Purification Of Antibodies 3 Essays Example

Biotechnological Approaches to purifying High Value Products (i.e. antibodies)
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Chromatographic process has been the best and efficient method of purification for decades. 6
Affinity Chromatography 7
Examples 8
1-Insulin 8
2-Antibodies 9
3-Protein A 10
Conclusion 11

References 12

Purification of Antibodies
Advancements in the field of Biotechnology has been boosted with the improvement in techniques of protein purification. Protein purification methods range from a simple process to a complex multiple step processes. An important aspect of successful results is the identification of the right techniques and their optimization to obtain maximum protein yield. Chromatography is an essential laboratory tool and is part of most of the purification techniques. Recombinant DNA has been another powerful technique that revolutionized mass production of protein (Protein Purification, 2001; Liu et al., 2010).
Why purification is required?
Many companies are using multiple antibodies as part of their product development, where these molecules are obtained from a common set of techniques or a framework. Most molecules have similar physiochemical properties that allow establishing a standard purification process. Many impurities such as endotoxins, DNA, endogenous and adventitious viruses, host cell protein must be removed to obtain a satisfactory protein yield. Purification is important in order to obtain a product that is fit for human consumption. Some impurities like resins extracts, leached Protein A, virus removal agents, are introduced during the purification process. Such impurities must also be removed to have a healthy yield (Liu et al., 2010; Matsudaira, 2012).
Purification Strategies
Purification process is a set of multiple step protocols that are used by most purification techniques. In the case of use of multiple techniques, conditioning steps are performed to transfer the product between techniques. Maximizing the yield is an example of using such strategy. In each step of a single technique, the yield is typically 80%. This means that increasing the number of steps would eventually decrease the overall yield. Conditioning adds another overhead. A healthy sufficient yield can be obtained by optimizing the steps for purification. It has been evident that an effectively optimized technique can produce high yield with good purity levels and in less than four steps. The conditioning steps can be reduced by choosing the correct sequence of techniques. Figure 1 depicts the relation between steps and percentage of yield produced and shows a typical recovery process (Protein Purification, 2001; Matsudaira, 2012).
Figure 1: Yields from multi-step purifications (Protein purification, 2001).
The phases of purification are three, Capture, Intermediate Purification and the Polishing of the produce. The target during Capture phase is to stabilize, concentrate and isolate the targeted product. Intermediate Purification phase is important to eliminate bulk impurities like nucleic acids, other proteins, viruses, and endotoxins. The Polishing phase ensures high purity by removing any trace of impurities and other related substances. Optimization of techniques with respect to these three phases is critical for obtaining a high producing and fast method (Protein Purification, 2001). Harvested Cell Culture Fluid (HCCF) procedure is the first step obtaining the antibody from mammalian cell culture. Centrifugation, sterile and depth filtration are the common approaches for this step. Chromatography is the industry accepted purification technique. The chemical and physical differences of biomolecules enable chromatography to separate the impurities. Protein-A based chromatography is the basis of most purification processes. Protein-A chromatography yields high purity product in relatively fewer steps. Additionally only one or two steps are required for polishing. The other types of chromatography that can be chosen for polishing purpose are hydrophobic interaction, hydroxyapatite, and mixed mode chromatography. Anion and cation exchange chromatography is used for polishing purpose. These steps remove unwanted variants and minor contaminants. Viral clearance can be achieved by viral filtration steps and low pH hold in case of Protein-A chromatography. Concentration and diafilteration of the final purified product are done. A robust strategy consists of developing an early stage process, efficiency and speed of processes and optimal resources utilization with sturdy viral clearance and validations (Liu et al., 2010; Protein Purification, 2001).
Selection and Combination of Purification Techniques
Different chromatographic techniques are available for achieving different performance levels with respect to speed, recovery, and resolution. Optimization of a technique can be done for any of the parameters for enhancing the performance or achieving a balance with other parameters. Optimization of any one of the parameters would leave an impact on the results differentiating in appearance from the other products. Chromatographic purification is the technique for Protein purification. Different chromatographic techniques are used depending upon the protein property. Table 3 below depicts the various techniques.
Methods of Purification
Primary Recovery Processes
The first step to purification is the elimination of cells and its debris from the culture chowder and cleansing of the cell culture supernatant that comprise of antibody product. There are several methods of purification in which present trend are to improve cell mass and productivity through enriching the culture medium. This process enables to increase the product broth. Primary Recovery Processes include Tangential flow micro filtration, Depth filtration, Centrifugation, Flocculation/Precipitation (Liu et al., 2010).
