:45 ®
To end, the diafiltration cup, a0:03:16
this100 0:03:16 accessory, was filled
Vivaflow The
Table2200
Table 2shows
showsmLthe solution
the time
time needed nowfor
needed contained
for diafiltration
diafiltration the correct
ofof200200mlmlsample buffer
sample
400
with
200 the 0:14:38 against 1000 ml exchange buffer, again using Vivaflow ® ® 200 with
200200 ml concentrated sample. Figure 2 shows the diafiltra-
0:06:50
0:06:50 to maintain
against 1000 ml the stability
exchange of the
buffer, again proteins
using Vivaflowof interest 200 with for
® a a55kDa PES membrane.
tion
500 set
300 300 up. The Vivaflow 200 system was
0:18:36
0:10:45 set up as before, howev-
the kDanext PESpart membrane.
of the protocol and had the correct pH
0:10:45
er
600attaching an additional tube to the diafiltration
0:22:43 lid and placing
400new400inlet tube into a 25 mM Sodium 0:14:38
0:14:38 and salt concentration for the ion exchange binding
this Acetate (pH 5.5) buffer
700
500 500to re-adjust the sample concentrate0:26:57
0:18:36 for the ionic starting
0:18:36 step. BCA protein quantification again showed a 100%
(needed
800
conditions
600 600 of the ion exchange chromatography0:31:14
0:22:43
0:22:43 step which was to protein recovery.
follow).
900 700This leads to the concentration 0:36:01of the sample in the reser-
0:26:57
700 0:26:57
voir
1000 and to the extent in which the0:40:50 original buffer is removed and Table 2 shows the time needed for diafiltration of 200
800 800 0:31:14
0:31:14
collected as waste (filtrate), new buffer (25 mM Sodium Acetate) Fig. 2: Diafiltration system set up for buffer exchange. Culture medium
1100 900 0:45:46
0:36:01 mL sample against 1000 mL exchange buffer, again
is900
sucked into the closed system, gradually 0:36:01 leading to a buffer concentrate can be seen in the center of the image. 1 L 25 mM Sodium Acetate
1200 1000 0:50:36
0:40:50 using Vivaflow
(exchange buffer) can be200
®
seen with a 5tokDa
connected PES on
the system membrane.
the left of the
exchange
1000 while keeping the sample 0:40:50 constant at 200 ml.
volume
Fig.
image. 2: Diafiltration system set up for buffer exchange. Culture medium
The
1300 system
1100 was run at 3 bar. Once 0:55:32 1 l 0:45:46
of buffer had been Fig. 2: Diafiltration system set up for buffer exchange. Culture medium
1100 0:45:46 concentrate can be seen in the center of the image. 1 L 25 mM Sodium Acetate
concentrate
Table can be seen in the
2: buffer)
Diafiltration center of the
of connected
200 mL image. 1 L 25 mM Sodium
concentrated Acetate
mple exchanged,
1400 1200 the filtration was stopped. 0:50:36
1:00:24 (exchange can be seen to the system on thecell culture
left of the
1200 (exchange buffer) can be seen connected to the system on the left of the
The 200 ml solution now contained0:50:36 the correct buffer to maintain supernatant
image. containing the proteins lysozyme and BSA
1500 1300 0:55:32
1:05:26 image. Volume (mL)
Filtrate Time taken (h:min:s)
the
1300stability of the proteins of interest for the next part of the
0:55:32 against 1000 mL 25 mM sodium acetate.
1600 1400 1:00:24
1:10:28 0 0:00:00
protocol
1400 and had the correct pH and salt
1:00:24 concentration for the ion
exchange1500binding step. BCA protein 1:05:26
quantification again showed Filtrate Volume (mL)
100 Time taken (h:min:s)
0:06:58
1700
1500 1:15:52
1:05:26 Filtrate Volume (mL) Time taken (h:min:s)
a 100% protein
1600 recovery. 1:10:28 0 0:00:00
1800 1:21:50 200
0 0:14:16
0:00:00
1600 1:10:28 100 0:06:58
1700 1:15:52 300 0:22:39
Table
170021:shows the time needed for1:15:52diafiltration of 200 ml sample 100
200
0:06:58
0:14:16
Table 1800 1:21:50
Vivaflow® 200, PES, 5 kDa MWCO concentration speed. 400 0:29:40
against
1800 1000 ml exchange buffer, 1:21:50 again using Vivaflow® 200 with 200
300 0:14:16
0:22:39
a 5 kDa PES membrane. 500
300 0:37:02
0:22:39
Table 1: Vivaflow® 200, PES, 5 kDa MWCO concentration speed. 400 0:29:40
Table 1: Vivaflow® 200, PES, 5 kDa MWCO concentration speed. 600
400 0:44:15
0:29:40
Figure 2: Diafiltration system 500 0:37:02
700
500 0:51:34
0:37:02
set up for buffer exchange. 600 0:44:15
Culture medium concentrate 800
600 0:58:54
0:44:15
700 0:51:34
can be seen in the center of 900 1:06:03
700
800 0:51:34
0:58:54
the image. 1 L 25 mM sodium
1000
800
900 1:13:02
0:58:54
1:06:03
acetate (exchange buffer) can
be seen connected to the 1000
900 1:13:02
1:06:03
system on the left of the image. Table 2: Diafiltration of 200 ml concentrated cell culture supernatant contain-
1000
ing 1:13:02
the proteins lysozyme and BSA against 1000 ml 25 mM Sodium Acetate.
Fig. 2: Diafiltration system set up for buffer exchange. Culture medium Table 2: Diafiltration of 200 ml concentrated cell culture supernatant contain-
26
concentrate can be seen in the center of the image. 1 L 25 mM Sodium Acetate ing the proteins lysozyme and BSA against 1000 ml 25 mM Sodium Acetate.
Table 2: Diafiltration of 200 ml concentrated cell culture supernatant contain-
(exchange buffer) can be seen connected to the system on the left of the
ing the proteins lysozyme and BSA against 1000 ml 25 mM Sodium Acetate.
with further 10 ml of 25 mM Sodium Acetate, discarding the
flow through, followed by an elution step with 5 ml of 1 M NaCl in
25 mM Sodium. A BCA test revealed a 95 % lysozyme recovery.
Part 3 – Purification of Lysozyme,

PartProtein
the 3 – Purification
of Interestof Lysozyme, the protein of interest The eluate was then con
The purification of lysozyme was performed using a Vivapure® MWCO), Figure 5., and c
The purification of lysozyme was performed using ®
cation exchange membrane adsorber devices (Vivapure Maxi H S). approximately 2 ml of c
a Vivapure cation exchange membrane adsorber
®
The membrane
device (Vivapure Maxi
® adsorber
H S). Thematrix
membrane holds the active ligands and per-
adsorber was then re-filled with 1
matrix
formsholdslikethe active ligands and
a traditional performs
cation like a
exchanger. Membrane adsorbers pH 7.2 to 20 ml for a fin
traditional cation exchanger. Membrane adsorbers
represent
represent a special
a special form
form of of chromatography
chromatography matrix. matrix. Unlike tradi- purified sample. The sam
tionaltraditional
Unlike chromatography
chromatography resins,
resins,they make use of convective trans-
they make sample volume of 2 ml h
use of convective transport to bring proteins to the ion
port to bring proteins to the ion exchange surface; hence, binding, 97 % lysozyme recovery
exchange surface; hence, binding, washing and elution
washing
is performedand elution
quickly, is performed
and high quickly
binding capacities are and high binding capaci-
ties achieved
even are even achieved
at high at This
flow rates. highallows
flowtherates.
use This allows the use of the
of the chromatography matrix in fast and convenient
chromatography matrix in fast and convenient centrifugal spin
Figure 4: Vivapure Maxi spin columns can be used in a
®
centrifugal spin columns (Figure 3).
columns (Fig. 3). Fig. 4: Vivapure Maxi spin columns can be used in a centrifuge for fast and
®
centrifuge for fast and easy protein purification.
easy protein purification.
The eluate was then concentrated in a Vivaspin® 20

