438 Opinion TRENDS in Biotechnology Vol.19 No.

11 November 2001

The bioseparation have accelerated biotech research and development,

in academia, industry and government-funded
laboratories.

needs for tomorrow ‘Bioseparation processes for life

sciences require different
Karsten Keller, Thomas Friedmann approaches from those used in
and Arthur Boxman traditional chemical industries.’

For example, after World War I, DuPont

Will we replace oil with wheat or corn as a feedstock for producing natural (Wilmington, DE, USA) transformed itself into a
plastic? The success of biotechnology for bulk product manufacturing will diversified chemical company as a result of
heavily depend on engineering solutions in the downstream processes in which fundamental research in material sciences. The
separation and purification have a crucial role with respect to commercial understanding of free radical polymerization,
development. Development of efficient bioseparation methods is important for condensation polymerization and polymer structure
a broad range of business areas including pharmaceuticals, nutrition and health led to the development of Neoprene synthetic
products, bio-based materials and crop protection chemicals. Depending on the rubber and nylon fibers. A new focus on biology,
value of the end product and the scale of production, the processing required chemistry and knowledge-intensive solutions has
varies significantly. Key factors that have an impact on the choice of separation taken place in the last ten years, representing a
strategy include process throughput, particle size of the product and impurities major corporate commitment. In September 1999,
and the desired end-product concentration. The development of efficient, Chad Holliday (DuPont’s CEO) announced ‘We will
economical and selective separation methods will be required for successful take important steps toward the use of renewable
commercialization of bioprocesses. Despite this well-recognized need, there are resources and energy’. The goal is to source 10% of
relatively few available methods for commercial implementations. Development our energy needs in the year 2010 from renewable
of novel mechanical systems for selective separation of solid and liquid energy sources and to derive 25% of our revenues in
mixtures must become a top priority for current research investment to reduce 2010 from areas other than those requiring finite
the reliance on expensive chromatographic and thermal separation methods. raw materials. We are studying the potential use of
plants to manufacture useful chemicals; for
Karsten Keller* Over the past decade, many traditional chemical example, one day we might be able to create a plant
Thomas Friedmann
industries have been undergoing a change in their that produces biosilk. We are also looking for the
Arthur Boxman
DuPont Central Research
orientation from conventional chemicals to life- genes that control the synthesis of natural rubber to
& Development, PARSAT science products. Despite discussion about what a life enable us to use plants that are commonly grown in
Experimental Station, sciences company should consist of, the required this country as a source of rubber. With these
E304/A207, Wilmington,
technology for the future will not change. Recent initiatives comes the need for more sophisticated
DE 19880-0304, USA.
*e-mail: karsten.keller@ developments in biochemistry, together with bioseparation methods and processes in a broad
usa.dupont.com recognition of the need for renewable resources, range of business areas (Fig. 1).

Removal of insolubles and/or Bioseparation – today’s technologies

sampling of cell-free liquid Bioseparation processes for life sciences require
different approaches from those used in traditional
chemical industries. For example, in the
Nutrition and Agriculture pharmaceutical industry, only a few kilograms of a
health A
protein might be produced per year with a total
selling price of millions of dollars. In industrial
B
C
biotechnology, thousands of tons of bio-based
polymers, such as Biopol or Xanthan gum, could be
Cell recovery
Sterilization
and/or
made per year, also yielding millions of dollars in
and/or
cell harvest sales but at a significantly lower price per unit mass.
filtration
Separation of biologically based products often needs
entirely new processes to handle unusual material
properties, such as the compressibility of a biomass.
Pharmaceuticals Performance The desired product might be a single component
materials present at low concentration that must be separated
from bulk water and other soluble components.
Separation technologies can be categorized
Isolation and/or
purification TRENDS in Biotechnology according to their fundamental principle (Fig. 2).
Many of these principal separation approaches are
Fig. 1. In the future, various bioseparation technologies are needed for a broad range of business described in bioseparation handbooks1. Often the
areas.