Chromatographic Processes
Chromatographic process has been the best and efficient method of purification for decades. Its capabilities have made it the most favored technique and constant improvements in the process for improving resins and sorbents; it continues to be on top. There are many types of Chromatography depending on the chemical nature of involved molecules and solvents.
Affinity Chromatography
Affinity separation is the most selective method of chromatography, applied in biotechnology. The main principle of its mechanism is based on a reversible interaction linking a protein with its cognate ligand that is covalently attached to the chromatography matrix (Liu et al., 2010).
The efficiency of affinity depends on the placement of the ligand at a distance from the matrix backbone (Cuatrecasas, 1972). Affinity separation requires an appropriate matrix that acts as a supporter for ligand. A uniform, macroporous, stable, hydrophilic, matrix is preferred that exhibit non-specific absorption capability and insolubility in the solvent. Most studied and widely used activation reagent is Cyanogen bromide, tresyl chloride, carbodiimide, glutaraldehyde and phosphoryl chloride. Ligands play the most important component of stability and specificity of the system. The affinity of a ligand to the target, its specificity and stability in harsh washing, elution and retention of binding capacity is the main features of a ligand.
Bacterial proteins are the best tool to detect and purify the antibodies due to its high affinity towards antibodies. Streptococcal protein G (SpG) and Staphylococcal protein A (SpA) are common ligands applied in full-length antibodies’ purification. The level of affinity varies according to the antibody subclasses (Protein purification, 2001; Liu et al., 2010; Degerman, Jakobsson & Nilsson, 2007).
Affinity chromatography involves four main steps: preparation of absorption media, sample adsorption, washing and elution to acquire maximum purity and yield.
A ligand is immobilized in the preparation of Absorption media, with matrix. A target range of 106 –1010 M, is favored for a ligand (Liu et al., 2010). Mechanisms of other types of chromatography are based on charge, size, hydrophobicity and types of ligands.
Examples
1-Insulin
For the separation and purification of Insulin, affinity chromatography is the most suitable choice. The source material is liver membranes that exhibit high-affinity insulin-binding activity of liver homogenates. The insulin receptor retrieved from these membranes in the liquid form with Triton X-100. Further purification is performed using diethylaminoethyl-cellulose chromatography with 60-fold purification. For the extraction of insulin bound protein, insulin agarose derivatives are prepared using detergent membrane extracts. Urea based buffers at low pH are used to retrieve the macromolecule from the affinity columns with high production (50-80%). This is a purified form of insulin obtained from detergent extraction and affinity chromatography. Insulin has a therapeutic use in treating Diabetes (Cuatrecasas & Jacobs, 2013; Cuatrecasas, 1972)
2-Antibodies
Three types of antibodies are formed using Biotechnological approach. B-lymphocyte lines produce a heterogeneous mixture of antibodies called polyclonal antibodies (pAbs), and they target different epitopes on antigens. A monoclonal antibody (mAb) is produced by B-lymphocytes clone and targets a single epitope on an antigen. The third type is Recombinant antibody fragments (rAbs) produced using various molecular techniques (Ayyar, Arora, Murphy & O’Kennedy, 2012). The purification process applied on antibodies exhibits the efficiency of high selectivity affinity chromatography approach. It is a two-step purification process that requires capture and polishing without intermediate purification. For polishing gel filtration, step is sufficient to achieve pure form of antibodies. For the purification of Antibodies, the chosen target molecule is monoclonal IgG1 antibodies of mouse (Protein Purification, 2001). For sample separation and clarification involves the adjustments of salt concentration and pH according to the binding buffer of the capture step. For such supernatant cell samples, ion exchange chromatography is the most appropriate method because of its binding capability concentrates the objective protein reducing the sample volume. For mAB purification, highly selective affinity chromatography medium is required for capture and a HiTrap rProtein A, column is used. Most mouse mAB that belongs to IgG1 subclass needs high concentration salt that help them bind with immobilized Protein-A. Salt concentration is chosen in such a way that it can produce the largest elution peak area and exploration to the best possible elution pH improve recovery of antibody (Protein Purification, 2001).