(PES, 5 kDa MWCO), shown in Figure 5, and centrifuged
at 5000 x g for 10 min or until approximately 2 mL
of concentrate had been collected. The device was
Part 3 – Purification of Lysozyme, the protein of interest then refilled
The eluate waswith
then18 mL 50 mM
concentrated in potassium
a Vivaspin® 20phosphate
(PES, 5 kDa
®
The purification of lysozyme was performed using a Vivapure MWCO), Figure 5., and centrifuged at 5000 xg for
buffer, pH 7.2 to 20 mL for a final buffer exchange 10 min. or until
cation exchange membrane adsorber devices (Vivapure® Maxi H S). and approximately 2 ml of concentrate had been
desalting of the purified sample. The samplecollected. The device
The membrane adsorber matrix holds the active ligands and per- was then re-filled with 18ml 50mM Potassium Phosphate buffer,
was again centrifuged until a final sample volume of
forms like a traditional cation exchanger. Membrane adsorbers pH 7.2 to 20 ml for a final buffer exchange and desalting of the
represent a special form of chromatography matrix. Unlike tradi- 2purified
mL had beenThe
sample. attained. A BCA
sample was againtest revealed
centrifuged a 97%
until a final
tional chromatography resins, they make use of convective trans- lysozyme
sample recovery.
volume of 2 ml had been attained. A BCA test revealed a
Figure 3: The electron microscopic image of
Fig.
port 3:
to The
bring electron
proteins to microscopic
the ion exchange
chromatography gel beads (upper right) in image
surface; of chromatography
hence, binding, 97 % gel beads
lysozyme (upper
recovery.
washinginand
right)
comparison elution
comparison
to is performed
a Q ion to a Qquickly
exchange and high binding
ion exchange
membrane capaci- adsorber (background)
membrane
ties are even
adsorber achieved at reveals
(background) high flow rates. This
100-fold allows the use of the
larger
reveals
pore
100 fold larger
sizes of thematrix
chromatography membranein fastpore sizes of the
adsorber.
and convenient membrane
centrifugal spin adsorber.
columns (Fig. 3).
® H S type devices (Figure 4) were
TwoVivapure
Two Vivapure ®
Maxi Maxi H S type devices (Fig. 4) were equilibrated
equilibrated with 10 mL of 25 mM sodium acetate,
with 10 ml of 25 mM Sodium Acetate, pH 5.5 each, by filling with
pH 5.5 each, by filling with 10 mL of this buffer and
10 ml of this
centrifuging for 5 buffer
min in a swingand centrifuging
bucket centrifugefor at 5 min. in a swing bucket
centrifuge
500 at 500 the
x g and discarding xgflowandthrough.
discardingUsing the the flow through. Fig. 5: Vivaspin® 20 ultrafiltr
concentrated and buffer exchanged sample from Part
Using the concentrated and buffer exchanged sample from Part
2, 10 mL sample were pipetted into each of these two
which allows pressurization o
Fig. 3: The electron microscopic image of chromatography gel beads (upper
2, 10in comparison
right) ml sample
equilibrated Vivapure ®were pipetted into each of these two equilibrated
to a Q ion devices and centrifuged
exchange membrane again
adsorber (background)
in a centrifuge.
for 5 min
Vivapure
reveals in ®alarger
100 fold swingporebucket
devices andcentrifuge
sizes of the at 500 xagain
centrifuged
membrane adsorber. g. The for 5 min. in a swing
Vivapure devices were washed with further 10 mL of ®
®
bucket
TwomM
25 Vivapurecentrifuge
sodium
®
Maxi atdevices
500(Fig.
H S typediscarding
acetate,
xg.4) The
the were Vivapure devices were washed
flow equilibrated
through,
with
with 10further
followed mlbyofan 10Sodium
25 elution
mM mlstepof 255mM
Acetate,
with pH
mL5.5ofSodium
each,
1 M by
NaCl
10 ml of this buffer and centrifuging for 5 min. in a swing bucket
Acetate,
filling with discarding
Figure 5: Vivaspinthe®
20 ultrafiltration device, on the right with
in
flow25 mM sodium.
through, A BCA
followed test revealed
by flow a 95%
an through.
elution step with 5 ml
a pressure of ® 1
cap M
that NaClpressurization
allows in on the rightofwith
the device as
centrifuge at 500 xg and discarding the Fig. 5: Vivaspin 20 ultrafiltration device, a pressure cap
lysozyme recovery. and buffer exchanged sample from Part well, and the regular utilization in a centrifuge.
Using the concentrated
25 mM Sodium. A BCA test revealed a 95 % lysozyme
2, 10 ml sample were pipetted into each of these two equilibrated
recovery.
which allows pressurization of the device as well and the regular utilization
in a centrifuge.
Vivapure® devices and centrifuged again for 5 min. in a swing
bucket centrifuge at 500 xg. The Vivapure® devices were washed
with further 10 ml of 25 mM Sodium Acetate, discarding the 27
flow through, followed by an elution step with 5 ml of 1 M NaCl in
1 ml sample taken during the experiment were diluted with 95µl 1 wor
reducing sample buffer, of which 20 µl were loaded onto a 12% strate
tris-HCl SDS gel (Fig. 6)
Figure 6: Coomassie stained 12% Task

Tris-HCl SDS gel loaded with
20 μL sample preparations. Lane 1: Vivafl
Marker (SDS Broad Range marker);
Lane 2: Original sample; Lane 3: Vivafl
Original sample filtrate (Part 1); Lane and ru
4: Marker; Lane 5: Buffer exchange
concentrate (Part 2); Lane 6: Filtrate Vivap
after binding (Part 3); Lane 7: Marker;
Lane 8: Filtrate after eluting (Part 3); Vivasp
Lane 9: Filtrate after concentrating conce
and desalting (Part 3); Lane 10:
Concentrate after concentrating Total
and desalting.
Produ
Fig. 6: Coomassie stained 12% tris-HCl SDS gel loaded with 20 µl sample
Part
preparations. Lane 1: Marker (SDS Broad range marker); Lane 2: Original sam- Vivafl
Part 44– – Analyzing
Analyzing the Samples
the samples The complete
The complete setsetup
up andand completion of protein
ple; Lane 3: Original sample filtrate (Part 1);completion of protein
Lane 4: Marker; Lanepurification takes
5: Buffer
The
The samples
samples ofofthethe individual
individual stepssteps were analyzed
were analyzed byusing purification
by SDS gel, approx. 3.45 htakes approx.
using this 3.45
method, h using
starting form this method,
a culture super- 500 m
reducing sample buffer (prepared by adding exchange
50 µl concentratenatant,
2-mercaptoe- (Part 2);
with Lane
high 6:protein
Filtrate after binding
recoveries in each (Part
step 3); Table
(see Lane 3)7:
SDS gel, using reducing sample buffer (prepared by starting from a culture supernatant, with high protein
thanol
Part 4 to 950µl Laemmli sample buffer). Marker; Lane 8: Filtrate after eluting (Part 3); Lane 9: Filtrate after concen-
adding –50Analyzing the samples
μL 2-mercaptoethanol to For
950allμL
steps, 5µl of the
Laemmli The
The total protein
complete
recoveries inset purification
up and
each step procedure
completion
(see can be purification
of protein
Table 3). The completed within
takes
total protein Vivap
1 mlsamples
The sample of
taken
the during the steps
individual experiment
were weretrating
diluted
analyzed by and
with
SDS desalting
95µl
gel, using 1(Part
approx.3);3.45
working Lane
day,
h 10: Concentrate
including
using this after concentrating
SDS gel analysis,
method, starting utilizing
form a andsaving
this time
culture super-
sample buffer). For all steps, 5 μL of the 1 mL sample purification procedure can be completed within 1
reducing
reducing sample
sample buffer, of which 20
buffer (prepared by µl
adding desalting.
were 50
loaded onto a 12%
µl 2-mercaptoe- strategy, when
natant, with adapted
high proteintorecoveries
individualinneeds.
each step (see Table 3) Vivasp
taken duringgel
tris-HCl the experiment were diluted with 95 μL working day, including SDS gel analysis, utilizing this
thanol toSDS (Fig.
950µl Laemmli6) sample buffer). For all steps, 5µl of the The total protein purification procedure can be completed within
reducing sample buffer, of which 20 were
1 ml sample taken during the experiment μL were loaded
diluted with 95µl time-saving
1 working day,strategy, when
including SDS geladapted to individual
analysis, utilizing needs.
this time saving
onto a 12% Tris-HCl SDS gel (Figure 6). Conclusion
reducing sample buffer, of which 20 µl were loaded onto a 12% strategy, when adapted to individual needs.
Task Time Recovery
tris-HCl SDS gel (Fig. 6) The overall result
shows
Vivaflow
Table
that
® a standard and straightforward 100%
3 200 set up and run through 1 h 25 min
procedure can be followed
Vivaflow® 200 toDiafiltration
concentrate,set up purify,
1 h isolate
20 min and100%
Conclusion Taskrun through
analyze a protein of interest
and Time
from a cell culturing device, using Recovery
The overall result shows that a standard and ®
Vivaflow ® 200 set up and run through 451 hmin
25 min 100%
Vivaflow® 200 tangential
Vivapure flow units for cell culture
purification supernatant
95%
straightforward procedure can be followed to ®
200 Diafiltration
Vivaflow® Lysozyme set| ®up 1
Vivaspin
concentration andand
diafiltration, desalting 30hmin
20 min 100%
97%
concentrate, purify, isolate and analyze a protein of run through Vivapure for ion exchange
concentration ®
chromatography
interest from a cell culturing device, using Vivaflow® 200 followed
Vivapure ® by Vivaspin 20 for final sample
purification 45
Total 3 h min
45 min 95%
92%
concentration andVivaspin
tangential flow units for cell culture supernatant desalting.
®
Lysozyme desalting | 30 min 97%
concentration and diafiltration, Vivapure ® for ion concentration
exchange chromatography, followed by In Vivaspin ®
20 for
many cases dialysis,
Total which is an overnight procedure
3 h 45 min would be
92%
final sample concentration and desalting.performed instead of the much quicker alternative ultrafiltration.
Here, we show how time saving and efficient ultrafiltration is
In many cases dialysis, which is an overnight procedure, Products used in this experiment Order No.
Fig. 6: Coomassie stained 12% tris-HCl SDS gel loaded for diafiltration
and desalting applications, as well as for protein
would be performed instead of the muchwith 20
quickerµl sample
preparations. Lane 1: Marker (SDS Broad range marker); Lane 2: Original sam-
concentration. Vivaflow® 200, PES, 5kDa VF20P1
alternative, ultrafiltration.
ple; Lane 3: Original sample filtrateHere,
(Part 1);we
Laneshow howLane
4: Marker; time-
5: Buffer
saving
exchangeand efficient
concentrate (Partultrafiltration
2); Lane 6: Filtrateisafter
for binding
diafiltration
(Part 3); Lane 7: 500 mL Diafiltration
Products used in thiscup
experiment VFA006
Order No.
Fig. 6: Coomassie
Marker; stained
Lane 8: Filtrate 12%
after tris-HCl
eluting SDS3);gel
(Part loaded with 20 µl sample
and desalting
preparations. Laneapplications,
1: Marker wellLane
as range as9:for
Filtrate after
protein concen-
Vivapure ®
S H Maxi
Vivaflow® 200, PES, 5kDa VS-IX20SH08
VF20P1
trating and desalting (Part 3);(SDS
LaneBroad marker);
10: Concentrate afterLane 2: Original and
concentrating sam-
concentration.
ple; Lane
desalting.3: Original sample filtrate (Part 1); Lane 4: Marker; Lane 5: Buffer
Vivaspin ®
20, 5 kDa cup VS2011
exchange concentrate (Part 2); Lane 6: Filtrate after binding (Part 3); Lane 7: 500 mL Diafiltration VFA006
Marker; Lane 8: Filtrate after eluting (Part 3); Lane 9: Filtrate after concen-
Conclusion Vivapure® S H Maxi VS-IX20SH08
trating and desalting (Part 3); Lane 10: Concentrate after concentrating and
The overall result shows that a standard and straightforward
desalting. ®
Vivaspin 20, 5 kDa VS2011
procedure can be followed to concentrate, purify, isolate and
analyze a protein of interest from a cell culturing device, using
Conclusion
28
®
The overall200
Vivaflow tangential
result flowa units
shows that for cell
standard andculture supernatant
straightforward
concentration and diafiltration, Vivapure® for ion exchange
Minimizing Syringe Filter Consumption