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 Opinion TRENDS in Biotechnology Vol.19 No.11 November 2001 439

sold for several hundred dollars per gram. Table 1

shows the processing variables that make it
Mechanical (forces) Physical (behavior) Thermal Chemical impossible to develop one generic separation
Separation tasks: Solid–Solid; Solid–Liquid; Solid–Gas; Liqui d–Liquid; Liquid–... approach for all biotechnology-derived products. In
addition, the required cleaning in place (CIP) and
Filtration Absorption Drying Chemi-sorbtion
Centrifugation
sterilization in place (SIP) standards make the
Adsorption Distillation Chemical reaction
Clarification Rectification … investment in equipment relatively costly, indeed, as
Crystallization
Membranes Evaporation
much as 30–70% of investment in a bioprocess is
Precipitation
Classification Extraction … related to separation. Given that expert systems are
Purification … unable to provide an economical solution, engineers
Agglomeration will have to develop customized strategies for each
Washing bioseparation process.
…
Products, behavior and demands
New life science products often need entirely new
methods of managing unknown material properties.
TRENDS in Biotechnology
For example, compressible sludge must be handled
in a different manner from crystals and other
Fig. 2. Separation effort involved in thermal and chemical separation well-known bulk materials.
technologies can be techniques is higher than that in mechanical
categorized according to
techniques. Although thermal and chemical
their fundamental
separation tasks will not change greatly when applied
‘The challenge for the future will be
principles. The bars
represent the relative cost to the field of bioseparation, mechanical separation to develop and adapt further current
of each process. applications will face new engineering challenges, for
example, in bioseparation, the handling of soft
technologies or to create innovative
bioparticles is particularly crucial2. A broader separation technologies to meet the
analysis indicates that there will be three major
challenges in the engineering of downstream
bioprocess requirements.’
processes: managing variables, product behavior and
selection of the proper separation technology. This type of product behavior issue must be
considered before bioprocesses are developed (Fig. 3).
Managing variables Another important issue is the interaction of the
The ‘particle size’ of the desired product to be microorganisms with the process environment.
separated can range from single molecules to During processing, the cells can be exposed to a
particles of nearly a millimeter in diameter. The variety of unfamiliar stresses, such as shearing
viscosity of the fluids can range over three orders of forces, heat, electromagnetic fields, hydrostatic
magnitude from 1 mPas to 1 Pas. Biological pressure or vibration. These severe conditions might
processing often involves a high throughput of influence the morphology or behavior of the
material containing a low volume of final product. microorganisms, perhaps causing lysis of the cell
The concentration of desired compounds can be as low membranes. Modifying these process conditions to
as a few parts per million or as high as 50% (Ref. 3). produce a desirable separation effect might aid the
The value of the compounds to be separated ranges downstream processing. This desirable effect could
from relatively low value compounds (cents kg1) include the agglomeration of particles to form
produced in large quantities to high-value materials flocculates, or the inactivation or loss of bioactivity

Table 1. Challenges in managing variables

Solid Liquid Viscosity Throughput Concentration Investment unit –1 Unit operation
5 mm Water 0.5 mPas 1 kg min –1 10 ppm US$ 1 Separation
1 mm Toxic 1 mPas 5 kg min –1 100 ppm US$ 2000 Cell disruption
100 m Fermentation 100 mPas 1 t h–1 1% US$ 100 000 Washing
10 m Flammable 1 Pas 20 t h –1 5% US$ 500 000 Precipitation
1 m Hazard 50 Pas 200 t h–1 20% US$ 1 000 000 Classification
0.01 m Solvent 2000 t h–1 50% Extraction
0.001 m Polymer Handling
Drying
Evaporation
Fouling
Control
CIP and SIP
Life science fields are in red type. Abbreviations: CIP, cleaning in place; SIP, sterilization in place. The wide range of proc essing and material
variables makes it impossible to develop one generic separation approach for all biotechnically derived products.

