Advances in Bioprocessing

A report by Group 2
CHL 291 IIT DELHI

Table of Contents
BIOPROCESSING ................................................................................................................................... 2 INTRODUCTION .................................................................................................................................... 2 BIOREACTORS ....................................................................................................................................... 4 ADVANCES IN CELL DISRUPTION..................................................................................................... 7 FILTRATION ............................................................................................................................................ 9 CENTRIFUGATION .............................................................................................................................. 12 MEMBRANE CHROMATOGRAPHY .................................................................................................. 14 ION-EXCHANGE CHROMATOGRAPHY (IEC) ................................................................................ 17 SINGLE USE (DISPOSABLE) TECHNIQUES .................................................................................. 200 MONITORING AND CONTROL OF BIOPROCESSES .................................................................... 222 REFERENCES...................................................................................................................................... 244

Bioprocessing
The use of biological materials such as organisms, cells and enzymes to carry out a process for commercial, medical or scientific reasons is termed as Bioprocessing. Some industries have a long tradition of enzyme use. In leather tanning, hides are softened and hair removed using the proteases in faeces. In brewing, amylases in germinating barley are used to convert starch to maltose. Very recently, the use of Glucose Isomerase for the production of fructose from glucose has come into picture. Due to the immense importance of bioprocessing in a multitude of fields, there is always an ongoing effort to improve the existing technologies, from the viewpoint of process efficiency, economics and ease. Dramatic improvements in cell culture titers, product quality constraints, new regulatory directives, and the emergence of biosimilars have necessitated the development of more efcient downstream bioprocesses for biopharmaceuticals. This has resulted in signicant improvements in traditional separation processes as well as the emergence of entirely new approaches. We highlight some of these recent advances in this report.

Introduction
Several recent changes have occurred that have had a profound impact on downstream bioprocessing. Major advances in upstream processes have led to dramatic improvements in cell culture titers, particularly for monoclonal antibodies (mAbs), placing increased demand on downstream bioprocessing technology. This has catalysed many recent advances in downstream bioprocessing such as the implementation of high-throughput process development techniques, improved unit operations, and the promise of continuous bioprocessing. Recent regulatory directives such as quality by design (QbD) and process analytical technology (PAT) have resulted in a noticeable shift in the perspective of the industry towards the implementation of new downstream bioprocessing strategies. There have also been significant efforts to develop generic processes, particularly for mAbs. A powerful example of this is the two-step process that includes weak partitioning ion exchange as a second step. The emergence of biosimilars has also brought entirely new bioprocessing challenges as well as a variety of product quality issues to the forefront. This report highlights some of the important recent developments in bioprocessing. Beginning with the bio-reactors, the report talks about the present designs and some of the problems that are faced with them. The report goes on to introduce some of the new designs like the continuous packed bed. We also talk about integrators and cell culture adaptors. A very important point to note is that, in most of the new developments, saving space and money have been the common aims. Cell disruption is one of the key steps in the recovery of bioprocessed products. Most of the microbial organisms used in fermentation express the desired proteins intracellularly. Cell disruption is the process by which desired biomolecules are released from the cell. Non mechanical methods of cell disruption and release kinetic theory are discussed. Filtration is a process of separating, fractionating or concentrating particles, molecules or ions within or from a fluid by forcing the material through a porous or semi-porous barrier. It is a highly important step in bio-processing. Present techniques include macro, micro, ultrafiltration and reverse osmosis. In all these techniques, membrane fouling is a common problem. To address this, we have talked about recent advancements to reduce this fouling. 2

Centrifugation, an important process often results in lysis, due to which undesirable intracellular proteins get released into the broth. Culturefuge system is a modification of the disk stack centrifuge. It is a hermetic cell culture centrifuge which is designed for gentle harvesting of cell culture having shear sensitive material. Hermetic means it eliminates any air entrainment and eliminate any air-liquid interface inside the separator bowl which can cause problems in downstream filtration process. So, its beneficial and more economical to use culturefuge system instead of ordinary centrifugation system. Chromatography is a very critical technology used in the downstream processing in the biopharmaceutical industries. Membrane chromatography seems to be a very promising option, particularly for processing large volumes, and many pharmaceutical companies have already started adopting this technique. Membrane chromatography columns, while being very effective are also cheaper to use, not to mention many other advantages over the traditional resin beads column and hence can prove to be very effective alternative the same. Polymeric monoliths can be used as ion exchangers in the chromatography column. The use of polymeric monoliths in ion-exchange chromatography applications is advantageous because of their typically high mechanical stability and tolerance of a wide range of pH conditions. In addition, the continuous structure, the porosity of the material and pressure stability are key features of these materials. In the biopharmaceutical industry, the term single use, also commonly known as disposable, refers to a product that is intended for one time use. Single use (Disposable) technology has emerged over the past decade as a cost effective and flexible basis for biopharmaceutical manufacturing. It has moved beyond the limited applications of culture bags, liquid storage bags, and sampling devices, and now includes more unit operation based capabilities such as cartridge filtration, ultrafiltration, chromatography, etc. For efficient and controlled functioning of any biological process at the industrial scale, it is necessary to implement a variety of monitoring systems and control measures. Mass spectroscopy, Biosensors and on-line sensors have been some of the recent areas, which have been researched in order to improve the control. The applications of bio-processing have become more widespread than ever. With the new requirements, the scales that we are operating on, the resources that we have with us, it is very important for us to understand the work that has been going on in to improve the entire process.