How tofor Monoclonal
Avoid Contamination Antibody
in Pipetting Harvest from
CHO Cell Culture Supernatants #05

Michael Grauf1*, Klaus Schöne2 Application #01
1. Sartorius Stedim Cellca, Erwin-Rentschler-Straße 21, 88471 Note
Laupheim
2. Sartorius Lab Instruments GmbH & Co.KG, Otto-Brenner-Str 20, 37079 Göttingen
*
Correspondence Practical methods #02
E-Mail: michael.grauf@sartorius.com for avoiding
contamination in
pipetting. #03
#04
Abstract
The clarification of cell culture supernatants with volumes < 25 mL for harvesting
monoclonal antibodies by using syringe filters is often a laborious and sometimes an
exhausting work step. Therefore, a proper selection of the suitable filter model could be
paramount. In this work, we compared two syringe filter models with a similar effective
filtration area from two suppliers regarding their clarification characteristics of CHO cell
culture supernatant samples. To obtain robust results we examined 10 combinations
of cultivation methods and monoclonal antibody products like IgG1, IgG2, fc fusion
proteins, and bispecific antibodies with regard to turbidity, mAb recovery, relative yield,
and throughput. As a result, we found that syringe filter model Minisart® High Flow shows
an average throughput of 18.0 mL compared to 9.3 mL of another premium brand at cell
densities between 38.3 and 163.6 x 105 cells/mL. For the other parameters, we could
not find any significant differences. This finding emphasizes the importance of carefully
selecting the syringe filter model that reduces the number of both devices needed and
thus the workload.
29
means of the parameters: turbidity, mAb recovery, relative yield, throughput, and filter consumption.
Y O13
In U Rcultivation
PR A C T I C A batches
L G U IDE 10
TOcombinations
BA SIC LA BORATORY
of targetTECHNIQUES
proteins and cultivation methods were used
(Table 1). In addition to 125 and 1000 mL shaking flasks, 5 L stirred tank reactor (UniVessel, Sartorius)
Introduction
were also used. The cell density and viability was examined with the Vi-CELL XR from Beckman
Coulter. As of
Clarification target proteins,
mammalian cell CHO cell
culture lines were
samples selected with
for preparative mAb from
or analytical different
purposes IgG1 types,
is a necessary IgG2,
step fc
to enable
both a subsequent purification step and a smooth operation of analytical instruments.
fusion protein and a bispecific antibody. The specific designation has been anonymized, due to The overall aim of the clarification
step is to removeagreements.
confidentiality cells, cellular debris and other particles from the cell culture while simultaneously allowing the target
product to be recovered with a sufficient yield. The conventional procedure for clarification of small volumes (approx.
<All
25cell
mL)culture
is a combination of centrifugation
batches were harvestedof the cell
after cultureFrom
14 days. sample followed
every bytwo
batch, a microfiltration
samples were of the supernatant
taken
obtained.
(max. 31 While centrifugation
ml per sample), one removes
samplecoarse and high-density
destined particles,
for clarification withmicrofiltration
Minisart® Highis frequently
Flow andnecessary
one for to
pull out small or low-density particles from the centrifugation supernatant. Microfiltration can serve as a simultaneous
another premium brand. The samples were clarified by centrifugation for 60 min at 4,000 g and the
sterilization step by using 0.2 or 0.22 μm rated sterile filters.
supernatants were filtered with the respective syringe filters (Figure 1).
Figure 1: Clarification and sterile filtration of cell culture supernatants under aseptic conditions by using the
syringe filter Minisart® High Flow with a pore size of 0.22 μm.
Even though centrifugation removes the vast majority Methods and Materials
of particles, clogging of filters is often a problem and In an attempt to facilitate the filtration of cell culture
Figure
can 1: Clarification
lead and sterile
to an increased filtration of cell
consumption culture
of filter supernatantssupernatants
devices under aseptic conditions by using theof
for the development syringe filter a
cell lines,
Minisart® High Flow with a pore size of 0.22 µm.
or to ergonomic handling issues. However, both the comparative study was performed. The study was
reduction of filter consumption and the associated executed by using CHO cell culture samples from real
The mAb titer was determined in the unharvested and harvested cell culture fluid using the Octet
operation time can be achieved by a well-considered projects spread over a period of 3 months. The syringe
QKe system equipped with a protein A Biosensor (ProA) from
choice of the right filtration device.
FortéBio without any interfering sample
filter models Minisart® High Flow (Sartorius, order no.
preparation. The recovery was calculated by the values determined.
16532-K, 0.22 μm PES membrane, EFA = 6.2 cm2) and
In the present work, we show that a proper choice another premium brand (0.2 μm PES membrane,
of the syringe filter device for the clarification of EFA = 5.8 cm2) and were examined regarding their
CHO cell cultures can improve sample throughput and filtration performance by means of the parameters:
filter consumption without having a negative impact on turbidity, mAb recovery, relative yield, throughput, and
turbidity, recovery of mAb product, and total yield. Two filter consumption.
common sterile syringe filters available in the market were
chosen with a pore size of 0.22 μm, slight difference in In 13 cultivation batches, 10 combinations of target
effective filtration areas, and a polyethersulfon membrane. proteins and cultivation methods were used (Table 1).
30
In addition to 125 and 1000 mL shaking flasks, 5 L stirred tank reactor (UniVessel, Sartorius) were also used. The
cell density and viability was examined with the Vi-CELL XR from BeckmanCoulter. As target proteins, CHO cell
lines were selected with mAb from different IgG1 types, IgG2, fc fusion protein and a bispecific antibody. The
specific designation has been anonymized, due to confidentiality agreements.
All cell culture batches were harvested after 14 days. From every batch, two samples were taken (max. 31 mL per
sample), one sample destined for clarification with Minisart ® High Flow and one for another premium brand. The
samples were clarified by centrifugation for 60 min at 4000 g, and the supernatants were filtered with the respective
syringe filters (Figure 1).
The mAb titer was determined in the unharvested and harvested cell culture fluid using the Octet QKe system
equipped with a protein A Biosensor (ProA) from FortéBio without any interfering sample preparation.
TheAs sample
recovery waswith different
calculated volumes
by the values between 25 and 31 ml were compared, the
determined. relative mAb
calculated (Equation 1).
𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣 [mL
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 ] × 𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑣𝑣𝑣𝑣 �mL �
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣
𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚 × 100% = yield [%]
[
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑠𝑠𝑠𝑠𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑠𝑠𝑠𝑠𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 mL ]
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 × 𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 �mL �
𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣
Equation 1: Calculation formula of the relative mAb yield [%].This was necessary to compare the results from d
Equation 1: Calculation formula of the relative mAb yield [%].This was necessary to compare the results from different
sample
sample volumes
volumes ranging
ranging from 24from
mL–3124 - 31
mL. ml.
CCF CCFculture
= cell = cellfluid
culture
(= cellfluid (= cell
culture culture broth).
broth).
As Turbidity
samples withvalues
differentwere measured
volumes between 25 before
and and after
titers (cell clarification
culture) betweenby
0.4 using
and 8.8the TurbiCheck
mg/mL. This WL
31 mL were compared, the relative mAb yield was diversity was the prerequisite for a robust statement in
turbidimeter from Lovibond (white light source). Afterwards the reduction of turbidity was
calculated (Equation 1). terms of the syringe filter suitability.
determined by calculation of the ratio of values from harvested and unharvested samples.
Turbidity values were measured before and after To determine the particle reduction, we examined the
clarification by using the TurbiCheck WL turbidimeter turbidity of the cell culture and the filtrate. We found that
Results and Discussion
from Lovibond (white light source). Afterwards the both filter models removed particles efficiently from the
reduction of turbidity was determined by calculation supernatant. The filtrate of the Minisart® High Flow showed
Goal of the study was to compare
of the ratio of values from harvested and unharvested the suitability
an average of oftwo
17.6 different
NTU, and other syringe
premium filter models for c
brands
of mAb supernatants in regard to particle reduction,
samples. showed an average mAbturbidity
recovery, yield,
of 17.7 NTU. and consumptio
Considering the
entire clarification process, including centrifugation and
units. and Discussion
Results filtration, this leads to a relative reduction in turbidity
The goal of the study was to compare the suitability between 93.8 and 98.8%. Remarkably, the turbidity in
Fordifferent
of two the experiments, we used
syringe filter models both various
for clarification of thecultivation
filtrate does notsystems
depend on and theexpression
initial turbidity vectors.
of the Wi
mAbapproach,
supernatants we generated
in regard a heterogeneous
to particle reduction, mAb range
cell of(Figure
culture characteristics
3). with respect to viable ce
recovery, yield, and consumption of filter units. The various cell culture samples had titers of monoclonal
viability, turbidity, mAb product, and titer (Table antibodies1). in aIn particular,
range of 0.2 to 8.8theg/L. turbidity
The mAb titersof the cell c
Forharvest ranged
the experiments, wefrom 457various
used both to 1431 NTU, theofviable
cultivation cell
the filtrate count
ranged fromranged
0.2 to 8.2from
g/L for 4 x 106 to 16 x 10
both
systems and expression vectors. With this approach, manufacturers, resulting in recovery rates between 89.9
weagenerated
viabilitya heterogeneous
from 48 to 89 range%,ofand mAb titers and
characteristics (cell culture)
103.9% between
(average: 97.7%) for0.4 and
Minisart ® 8.8 mg/ml. This d
High Flow and
was
with thetoprerequisite
respect viable cell count,for a robust
viability, statement
turbidity, mAb 86.9inandterms
107.3% of(average:
the syringe
98.2%) forfilter suitability.
the other premium
product, and titer (Table 1, next page). In particular, the brand. It should be emphasized that the recovery was
turbidity of the cell culture at harvest ranged from 457 independent of the cell culture titer (Figure 3). This is
To determine the particle reduction, we examined the turbidity of the cell culture and the
to 1431 NTU, the viable cell count ranged from 4 x 10 6 to important when regularly monitoring mAb titers during
16 We
x 10 6 found
cells/mL,that bothfrom
a viability filter
48 tomodels
89%, andremoved
mAb particles
cultivation withefficiently
different levelsfrom the concentrations.
of product supernatant. The f
the Minisart® High Flow showed an average of 17.6 NTU and other premium brand31showe
average turbidity of 17.7 NTU. Considering the entire clarification process, including centri
The relative yield Table 1: Overview of various sample types (expression vectors| mAb products) and their parameters
per sample was such as cultivation system (STR = stirred tank reactor and SF = shake flask), viable cell count (VCC)
and viability after 14 days, and turbidity of the cell culture at harvest. Clarification tests were run
the same for both
with both syringe filters, so that the respective volume was clarified with both variants to obtain an
syringe filter models, objective comparison.
despite differences [105 cells/mL] cell culture cell culture volume
[NTU] [mg/mL] [mL]
in housing design
V1 | IgG1 STR (5 L) 86.9 58% 1431 7.8 31
and number of filters
V1 | IgG1 STR (5 L) 155.2 78% 1355 6.0 31
used per sample
V1 | IgG1 STR (5 L) 163.6 89% 828 8.8 31
(Figure 4).
V2 | fc fusion protein SF (25 mL in 125 mL) 121.0 71% 1031 0.2 25
V3 | IgG1 SF (25 mL in 125 mL) 73.0 64% 508 0.9 25
In terms of
V4 | fc fusion protein SF (25 mL in 125 mL) 47.7 67% 457 0.4 24
throughput and filter
V5 | IgG2 SF (25 mL in 125 mL) 112.6 67% 873 0.7 23
consumption, we
V6 | fc fusion protein SF (300 mL in 1 L) 42.2 69% 701 1.8 25
determined for each
V6 | fc fusion protein SF (300 mL in 1 L) 43.5 62% 834 1.2 25
sample the volume
V7 | IgG2 SF (300 mL in 1 L) 38.3 48% 821 0.4 25
of the supernatant,
V8 | IgG1 SF (300 mL in 1 L) 69.9 73% 558 1.6 25
the volume of the
V9 | IgG1 SF (300 mL in 1 L) 52.3 59% 669 0.3 25
total filtrate, and the
V10 | bispecific an body SF (300 mL in 1 L) 46.1 69% 671 0.6 25
required number
of filter units. The
average throughput for Minisart® High Flow was 18.0 mL and for the other premium brand was 9.3 mL and (Figure 5).
This 100% discrepancy cannot be explained by the small difference in the effective filtration area (Minisart® High Flow:
6.2 cm2, other premium brand: 5.8 cm2). More likely, differences in the structural design of the polyethersulfon membrane
utilized in the devices could be the reason for this observation. In consequence, we found an average filter consumption
per sample of 2.5 pcs for Minisart® High Flow and 1.4 pcs for the other premium brand.
These results demonstrate that the Minisart® High Flow allows the filtration of larger volumes before clogging, while other
parameters like turbidity, mAb recovery, and relative yield showed the same high performance.
Conclusion supernatant clarification can be improved substantially