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440 Opinion TRENDS in Biotechnology Vol.19 No.11 November 2001

common unsolved issues are membrane fouling,

(a) Products
degradation and issues related to the handling of
Cell r = 1–10 m
Polysaccharide  = 0.0–0.1 kg l–1 compressible filter cakes.
 = 10–3–100 Pas Solids The concept of using biological pathways to
MicroStructure produce a wide variety of valuable compounds offers
FS many opportunities for innovative products in the
Protein
areas of nutrition, pharmaceuticals, agriculture and
Crystals Low molecular
products performance materials. The challenge for the future
will be to develop and adapt further current
(b) Demands (c) Behaviors technologies or to create innovative separation
technologies to meet the bioprocess requirements.
Consumer acceptance
 natural P Researchers might even change biological pathways
 no solvents
Denaturation and in advance of demanding downstream processes.
 no additives
degradation
Functionality Selection
Activity of a product
Fouling Compressible filter cake Separation technologies available for bioseparation
include mechanical techniques such as micro-, ultra-
TRENDS in Biotechnology and nanofiltration, deep bed filtration, cross-flow
filtration (both static and dynamic), electrofiltration
Fig. 3. Material properties have to be considered for the development of bioseparation processes. and centrifugation (filtration and sedimentation)5.
Using a variety of polymer membranes or ceramics
Cell separation can often cause plugging and fouling. Several
Sterilization and washing chemical and thermal separation techniques are used
Filtration
Cell disruption
e.g. Filtration, for bioprocesses including reverse osmosis, ion-
centrifugation exchange, chromatography, electrophoresis,
CIP and SIP
crystallization and extraction6–9. The challenge is to
Low
Screening find and adapt the relevant types of the multiple
molecular e.g. M,S,E
products available separation technologies throughout the
Cell recovery
process (Fig. 4).
Filtration and
High molecular products One example that incorporates the challenge in
centrifugation bioseparation is the development of the DuPont
bio-based 1,3-propanediol (3G) process. In nature,
Crystallization two separate microorganisms are required to convert
glucose to glycerol and then convert glycerol to 3G.
Sampling of To make the fermentation process more efficient,
cell-free liquid Product Centrifugation however, a genetically engineered microorganism has
been developed to convert glucose to 3G. The bio 3G is
TRENDS in Biotechnology used as a monomer for the production of polyester9.
However, to decrease the high cost of the downstream
Fig. 4. In bioseparation, the challenge is to find and adapt the relevant types of separation process remains a challenge.
technologies. Abbreviations: E, evaporation separation process; M, membrane separation process;
S, sorption (ab/adsorption) separation process.
Bioseparation – future challenges
Figure 5 presents a common view of bioseparation
through mechanical stress, heat or electrical field needs. In the future, selective separation will be
pulses4. Changes in pH and temperature are known important and thus, despite the high costs involved,
to have a marked influence on the separation it should be given top priority for current investment.
kinetics of colloidal suspensions and could be used to The specific needs of each application will strongly
enhance bioseparations. Presence of biomass in the indicate the separation technology to be used.
separation process will require a basic However, the question arises as to whether current
understanding of how cell physiology changes during technologies are adequate for the biotechnology
the process, including an assessment of the expected industry of tomorrow. A review of available processes
range of process parameters. For example, the design and their limitations suggests that new techniques
of the vessel and agitator in a bioreactor, or the are needed to develop bioseparation technology
transfer piping between two unit operations, will further to optimize yields and minimize costs. Many
determine the peak shear and elongational rates applications are currently undergoing laboratory-
experienced by the product and the cells. Do we scale testing only. For example, in the handbook of
know the limiting shear stress on the cell? Are bioseparations1 only 15 out of 750 pages are related
microstructural changes of the product probable? to process-scale separation. Some of these
Can we influence the functionality of a product? developments might prove commercially promising,
Questions such as these must be explored. Other even for large-scale operations.