BIOREACTORS
Introduction A Bioreactor is a vessel in which a chemical process is carried out with the help oforganismsor biochemically active substances, and is the fundamental unit in any bioprocess.Bioreactor designs are commonly cylindrical, ranging in size from liters to cubic meters and are often made of stainless steel. They can be used for both aerobic and anaerobic processes though the specific parameters in their design are dependent on this decision and other process parameters [1]. Types of Bioreactors Typical configurations of a bioreactor include stirred tank reactors, air-lift bioreactors, fluidized bed reactors, packed bed reactors, and trickle bed reactors. We will explore a bit on each of those below. Stirred Tank Mixing Method: Mechanical agitation Application: Free and immobilized enzyme reactions[2] Design:This is the conventional mixing reactor which is made of either glass or stainless steel.The dimensions of the reactor depend on the amount of heat to be removed from the vessel.The stirrer can be either at the top or bottom of the reactor.Baffles in the center of the tank prevent formation of vortex and effective mixing of the ingredients [3]. Advantages: Low investment needs Low operating costs Disadvantages [4]: Foaming High shear forces may damage cells Require high energy input

Recommendation:Foaming can be overcome using proper anti-foaming agents [2]. Air Lift Bioreactor Mixing Method: Air lift [2] Design: Entire reactor is divided into 2 halves by a Draft tube: The inner gassed region (Riser) and the outer un-gassed region (Down comer). Riser has gas injection, connected-air moves upwards. Down comer region has degassed media and cells. Mean density gradients between riser and down comer regions cause continuous circulation [3]. Advantages [4]: Low friction and energy requirements The mechanical parts are easy to construct. There is no need of special aseptic seals. Scaling up is easier Fig. 1: Air Lift Bioreactor 4

Metabolic performance does not drastically reduce on scale up.

Disadvantages[4]: Capital needed is more Difficulty of sterilization Efficiency of mixing is low Fluidized Bed Reactor Design:In this method there are two liquid steams: up-flowing and down-flowing steams. Liquid circulates in an airlift reactor as a result of density difference between riser and down comer [2]. Advantages: Heat and mass transfer are efficient The mixing of the media between the liquid, solid and gaseous phases are effective. The reactor requires less energy. Low shear rates and hence suitable for cells which are more sensitive to friction like the plant cells and mammalian cells. Good uniformity of temperature. Catalyst can be continuously regenerated with the use of an auxiliary loop[3]. Disadvantages: Bed-fluid mechanics not well known. Severe agitation can result in catalyst destruction and dust formation. Uncertain scale-up[3]. Fig. 2: Fluidized Bed Reactor

Design: Packed-bed reactors are used with immobilized or particulate biocatalysts.Medium can be fed either at the top or bottom and forms a continuous liquid phase [2]. Advantages: High conversion per unit mass of catalyst Low operating cost Continuous operation[3] Disadvantages: Undesired thermal gradients may exist Poor temperature control Channeling may occur Unit may be difficult to service and clean[3]

Fig. 3: Packed Bed Reactor

Trickle Bed Reactor Design:The trickle-bed reactor is another variation of the packed bed reactor.Liquid is sprayed onto the top of the packing and trickles down through the bed in small rivulets[2]. 5

Advantages: Due to its relatively simple design, it is extensively used in processing plants [5]. Disadvantages: The hydrodynamics involved in its design is extremely complex. So, further development leads to some issues [5]. Practical Issues in Bioreactor Agitation An agitator is a device or mechanism to put something into motion by shaking or stirring.Biological reactions almost invariably are three-phase reactions (gas-liquid-solid). Effective mass transfer between phases is often crucial. For example, for aerobic fermentation, the supply of oxygen is critical [2].Suggestions for improvement: Use Mechanical stirring for small reactors, and/or viscous liquids, low reaction heat. Use Air-driven agitation for large reactors and/or high reaction heat. Recent Developments in Bioreactors Continuous Packed Bed[6]: A continuous packed bed has the following improvements over a batch packed bed reactor: Easy, automatic control and operation Stabilization of operating conditions Reduction of labor costs Easy quality control of products Integrator [7]: In order to know about the amount of acid or base which has been added to keep the pH constant electronic balances were used, which are now replaced by integrator. Electronic Balances: Integrator: Expensive Simple and Precise Occupy a lot of space Does not require additional bench space Can be used to measure enzyme activity Cell Culture Adaptor[7]:Aerating spiral has been developed to provide stirring for very sensitive cell lines. Due to the up and down movement of the spiral, layers of oxygenated medium are rapidly displaced from the tubing surface. pH control in Cell Cultures [7]: A new controller system based on mass flow measurements of the gas flow has been developed in order to maintain the pH of the culture by addition of CO2 (g). PC Control [7]: In order to control the bio reactor system better, new and better softwares are continuously being developed which help us monitor and control the system both manually and automatically.