Microfiltration is most commonly an indispensable step after without impairment of other relevant parameters.
centrifugation of a cell culture sample. When processing a
small number of samples with volumes < 25 mL, syringe Acknowledgment
filters are often the perfect choice. Using the right filter Special thanks go to Dr. Noushin Delmdahl and
can significantly facilitate the task and reduce the number Dr. Andrea Friße for reviewing the manuscript and
of devices needed. In this study, we compared two for their constructive discussion on this subject.
different filter models with slight differences in filtration
areas (Sartorius Minisart® High Flow: EFA = 6.2 cm2 Abbreviations
and other premium brand: EFA = 5.8 cm2). However,
CHO Chinese hamster ovary
no significant difference in terms of turbidity, recovery,
and yield per sample could be found. What could be mAb monoclonal antibody
observed is a clear impact of the filter model on the EFA effective filtration area
filtration performance. With its 7% larger filtration area, the
PES polyethersulfon
Minisart® High Flow achieved a 94% higher filtrate volume
per device and thus halved the number of filter units per NTU nephelometric turbidity units
sample. This finding emphasized that the efficiency of
32
Figure 3: The recovery [%] of mAb products in relation to the mAb titer
These results demonstrate that the Minisart® High Flow allows the filtration of larger volumes before
influenced by the syringe filter modelAPPLICATION
and was on average at 97.7 % for
N OT E S
clogging while other parameters like turbidity, mAb recovery, and
premium relative yield
brand. No showed
impact of thethe same
syringe filter used on the mAb recove
high performance. culture titer.
110
Turbidity 100
Rel. Yield
90
100 80
Relative yield mAb [%]

Turbidity Reduction [%]
70
90 60
50
40 82% 83%
80
30
20
70 Minisart High Flow
10
100% Reduction
0
60 Other premium brand Minisart High Flow
400 900 1400
Turbidity unharvested cell culture [NTU] Figure 4: Average values of the relative yield [%] of
Figure
various4:mAbAverage values The
products. of the relative
relative yieldof[%]
yield of various
a sample is mAb produc
Figure 2: Turbidity reduction [%] in relation to the total mAb amount
the relation of the in themAb
total filtrate and inin
amount the
theunharvested
filtrate andcell culture. As
Figure 2: Turbidity
turbidity reduction cell
of the unclarified [%] in relationThe
culture. to the turbidity of the unclarified
clarification cell culture.
models The clarification
irrespective of procedure
theculture.
syringe filter designwe andfound
consumption.
in the unharvested cell As a result, no
comprises
procedureacomprises
centrifugation and a microfiltration
a centrifugation and a step.
micro-The reduction of the turbidity does not depend on the turbidity of the
difference between both filter models irrespective
cell culture.
filtration ThisThe
step. is valid for the Minisart®
reduction High Flow
of the turbidity does asnot
well as for the other premium brand (figure not shown because the
of the syringe filter design and consumption.
data points are virtually the same).
depend on the turbidity of the cell culture. This is valid
for the Minisart® High Flow as well as for the other
premium brand (figure not shown because the data
points are virtually the same). 24
22
Throughput
Throughput per filter unit [mL]
20
18.0 mL
120 Recovery 18
+ 94%
16
14
100
12
9.3 mL
10
Recovery mAb [%]
80
8
6
60
4
2
40 Other premium brand
0
Minisart High Flow Other premium brand Minisart High Flow
20 100% Recovery
Figure 5: CHO cell culture supernatants were filtered
Figure 5: CHO
through cell culturesyringe
two different supernatants were filtered
filter models: through two different sy
Sartorius
0 Minisart High Flow and other premium brand. Theper filter unit was d
Flow and ®other premium brand. The average throughput
models in throughput
average throughput were probably
per filter unitnot
wascaused by the deviation in effective
determined.
0 1 2 3 4 5 6 7 8 9 10
other premium brand: 5.8 cm 2) but most likely by differences in structural me
mAb titer unharvested cell culture [g/L] Differences between both filter models in throughput
were probably not caused by the deviation in effective
Figure 3: The recovery [%] of mAb products in relation to
Figure 3: The recovery [%] of mAb products in relation to the mAb titer of Conclusion
filtration area (Minisart® High Flow: 6.2 cm2, other
the unclarified
premium brand: cell
5.8 culture.
cm2) butThemost recovery
likely was not
by differences in
the mAb titer of the unclarified cell culture. The recovery
influenced
was by the syringe
not influenced filter
by the modelfilter
syringe and model
was onand average Microfiltration
was at 97.7 % forstructural
the Minisart® High
membraneis most
Flow commonly
and
design. at 98.2 %an for indispensable
the other step after c
premium
on averagebrand. No impact
at 97.7% of Minisart
for the the syringe
® filterFlow
High usedand
on the
at mAb recoveryWhenwas observed
processing in aarange
smallofnumber
0.3 to 8.8ofg/lsamples
of the cell
with volumes < 2
culture for
98.2% titer.
the other premium brand. No impact of the perfect choice. Using the right filter can significantly facilitate t
syringe filter used on the mAb recovery was observed in
a range of 0.3 to 8.8 g/L of the cell culture titer. devices needed. In this study, we compared two different filter
100
Rel. Yield filtration areas (Sartorius Minisart® High Flow: EFA = 6.2 cm2 a
90 cm2). However, no significant difference in terms of33turbidity, r
80 be found. What could be observed is a clear impact of the filte
[%]
Correlation Between Colony Forming Units and

Genome Copies of 9 Different Mycoplasma
Species
How Using
to Avoid Quantified CFU
Contamination in and GC Standards
Pipetting
for Validation
#05
Correlation between colony forming units and
Caroline Paeschke1, Dr. Alexandra Mueller-Scholz1*, Dr. Susanne Roederstein2, Kai Nesemann2
Application
genome copies of 9 different Mycoplasma#01 specie
1.Research and Development Department, Sartorius Stedim Biotech, Göttingen, Germany
Note
using quantified CFU and GC standards #02 for valid
2. Department Microbiology, Sartorius Lab Instruments, Göttingen, Germany
*
Correspondence:
Practical methods
E-mail: PCR@Sartorius.com
for avoiding
contamination in
pipetting. #03
#04
Applica
Note
34
Abstract using Sartorius’ quantified CFU and GC standards to

