Therapies Based On Monoclonal Antibodies Biology Essay

Introduction

Therapies based on Monoclonal antibodies (mAbs) are one of the fastest growing and most lucrative segments of the Biopharmaceutical industry (Azavedo et al 2010).Immunoglobulin have a well-defined biochemical structure and consists of four covalently linked polypeptide chains with varying degree of glycosylation. The chains consist of two heavy and two light joined together to form a Y shape by disulphide bonds. They also have two functional regions known as the fragment of antigen binding (fab) and constant fragment (Fc) The first approval of a monoclonal antibody was in 1986 for the treatment of acute kidney transplant rejection and ever since, several mAbs have been approved for the treatment of a varying number of diseases. There has since been an increase in demand for mAbs as many indications require high doses, which has challenged Biotechnologists to increase production capacity and improve the purification technologies involved. Improvements in cell culture technology such as the construction of vectors for high level expression has shifted the bottlenecks normally experienced upstream to downstream, as a result of the time it takes to process a single reactor harvest considering flow rate and capacity limitations or cost of the affinity media in the capture phase (Shukla et al 2007) .Efficient recovery and purification of antibodies has thus become a very critical aspect of the production process. This can impact a great deal on the total cost and can also be construed as being a "cine qua non" to quality and efficacy of monoclonal antibodies in meeting regulatory requirements. The challenges faced in the purification of antibodies include several ranges of sources or contaminants, such as endotoxin, DNA, cell culture supernatants coupled with other amounts of proteins and impurities. The need for high purity as a result of regulatory requirements and the need to reduce the overall manufacturing cost have resulted in the need to optimise the purification process (Kim et al 2005). Chromatography techniques based on immobilised antibody binding proteins such as protein A, G and L are widely utilised in the capture stage of the purification process (Huse et al 2002).Several recombinant forms geared towards increasing the binding specificities and addressing the limitation associated with the natural forms of these ligands have also been developed. Considerable efforts have been spent in the development of ligands with variable complexity and specificity .Affinity ligands have thus evolved from naturally binding biospecific molecules such as Protein A and G to bioengineered and fully synthetic ligands (Roque et al 2007).

Affinity chromatography purification of antibodies relies on the specific recognition or attraction between a ligand attached to a solid matrix and the antibody. This type of complementary interaction can be likened to a lock and key mechanism. Native and engineered antibodies possess high affinity towards specific target and are thus exploited in purification strategies for antibodies(Roque et al 2007).The dissociation technique to recover purified product is often dependent on the type of resin or ligand employed in the purification strategy. The affinity ligand is commonly coupled or immobilised unto a solid support which is often an agarose or its derivative and the adsorption of the antibody can be either positive or negative, although more often than not a positive interaction is always the choice as it allows the concentration of the target molecules (Hage 2005). The interaction is a reversible one that allows the recovery of products which is often by change in pH, ionic strength or the addition of chaotropic agents that serve to either disrupts or cause denaturation. The interaction of the bound antibody to the complementary resin is often as a result of electrostatic, hydrophobic, hydrogen bonds and Van der Waals forces.

There are numerous support matrices in use for affinity chromatography and they include natural polymers such agarose, cellulose and dextran. Synthetic polymers in use for affinity chromatography include hydrophobic vinyl polymers, polyvinyl styrene and polyacrylamide and they share the advantage of greater resistance to pH in comparison to the natural polymers. It should however also be noted that inorganic media made of materials such as glass, silica and ceramic are also finding increasing use in affinity chromatography.

Figure Schematic Representation of Affinity Chromatography Steps for Antibody Purification (Roque et al. 2007)

Protein A. affinity resin is widely used in the purification of antibody due to its wide selectivity characteristics i.e. avidity as mentioned above and has resulted in it being considered as the "Gold Standard" by several scientists in the Biotechnological industry. The use of Protein A. in affinity chromatography for mAbs has been shown to produce a yield of >95% in a single step (Gannon 1995). It should be emphasised however that protein A. has its limitations with regard to cost, life cycle and leachability .In meeting the demand for increased bioreactor volumes, an optimum use of the resin can cost in excess of €6000 - € 9000 per litre which can be really expensive, although the benefit is significant (Swinnen et al, 2007). The limitations of affinity chromatography in keeping up with the increased culture expression due to improvement in upstream technology as mentioned earlier has resulted in scientist looking for ways to optimise the functionality of the resins used in the purification process. These innovative technologies are geared towards, reducing cost, making the resins long lasting and increasing binding capacity. Some of the conventional resins that have been used as a substitute for protein A. resins have been known to offer improved qualities such as, alkali compatibility, reduced leachability, better recycling potential and higher binding (Arunakumari et al 2007) .Mixed mode resins and mimetic ligands have found uses in this area.

Protein A. Background

Protein A is a 56KDa surface protein that is originally found in the cell wall of the gram positive bacterium Staphylococcus Aureus and has found a wide use in Biotechnology due to its ability to bind to the fc regions of most immunoglobulin. The fc interaction has been suggested to come about by the presence of eleven amino acids which are situated in the two helices of each immunoglobulin binding domain (Uhlen et al 1984). The interaction between protein A. and immunoglobulin (lgG) can be said to be one of the most studied protein-protein interactions (Delano et al 2000). Staphylococcal protein A. consist of five domains which are arranged in an helical 3D helical structure that is stabilised by an hydrophobic core (Hober et al 2004).These domains have the propensity to bind to the Fc part of IgG1, IgG2 and 1gG3 with an estimated affinity constant of 108 (M−1) . .It is this binding capability that has made it a standard in the purification of antibodies. The biotechnological applications of Protein A. stretches beyond the purification of immunoglobulin and has been known to activate TNFR1 which is a receptor for tumour necrosis factor-α (TNF-α) that results in pneumonia (Gomez et al 2004). There are various variants of Protein A. which differ to a greater extent in terms of their sequence length but they however still show great homology in terms of their binding capability to immunoglobulin of different subclasses (Olsson et al. 1987). Recombinant protein A has been produced using the Escherichia Coli bacterium e.g. MabSelect and often used by being coupled to magnetic, agarose, latex beads, etc. It should however be emphasised that the affinity of antibodies to bind to the resin is both species and subclass dependent.

There are several limitations associated with the use of protein A in the capture stage of antibody purification. Apart from the primary disadvantage associated with the high cost of the resin which can be several fold more expensive than other conventional resin, atypical protein A. column is cycled several times in the purification of antibody supernatants thus increasing the possibility of the occurrence of leachability. The loading of the column for protein A. affinity chromatography is thus rate limiting, as high volume of cell culture supernatant is utilised comparison to a small column (Shukla et al 2006). The low pH elution of the resin is also a debilitating factor as this makes the antibody in question more prone to the formation of soluble and insoluble high molecular weight aggregates and precipitates

Table 2 : Binding strengths of Protein A. to immunoglobulin from different species.

Aims / Objective:

The main objective of this work is to research alternative resins to Protein A in relation to their limitations and advantages as utilised by the Biopharmaceutical industry for the capture stage of Monoclonal antibody in affinity chromatography applications.

My first goal would be to give an overview of affinity chromatography and the rationale in the selection of an affinity chromatography resin for the capture stage of the purification of an antibody.

My second goal would be to explore other alternative resins such as Lectin, Protein G, protein A/G mixed mode ligands, mimetic, dye binding ligands including other resins applicable to mAbs purification taking process economics and optimisation into consideration. A Comparative study in terms of their limitations and advantages will be researched and inferences drawn on the future trends.

My third goal will be to give a brief overview of other forms of chromatography that have the potential in their use in the capture phase of the purification of mAbs.