Fig. 4: Cell Culture Adaptor 6

ADVANCES IN CELL DISRUPTION

Cell disruption is one of the key steps in the recovery of bioprocessed products. Most of the microbial organisms used in fermentation express the desired proteins intracellularly. Cell disruption is the process by which desired biomolecules are released from the cell. It makes the intracellular proteins accessible for extraction and solubilisation. Broadly there are two ways of going about protein release. They are: 1. Mechanical method (or the harsher method). 2. Non-mechanical method (or the gentler method) Mechanical method includes high speed bead mills, high pressure homogenisation, french press, impingement etc. Non-mechanical method includes chemically/enzymatically induced cell lysis, osmotic shock, freeze thaw etc. Over the years, cell disruption techniques have been more or less the same with major breakthroughs such as homogeniser, french press etc. coming late in the 20th century and early 21st century. The past ten years have seen these methods evolve with better research methodology and better understanding of the processes. Mechanical methods are the most widely used to achieve microbial cell disruption on industrial scale. However, it has several disadvantages such as: 1. Unlike the non-mechanical gentle techniques, mechanical disruption shows no selectivity which makes separation and purification process challenging. 2. Repeated passing through disrupting equipments lead to considerable decrease in particle size making the downstream processes even more difficult. 3. Mechanical processes are generally energy inefficient. On the other hand scale up of a Nonmechanical process is also not possible. One way to deal with this is by combining both the aspects of mechanical and non-mechanical process.This results in decreased exposure to mechanical disruptor and higher intracellular release. In a study by H. Anand and coworkers in 2007 [8], conducted on intracellular protein from E.coli, it was found that pretreating the cell culture with an optimised amount of EDTA resulted in less pressure requirement in high pressure homogenisation (HPH). Significant reduction in energy input was observed for this combination method. Release Kinetics Theory Microbial disruption kinetics for HPH has been extensively studied. Protein release from this method is follows first order kinetics and is described as:

Where Rm is the maximum amount of protein available for release, R is the amount of protein released after N passes from the disruptor, P is the operating pressure of homogeniser and k is a constant. For a pretreated culture, the equation can be modified as:

where R0 is the amount of protein released during the pretreatment. In this study since the pretreated cell culture was washed off the chemicals and released protein, and then homogenised the amount of protein left for release in the homogeniser was (Rm - R0), so the modified kinetics equation is:

Results Chemical pretreatment was successful with: 1. Increasing the amount of inclusion bodies released: It was observed that for EDTA concentration of 0.04M, protein release was maximum. 2. Efficient use of energy: EDTA was successful in permeabilising bacterial cells, bringing down the optimal pressure of homogeniser to 13.8MPa from 34.5 MPa, resulting in 60% energy savings. The experiment was also conducted for chemicals Guanidium Hydrochloride (G-HCI) and Triton X100. The above table tabulates the obtained data. The extent of release measured is in comparison with Rmax. Rmaxwas calculated by measuring protein release from untreated culture under high pressure homogeniser (pressure varying from 13.8MPa to 69MPa). Even though the use of pretreatment techniques have been successful in increased yield of intracellular release and reduced exposure to mechanical disruption and resulted in increase in energy efficiency of the process, the quantity of chemical added in pretreating operation and the operating conditions of the homogeniser needs to be optimised. The chemicals were found to have minimal interference with protein release. Still the removal of chemicals after pretreatment is necessary to avoid product denaturation as failure to do so will result in sub-optimal yield. Table 1: Protein release following pretreatment and homogenization of E. coli at 13.8 MPa [8]

FILTRATION
Filtration is a process of separating, fractionating or concentrating particles, molecules or ions within or from a fluid by forcing the material through a porous or semi-porous barrier. Types of Filtration Techniques Membrane filtration techniques use membranes of defined pore size and structure. Based on the driving forces and size of molecules retained, there are broadly four different types of membrane filtration techniques:a) Pressure driven operation 1) Macro filtration: It is the process involving separation of two differently-sized particles in which one particle is at least 5 m in size. 2) Micro filtration: It functions primarily on the basis of surface capture or rejection of matter of rated pore size. It separates in the range of approximately 0.05-5 m. 3) Ultra filtration: It is fundamentally the same as microfiltration but it separates in the range of approximately 0.001-0.05 m. 4) Reverse osmosis: Osmosis is a natural phenomenon that takes place when water passes from a less concentrated solution through a semipermeable barrier to a more concentrated solution. Reverse osmosis occurs when pressure more than the osmotic pressure is applied to the side containing the more concentrated solution, leading to reverse flow taking place. It separates particles less than 0.001 m.

Fig. 5: Comparison of different filtration techniques [9]

Fig. 6: Reverse Osmosis Schematic [10]

b) Concentration driven operation 1) Dialysis: It is a separation of solutes in based on size exclusion across a semi permeable membrane where difference in concentration drives the process.Particles from a region of higher concentration move towards a region of lower concentration.

Fig. 7: Depiction of Dialysis [10] 2) Osmosis: It a separation from a region of higher concentration to the region of lower concentration across asemi-permeable membrane. c) Operation in electric potential gradient Electro dialysis:It is defined as the transportation of ions through a semipermeable membrane as a result of an electrical driving force. Most frequent use of electro dialysis is removing salt from water.