In this study, the correlation between genome copies (GC) facilitate implementation and approval of qPCR-based
and colony forming units (CFU) of 9 different Mycoplasma Mycoplasma detection methods.
species has been investigated using Sartorius’ quantified
CFU and GC standards. As PCR technology only detects Materials and Methods
genome copies (GC), a correlation between colony DNA Extraction of Microsart ® Validation Standard
forming units (CFU) and GC is required by different Each package of Microsart® Validation Standard contains
authorities, i.e., the Korean Food and Drug Administration 3 vials, each containing 10 CFU of the chosen Mycoplasma
(KFDA) or the Pharmaceuticals and Medical Devices species. 250 μL Dulbecco’s Modified Eagle Medium
Agency (PMDA), Japan, before an assay is accepted to be (DMEM) + 10% fetal bovine serum (FBS) were added to
used in quality control of cell culture products. The results two vials to prepare a suspension with a concentration of
of this study show that the number of genome copies vary 40 CFU/mL. 500 μL DMEM +10% FBS were added to one
between Mycoplasma species, but have successfully been vial to prepare a suspension with a concentration of 20
correlated to 20 CFU and 40 CFU respectively. CFU/mL. The DNA of the cell suspensions was extracted
with Microsart® AMP Extraction Kit in duplicates according
Introduction to the protocol. The eluate was used directly for Microsart®
Mycoplasma are the smallest free-living organisms. ATMP Mycoplasma qPCR.
They belong to the bacterial class Mollicutes, which are
distinguished by their lack of cell wall. For that reason they Microsart ® ATMP Mycoplasma qPCR
are unaffected by many commonly antimicrobial All lyophilized components were rehydrated. For one
agents such as beta-lactam antibiotics [1]. Mycoplasma reaction, 15 μL of Mycoplasma Mix were mixed with
are widespread contaminants in cell culture. In fact, 1 μL Internal Control DNA. 15 μL of this mix were added
depending on the laboratory, 10% to 85% of cell lines to each PCR tube. Each test was carried out with at
may be contaminated [2]. Due to their extremely basic least two Non-Template Controls (NTC) and samples in
genomes, mycoplasma live as parasites. They compete duplicate. Therefore 10 μL of sample or NTC were added
with host cells for biosynthetic precursors and nutrients to the PCR tubes with Master Mix respectively.
and can alter DNA, RNA, and protein synthesis and induce
chromosomal alterations [2]. Given their tiny size (~0.3 μm– Standard Curve with Microsart® Calibration Reagent
0.8 μm) mycoplasma contamination cannot be visualized To quantify the DNA extracts of Microsart ® Validation
with a light microscope [1]. Moreover, altered growth rates Standards, it is necessary to generate a standard
and morphological changes in infected cell cultures can curve with known concentrations of genome copies
be minimal or unapparent. Furthermore, mycoplasma- (GC). Therefore Microsart ® Calibration Reagents were
contaminated products represent a human health risk [2]. used. The calibration reagents contain 10 6 GC/μL of
All these facts show clearly the high demand of routine the specific organism after rehydration. Dilution series
mycoplasma detection. havebeen prepared in Tris-buffer to achieve final
concentrations of 5 GC/10 μL to 500 GC/10 μL.
Microsart® ATMP Mycoplasma enables a reliable and
sensitive detection of mycoplasma DNA in cell cultures and Microsart ® ATMP Mycoplasma qPCR was performed
cell culture derived biologicals, like autologous transplants, on the CFX96 Touch Cycler (Bio-Rad; 45 cycles,
according to European Pharmacopeia 2.6.7. Regulations 3 min 95°C, 30 s 95°C, 30 s 55°C, 45 s 60°C). The
require comparability studies with compendial growth based mycoplasma DNA is indicated by an increasing
methods. As PCR technology only detects genome copies fluorescence signal in the FAM ® channel. Internal
(GC), a correlation between colony forming units (CFU) and Control DNA is detected in the ROX® channel in the
GC is required by different authorities, i.e., the Korean Food same tube to indicate a successful reaction in every
and Drug Administration (KFDA) or the Pharmaceuticals and individual PCR tube. The analysis of the reaction was
Medical Devices Agency (PMDA), Japan. done with the CFX Manager Software (Bio-Rad). The
limit of detection of all Mycoplasma species listed in
In this study, the correlation between CFU and GC the EP/USP is ≤ 10 CFU/mL.
of 9 different Mycoplasma species was investigated
35
the protocol. The eluate was used directly for Microsart® ATMP
Mycoplasma qPCR.
Table 1: Product overview of Microsart® Validation Standards and Microsart® Calibration Reagents for different mycoplasma species.
Table 1: Product overview of Microsart® Validation Standards and Microsart® Calibration Reagents for different Mycoplasma
The Validation Standard contains 10 CFU per vial, the Calibration Reagent contains 106 GC/µl after rehydration.
species.The Validation Standard contains 10 CFU per vial, theResults Calibration Reagent
and contains 106 GC/μL after rehydration.
Discussion
Figure 2 andCatalog
3 show No.
exemplary amplification
Catalog plots
No. of
A. laidlawii. On basis of® the ct values (FAM™ channel) and
Mycoplasma
Mycoplasma species NCTC code ATCC code Microsart Validation Microsart® Calibration
concentrations of
Standard
the standards the CFX Manager
Reagent
software
created a standard curve (Fig. 1) with a linear equation.
Mycoplasma arginini 10129 23838 Results
A and
regression Discussion
SMB95-2011
coefficient
SMB95-2021
of 0.983 is an indication for a good
Mycoplasma orale 10112 23714 Figure 2 and
standard SMB95-2012
3 show
curve. SMB95-2022
exemplaryofamplification
The efficiency an optimal plotsisof100 %.
qPCR
Mycoplasma gallisepticum 10115 19610 A. that
In laidlawii
case. On basis
SMB95-2013
the of theDNA
amplicon ct values
will be(FAM™ channel)
SMB95-2023
doubled in eachand
cycle.
Mycoplasma pneumoniae 10119 15531 concentrations
According to of
the the standards
analysis
SMB95-2014 of the the
CFX CFX Manager
Software
SMB95-2024the software
exemplary
created
qPCR runa standard curveran
A. laidlawii
of SMB95-2015 (Fig. 1) with
highly a linearwith
efficient equation.
an efficiency
Mycoplasma synoviae 10124 25204 SMB95-2025
A regression
of 101 % (seecoefficient
efficiency of
in 0.983
Fig 1). is an indication for a good
Mycoplasma fermentans 10117 19989 standard curve.SMB95-2016 SMB95-2026
The efficiency of an optimal qPCR is 100 %.
Mycoplasma hyorhinis 10130 17981 In that case the amplicon DNA will beSMB95-2027
SMB95-2017 doubled in each cycle. Figure
Acholeplasma laidlawii 10116 23206 According toSMB95-2018
the analysis of the CFX Software
SMB95-2028the exemplary with M
Spiroplasma citri 10164 27556 A. laidlawii ran highly efficient
qPCR run of SMB95-2019 with an efficiency
SMB95-2029 channe
of 101 % (see efficiency in Fig 1). Blue: 1
Results and Discussion 20 CFU

Figure
Figures 2 and 3 show exemplary amplification plots detect
with M
of A. laidlawii. On basis of the ct values (FAM ® determ
channe
Microsart ®
ATMP Mycoplasma qPCR Blue: 1
channel) and concentrations of the standards, the
All lyophilized components were rehydrated. For one reaction Microsart® ATMP Mycoplasma qPCR was performed on the CFX96 Based
CFX Manager software created a standard curve
15 µl of Mycoplasma Mix were mixed with 1 µl Internal Control Touch Cycler (Bio-Rad; 45 cycles, 3 min 95 °C, 30 s 95 °, 30 s 55 °C, 20 CFU
calcul
(Figure
DNA. 15 1) with
µl of thisa mix
linear
wereequation. A regression
added to each PCR tube. coefficient
Each test 45 s 60 °C). The mycoplasma DNA is indicated by an increasing detect
(20 CF
of 0.983
was carriedisout
an with
indication
at leastfor
twoaNon
good standard
Template curve.
Controls The
(NTC) fluorescence signal in the FAM™ channel. Internal Control DNA determ
CFU ra
efficiency
and samplesofinan optimalTherefore
duplicate. qPCR is10100%. In thatorcase
µl of sample the
NTC were is detected in the ROX™ channel in the same tube to indicate a
Figure 1: Exemplary standard curve of Acholeplasma laidlawii (Microsart®
added
ampliconto the PCR will
DNA tubesbewith MasterinMix
doubled respectively.
each cycle. According successful reaction in every individual PCR tube. The Analysis of Based
Calibration Reagent), using final Genomic Copy (GC) concentrations of
to the analysis of the CFX Software, the exemplary 5 the reaction
GC/10 was
µl to 500 doneµl.with
GC/10 the CFX
generated Manager
with Software
Microsart® (Bio-Rad).
ATMP Mycoplasma. calcul
Table 2
The limit of detection of all mycoplasma species listed in the (20 CF
qPCR run of A. laidlawii ran highly efficient with an EP/USP is ≤ 10 CFU/ml.
efficiency of 101% (see efficiency in Figure 1). CFU ra
Mycop
Each sample and Non-Template Control (NTC) showed an amplifi-
Figure 1:1:Exemplary
Figure Exemplary standard Acholeplasma
curve ofcurve
standard laidlawii (Microsart
®
of Acholeplasma
cation of(Microsart
the internal Mycop
Calibration Reagent), ® control
using DNAReagent),
final Genomic and
Copyconsequently
(GC)using fluorescence
concentrations of
Each sample and Non-Template Control (NTC) showed laidlawii Calibration final genome
5 GC/10in
signal µl the
to 500 GC/10
ROX™ µl. generated
channel withdata
(Ct<40; Microsart
not
copy (GC) concentrations of 5 GC/10 μL to 500 GC/10 μL
ATMP Mycoplasma.
shown).
®
A success- Mycop
Table 2
an amplification of the internal control DNA and fully PCR without inhibition
® was indicated. The NTC did not show
generated with Microsart ATMP Mycoplasma. Mycop
consequently fluorescence signal in the ROX® channel a fluorescence signal in the FAM™ channel, as expected (see Fig. 2 Mycop
Mycop
(Ct<40; data not shown). A successful PCR without Each sample
and 3). and Non-Template
Consequently Control
a mycoplasma free(NTC) showedofanthe
preparation amplifi-
PCR
cation of the internal control DNA and consequently fluorescence Mycop
Mycop
inhibition was indicated. The NTC did not show a reactions without cross-contamination was indicated.
fluorescence signal in the FAM® channel, as expected signal in the ROX™ channel (Ct<40; data not shown). A success- Mycop
Mycop
(see Figures 2 and 3). Consequently a mycoplasma-
fully PCR without inhibition was indicated. The NTC did not show Mycop
Mycop
a fluorescence signal in the FAM™ channel, as expected (see Fig. 2
free preparation of the PCR reactions without cross- Mycop
Achole
and 3). Consequently a mycoplasma free preparation of the PCR
contamination was indicated. reactions without cross-contamination was indicated. Mycop
Spirop
3 Mycop
20 CFU/mL and 40 CFU/mL of each Mycoplasma Mycop
species have been detected successfully in all samples The st
Achole
specie
(Assay LOD is ≤ 10 CFU/mL; determined during kit
Spirop
DNA e
validation).
Based on the linear equation of the standard curve, The st

