Monoclonal antibodies have emerged as one of the most interesting therapeutic mode in the biopharmaceutical industry. Nineteen monoclonal antibodies have been approved for therapeutic purpose and several of these molecules serve significant medical needs (Shukla, Hubbard et al. 2007). Some of important are mentioned in Table 1.
Crucial properties of monoclonal antibodies for biological uses include their specificity of targets in vivo diseases. Therapeutic antibodies are reprenseted as IgGs with IgG1 and IgG2 being the most common subclasses . Biochemical structure of IgGs consists of two heavy and two light chains held together by intra-molecular disulfide bonds. Each chain comprises of fixed and variable regions (heavy chain: CH1, CH2, CH3 and VH; light chain: CL and VL). Fig1 describes a schematic structure of a monoclonal antibody.
Fig. 1. Structure of a monoclonal antibody. VH, variable region, heavy chain; CH, constant domain, heavy chain; VL, variable region, light chain; CL, constant domain, light chain. Adopted from (Shukla, Hubbard et al. 2007)
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The development of monoclonal antibodies has significantly increased the demand of production for biopharmaceuticals. Most biotechnology products including a variety of vaccines, hormones and growth factors required very small quantities of purified product. In contrast, due to the high doses and the large patient populations in the indications they have been approved for, monoclonal antibodies require large sums of annual production of bulk drug substance. Thus, the considerations for large-scale production of pharmaceutical grade antibodies are quite different from their routine laboratory scale purification .
Monoclonal antibodies are generally large, glycosylated molecules, they are usually produced commercially by deep tank mammalian cell culture . Recent increases in cell culture titers to >2 g/L  have modified this production technology to stave off immediate competition from transgenic sources of production although these production methods might still find broad applicability in the future . Our report mainly deals with the recovery and purification of monoclonal antibodies by techniques like chromatography.
This report presents an overview of large-scale downstream processing of monoclonal antibodies. The recent research and Phase I trial has shown therapeutic modality of monoclonal antibody for a range of critical sicknesses. Our downstream processing incurs several hurdles due to significant biochemical differences and different challenges, making downstream purification as an impractical process. Here, we describe the key components of a flexible downstream process that we can adopt for future use. The process consists of well-defined sequence of operations with most predefined parameters. The process relies on the Protein A chromatography, a highly effective step in downstream processing of monoclonal antibodies. Key elements of each step with the description of process validation and characterization will be discussed below.
As Antibodies are secretions of cells so cell culture is basic step in the production of antibodies. Effective recovery and purification of antibodies from cell culture media is a vital part of the downstream production process and can significantly effects proportion of the total output quantity and manufacturing costs . The primary condition during downstream process is purity. Another important consideration is the speed of process. Overall yield and process throughput are the key consideration of the whole downstream process. In addition, the process must be reliable, cost effective and meet several manufacturability standards including robustness.
Product should be freed from process related contaminants like endotoxins, DNA, cell culture media additives, host cell protein and as well as product related impurities e.g. aggregates of high molecular species with low molecular weight species. The process should also validate extra care in cleaning of viruses.
For the process-scale purification of monoclonal antibodies, a variety of preparative modes of chromatography have been utilized. Most of them involved the use of Protein A affinity chromatography. Protein A chromatography employs the principle of tapping the specific interactions between the Fc region of monoclonal antibodies and immobilized Protein A, a cell wall component of Staphylococcus aureus  . Protein A affinity chromatography has been shown to be highly effective for monoclonal antibodies downstream processing, resulting in more than 95% purity in a one-step starting from complex cell culture media .
Various other modes of chromatography have been merged with Protein A chromatography to increase pharmaceutically acceptable purity levels. These steps are generally chosen to provide orthogonal modes of interaction with the product with an aim to achieve effective separation from cell proteins and other contaminants. Protein A chromatography is followed by anion-exchange chromatography and size exclusion chromatography is further applied for purifying a monoclonal antibody expressed by hybridoma cell culture . DNA and endotoxins are cleared during the second step of Anion-exchange, while size-exclusion is used as the last step for remotion of aggregates and degradation products. To concentrate the product an ultrafiltration/diafiltration (UF/DF) buffer exchange step is introduced prior to size exclusion chromatography. Other modes of chromatography including hydroxyapatite and immobilized metal affinity chromatography (IMAC) can also been employed for antibody purification . Protein A chromatographic capture followed by cation-exchange chromatography (CEX) and anion-exchange chromatography (AEX) operated in the flowthrough mode  helps to remove host cell proteins, aggregates leached protein A, DNA and further impurities respectively. This sequence of steps has been adopted as a generic purification scheme  for a number of monoclonal antibody products.
