Tools & Techniques

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.

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.

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

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

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

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 10m 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 nonmembrane 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

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Tools & Techniques

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. 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 rate and concentration of retained molecules increase until the accumulation of retained molecules form a concentrated, dense gel layer. The gel layer becomes the limiting factor to filtrate flow rate and further pressure increases will have little to no effect. This type of filtration is referred to as gel limited ultra filtration which is a pressure driven

membrane process used to concentrate, separate or purify macromolecules. The separation is based on molecular weight of the macromolecule. The membrane is a thin semipermeable 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 inprocess quality control test. During this process the desired proteins and their allied products are separated by their molecular weight, and the volume is reduced thereby increasing the purity considerably compared to the starting volume.

Figure - 3 Schematic representations of filter modules (a) Hollow fiber, plate, and spiral-wound membrane modules (b) A rotating cylinder module (Zeman et. al)

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Tools & Techniques

Table 3 Comparison of various systems from different manufacturers
Feature Crossflow cassettes Low Low Excellent Excellent Yes Tubular Modules High High Good Excellent Depends on the material Poor Hollow-fiber Modules Average High Average Average Depends on the configuration Poor Plate and frame system High High Poor Good Not for ultrafiltration Poor Spiral wound Modules Average Average Average Good No

Nanofiltration Removal of virus and virus like particles from the mammalian cell culture by this method is mainly on the basis of size difference between proteins and viruses. It was an advantage over chemical treatment (Horowitz, and Highsmith). For reducing the virus load in biological products since it does not entail the use of toxic reagents that subsequently have to be eliminated from the proteins. The procedure also ensures the least disturbance to the therapeutically active protein composition. It makes use of tangential flow across a nanofilter with nominal cutoff values of 70 and 160 KDa and15 to 75 nm (Dileo). Membrane Chromatography

Hold up volume Energy consumption Easy to cleaning Pressure Resistance Steaming-inplace Scalability

Excellent

Poor

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

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 l l l

Inlet and outlet pressures Crossflow (or tangential )velocity Flow channel sizes (height and width) Feed stream properties test slurries should be representative of the actual process streams. Membrane type and configuration-test data from one design directly be used to design another geometry.

Table 4 Comparision of key characteristics of Crossflow modules (Zeman et al)

Module type Channel spacing (cm) Packing density (m2/ m3) Energy costs Particulate Plugging Ease of Cleaning

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, lowpressure 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) High-performance tangential flow filtration (HPTFF) is a new membrane technology that

Hollow fibre Tubular Flat plate Spiral wound Rotating

0.02-0.25 1.0-2.5 0.03-0.25 0.03-0.1 0.05-0.1

1200 60 300 600 10

Low High Moderate Low Very high

High Low Moderate Very high Moderate

Fair Excellent Good Poor-fair Fair

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Tools & Techniques

Table 1 Various product purification's features processed by the technique of HPTFF

Product (mw) Impurity (mw) Purification Factor Yield(%) Reference

containing very small pores next to the filtering surface. However, the thin and thick layers of such membranes are made up of two different types of material. Filtration membranes are made from wide variety of polymers and inorganic materials. The polymers that are used include cellulose acetate, polyamide, polyether, polycarbonate, polyester, polyethylene, regenerated cellulose, poly vinyl chloride, poly vinylidene fluoride, PVDF, poly tetra fluoro ethylene (PTFE), acrylnitrile copolymers, and polysulfone. The inorganic materials used include ceramics, zirconium oxide, borosilicate glass, stainless steel, and silver. Cellulose Acetate and Triacetate Membrane The cellulose triacetate membrane is a membrane polymer that is well established in the biotechnological and pharmaceutical industries. It is extremely hydrophilic; virtually non-protein binding (Fig 4).These features make cellulose triacetate membranes ideally suited for biotechnological applications. It shows minimal adsorption of protein, viruses etc. and also its retention is unaffected by repeated re-use. They are commercially available in a choice of the following nominal molecular weight cutoffs like 5K, 10K and 20K. They are characterized as least protein binding.