Figure 2:2:Exemplary
Figure Exemplaryamplification plot of Acholeplasma
amplification laidlawii, generated
plot of Acholeplasma
the software calculated the GC concentrations of the with Microsart® ATMP Mycoplasma qPCR. Fluorescence signals in FAM™ specie
laidlawii, generated with Microsart ATMP Mycoplasma qPCR.
®
mycoplasma samples (20 CFU/mL and 40 CFU/mL channel. Black Lines: Non template Control (NTC). Blue Lines: 20 CFU/ml of DNA e
Fluorescence signals in FAM® channel. Black Lines:
extracts). In Table 2, the average GC to CFU ratios of A. laidlawii. Red Lines: 40 CFU/ml of A. laidlawii.
Non-Template Control (NTC). Blue Lines: 20 CFU/mL of
9 different Mycoplasma species are shown. A. laidlawii. Red Lines: 40 CFU/mL of A. laidlawii.
Figure 2: Exemplary amplification plot of Acholeplasma laidlawii, generated
36 with Microsart® ATMP Mycoplasma qPCR. Fluorescence signals in FAM™
channel. Black Lines: Non template Control (NTC). Blue Lines: 20 CFU/ml of
A. laidlawii. Red Lines: 40 CFU/ml of A. laidlawii.
The study indicated that the GC/CFU ratio varied from approval. These results demonstrate a good data
species to species and lies within a range of 9 GC/CFU basis. Nevertheless it should be kept in mind that
to 68 GC/CFU after DNA extraction. significant variations within the GC:CFU ratio might be
observed if other media | matrices are used for the CFU
spikes, which affects the DNA isolation efficiency, or if
other conditions are used for correlation. The results
of this study indicate a higher GC than CFU number, as
y expected. The theoretical GC:CFU ratio should be
1:1, as 1 GC per cell should ideally be detected.
Practically, this ratio is not realizable even if
Figure 3: Exemplary amplification plot of Acholeplasma laidlawii, generated
with Microsart® ATMP Mycoplasma qPCR. Fluorescence signals in FAM™ mycoplasma cultures are harvested during early
channel. Black Lines: Non template Control (NTC). Red: 5 GC/PCR. logarithmic growth to prevent detection of DNA
y Blue: 10 GC/PCR. Green: 50 GC/PCR. Yellow: 100 GC/PCR. Brown: 500 GC/PCR.
from dead cells in the preparation. This non-equal ratio
20 CFU/ml and 40 CFU/ml of each mycoplasma species have been arises because a significant number of the mycoplasma
detected successfully inamplification
all samples
plot of (Assay LOD islaidlawii
≤ 10 CFU/ml; cells would not grow to a colony in culture and remain
Figure 3:3:
Figure Exemplary amplification
Exemplary plot of Acholeplasma
Acholeplasma , generated
with Microsart
laidlawii,
determined ®
ATMP Mycoplasma
during
generated kit with qPCR. Fluorescence
validation).
Microsart ® signals in FAM™
ATMP Mycoplasma undetected (i.e., stressed or viable but non-culturable
channel. Black Lines: Non template Control (NTC). Red: 5 GC/PCR.
qPCR. Fluorescence signals in FAM® channel. Black Lines: cells). Furthermore, mycoplasma cells tend to form
Blue: 10 GC/PCR. Green: 50 GC/PCR. Yellow: 100 GC/PCR. Brown: 500 GC/PCR.
Non-Template
Based Control
on the linear (NTC).
equation Red:
of the 5 GC/PCR.
standard curveBlue: 10 GC/
the software agglomerates, which would be detected as 1 CFU,
PCR. Green:
calculated the 50
GCGC/PCR. Yellow:
concentrations 100mycoplasma
of the GC/PCR. Brown:
samples
20 CFU/ml and but in fact combine several cells and therefore
500
(20 GC/PCR.
CFU/ml and 40
40 CFU/ml
CFU/ml of each mycoplasma
extracts). species
In table 2 the have
average GCbeen
to
detected
CFU ratiossuccessfully in all
of 9 different samples (Assay
mycoplasma LOD
species areisshown.
≤ 10 CFU/ml; several GC. Both scenarios lead to a significant
®
determined during kit validation). underestimation of the realistic mycoplasma cell
number in the sampled cell culture, as only a portion
a. Table
Based2:2: Average
onAverage GC
the linear to CFUofratio
equation of 9 different
the9 standard curve the software
Table GC to CFU ratio of different mycoplasma species of the cells would grow to form a CFU. Non-culturable
Mycoplasma
calculated thespecies.
GC concentrations of the mycoplasma samples
(20 CFU/ml and 40 CFU/ml extracts). In table 2 the average GC to species or viable but non-culturable cells could lead to
Mycoplasma Species Average GC to CFU ratio false-negative results using a growth-based method.
plifi- CFU ratios of 9 different mycoplasma species are shown.
®ence Mycoplasma arginini 1.1 × 10 GC/CFU Undetected mycoplasma contamination because of
ss- Mycoplasma orale 3.5 × 10 GC/CFU false-negative results in growth-based methods can
a.
how Table 2: Average GC to CFU ratio of 1.7
9 different mycoplasma species
Mycoplasma gallisepticum × 10 GC/CFU result in unsafe products with potential infection risks,
Fig. 2 especially for patients with immunodeficiency. This
Mycoplasma pneumoniae 4.3 × 10 GC/CFU
CR Mycoplasma Species Average GC to CFU ratio
plifi- Mycoplasma synoviae 0.9 × 10 GC/CFU study shows that the correlation between GC and
ence Mycoplasma fermentans
Mycoplasma arginini 1.1××10
1.2 10GC/CFU
GC/CFU CFU can successfully be demonstrated and easily be
ss- Mycoplasma hyorhinis
Mycoplasma orale 3.5××10
0.9 10GC/CFU
GC/CFU implemented during validation. Furthermore, detection
how of GC by PCR shows a more realistic result of the
Acholeplasmagallisepticum
Mycoplasma laidlawii 1.7 × 10 GC/CFU
5.6 × 10 GC/CFU
Fig. 2 real contamination level in the respective sample and
Mycoplasma pneumoniae
Spiroplasma citri 4.3××10
6.8 10GC/CFU
GC/CFU
CR
Mycoplasma synoviae 0.9 × 10 GC/CFU therefore directly contributes to drug safety.
Mycoplasma fermentans 1.2 × 10 GC/CFU
The study indicated that the GC/CFU ratio varied from species to
Mycoplasma hyorhinis 0.9 × 10 GC/CFU
Discussion
species and lies within a range of 9 GC/CFU to 68 GC/CFU after References
Acholeplasma
DNA extraction.laidlawii 5.6 × 10 GC/CFU
In this study, the correlation between genome copies 1. Drexler HG, Uphoff CC. Mycoplasma contamination
Spiroplasma citri 6.8 × 10 GC/CFU
(GC) and colony forming units (CFU) of 9 different of cell cultures: Incidence, sources, effects, detection,
Mycoplasma species has been investigated using elimination, prevention. Cytotechnology. 2002;39(2):75-90.
ted
Sartorius’
The quantified
study indicated GCGC/CFU
that the and CFU ratiostandards.
varied from A
species to doi:10.1023/A:1022913015916.
of correlation
species between
and lies within a CFU
range and GC is required
of 9 GC/CFU by after
to 68 GC/CFU 2. Olarerin-George AO, Hogenesch JB. Assessing the
different
DNA authorities, i.e., the Korean Food and Drug
extraction. prevalence of mycoplasma contamination in cell culture via
Administration (KFDA) or the Pharmaceuticals and a survey of NCBI’s RNA-seq archive. Nucleic Acids Research.
Medical Devices Agency (PMDA), Japan, for assay 2015;43(5):2535-2542. doi:10.1093/nar/gkv136.
ted
of
37
Scouting Protein
How to Avoid Purification
Contamination in PipettingConditions
Using Vivapure Centrifugal Ion Exchange
#05
Membrane Absorbers
Application #01
C. Naumann and N. Kashani-Poor
Note
for avoiding
contamination in
pipetting. #03
Introduction
#04
For separation and purification of proteins from biological samples, different
characteristics of the target protein, e.g., its size, charge, hydrophobicity, or
specifically engineered tags, are exploited.
With ion exchange chromatography, separation is achieved on the basis of

different charges of biomolecules. This makes it a versatile method often
used for prefractionation or purification of a target protein from crude protein
mixtures. To optimize the purification procedure for an individual target,
several binding and elution conditions have to be tested on cation and anion
exchange matrices.
38
trifugal Ion Exchange Membrane Absorbers APPLICATION N OT E S
mann and N. Kashani-Poor