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While Protein A chromatography is highly selective for mAbs, the use of an immobilized protein as a ligand also lends its own share of challenges to this mode of chromatography. The ligand is prone to proteolysis and the cleaved domains can adhere to product molecules creating a separation challenge. Conventional Protein A ligands cannot be exposed to alkaline conditions that are commonly employed to sanitize other column modes thus necessitating the use of high concentrations of chaotropes such as urea for column regeneration and sanitization. The use of high concentrations of chaotropes creates a cost issue as well as a disposal challenge. The need to elute the column at a low pH can induce product aggregation for some mAbs. Most significantly, the cost of Protein A resins is nearly an order of magnitude higher than conventional chromatographic resins. Clearly, there is a significant driver for the development of small molecule ligands that can match the selectivity of Protein A for binding to mAbs.
Hydrophobic Charge Induction Chromatography (HCIC) utilises a heterocyclic ligand such as 4-mercaptoethanol (MEP) that takes on an inducible positive charge at low pHs. This resin has been described to be selective for antibody separations   W. Schwartz, D. Judd, M. Wysocki, L. Guerrier, E. Birck-Wilson and E. Boschetti, J. Chromatogr. A 908 (2001), p. 251. Article | PDF (740 K) | View Record in Scopus | Cited By in Scopus (55). However, more recent findings  have suggested this mode of chromatography to be based on non-specific hydrophobic interactions with electrostatic repulsion at low pH being responsible for product elution. In the capture mode, this resin was nearly an order of magnitude less selective for mAbs over host cell proteins as compared to Protein A chromatography but was found to be a potentially useful polishing step. Ligands that can mimic the binding pocket of Protein A for the Fc region of mAbs have been found  and developed into Protein mimetic resins marketed as MAbSorbent A1P and A2P . In internal investigations, these resins have also been found to possess lower selectivity than Protein A. Thus, at this point none of the small molecule ligands can universally match the selectivity offered by Protein A chromatography for mAb separations. However, they might be useful additions to the downstream process sequence due to their orthogonal selectivity with conventional modes of chromatography.
Similar to what is done for other proteins, it is conceptually possible to design a downstream process for mAbs without a Protein A affinity step by employing combinations of conventional chromatographic modes. Three-step combinations of cation-exchange, anion-exchange flowthrough, hydrophobic interaction chromatography and mixed mode cation-exchange chromatography were found to deliver adequate clearance of host cell protein contaminants for a CHO derived monoclonal antibody . However, such purification schemes by-and-large have not caught on in commercial downstream operations due to the need to design the purification sequence separately for each mAb. Given the almost universal applicability of Protein A chromatography and the development of workarounds for most of its limitations (described in Section 4.2), it appears that this ligand will continue to be employed for commercial scale mAb purification at least in the foreseeable future.
The Protein A chromatographic step is typically utilised for direct capture of the product from cell culture supernatant after harvest operations planned to remove cells and cell debris. In a few cases, the Protein A step is the second step in the process following capture on a conventional mode of chromatography . This was done to protect the expensive Protein A resin from possible fouling through direct exposure to cell culture harvest media. However, the development of effective column regeneration schemes usually allows Protein A resins to be employed for over 100 cycles with direct load of the cell culture supernatant. This also eliminates the need for concentration or buffer exchange of the harvest prior to chromatography. For the vast majority of commercial mAb processes, Protein A chromatography appears to be firmly ensconced as the primary capture step that also delivers a high purification factor.
This report mainly gives an introduction to chromatography focusing further on affinity chromatography that is used to purify monoclonal antibodies. Protein A chromatography is widely used to purify monoclonal antibodies mainly because of its high selectivity. To meet the demands with the increasing bioreactor volumes and cell culture expression levels an optimum use of expensive Protein A affinity resin (€6000-€9000/L resin) can have significant benefits (Swinnen et al., 2007).