Fig. 8: Electro Dialysis [10] d) Polymeric Membrane Extraction It is used for selective extraction and concentration of organic compounds from dilute aqueous solution. The pores of these membranes are impregnated with polymeric liquid having an affinity for the organic compound of interest.

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Advances in Filtration The major problem in an ultra-filtration process is membrane fouling due to the accumulation, denaturation and aggregation of proteins at the membrane-solution interface. This fouling is usually attributed to the non-specific protein adsorption on the membrane surface. Various techniques have been suggested to reduce this fouling. One of the methods is to improve the hydrophilicity of membrane. This is so because hydrophilic surfaces have the capacity to mitigate the adsorption of non-specific proteins. Thus a lot of effort has gone into enhancing the hydrophilicity using physical and chemical methods. This, however, reduces the pore size of the membrane sometimes, which is still a problem to be tackled. Thus, there is always a trade-off between permeability and selectivity for UF membranes. This problem can be resolved by yet another alternative technique, in which anionic or cationic groups are anchored in the barrier layer of membranes. This helps in improving the performance of UF membranes by controlling the surface charge density because the charged membranes provide high retention of protein with the same charge by exploiting electrostatic phenomena. Conclusions Different process industry deals with different types of particles having different size or structure having different characteristics of filter media and different operating condition so a single method cannot be applied. Hence it is always essential to select the best technique for a particular case.

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CENTRIFUGATION
Modification of centrifugation system: Culturefuge
Introduction Harvesting is performed by separating the cell culture from the growing medium and several techniques are used to perform this delicate operation. Most industrial applications use disk stack centrifuges to remove cells and cell debris from the nutrient broth but acceleration of the product rich feed material causes damage to the product. The highly shear-sensitive cell wall membranes are destroyed and cause lysis due to which undesirable intracellular proteins get released into the broth. Culturefuge system is a modification of the disk stack centrifuge. It is a hermetic cell culture centrifuge which is designed for gentle harvesting of cell culture having shear sensitive material. Hermetic means it eliminates any air entrainment and eliminate any air-liquid interface inside the separator bowl which can cause problems in downstream filtration process. Using culturefuge technique, we can prevent additional lysis during acceleration; also it is possible to increase the separator's capacity while still achieving the required separation result. Downstream purification of target proteins is also simplified,thus generating significant savings in the process. This technique gives maximum separation efficiency with minimal product disruption. Design principle Research has shown that the breaking of cell membrane can be avoided by using hermetic conditions. In a mathematical model for feed zone breakage of shear sensitive particles in centrifugal separators, the breakage was found to be independent of flow-rate. When air is not present in feed zone, the maximum energy dissipation is half of that when air is present in feed zone.Higher energy dissipation leads to more breakage of membranes, hence more lysis.Also there are other problems like foaming due to contamination by air which cause degradation of products. Design of culturefuge system It is a skid-mounted system consisting of a disk-stack centrifuge mounted on a fixed base frame with horizontal drive shaft, worm gear, lubricating oil bath and hollow vertical bowl spindle, and piping for service liquids and process liquids. It includes an integrated electrical system with starter, programmable logic control (PLC) system and pneumatic unit. Working of culturefuge system Feed material enters the culturefuge through a hollow spindle feed inlet and accelerates gradually as it moves upwards, thereby minimising the shear forces on the liquids and preventing celllysis. To prevent the risk of mixing with air, the feed zone is completely filled with rotating liquid. The provision of this completely hermetic outlet eliminates the possibility of materials coming into contact with the air or the external environment,thus avoiding foaming and denaturisation of the product. The biological material to be separated enters the centrifuge through a hollow spindle feed inlet and gradually accelerates as it moves upward to the disc stack, where the separation takes place in the rotating centrifuge bowl.The 12

separated liquid phase leaves through the liquid outlet at the top of the bowl. The collected solids in the solid space are intermittently discharged from the periphery of the bowl. During normal production the operating water keeps the sliding bowl bottom closed against the bowl hood. During discharge the sliding bowl bottom drops for a short time (less than a second) and the solids are ejected through the discharge ports. The high velocity of the ejected solids is reduced in the cyclone. The design of a culturefuge is shown in Fig. 9:

Fig. 9: Design of a Culturefuge [19] Description of figure 1. Hollow spindle in bottom through which feed is introduced to the rotating centrifuge bowl. 2.Distributor in which feed is accelerated. 3.Disc stack in which separation takes place. 4.Liquid outlet at the top of bowl through which separated liquid phase leaves. 5.Solid space in which collected solids are discharged from periphery of bowl. 6.Sliding bowl bottom which remains closed against the bowl hood by the operating water during the normal operation. 7.Discharge ports through which solids are ejected when sliding bowl bottom drops. Conclusion It is found that the culturefugeprovides a 2.5-fold increase in throughput for the same clarification performance when compared to the simple centrifugation. Downstream processing become much easier since the impurities are much less in this system.Due to use of hermetic enviroment, the problem of degradation of product is also eliminated.So its beneficial and more economical to use culturefuge system instead of ordinary centrifugation system.