In contrast to traditional column chromatography
methods, Vivapure IEX centrifugal columns allow
tion In the first step of this protocol, binding con- Buffers used
scouting of
ation and purification ofseveral
proteins chromatography conditions
ditions are evaluated in the sample
by loading Buffer A: 25 mM citrate, pH 4
parallel,
ogical samples, leading
different quickly to
character- ondifferent
Vivapure Qfractions which
and S columns at various pH-
he target protein
can bee.g. its size,analyzed
further charge, forvalues, eluting or
enriched bound
even proteins
already with a high Buffer B: 25 mM potassium phosphate,
bicity or specifically engineered tags salt concentration buffer and analyzing all pH 6
purified target protein.
it. fractions for the target protein. This step Buffer C: 25 mM HEPES, pH 8
results in the optimal binding pH and the best
Here, we demonstrate the
exchange chromatography, ionperformance
exchange chemistry of Vivapure IEX
for the purification. Buffer D: 25 mM sodium bicarbonate,
n is achieved on the basis of pH 10
Mini spin columns for evaluation of optimal purification
charges of biomolecules. This makes In a second step, the best elution method is Buffer E: 25 mM citrate, pH 4, supple-
conditions
satile method often used offor
cloned
pre- SH2evaluated
domains byfrom
applyingan increasing
E. coli lysate
salt concen- mented with 1 M NaCl.
in a two-step
tion or purification procedure.
of a target pro- This protocol
trations to columns can generally
which be to
were shown
25 mM potassium phosphate, pH 6,
crude protein mixtures.for
employed Tofinding bind the target
optimizea purification proteinbased
method in stepon leading to Buffer F:
one,ion
supplemented with 0.2 M, 0.4 mM,
cation procedure for an individual a complete purification protocol in less than
exchange
veral binding and elution chromatography
conditions onefor a given target protein, as
hour.
0.6 mM, 0.8 mM,
it is fast and only uses up small amounts of the sample. & 1 M NaCl, respectively.
e tested on cation and anion
matrices. Experiment Buffer G: 25 mM HEPES, pH 8,
Using the described scouting procedure, supplemented with 1 M NaCl
In the first step of this protocol, binding conditions are
st to traditional column chromatog- a purification method for a SH2 domain Buffer H: 25 mM sodium bicarbonate,
evaluated
thods, Vivapure by loading the expressed
IEX centrifugal sample on in E.Vivapure Q and S In a first
coli was developed. pH 10, supplemented with
columns
allow scouting at various
of several chro- pH values, eluting
step, proteins bound
were bound proteins
to the Vivapure IEX 1 M NaCl
phy conditions
withinaparallel,
high salt leading membranes
concentration buffer at and
different pH values, then
analyzing
o different fractions which can be eluted with high-salt buffer. In Step Two a
nalyzed forall fractions
enriched for already
or even the targetfresh
protein.
sampleThis
was step results
adjusted to the respective
in the optimal binding pHpH
arget protein. and the best
elucidated ion exchange
previously as the best choice
chemistry for the purification. for binding the protein and was loaded onto
demonstrate the performance of
IEX Mini spin columns for evaluation conditions.
a new column for refining optimal elution
Procedure
In the
al purification second
conditions of step,
clonedthe best elution method is evaluated
ains from an byE.applying
coli lysateincreasing
in a two salt concentrations to columns
Materials Step One: Scouting for Binding Conditions to
edure. Thiswhich
protocol can generally be – Vivapure Mini
were shown to bind the target protein in stepQ H spin columns the Appropriate Ion Exchange Chemistry
d for finding a purification method – Vivapure Mini S H spin columns
one, leading to
ion exchange chromatography for a a complete purification protocol in
– Minisart syringe filter (0.45 µm CA,less
get proteinthan one
as it is fasthour.
and only uses Sartorius AG) Expression of Target Protein
amounts of the sample. – Centrifuge, 45°-fixed-angle rotor; 2000 300 + g mL LB media were inoculated with 4 mL of an
Experiment overnight culture and incubated at 37°C, shaking at
Using the described scouting procedure, a purification 150 rpm until an OD600 of 1.0 was reached. IPTG was
method for a SH2 domain expressed in E. coli was added to a final concentration of 1 mM and incubated for
developed. In Step One, proteins were bound to further 4 h with shaking at 150 rpm. Cells were harvested
the Vivapure IEX membranes at different pH values, by centrifugation at 4000 x g for 30 min at 4°C. The
then eluted with high-salt buffer. In Step Two, a fresh pellet was resuspended in 35 mL PBS (150 mM KPi, pH
sample was adjusted to the respective pH elucidated 7.3) and cells were lysed by addition of lysozyme to a
previously as the best choice for binding the protein final concentration of 0.1 mg/mL and incubation for 1 h
and was loaded onto a new column for refining optimal at 37°C. Insoluble particles as cell debris were removed
elution conditions. by centrifugation at 10000 x g for 30 min at 4°C.
Materials Sample Preparation

– Vivapure Mini Q H spin columns 4 x 200 μL of the cell lysate were diluted with 1.8 mL
– Vivapure Mini S H spin columns binding buffer A to D each, to adjust the sample to the
– Minisart syringe filter (0.45 μm CA, Sartorius AG) respective pH conditions. In order to avoid clogging
– Centrifuge, 45°-fixed-angle rotor; 2000 x g of the membranes in the Vivapure Mini spin columns,
samples were clarified by passage through Minisart
syringe filters.
39
re
Column Equilibration
e: Scouting pH = 6 pH = 8 pH = 10
4 x for binding
Q and 4 x Sconditions
Vivapure
ppropriate ion exchange
Mini spin columnschemistry.
were
labeled 4, 6, 8, and 10,
on of target protein
corresponding
B media were inoculated to withthe4 pH of
ml of 66 kDA
theand
ight culture buffer to be used.
incubated To
at 37°C,
at 150 rpm untilspin
each an OD600
column, of 1.0 was
IPTG was400added to a
μL of the final concentra- 45 kDA
mM and corresponding
incubated for further 4h Target
binding protein
king at 150 rpm. Cells were harvested
ifugation buffer
at 4000were added and
+ g for 30 min at
pellet was resuspended inat352000
spun for 5 min x g.
ml PBS 31 kDA
M KPi, pH 7,3) and cells were lysed by
Binding
of lysozyme and
to a final Washing
concentration
g/ml and incubation
400 μL of the 1clarified
for h at 37°C.
e particlessamples
as cell debris were
adjusted to
removed
ifugation at 10000 + g for 30 min at 22 kDA
pH values 4, 6, 8, and
10 were applied each
to the correspondingly
preparation
µl of the cell lysate wereVivapure
equilibrated diluted with Sample Sample Volume loaded
nding buffer A toSDspin
each,columns.
to adjust volume (L) on the gel (L)
Q and
ple to the respective pH conditions. In M = Broad range marker
Columns were spun for s = Sample before application 800 4
avoid clogging of the membranes in
pure Mini5spin
mincolumns,
at 2000samples
x g. were f = Flow-through 800 4
by passage through Minisart syringe w = Wash fraction 400 4
Afterwards, Vivapure e = Elution with 1 M NaCl 200 4
Mini spin columns were
equilibration
reloaded with 400 μL Figure 1: Scouting for optimal binding conditions of a SH2 domain expressed in E. coli. SDS
d 4 + S Vivapure Mini spin columns gel1:(reducing,
Fig. 12%), binding
Scouting for optimal silver stained. Shown
conditions of are sample
a SH2 domain expressedbefore
in E. coliloading, flow-through,
. SDS gel (reducing, wash,
12%), silver stained.
sample and spun again Shown are sample before loading,
eled 4, 6, 8 and 10 corresponding to and elution fractions (1 Mflow-through,
NaCl) fromwash, and elution
Vivapure Q andfractions
S Mini(1 Mspin
NaCl)columns,
from Vivapure
at Qthe
andvarious
S Mini spinpH
for to
f the buffer 5 min at 2000
be used. x g.spin
To each
columns, at the various pH values tested.
values tested.
400 µl of Loosely bound proteins
the corresponding binding
ere addedwere washed
and spun for 5away withatthe application of 400 μL of
minutes in this experiment. As can be seen on the SDS gel in
g. the respective binding buffer to each of the columns Figure 1, the target protein was present in the eluate
and spun for 5 min at 2000 x g. Flow-through and wash of the Vivapure Q Mini spin column at all pH values
and washing Analysis Differences could be detected in the binding
fractions were collected for subsequent detection of the tested together with most of the E. coli proteins
f the clarified samples adjusted to pH 4 µl of flow-through, wash, and elution efficiency of the target protein as at pH 8
target
6, 8 and 10 wereprotein.
applied each to the fractions from each column were analyzed (Lanes Q “e”). tracesIn contrast, usingprotein
of the target the Vivapure
were already S Mini
ndingly equilibrated Vivapure Q and S on reducing SDS-PAGE followed byspin column, at
silver all pH-values
found tested, most
in the flow-through, E. coli high-
with slightly
Complete
umns. Columns were spun Elution of at
for 5 min Bound Proteins
staining. proteins did not bind to the
er amounts at pH membrane and At
10 (Lane S "e"). werepH 6, found
the
g. 200 μL of elution buffer E, F, G, and H were applied to most efficient binding of
in the flow-through (Lane S “f”), thus resulting in purethe target protein
the washed columns and spun Result
for 3of minStep
at One
2000 x g. target protein to in the S membrane
all elution was observed.
fractions (Lane SNow “e”).that
rds, Vivapure Mini spin columns were Dilution of the E. coli lysate with binding the binding conditions, i. e. binding pH and
with 400Eluates
µl samplewere saved for subsequent analysis.
and spun again buffer A (25 mM Citrate, pH 4) lead to com- the best suited ion exchange chemistry, were
n at 2000 + g. Loosely bound proteins plete precipitation of sample proteins. Differences
Thus, could
found, bethe
detected in the binding
elution protocol of the target
Analysis
shed away with the application of pH 4 could not be tested in this experiment.efficiency of the target protein,
protein was optimized asinata pH 8, traces
second step.
f the respective binding buffer to As can be seen
4 μL of flow-through, wash, and elution fractions from on the SDS gel in figure 1, the
of the target protein were already found in the flow-
the columns eachandcolumn
spinningwerefor 5analyzed
min at on target proteinSDS-PAGE,
reducing was present in the eluatethrough,of with slightly higher amounts at pH 10 (Lane
g. Flow-through and wash fractions
followed by silver staining.
the Vivapure Q Mini spin column at all pH
S “e”). At pH 6, the most efficient binding of the target
lected for subsequent detection of the values tested together with most of the
rotein. E. coli proteins (Lanes Q "e"). In contrast,protein to the S membrane was observed. Now that
Result of Step One using the Vivapure S Mini spin column, the binding
at conditions, i.e., the binding pH and the
te elutionDilution
of boundofproteins
the E. coli lysate with bindingtested,
all pH-values buffermostA E. coli proteins
best suited ion exchange chemistry, were found, the
f elution buffer
(25 mM E, F,citrate,
G and H,pHwere did not bindprecipitation
4) led to complete to the membrane andelution were protocol of the target protein was optimized in
o the washed columnsproteins.
of sample and spun Thus,
for pHfound4 couldin the flow-through
not be tested (Lane Lane S "f"), step.
a second
2000 + g. Eluates were saved for thus resulting in pure target protein in all
ent analysis.
40 elution fractions (Lane S "e").
ns
pH = 6 Loosely bound proteins were washed away by
e, application of 400 μL binding buffer to the column and
ml spun for 5 min at 2000 x g. Flow-through and wash
rder
he fraction were saved for analysis.
sted 66 kDA
ha
Stepwise Elution
45 kDA
Target 100 μL elution buffer F, supplemented with 0.2 M NaCl,
one protein were applied to the Vivapure S Mini spin column and
or
spun for 3 min at 2000 x g. The eluate was collected. In
31 kDA
the next step, 100 μL of elution buffer F, supplemented
ed with 0.4 M salt, were applied and again spun for 3 min at
nd 22 kDA 2000 x g. Elution was continued until the entire gradient
the
ed had been tested, saving the eluates from each step.
min
Sample Sample Volume loaded
volume (L) on the gel (L) Analysis
ay by M = Broad range marker 4 μL of flow-through, wash, and elution
he s = Sample before application 800 16
+ g. f = Flow-through 800 16 fractions from each column were analyzed on reducing
aved w = Wash fraction 400 16 SDS-PAGE, followed by silver staining.
e1 = 25 mM KPi, pH 6, 200 mM NaCl 100 8
e2 = 25 mM KPi, pH 6, 400 mM NaCl 100 8
e3 = 25 mM KPi, pH 6, 600 mM NaCl 100 8 Result of Step Two
ith e4 = 25 mM KPi, pH 6, 800 mM NaCl 100 8
eS e5 = 25 mM KPi, pH 6, 1 M NaCl 100 8 The target protein started to elute with 200 mM NaCl,
however the main fraction eluted with 400 mM NaCl.
e next Figure 2: Scouting for optimal elution conditions of a SH2 domain
ented Fig. 2 Scouting for optimal elution conditions of a SH2 domain expressed in E. coli. SDS gel (reducing, 12 %), silver stained.Traces of the target protein were also found in the next
expressed
Sample before loading, E. coli. SDS
inflow-through, gel
wash, and (reducing,
elution fractions from12%),
Vivapure Ssilver
Mini spinstained.
column at pHSample
6 are shown.
pun elution step with 600 mM NaCl, but this might be dueto
nued before loading, flow-through, wash, and elution fractions from
d, Vivapure S Mini spin column at pH 6 are shown. the low elution volume.
Discussion
Step Two: Optimizing Elution Conditions A two-step procedure was used to rapidly scout optimal
ed
r purification conditions for a target protein (a SH2 domain
Sample Preparation from E. coli lysate) with ion exchange chromatography. In
Taking account of the results of Step One, 200 μL cell the first step, the most suited buffer pH for binding the target
lysate were diluted with 1.8 mL binding buffer B (25 mM protein to the most adequate ion exchanger was verified.
n
target KPi, pH 6). In order to avoid clogging of the membrane In the second step, the elution condition was optimized,
ion in the Vivapure Mini spin column, the pH adjusted building on the results gained in Step One of this protocol
be due
sample was clarified by passage through a Minisart (elution optimization after optimal binding of the target to the
syringe filter. proper ion exchanger). With the scouting procedure described
here, it was possible to quickly and conveniently purify the
Column Equilibration target protein to homogeneity. The results obtained in this
400 μL binding buffer B were applied to one experiment can be used for various ends, e.g.:
Vivapure S Mini spin column and spun for
– polishing a specific protein after a first chromatography step
5 min at 2000 x g.
with another chemistry
Binding and Washing – establishing quickly a FPLC method for a new protein
400 μL of the clarified sample were applied to the – finding a purification method for a new protein for
equilibrated Vivapure S column and spun for 5 min at upscaling with Vivapure Maxi or Mega.
2000 x g. Afterwards, the Vivapure S Mini spin column
was reloaded with 400 μL sample and spun again for For these purposes, Vivawell 96-well plates, Vivapure Maxi,
5 min at 2000 x g. and Sartobind membrane adsorber units with FPLC
connectors are available.
41
Additional Resources
Trainings
Customized Training for Lab Professionals. With our Lab Academies we strive to offer a beneficial
selection of training courses in pipetting, filtration, lab weighing and lab water purification. For the most
How to Avoideffective learning,inour
Contamination training sessions are custom-tailored to target your specific needs and to enable
Pipetting
you to excel in your daily tasks. Contact us today to advance your expertise. https://promotions.sartorius.com/
passionforscience/trainings
#05
Sartorius Pipetting Academy. Sartorius Pipetting Academy offers training and content aimed at not only
beginners to pipetting but also to experienced lab professionals.
Application Pipetting Academy modules are a balance
#01
of theory and hands-on sessions. They are packed with tips
Note and tricks that help you get the most out of your
pipetting. Join the Pipetting Academy now. https://promotions.sartorius.com/pipetting-academy
for avoiding
contamination in
Life Science & Biopharma Training Courses.
pipetting. #03 At different training sites we designed methodical training
courses in the life sciences and biopharma sectors. The theoretical part will help you better understand the
background on your application and Sartorius products. In the practical part, you can apply what you have
#04 methods. https://www.sartorius.com/en/knowledge/trainings
just learned and quickly assimilate procedural
The Knowledge Source

