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Filtration

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Filtration Technique in
Vaccine Manufacturing
S.Jagan Nathan1*,K.C.Shivanandappa2 B.Sundran3 ,K.N.Venkataramana4 ,K.R.Mani5
Abstract
The Pharmaceutical products must conform
to well defined quality standards. Vaccines
play a vital role in humanity, vaccines
safeguard the children from life threatening
diseases and in vaccine production and
filtration is one of the most imperative steps.
The various filtrations like microfiltration,
ultrafiltration, membrane chromatography,
crossflow filtration are important play an
important role during the production of
immunobiologicals. All the filtration
techniques in same principle and they are
differentiated from their principle only. The
techniques of filtration are same for bacterial
and viral vaccines. In the complex of
manufacturing environment of vaccines for
fulfilling all necessary processing
requirements under validation control and
good manufacturing practices.
Keywords: Vaccine, Microfiltration,
Crossflow filtration and hollow fiber.
Introduction
Vaccines play the vital role for humanity as
their use safeguard the children from life
threatening diseases like tuberculosis, polio,
whooping cough, tetanus, encephalopathy,
yellow fever, measles etc. The national
governments are responsible for
immunization and they are adhering with
WHO immunization project, and their
guidance. The UNICEF plays an important
role in this regard. The Pharmaceutical
products must conform to well defined quality
standards. The production of infusion and
injectable solutions, or those which come in
contact with open wounds, are regulated by
such standards. Quality results can only be
achieved by effectively safe guarding an
37 | Advanced Biotech | August 2008
entire process against contamination.
Membrane technology is critically reviewed
in international pharmacopeias, with a
concentration on sterilizing grade filters. In
the vaccine production the process namely
filtration is an important manufacturing
process and this article deals with various
filtration technologies.
Figure 1-A General flow diagram of a purification train in the
vaccine process (Paul K Ng. et. al)
Filtration
Filtration is a process for separating two
substances of two different physical states. It
is used for separating solids from turbid
liquids (filtrate), pure gases or solids.
Filtration is classified in two ways based on
the principle of operation as follows (Fig2).
1. Dead end filtration
2. Tangential Flow Filtration
In the Dead end filtration, all the flows are
directed through the membrane with material
building up on the surface of filter. As these
particles build up, flow through the filter is
quickly reduced and finally it ceases
completely. In tangential flow filtration, the
Tools & Techniques
flows are directed across the membrane
surface. This sweeping action helps to keep
the retained material from settling on the
membrane surface and thus will help the
membrane to perform effectively for long
periods. They are so-called depth filters
whose effectiveness is influenced by the
whole complex of the following
characteristics: mechanical retention of
particles, absorbability, pH values, surface
quality, thickness and strength of the filter
paper and the shape, density and quantity of
particles to be filtered.
Microfiltration
Micro filtration is defined as the process of
removing contaminants in the 0.25 to 10µm
range of particles present in the process fluids,
by passage through a microporous medium,
such as membrane filter. Although micron
sized particles can be removed by use of non-
membrane or depth filters, particles found in
fibrous media can be removed only by the
membrane or screen filters having a precisely
defined pore size. The retention boundary
 Tools & Techniques

membrane process used to concentrate,

Figure -2 Schematic diagrams for (a) dead-end or conventional
filtration and (b) cross-flow filtration (R.G.Harrision et al)
which a membrane filter defines can also be
used as an analytical tool to validate the
integrity and efficiency of a system. These
filtration techniques are applied in
clarification of viral harvest as well as the
bacterial cell mass those are produced by the
fermentation techniques or the roller bottle
culture. During this process the desired
proteins and products are separated from the
media components which are a part of
cultivation.
separate or purify macromolecules. The
separation is based on molecular weight of the
macromolecule. The membrane is a thin semi-
permeable polymeric material that will retain
macromolecules and allow smaller dissolved
solutes to pass through the membrane. The
Table 3 shows the various companies dealing
with the hollow fiber technology. The
retentive abilities of UF membranes are
described by the nominal weight limit
(NMWL). UF membranes for spiral
ultrafiltration cartridge are available in the
NMWL ranges of 1000 to 3, 00,000. These
filtration techniques are applied in
concentration of virus and bacteria. Before
concentration process the input materials are
inactivated/detoxified by the chemical
method and it should have passed the in-
process quality control test. During this
increase until the accumulation of retained
process the desired proteins and their allied
molecules form a concentrated, dense gel
products are separated by their molecular
layer. The gel layer becomes the limiting
weight, and the volume is reduced thereby
factor to filtrate flow rate and further pressure
increasing the purity considerably compared
increases will have little to no effect. This type
to the starting volume.
of filtration is referred to as gel limited ultra
filtration which is a pressure driven

Ultrafiltration (UF)