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Despite Protein A affinity chromatography being a predominant mAb capture step in bioprocessing it has several limitations. Firstly the high cost of the resins. Secondly the differences in the dynamic binding capacity at various flow rates and in the pressure-flow characteristics of various Protein A chromatographic media can result in wide variations in throughput (Shukla et al., 2007). A Protein A step also adds impurity in the process in the form of leached Protein A ligands. Another major limitation is the inability to increase large-scale column diameter to beyond 2m
without encountering significant issues with flow distribution and packing (Shukla et al., 2007). There is a ready market for the company that can produce a Protein A resin with a dynamic binding capacity of >50 g/L at an even lower residence time (Low et al., 2007). Some of the options that are currently being investigated include higher ligand densities on their resins, ligand orientation/accessibility, particle size, pore size and distribution, alternative protein A mimic synthetic ligands and more stable support matrices with increased mass transfer.
1.2 Affinity Chromatography
Affinity chromatography involves the use of ligands that attach to the media and that have binding affinity to specific molecules or a class of molecules.
Ligands can be bio-molecules, like protein ligands or can be synthetic molecules. Both types of ligand tend to have good specificity. But protein ligands have the disadvantage that they are expensive and mostly denature with the use of cleaning solutions, whereas synthetic ligands are less expensive and more stable.
The most commonly used protein ligand in production is the protein A ligand, which is specific to the IgG antibody.
In affinity chromatography when the solution is introduced to the column the target protein is adsorbed while allowing contaminants (other proteins, lipids, carbohydrates, DNA, pigments, etc.) to pass through the column (Fig 1.3). The adsorbent itself is normally packed in a chromatography column; though the adsorption stage can be performed well by using the adsorbent as stirred slurry in batch binding mode. The next stage after adsorption is the wash stage, in which the adsorbent is washed to remove residual contaminants. The bound protein is then eluted in a pure form. Elution is normally achieved by changing the buffer or salt composition so that the protein can no longer interact with the immobilized ligand and is released. Affinity chromatography can be performed in a fixed bed or a fluidised bed.
Fig 1.3 - Stages during the affinity chromatography process that includes adsorption, wash and desorption
1.3 Types of media
The support to which the ligands can be attached to can be of different materials.
1.3.1 Natural polymers
Natural polymers can be of agarose, cellulose or dextran. Their most beneficial point is that these materials have very low non-specific adsorption. This is because the polymer chains are very hydrophilic due to the presence of many hydroxyl groups and hence the proteins do not adhere to them.
Dextran and agarose have better flow properties than cellulose. Fibrous cellulose is extremely hard to pack (Jaunbauer et al., 2005). Its usefulness is limited by its fibrous and non-uniform character. The main disadvantage of dextran is its low degree of porosity. Agarose beads on the other hand are extremely soft and therefore cross-linked to increase the strength. However due to their loose structure it allows ready penetration by macromolecules with molecular weights in the order of several millions (Lowe et al., 1974). The uniform spherical shape of the particles in particular gives good flow properties.
1.3.2 Synthetic polymers
The three synthetic polymers of importance are hydrophobic vinyl polymers, polyacrylamide and polyvinylstyrene.
All of these are relatively hydrophobic and hence needs to be modified by coating the surface with a hydrophillic polymer to avoid low recovery (Jaunbauer et al.,
2005). An advantage of the synthetic polymer-based media is their resistance to extreme chemical conditions, such as pH.
The main disadvantage with polystyrene adsorbents is their relatively low porosity. Polyacrylamide beads on the other hand are superior to many polymeric supports due to their polyethylene backbone, which increases chemical stability. They tend to have a more uniform physical state and porosity, permitting the penetration of macromolecules with molecular weights of upto 500,000 (Lowe et al., 1974). Another advantage is that they posses many modifiable groups which enables the covalent attachment of a variety of ligands.
1.3.3 Inorganic media
The commercially available inorganic media are made of ceramic, silicate or glass. The silicate media are coated with several other materials. The most common glass media used consists of irregularly shaped controlled porous glass (CPG) particles. These adsorbents are rigid and can work at high flow rates. Due to their controlled pore size they tend to generate sharp exclusion limits (Lowe et al., 1974).