Concentrated Scientific Knowledge at a Glance. Take your tour through “SCIUS” and take advantage
of our comprehensive source of scientific expert knowledge. Discover scientific publications, application
notes, expert lectures, webinars, and many more valuable resources to support your big ideas and
small daily routines alike. Start your SCIUS search now. https://promotions.sartorius.com/passionforscience/
scius-the-knowledge-source
Prize
The Sartorius & Science Prize for Regenerative Medicine & Cell Therapy. The Sartorius & Science Prize
for Regenerative Medicine & Cell Therapy is an annual prize aimed at supporting and encouraging scientists
focused on basic or translational research that advances medical progress in regenerative medicine and
cell therapy. Are you a representative of the upcoming generation of thought leaders in your field? Then
go ahead and apply in April 2019. https://promotions.sartorius.com/passionforscience/sartorius-and-science-prize
Guides
Sartorius Cubis® Premium Balances. In this guide you will find an overview of various weighing
applications from the fields of biopharma, pharma, experimental medicine and chemical engineering
published in peer-reviewed academic journals. https://goo.gl/JneXLA
Products
Avoid Sample Transfer and Loss with Cubis® High-Capacity Micro Balances. Weigh minimum
amounts of sample directly into heavy flasks: With Cubis® High-Capacity Micro Balances, you can
add your small samples directly to large containers of up to 250 mL and avoid sample losses and
errors in your experiments. https://promotions.sartorius.com/cubis-high-capacity-micro-balances
Speed Up Your Clarification and Sterile Filtration up to 70%. Sartoclear Dynamics® Lab kits have been
designed for harvesting 15 mL to 1000 mL volumes of cell cultures in the lab. They enable clarification
and sterile filtration to be performed in one step, quickly and easily. https://promotions.sartorius.com/
sartoclear-dynamics-lab
42
arium® mini:
The Only Ultrapure Water System with
Integrated Bagtank Technology
Easy to operate with

touch screen activated
color display, the
compact arium® mini
delivers reliable and
reproducible results.
Its unique bagtank technology
can save valuable time in
cleaning and prevent permanent
biofilm, ensuring the highest
water quality for your
applications.
To find out more, visit:

www.sartorius.com/arium-mini
Take Your Microwell Plate Pipetting
Back to the Comfort Zone
Looking for ways to speed up

Microwell plate pipetting
without errors?
Sartorius AD
Experience the Picus® with the unique
Plate Tracker feature that keeps track
of where you need to pipette next. In
combination with the multi-dispensing
mode, plate work has never been easier
and faster!
To find out more, visit:

www.sartorius.com/picus

Your Practical Guide To Basic Laboratory Techniques: A Sponsored Supplement To Science

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Your Practical Guide To Basic Laboratory Techniques: A Sponsored Supplement To Science

Uploaded by

Copyright:

Available Formats

A Sponsored Supplement to Science

Your Practical Guide to

Pushing the Boundaries of Knowledge

Practical 3 Taking the First Steps

Laboratory Feature Articles

7 The Paperless Lab

10 How to Avoid Contamination in Pipetting

13 Concentration to a Defined Final Volume with

20 How to Achieve Optimal Weighing Performance

Cover Image: © abscent/shutterstock.com

© 2018 by The American Association

In a scientific laboratory, there are also fundamental skills that

Gaining competence in accurately weighing dry reagents is a

researchers to ensure Filtration is a foundational technique used ubiquitously in the

With increasing focus in the scientific community on reproducibility

Sean Sanders, Ph.D.

“A Journey of a With such a strong focus on exciting discoveries—like the next

Sartorius, a global laboratory products and services supplier for the

Ferencz Paldy, Ph.D.

Fiona Coats, Ph.D.

Building Skills in Basic Lab Techniques:

Others agree on the value of the right attitude with this

Keeping cultures healthy

Increasingly, scientists must ensure the integrity of cultures.

The Paperless Lab of software development for the Accelrys

The Paperless Lab

468 sciencemag.org/products SCIENCE

kept a homemade database of ministration encourage electronic documentation, Dathe says,

Originally published 25 July 2014 in SCIENCE

of scientific informatics Featured Participants Santa Cruz has traveled

Originally published 25 July 2014 in SCIENCE

Introduction tips manufactured from 100% virgin polypropylene in

Pipette-to-Sample Contamination Sample-to-Pipette Contamination

Sartorius Lab Instruments GmbH

Sartorius Biohit Liquid Handling Oy Sartorius Biohit Liquid Handling

How Concentration toinaPipetting

– Vivaspin® Turbo 4 10kDa MWCO in the3.centrifuge.

Results for Vivaspin® Turbo 4

Results for Vivaspin® 500 in 40° fixed-angle rotor

USA Toll-free +1.800.635.2906 Specifications subject to change without notice.

Cell Culture Expansion in Fully Closed

Tubing materials of the cap closure are

pH probe in the side wall so that the probe

Time (hours) would be in direct contact with solution to

Change in pH of the solution indicates gas

The pH change of the solution on flasks with

Fig. 1: Cell Growth Study Process Diagram incubator

Traditional Flask MYCAP CCX Flask

Conclusion Carefully controlled conditions for cell growth in a

Recommendations MYCAP™ CCX Validation Template Tool:

Growth Rate at each Passage Overall Growth Rate

MYCAP™ CCX 0,0200

Overall Doubling Time

MYCAP™ CCX and Opta ® are registered trademarks of Sartorius-Stedim Biotech

Kleenpak ® is a registered trademark of Pall Corporation

External environmental influences or improper handling

Work in the Lab under Consistently

4. Use the Sartorius ionizer option to eliminate

2. Moreover, routinely check the balance using an

During the Measuring Sequence,

4. Avoid touching a vessel with your bare fingers at

5. When weighing, ensure that no powder falls

6. Avoid the complete interchange of air when