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MEMBRANE CHROMATOGRAPHY
An alternative to Resin Chromatography
Introduction Chromatography is a very critical technology used in the downstream processing in the biopharmaceutical industries. It is one such technique which utilizes both the physical and chemical differences of the biomolecules to achieve a satisfactory level of separation. The major objective to be achieved via this process is to obtain highly purified target biomolecules that are completely free of contaminating host-cell protein, viruses, nucleic acids, enzymes, and endotoxins. Traditionally, this has been achieved through small resin beads packing in a column, with very good results [20]. The recent developments in the upstream stage of bio-processes, as have already been discussed above, have shifted the complete weight of the capital investments onto the downstream purification stage. On the other hand, the ever-increasing requirements of a high-throughput, single-use (i.e. disposable) continuous operation have driven recent researches in developing a cost effective alternative to resin chromatographic columns [21]. One of the possible alternatives was to use larger size resin beads to obtain high-throughput. However, this not only increased the capital cost way too much, but also led to a compromise in the resolution of the final product. Additionally manufacturing limitations exists regarding the size of high-pressure columns [20]. Having listed the various drawbacks which led to a shift of the technology from the resin beads columns, an alternative is being suggested in this section. Membrane chromatography seems to be a very promising option, particularly for processing large volumes, and many pharmaceutical companies have already started adopting this technique [21]. One of the most significant advantages for membrane chromatography is its reduced mass transfer resistance as compared to column chromatography. This results in a fast binding behavior with a high linear velocity of the mobile phase. Thus, the flow rate becomes a critical determinant for cost-effective operation of this technology. Other benefits include reduced buffer usage due to a low void volume; lowered pressure drops, compression, and channeling which effectively simplify the operational facility; and high scalability for bioprocess development. Furthermore, membranes are simple to manufacture and, therefore, the cost of the stationary phase is reduced. Finally, while membrane adsorbers can be reused, they are often promoted as disposable, eliminating the need for lengthy cleaning and regeneration [21]. Despite of the many advantages over the traditional resin bead type column, membrane columns are still in a very primitive stage of development and requires a lot of work to be put in areas to address certain drawbacks such as poor binding capacity, ineffective design(scale up), as well as irregular physical characteristics of the membrane such as pore size distribution, membrane thickness and ligand density [21]. Limitations of the traditional resin based column chromatography and where does membrane chromatography come into picture: a comparative study. 14

Many limitations of the traditional resin based column chromatographic techniques have been identified which has led to the need for developments in the field of membrane chromatography, some of which have already been highlighted in the introduction of the discussion. The major drawbacks have been discussed in details henceforth [20]: 1. Throughput: The resin based chromatographic columns suffer from a big disadvantage of its capability of handling small volumes of the feed stream. This happens because, to improve the resolution of the final product, smaller resins have to be used, which ultimately limits the order of flow rates that can be handled by the column. While, on the other hand a comparatively smaller membrane chromatography unit can do the workload of a large chromatography column. In fact, it offers about 100 times better throughput, with very good efficiencies when compared to the traditional column chromatography units. 2. Residence time of the chromatography step: Resin-bead chromatography presents several challenges to fast purification, and this step usually tend to become the rate limiting step of the whole bio-process. This can be attributed to the fact that the resin beads have long and tortuous internal pores, which lead to long and restrictive path of motion for the biomolecules. This problem can be addressed in the membrane columns because it the surface for the motion of the molecules is readily accessible in case of membranes thus resulting in a low mass transfer as compared to the column chromatography. 3. Cost Effectiveness: Due to the bulky nature of the resins used the column chromatography becomes a very capital intensive unit. Now, if we decide to process larger volumes of the streams through the column, larger columns will be required and the cost input will shoot up very sharply. Apart from this, a very good amount of money and energy also goes into the regular cleaning and maintenance of the unit. This is where membrane chromatography units prove their real worth. A study shows that the cost of a membrane chromatography unit required to achieve the same throughput as the column chromatography is nearly half that of the latter case. Though the membranes can be reused very easily and cleaning is not that big an issue for this case, but these days single use membranes are being promoted to completely do away with issues of cleaning. Even the single use units are cost effective when compared to the resin beads chromatography column [22]. Table 2: Key Factors between the Two Chromatography Methods [22]

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Design Considerations for a Membrane Chromatography Column

Fig. 10:A typical Membrane Chromatography Unit [23] 1. Membrane support: The pore size, structure and distribution are typically designed for operations in the microfiltration range with high flow rates. Pore size is designed keeping in mind that sufficient access of the ligands is allowed to the large biomolecules, with minimum exclusion at the pore entrance. While very large pores can diminish operational flow. Membrane should also be capable of withstanding harsh operating conditions. Organic support such as regenerated cellulose (RC) is most popular choice as a support [21]. 2. Membrane Ligands: Affinity is the most prevalent chemistry for chromatographic membrane. Protein A for purification of immunoglobulins, immobilized metals for purification of his-tagged proteins, dye affinity and specialized ligands are some common ligands used in the industries [21]. 3. Membrane geometry: Common geometries include stacked disk type column, cross-flow sheet cassette, hollow fiber, spiral wound and pleated sheet type column. This is another advantage of having membrane chromatography that flow pattern can be varied by using some unconventional type of geometric format, which is very difficult to implement for resins [21]. Conclusions In the above discussion we saw the major factors which are driving innovations in the field of a very important bio-process of chromatography. The discussion focused on the need for a change required from the resin beads chromatography column and what can be a cost effective yet an efficient alternative to the traditional ways. We saw that membrane chromatography columns, while being very effective are also cheaper to use, not to mention many other advantages over the traditional resin beads column and hence can prove to be very effective alternative the same.