Ultrafiltration is a technique for separating

dissolved molecules in solution on the basis of
size rating the particles will be retained at the
surface of the membrane and not in the
polymer matrix as they are in micro porous
membranes (Fig 3). The accumulation of
retained molecules may form a concentrated
gel layer which can significantly alter the
performance characteristics of the membrane.
This phenomenon is commonly referred to as
concentration polarization. Understanding
the effects of concentration polarization and
the operating conditions that can minimize
this phenomenon will lead to optimal flux and
retention properties in separation process. As
the gel layer gets formed the filtrate flow rate
is controlled by the permeability of the
membrane and the applied pressure, this
condition is said to be membrane controlled.
As the pressure is increased, the filtrate flow Figure - 3 Schematic representations of filter modules (a) Hollow fiber, plate,
rate and concentration of retained molecules and spiral-wound membrane modules (b) A rotating cylinder module (Zeman et. al)

38 | Advanced Biotech | August 2008

 Tools & Techniques

Table 3 Comparison of various systems from different Nanofiltration

manufacturers Removal of virus and virus like particles from
Feature Crossflow Tubular Hollow-fiber Plate and Spiral the mammalian cell culture by this method is
cassettes Modules Modules frame wound mainly on the basis of size difference between
system Modules proteins and viruses. It was an advantage over
Hold up volume Low High Average High Average chemical treatment (Horowitz, and
Energy Highsmith). For reducing the virus load in
consumption Low High High High Average biological products since it does not entail the
Easy to cleaning Excellent Good Average Poor Average use of toxic reagents that subsequently have to
Pressure be eliminated from the proteins. The
Resistance Excellent Excellent Average Good Good procedure also ensures the least disturbance to
Steaming-in- Yes Depends Depends Not for No the therapeutically active protein
place on the on the ultrafiltra- composition. It makes use of tangential flow
material configuration tion across a nanofilter with nominal cutoff values
Scalability Excellent Poor Poor Poor Poor of 70 and 160 KDa and15 to 75 nm (Dileo).

Tangential Flow Filtration
The cross flow technology is commonly used
during the downstream processing of
proteins. Various applications such as product
clarification and concentration are best
achieved by the use of cross flow filtration.
For many products cross flow technology is
often the last processing step prior to final
formulation for heat labile products. There are
two types of filtration in separation. Normal
Flow filtration (NFF) and Tangential Flow
Filtration (TFF). In normal filtration all flows
is directed to the membranes with retained
material building up on the surface of the
filter. As these particles build up, the flow
through the filter is slowly reduced until it
ceases completely. In tangential Flow
Filtration, flows are directed across the
membrane surface. The sweeping action of
the fluid restricts retained material from
settling and eventually reduction flow. Some
of the key characteristics are detailed in the
Table 4 of the cross flow modules. Tangential
Flow Filtration is a pressure driven membrane
Table 4 Comparision of key characteristics of Crossflow
modules (Zeman et al)
Module type Channel Packing
spacing density
(cm) (m2/ m3)
Hollow fibre 0.02-0.25 1200
Tubular 1.0-2.5 60
Flat plate 0.03-0.25 300
process used to concentrate separate or purify
macromolecules. Membrane selection for
Crossflow filtration is determined by the
properties of the target molecule and the aim
of the step. Cross flow filtration modules are
available from manufacturers for carrying out
laboratory or pilot plant test. The
determination of the size of a plant unit can be
done by a direct scaleup of the filtration area
based on the feed or output flow rate. For this
scale up, however, it is important that the
following variables be kept constant
(Dater et al).
l Inlet and outlet pressures
l Crossflow (or tangential )velocity
l Flow channel sizes (height and width)
l Feed stream properties test slurries
should be representative of the actual
process streams.
l Membrane type and configuration-test
data from one design directly be used to
design another geometry.
Energy Particulate Ease of
costs Plugging Cleaning
Low High Fair
High Low Excellent
Moderate Moderate Good
Membrane Chromatography
Membrane chromatography for protein
purification is a recent technology that has
been used successfully in a wide range of
applications with a variety of membrane
geometrics and interaction modes (Zeng
et.al).For this particular application, the
apparent advantages of membrane absorbers
at this stage of development are still rather
qualitative. The Sartorius S15 membrane
absorbers were determined to be an
alternative to conventional adsorption column
chromatography for the isolation of
recombinant immunofusion protein. The
membrane in chromatography is having
advantages like short set up time, low-
pressure operability, high capacity and high
volumetric velocity capabilities. These
features are likely to be transferable to the
isolation of other proteins by ion exchange.
Significant time savings result from the quick
setup, which amounts to connecting tubing
and loading a peristaltic pump head, and the
high volumetric throughput of these
membranes. It requires sufficient chemical
and thermal stability for repeated sanitization,
of course, a critical requirement for
economical scaleup in the biopharmaceutical
industry. These techniques are applied in the
final process and it plays a major role in the
purity. The major impurities like residual
cellular DNA and bacterial endotoxins are
filtered.
High-Performance Tangential-Flow
Filtration (HPTFF)

Spiral wound 0.03-0.1 600 Low Very high Poor-fair High-performance tangential flow filtration
Rotating 0.05-0.1 10 Very high Moderate Fair (HPTFF) is a new membrane technology that

39 | Advanced Biotech | August 2008

 Tools & Techniques

Table 1 Various product purification's features containing very small pores next to the
processed by the technique of HPTFF filtering surface. However, the thin and thick
layers of such membranes are made up of two
Product (mw) Impurity (mw) Purification Yield(%) Reference
Factor
different types of material.