BSA(68,000) Fab (45,000) BSA (68,000) IgG (155,000) BSA (68,000)

Fab(45,000) BSA (68,000) Hb (67,000) BSA (69,000) BSA dimer (136,000)

990 830 100 30 9

94 69 68 84 86

Van Reis et al 1 Van Reis et al 1 Van Eijndhoven et.al Van Reis et al Van Reis et al

Table 2 Comparison of the various separation methods

Traits Cross flow Centrifugation Sedimentation Precipitation

Yield Energy consumption Scalability Time required Service costs Degree of purity

High Low Excellent Little Low High

Average High Average Average High Average

Low Low Not merely Considerable Low Low

Average Low Not merely Considerable High Low

can be used for the separation of protein mixtures without limit to their relative size (Van Reis et al) HPTFF could potentially be used throughout the downstream purification process to remove specific impurities (e.g., proteins, DNA or endotoxin), clear virus and eliminate protein oligomers or degradation products. HPTFF can also be used for the 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.

asymmetric structure, there is a thin layer next to the filtering surface that has very small pores. Below this thin layer is a much thicker layer that has much larger pores and serves as structural support for the membrane. The composite membrane is similar to the asymmetric membrane in having a thin layer

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)

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Tools & Techniques

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, 2m. 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.

These biological structures derived mainly from the outer membranes of gram-negative bacteria are composed of complex lipopolysaccharides which may cause nonspecific, generally weak toxic reactions such as fever, redness or swelling at the injection site. But when contained in larger amounts in pharmaceutical preparations, the reaction may be severe. Their elimination from biological products, which are derived from the cultivation of bacteria, through appropriate purification procedures represents a major task of the manufacturing process. This task is rendered even more difficult as this endotoxin is chemically and thermally very stable.

R.G.Harrision Paul Toda, Scott R Rudge, and Demetris P. Petrider, Bioseperaions Science and Engineering, New York, Oxford University Press, 2003: 104-106. VanReis R,Gadam S, Frautschy LN, Orlando S, Goodrich EM Saksena S, Kuriyel R, simpson CM, Pearl S, Zydney AL. 1997 High performance tangential flow filtration. Biotechnol Bioeng.56: 71-82. Van Reis R, Brske JM, Chsrkoudisn J, burns DB, Zydney AL. 1997 High performance tangential flow filtration using charges membranes. J.Membr Sci 159:133-42. Van Eijndhoven HCM, seksena S, Zydney AL. 1995 Protein fractionation using membrane filtration: role of electrostatic interactions. Biotechnol Bioeng 48: 406-14. Wang WK, 2001,Membrane separations in biotechnology, Marcel Dekker Inc, New York. Zeng X,Ruckenstein E, 1999 Membrane chromatography: preparation and application to protein seperation. Biotechnlo. Prog 15:1003-19. Zeman, L J,and Zydney A L 1996. Microfiltration and Ultrafiltration, Dekker, New York.

Reference
Harrision R,Todd P,Rudge SR,Petrides DP, 2003 Bioseparation science and engineering, Oxford University Press. New York. Highsmith F,Xue H,Che L,Bende L, Owens J, Shanbron, Drohan W 1995 Iodine mediated inactivation of lipid and nonlipid enveloped viruses in human antithrombin III concentrate Blood: 86:791-96. Horowitz B, Wiebe ME, lippin A, Strykar MH 1985 .Inactivation of viruses in labile blood derivetis. I Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations. Transfusion 25: 516-22. www 2.dupont.com/Teflon_industrial/ en_us/tech_onfo/techinfo_compare.html. DiLeo AJ,Allegrezza AE, Jr.Builder SE. 1992 High resolution removal of virus from protein solutions using the membrane of unique structure. Biotechnology 10:182-88. Dater, R V and Rosen, C.-G 1993. Cell and cell debris removal: Centrifugation and Crossflow filtration. In Biotechnology, vol 3. Bioprocessing H-J Rehm and G. Reed eds.VCH publishers, Weinheim, p-486. Paul K ng,Alfred C,Dadson Jr,G.Micheal conneww and Bernard P Brutisk. Bayer corporation California, Filter applications in p r o d u c t r e c o v e r y, f r o m M e m b r a n e separations in Biotechnology, Edited by William K. Wang. New york Mercel Dekker inc.2001: 215-216.

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.

Author For Correspondence

S. Jagan Nathan Assistant Research Officer, Pasteur Institute of India, Coonoor, The Nilgiris, TamilNadu. Email: seljag2005@yahoo.com, Mobile: 94860 81990

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