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ION-EXCHANGE CHROMATOGRAPHY (IEC)

Introduction Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. Thus, the two main controlling factors in ion exchange chromatography are the ionic charge (z) and the ionic radius (r). These two are combined in the ionic potential z/r that can be used to compare relative electrostatic bond strengths. Ions of high charge (trivalent or greater) and small radii have high electrostatic bond strength, and therefore are likely to attach strongly to an oppositely charged solid surface, whereas ions of low charge and large radius form relatively weaker electrostatic bonds. By manipulating the chemistry of the solution, ions can be separated from each other using this contrast in bond strength. Technique for separation by IEC In an ion exchange column, the stationary phase is a resin on which a usually organic coating provides a charged surface. The resin typically consists of inert (commonly polystyrene), spherical beads; these are coated with any range of polar molecules, some of which provide negatively charged surfaces (for separation of cations), and others of which provide positively charged surfaces (for separation of anions). The most common properties of all ion exchange resins are: A. They are generally insoluble in water and organic solvent such as benzene, ether and carbon tetrachloride (CCl4). B. They are complex in nature i.e. they are polymeric. The most important resins are polysterene resins formed by condensation of styrene and divinyl benzene. C. They have active counter ions that will exchange reversibly with other ions in a surrounding solution without any change in material. 1.Preparation of Column:The ion exchange chromatography is carried out in a chromatographic column which usually consists of a burette provided with a glass wool plug at the lower end. Generally a ratio of 10:1 or 100:1 between height and diameter is maintained in most of the experiment. Too narrow or too wide column give uneven flow of liquid and sometimes poor separation. 2.Preparation of Ion Exchange: Ion exchange materials are first allowed to swell in buffer or in HCl or NaOH solution for 2-3 hours or sometimes overnight. Almost all ion exchange resin swells when placed in buffer or distilled water and this is due to hydration of their ions. In dry condition, the pore of resins is restricted so in order to swell the pore of resin. Resins are suspended in buffer solution or in distilled water. 3.Washing of Ion Exchangers: The ion exchange material is obtained in required ionic form by washing with appropriate solution. For e.g. the H+ form of cation exchange resins is obtained by washing the material with HCl then with water until the washings are neutral. 17

Anionic exchangers are generally supplied in the form of salt and amines. Similarly, Na+ form is prepared by washing the resins with NaCl or NaOH solution and then with water. 4. Packing of Column: This is one of the most critical factors in achieving a successful separation. The column is held in vertical position and the slurry of resins is poured into the column that has its outlet closed. The column is gently tapped to ensure that no air bubbles are trapped and that packing material settles evenly. 5. Sample Application: Sample can be loaded by using pipette or syringe. The amount of sample that can be applied to acolumn is dependent upon the size of the column and the capacity of resins, if the starting buffer is to be used throughout the development of column, the sample volume is 1 % to 5 % of bed volume. 6. Development an Elution of bound ions: Bound ions can be removed by changing the pH of buffer. E.g. separation of amino acid is usually achieved by using a strong acidic cation exchanger. The sample is introduced onto the column at pH of 1-2, thus ensuring complete binding of all of the amino acids. Gradient elution used in increasing pH and ionic concentration results in the sequential elution of amino acid. Then acidic amino acid such as aspartic acid and glutamic acid are eluted first. The neutral amino acid such as glycine and valine are eluted. The basic amino acid such as lysine and arginine retain their net positive charge at pH value of 9 to 11 and are eluted at last. 7. Analysis of eluate: Equal fraction of each elute are collected at different test tube keeping the flow rate at 1 ml perminute. The eluate collected in each fraction is mixed with ninhydrin color reagent. The mixture is then heated to 105C to develop the color and intensity of color is determined by colorimeter method or spectrophotometer method at 540 to 570 nm.

Fig. 11: Ion exchange chromatography [29]