BSA(68,000) Fab(45,000) 990 94 Van Reis et al 1 Filtration membranes are made from wide
Fab (45,000) BSA (68,000) 830 69 Van Reis et al 1 variety of polymers and inorganic materials.
The polymers that are used include cellulose
BSA (68,000) Hb (67,000) 100 68 Van Eijndhoven
et.al acetate, polyamide, polyether, polycarbonate,
polyester, polyethylene, regenerated
IgG (155,000) BSA (69,000) 30 84 Van Reis et al
cellulose, poly vinyl chloride, poly vinylidene
BSA (68,000) BSA dimer fluoride, PVDF, poly tetra fluoro ethylene
(136,000) 9 86 Van Reis et al
(PTFE), acrylnitrile copolymers, and
polysulfone. The inorganic materials used
include ceramics, zirconium oxide,
Table 2 Comparison of the various separation methods borosilicate glass, stainless steel, and silver.
Traits Cross flow Centrifugation Sedimentation Precipitation
Cellulose Acetate and Triacetate
Yield High Average Low Average
Membrane
Energy consumption Low High Low Low
Scalability Excellent Average Not merely Not merely The cellulose triacetate membrane is a
membrane polymer that is well established in
Time required Little Average Considerable Considerable
the biotechnological and pharmaceutical
Service costs Low High Low High industries. It is extremely hydrophilic;
Degree of purity High Average Low Low virtually non-protein binding (Fig 4).These
features make cellulose triacetate membranes
can be used for the separation of protein asymmetric structure, there is a thin layer next ideally suited for biotechnological
mixtures without limit to their relative size to the filtering surface that has very small applications. It shows minimal adsorption of
(Van Reis et al) HPTFF could potentially be pores. Below this thin layer is a much thicker protein, viruses etc. and also its retention is
used throughout the downstream purification layer that has much larger pores and serves as unaffected by repeated re-use. They are
process to remove specific impurities (e.g., structural support for the membrane. The commercially available in a choice of the
proteins, DNA or endotoxin), clear virus and composite membrane is similar to the following nominal molecular weight cutoffs
eliminate protein oligomers or degradation asymmetric membrane in having a thin layer like 5K, 10K and 20K. They are characterized
products. HPTFF can also be used for the as least protein binding.
purification of natural protein products from
whey. HPTFF is unique among available
separation technologies in that it can affect
simultaneous purification, concentration and
buffer exchange, allowing several different
separation steps to be combined into a single
scalable unit operation. Although HPTFFis
still an emerging technology, a number of
recent studies have clearly demonstrated the
potential of this separation technology. The
results of several such applications are
summarized in Table-1.

Membranes
Three basic structures are commonly used for
membranes: homogeneous, asymmetric and
composite. The homogeneous structure has
no significant variation in pore diameter from
the filtering surface to the other side. In the Figure - 4 Cellulose acetate membrane. Electron micrograph
(1.00 kx, 15 kv 219) (Millipore)

40 | Advanced Biotech | August 2008


POLYTETRFLUOROETHYLENE (PTFE)
It is also called as TEFLON, it is a synthetic
fluropolymer and it's melting point is 327oC
but its properties degrade above 260 oC (50oC
F (7) membrane are stable in the pH range in
between 2 to14, it also steam sterilizable.
Polypropylene-reinforced membranes with
pore size 0, 2µm.
POLY ETHER SULFON (PES) It is
hydrophilic and constructed from pure
polyether sulfon polymer membrane. And it is
low protein and drug binding characteristics.
It also wide range of pore sizes and higher
wick rates with lateral flows, lot to lot
consistency. are steam sterilizable, their pH
are stable in between 2-14.
POLY VINYL CHLORIDE (PVC) It is
hydrophilic and constructed from pure
polyester having low protein binding
capacity, and stable pH range between 2-14, it
is steam sterilizable, has a long life and high
flux rate and is easy to re-cycle.
Discussion
Largescale industrial downstream process of
biotechnological products has utilized
ultrafiltration/diafiltration process since the
mid 1970s, when conventional separation
techniques, such as salt precipitation,
centrifugation, evaporation, lyophilization,
and membrane dialysis were
supplemented/replaced with alternative
industrial-grade ultrafiltration membranes.
Two primary objectives have been addressed:
concentration of biological products from
dilute solutions (ultrafiltration), and removal
of small molecules via constant-volume
buffer exchange (diafiltration). The
availability of membranes in the NMWL of
500 to 1,000,000 Daltons (Da) and
manufactured with a wide range of polymers
provide a reasonable degree of choice to the
p r o c e s s e n g i n e e r. I n d u s t r i a l g r a d e
ultrafiltration membranes are agreeable to
cleaning in place for reuse and the process of
which is extensively validated.
Validation of the elimination of
endotoxin
Endotoxin represents a major potential
contaminant in pharmaceutical products.
41 | Advanced Biotech | August 2008
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