1.4 Modes of Chromatography
Elution is carried out by first introducing a small sample of the mixture on to the column. It is then eluted with a mobile phase, which has a lesser affinity to the stationary phase than the sample components. The components then move along the column depending on their relative affinity for the stationary phase but at a slower rate than the eluent. Hence the components can be completely separated with a zone of mobile phase between them. This mode of chromatography is commonly used for analytical purposes (Braithwaite et al., 1985).
1.4.2 Frontal Analysis
Frontal analysis is executed by continuously adding the sample onto the column. The component with the least affinity for the stationary phase will pass along the
column while the component with the greater affinity will get adsorbed to the stationary phase. Eventually this component will pass along the column, when the capacity limit of the stationary phase is exceeded. This mode of chromatography is used to achieve breakthrough curves (Section 1.8.2).
Displacement is carried out by first introducing the sample mixture onto the column. Elution then occurs when a displacing solvent is passed through the column, which has a greater affinity for the stationary phase than the sample components.
Antibodies are protein molecules that play a crucial role in the immune system, which defends the body against toxins that enter the bloodstream and counters the disease threat posed by invading microbes and viruses. Antibodies are made in white blood cells, B-lymphocytes. Each B-lymphocyte makes copies of its own unique antibody, which are then displayed on its outer surface. Immunoglobin G is the major antibody in serum. It has a Y-shaped structure composed of four protein subunits which comprises of two identical light chains and two identical heavy chains as shown in Fig 1.4.
N N N N
Fig 1.4 - Structure of IgG molecule (L. Stryer 1995)
Monoclonal antibodies can be used therapeutically, to protect against diseases; they can also help to diagnose a wide variety of illnesses, and can detect the presence of drugs, viral and bacterial products, and other unusual or abnormal substances in the blood. Their specificity makes monoclonal antibody technology so valuable. Given such a diversity of uses for these disease-fighting substances, their production in pure quantities has long been the focus of scientific investigation. The monoclonal antibody (mAb) market has grown rapidly in recent years, reaching sales of $14bn in 2005, an increase of 36.5% from 2004 sales of $10.3bn. Monoclonal antibodies are homogenous because they are synthesised by a population of identical cells (a clone). Each such population is descended from a single hybridoma cell formed by fusing an antibody producing cell with a tumour cell that has the capacity for unlimited proliferation. Hence it possesses a structure that can only bind to one epitopal group on one antigen. Polyclonal antibodies, in contrast with monoclonal ones are products of many different populations of antibodies-producing cells and hence differ somewhat in their precise specificity and affinity for the antigen. Hence a polyclonal antibody mixture has typically most or all of the antibodies acting against all epitopal groups for antigens on the molecule.
1.6 Protein A affinity chromatography
1.6.1 Protein A
Protein A affinity chromatography is widely applied in the commercial production of IgG. Protein A, which has a molecular weight of 42,000, is a cell wall protein from Staphlococcus aureas with affinity for the Fc region of IgG. The amino-terminal region contains five highly homologous IgG-binding domains (Fig 1.5). All five IgG- binding domains of SpA bind to IgG via the Fc region (Jansson et al., 1993). Binding of the antibody is normally done at pH 7 and elution at pH 2-3 (Jungbauer et al., 2005). The two binding domains at the C-terminal are non-immunoglobulin binding regions and are thought to be responsible for the binding of Protein A to the
bacterial cell wall.
Fig 1.5 - Schematic drawing of regions encoded by the gene for Staphylococcal protein A. S is the signal sequence. E, D, A, B and C are the immunoglobulin binding regions. Xr and Xc are C-terminal located, non-immunoglobulin binding regions (Uhlen et al., 1984).
1.6.2 Commercially available Protein A media
There are various commercially available protein A chromatography media. The main differences between these media are the support matrix type, protein A ligand modification, the pore size and the particle size. The differences in these factors give rise to differences in compressibility, chemical and physical robustness, diffusion resistance and binding capacity of the adsorbents (McCue et al., 2003). Products of different manufacturers were researched to arrive at this table.