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Advancements
Polymeric monoliths can be used as ion exchangers in the chromatography column. The use of polymeric monoliths in ion-exchange chromatography applications is advantageous because of their typically high mechanical stability and tolerance of a wide range of pH conditions. In addition, the continuous structure, the porosity of the material and pressure stability are key features of these materials. Polymeric monoliths A polymeric monolith is a continuous porous polymer. Polymeric monoliths are made from a mixture of initiator,monomers (including crosslinking monomer), and pore forming solvents that are polymerized in a mould, for example a column, capillary or the channel of a microfluidic device. The formation of a monolith starts with a homogeneous polymerization mixture where both initiator and monomer are dissolved but, as the polymerization proceeds, polymer chains form that will certainly not be dissolved in the polymerization mixture. The resulting phase separation causes the continuous porous polymer to precipitate out and the monolith is formed, in the shape of the mo uld in which it was made. Introduction of ion-exchange functionalities As is the case for particles, there are a number of ways in which ion-exchange functionality can be introduced in a polymeric monolithic column, relying either on the chemistry of the support material itself or on the introduction of functionality to the surface of pre-prepared support materials. In general there are two ways of achieving this: 1. Functionality by co-polymerization: The chemistry of the monolithdepends on the monomer and cross linker used in itspreparation. Therefore, choosing monomers with ionisable features will result in a final material having ionisable groups. Incorporation of functionality can be achieved by alterations of either the non-crosslinking monomer or the crosslinking monomer used.Photo initiated polymerization of meth-acrylic acid (MAA) and ethylene glycol di-methacrylate(EDMA) produces a weak cation exchange monolith. 2. Post polymerization modification:Another approach to obtaining ion-exchange functionality isthe use of post-polymerization modification. The generaladvantage of performing a postpolymerization modificationis that the support monolith can be optimized independently ofthe desired final surface chemistry. Optimization of porousproperties thus needs only be performed once and, by careful selection of the post-polymerization approach, a range ofmaterials can be prepared from the same support monolith.Somepost-polymerization modification strategies require an initiator to be present on the surface ofthe monolith whereas others rely on anchor points such asvinyl groups and the use of an external initiator. As is thecase for particles, the reactive epoxy, tosyloxyl, and halogen groups provide for possible reaction sites or handles forincorporation of functionality. Examples include reaction of different reagents with functional groups onthe monolith surface, grafting of monomer to orfrom the surface, and coating procedures includingthe use of latex particles.

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SINGLE USE (DISPOSABLE) TECHNIQUES

Introduction In the biopharmaceutical industry, the term single use, also commonly known as disposable, refers to a product that is intended for one time use. Generally such objects are made from a plastic (polyamide, polycarbonate, polyethylene, polyethersulfone, polypropylene, polytetrafluoroethylene, polyvinylchloride, etc.) and are disposed after use. Single use/Disposable technology has emerged over the past decade as a cost effective and flexible basis for biopharmaceutical manufacturing. It has moved beyond the limited applications of culture bags, liquid storage bags, and sampling devices, and now includes more unit operation based capabilities such as cartridge filtration, ultrafiltration, chromatography, etc. Traditional vs Disposable A key aspect in traditional manufacturing plants is cleaning between runs of vessels and other equipment that comes into contact with products, this is a laborious and time consuming requirement that means manufacturing process must be taken offline and cleaning procedure must be extensively validated and documented to demonstrate elimination of bio-burden and residual products and to prevent cross contamination in multiproduct manufacturing plants. With these disadvantages in mind, Single use technologies were first introduced as a means to avoid cleaning and validation requirements and simultaneously reducing the risk of contamination and also reduced the need for utilities such as steam used to sterilize product contact equipment. The amount of liquid waste generated is high in traditional systems and the amount of solid waste generated in single use systems is higher, but the environmental benefits of reduced energy demand can be said to outweigh the increase in solid waste generated in disposal of single use devices. The disposable technology fully eliminates the traditional glass flasks and stainless steel reactors and employs fully disposable plastic products, which cuts down the costs not only of the material but also of the water used for cleaning purposes.Factors driving growth of single use systems are summarized in Table 3. Some breakthroughs in single use technologies are: 1. Cell culture in single use bio reactors 2. WAVE bioreactors 3. Orbitally shaken bioreactors 4. Pneumatically mixed bioreactors 5. Stirred tank bioreactors.

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Table 3: Factors driving the Growth of Single Use Technologies (adapted from [30])
Market Factors Advantages Current Limitations

Emphasis on production costs Flexible, multiproduct manufacturing facilities Biosimilars Multiple, smaller manufacturing plants collocated with markets Increasing number of low-volume biopharmaceutical products

Reduced capital costs for plant construction and commissioning Reduced risk for product cross-contamination in a multiproduct facility Rapid changeover Lower utility costs due to reduced need for SIP Reduced need for cleaning validation

Leachables and Extractables Prior investment in fixed equipment Scales limited by current 2000 L cell culture bioreactor capacity Limited number of vendors High cost of disposables Lack of universal standards for vendors

Challenges Faced by Single Use Technologies One of the key challenges faced by these technologiesis that of limited scale: the scales are limited compared to conventional technologies but it should be emphasized that it is a matter of time and in future this limitation will likely be overcome. Restricted diversity of options is another limitation, though it is less prevalent at laboratory scale but increasingly relevant as we move towardsprocess scale manufacturing. Lack of standardization and regulation of quality of materials used is also one of the key reasons cited by manufacturers for not taking up disposable technologies due to an inability to determine the nature, quantity and risk associated with leachables and extractables from the disposable plastics, which could potentially contaminate product intermediates.The issue of organic compounds leaching out of plastic surfaces is often cited as a concern because these leachables could interfere with cell growth and activity. Other performance issues and the disposal of plastic equipments after their use are also areas of concern. Future directions The next frontier in single use technologies will be expansion to large scale production, making them a viable replacement for stainless steel in a wider segment of production space and in turn will increase the throughput. In future it will also be necessary to develop sensor and monitoring technologies in compatible with single use facilities. Current single use microbial fermenters are limited to 50L scale and absence of higher volume fermenters is a critical unmet need. Further innovation is required to increase number of suppliers, standardization of vendor support packagesand integration of biosensor technologies for non-invasive process control. Conclusion Single use (disposable) technologies offer cost effective and flexible manufacturing alternatives for biopharmaceutical industry. This cost and flexibility advantage is most pronounced for clinical trial, initial product launch and small commercial scale manufacturing. The lower water requirements for this technology can mean a difference between feasible and infeasible projects with water supply or waste water generation sites. The industry which requires large number of smaller volume products is expected to present additional opportunities for this technology. 21