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 Opinion TRENDS in Biotechnology Vol.19 No.11 November 2001 441

(a) (b) Box 1. Networking for answers

Novel
separation Networking is fast and direct, it conserves the
Reverse micelle Mixer resources of all involved parties and stimulates
Settler theflow of advanced knowledge. The mission of
Product the International Network for Separation is set
down in the solid–liquid separation (SLS)
roundtable website as follows: ‘The SLS
Roundtable website has been set up as a
platform to facilitate
(c) (d)
Decanter with electrical field knowledge exchange between industry users in
thesolid liquid separation disciplines. “ User ”
means anyone conducting engineering or
research and alsocieties and consortia in the
TRENDS in Biotechnology field. SLS Roundtable members have the
opportunity to contribute to a growing database
Fig. 5. The bioseparation needs of tommorrow: (a) selective separation methods; (b) scale-up of knowledge and experience b ysubmitting
ofseparation technologies to meet technical and economic requirements; (c) the non-proprietary case studies, research topics,
combination of traditional and novel methods and (d) cost-effective large-scale processes.
courses, classes and other information intended
to educate experts throughout the SLS
It is evident that many traditional separation disciplines. The SLS Roundtable aims to
technologies are available and practiced at a range of promote separation technology on educational,
scales. Most large-scale operations still typically use research and industrial levels in the US and
the classical mechanical solid–liquid separation globally.’
equipment, such as decanter centrifuges and Every member (membership is free of charge
membrane filters. The combination of these and sponsored by DuPont) gains access to reports,
well-tested methods with novel techniques, such as information about SLS meetings, member profiles,
superimposed thermal treatment, electric fields, a message board, a newsletter and more. The
self-assembling membranes, magnetic fields and the message board distributes questions from one
rapidly advancing field of microfabrication member to all other members and answers from
techniques, are the foundation for new concepts in members appear on the message board on a
bioseparation. website. (For further information see
government and industry for sustainable growth and
http://www.slsroundtable.com)
Future prospects use of renewable resources, an integrated,
Beginning with an initiative from DuPont in April interdisciplinary effort of biological scientists and
1999, 50 international companies and universities engineers is required. The newly founded separation
started to discuss the challenges in separation network is making progress in the search for answers.
technology and education (Box 1). At four consecutive The complexity of new life science processes will
sessions (held at DuPont, Dow, Aventis and Degussa), require a strategy that emphasizes the importance of
professionals and academics from all over the world separation as a major component of unit operations.
assigned the priorities for future research in We are now in a new era of separation that requires
separation. To meet the ambitious goals set by novel and imaginative engineering solutions.
References AIChE Annual Meeting, Miami Beach, Florida, 7 Scott, K. (1995) Handbook of Industrial
1 Ahuja, S. (2000) Handbook of Bioseparations, Elsevier Membranes, Elsevier, Oxford
Academic Press, San Diego 4 Knorr, D. et al. (2000) Membrane permeabilization 8 Belter, P.A. et al. (1988) Bioseparations –
2 Schurr, G. et al. (2000) Focusing powder and inactivation mechanisms of biological systems Downstream Processing for Biotechnology,
technology on processing biologically produced by emerging technologies, 8th Int. Congress on John Wiley & Sons, New York
products, 14th Int. Congress of Chemical and Engineering and Food, Puebla, Mexico, IAEF 9 Laffend, L.A. et.al. (1997) Bioconversion
Process Engineering, Praha, Czech Republic 5 Dickenson, T.C. (1997) Filters and Filtration of a fermentable carbon source to
3 Keller, K. (1998) Thousand Ways of Solid/Liquid Handbook (4th edn), Elsevier, Oxford 1,3-Propanediol by a single microorganism.
Separation: How Can We Handle This? Selection 6 Singh, R.K. and Rizvi, S.S.H. (1995) Bioseparation US patent 5686276, E.I. DuPont de Nemours
Criteria and Required Technology in the Future, Processes in Food, Marcel Dekker, New York and Company

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