In this process, the intermediate purification is not need because the high selectivity of the capture step sufficiently eliminated the contaminations and provided a highly competent purification. It is observed that mostly antibody preparations contain IgG aggregates and dimmers. So it is necessary to elucidate them and for this process a gel filtration polishing step is the most suitable high the degree purity solution. The polishing step eliminates all contaminants ven al least level. For gel filtration, Superdex 200 prep grade media is the best choice due to its proper molecular weight separation range for IgG antibodies. For analytical assay, separation of collected fractions is done by SDS-PAGE. PhastSystem is used for silver staining (Protein Purification, 2001).
3-Protein A
Protein A is used in affinity chromatography as a ligand to purify the immunoglobulins from different sources and species. Protein A is a cell wall bound protein retrieved from Streptococcus and Staphylococcus bacteria. It shows strong affinity towards the IgG regions. Binding occurs at pH value of around three that is necessary for elution inducing denaturing (partial or complete) of IgG. A proper selection of stable monoclonal IgG’s clone can avoid denaturing and increase the pH as well. Protein A is a strong ligand with 5 Ig-binding domains (designated A, B, C, D and E). Crystallographic studies have shown that protein A attaches at Fc region of IgG, in the mid of CH2 and CH3 domains (Janson, 2012).4-DARPins
Designed ankyrin repeat proteins (DARPins) are binding proteins that have broadened the therapeutic approaches to an advanced level. These are small single domain proteins providing more opportunities than mAbs. Their high affinity and specificity provides them a potential of binding with any target protein. They act as a carrier for various effector functions, and enables new drug formats. These are next generation of protein that has exceeded the current antibody approach (Stumpp, Binz & Amstutz, 2008). Its purification is straightforward and IMAC (Immobilized metal affinity chromatography) is the most suitable method of purification. Initially a small amount in milligrams is required and overloading of IMAC column with small capacity produces pure protein in just a single step. Most of the contaminants (E. coli) are eliminated. For separation of PEGylated from non-PEGylated DARPins, Anion exchange method is applied (Tamaskovic, 2012).
Conclusion
Advancement in purification methods applying biotechnological approaches promises highly improved, stable and purified form of therapeutic proteins. Chromatography has been the most suitable and efficient technology of purification that yields pure products solutions. In future it will incorporate high capability chromatography resins, non-chromatographic processes such as membrane adsorbers, flocculation, etc. Through affinity chromatography antibodies, protein A, hormonal proteins and DARPins will give rise to a new cohort of therapeutic agents.
References
Ayyar, B. V., Arora, S., Murphy, C., & O’Kennedy, R., 2012. Affinity chromatography as a tool for antibody purification. Methods, 56(2), 116-129.
Cuatrecasas, P., 1972. Affinity Chromatography and Purification of the Insulin Receptor of Liver Cell Membranes. Proceedings Of The National Academy Of Sciences, 69(5), 1277-1281.
Cuatrecasas, P., & Jacobs, S., 2013. Insulin Receptor Structure and. In Neurotransmitters, Receptors: Proceedings of the 8th International Congress of Pharmacology, Tokyo, 1981 (p. 209). Elsevier.
Degerman, M., Jakobsson, N., & Nilsson, B., 2007. Modeling and optimization of preparative reversed-phase liquid chromatography for insulin purification.Journal of Chromatography A, 1162(1), 41-49.
Janson, J. C. (Ed.)., 2012. Protein purification: principles, high resolution methods, and applications (Vol. 151). John Wiley & Sons.
Liu, H. F., Ma, J., Winter, C., & Bayer, R., 2010. Recovery and purification process development for monoclonal antibody production. In MAbs(Vol. 2, No. 5, pp. 480-499). Taylor & Francis.
MarketWatch, 2015. Global Insulin Market Expected to Reach USD 32.24 Billion Globally in 2019: Transparency Market Research. [online] Available at: http://www.marketwatch.com/story/global-insulin-market-expected-to-reach-usd-3224-billion-globally-in-2019-transparency-market-research-2014-07-25 [Accessed 24 Feb. 2015].
Matsudaira, P. T. (Ed.)., 2012. A practical guide to protein and peptide purification for microsequencing. Elsevier.
Protein Purification: Handbook. 2001, Amersham Pharmacia Biotech.
Stumpp, M. T., Binz, H. K., & Amstutz, P., 2008. DARPins: a new generation of protein therapeutics. Drug discovery today, 13(15), 695-701.
Tamaskovic, R., Simon, M., Stefan, N., Schwill, M., & Plückthun, A., 2012. Designed ankyrin repeat proteins (DARPins) from research to therapy. Methods Enzymol, 503, 101-134.