MONITORING AND CONTROL OF BIOPROCESSES

Introduction For efficient and controlled functioning of any biological process at the industrial scale, it is necessary to implement a variety of monitoring systems and control measures. There are a number of parameters that can greatly affect the product yield, selectivity, and purity such as temperature, pressure, pH, stream flow rates, system weight, extent of foaming, power delivered, nutrient and product concentrations etc. Sensing the levels of these parameters within a working system and ensuring that they lie within an optimum range is crucial for any practicable bioprocess. The need for precise control of the bioreactors functioning has been recognized since long ago, with many devices and sensors usually inbuilt into the system. Thermocouples, thermistors, and many thermometer designs have been developed for temperature measurement, and pressure gauges incorporated both from the systemic optimality and safety standpoints. Flow rate measurements can be carried out through a variety of flow meters, while foaming can be quantified using conductivity probes, with electrodes used for pH control. While these have been standard practice for far longer than a decade, there have been many recent advances in the sensing and monitoring of cells and specific substrates and products. This is particularly challenging due to the wide array of substances within the milieu, and older methods usually relied on either downstream or off-line analyses for detection and quantification of the specific species. However, with recent focus being on developing on-line methodologies to achieve the same, many new developments have happened in this area, some of which we will explore below. Recent Advances 1. Mass Spectrometry One of the promising recent developments is that of advances in mass spectrometry that allow for on-line estimation of volatile species concentrations as well as that of dissolved gases. For instance, Proton-Transfer Reaction mass spectrometry (PTR-MS) allows for probing concentrations in the range of parts per billion by exploiting proton transfer events that occur during collisions [33]. Another new technique is that of surface enhanced laser desorption ionization mass spectrometry (SELDI-MS). One interesting study using the latter involved that of production of ApolipoproteinA-IM (ApoA-IM), produced using an E.coli host [34]. It was found that SELDI-MS techniques could be used even at small times after the start of the separation, which was a limitation of priorly-existing techniques. These methodologies are however highly expensive, and hence have limitations in their application. 2. Biosensors Biosensors convert a systemic parameter value into an electrical or electronic signal, and show high specificities and sensitivities when used for analyses. The fast progress and developments in DNA 22

recombinant technology has allowed the evolution of numerous biosensors of late, which can be made to target specific reporter proteins [35]. The green fluorescent protein (GFP) is one of the common targets in research studies for understanding fundamental phenomena. Industrially, biosensors have not achieved a great deal due to difficulty in sterilization (especially in the case of enzymatic biosensors) and also the unstable nature of biosensor-intermediates. Much work is being done on these aspects, and some promising results have also been obtained. 3. Miniaturized Sensors There has been much effort expended into developing small miniaturized sensors to measure the various parameters in any operation. For example, miniaturized calorimeters have been developed [36] to monitor the systems temperature. The growing field of microfluidics has also impacted this area, especially for fine processes that are on a smaller scale. 4. On-line Analyzers Continuous Air Segmented Flow Analyzers and Flow Injection Analyzers were the primary ex-situ on-line analyzers available for flow systems for most of the latter half of the previous century. Modern-day methodologies rely on combining them with techniques such as flow cytometry for bioprocess monitoring, with one recent study using this technique to monitor the products synthesized by Pichia pastoris [37]. 5. Optical Methods There are a number of optical methods that have been developed for bioprocess monitoring aspects [38]. These could be based on optical density, or spectroscopic or spectrophotometric measurements. Image analysis after microscopic techniques is also an area that is being explored. Fluorescence-based FRET biosensors have also been developed recently [39]. 6. Computational Methodologies There have been many computational tools developed for design of a bioprocess. For instance, metabolic flux analysis (MFA) utilizes a graphical map developed on the basis of extensive databases to find the fluxes through the sections of the reaction network. This is done on the basis of an optimization technique and some studies have recently been carried out with promising results [40]. Another recent work [41] used artificial neural network concepts to estimate biomass concentrations, showcasing the power of computational methodologies in modelling and monitoring. Future Directions It is certain that improved sensing techniques will play a major role in improving bioprocess technologies in the future. All of the above-mentioned techniques are being actively researched and explored, with an attempt to remove any drawbacks of these techniques such as issues with scaling up, and high cost. New methodologies to detect a variety of parameters, especially cell, substrate and product concentrations, will no doubt be developed, with the progress in this area surely followed with much interest and avidity. 23

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