Monoclonal Antibody Purification Common Problems And Solutions

1. What is the introduction of protein A and its interaction with IgG antibodies?

Antibody production and purification are achieved through highly standardized platforms including centrifugation, microfiltration, ultrafiltration, protein affinity chromatography, virus inactivation, ion exchange chromatography, and hydrophobic interaction chromatography. Protein A affinity chromatography uses natural or recombinant protein ligands obtained from Staphylococcus aureus or Escherichia coli, coupled to natural or synthetic substrates. Protein A has five homologous domains (E, D, A, B, and C) that can bind to the Fc portion of immunoglobulin G (IgG). In addition to mAb, protein A interacts with polyclonal antibodies (pAbs) produced by different cell lines with different antigen-binding properties.

Protein A affinity chromatography is a well-known process in the pharmaceutical industry because of its high binding affinity and the purity level that can be obtained. The purification process involves loading A clarified cell culture containing target antibodies at neutral pH onto an immobilized protein A carrier, allowing for ligand interaction. The protein is then isolated from impurities such as host cell protein (HCP) and the product desorption is achieved by reducing the mobile phase pH. The resin is then regenerated and an in situ cleaning program is performed.

Protein A can be covalently bound to natural (agarose or cellulose) or synthetic (polyethylene ether, polystyrene divinylbenzene, porous glass or polymethacrylate) substrates. In addition to this, another obstacle to protein A technology is resin scaling, which is due to non-specific adsorption, resulting in reduced adsorption capacity and shortened resin life. In response to this concern, several studies have attempted to develop enhanced protein A resins: stationary phases that can tolerate high concentrations of NaOH (up to 0.5M), for example, to improve their cleaning and disinfection efficiency and extend their service life.

2. What are the strategies to improve the stability of protein A purification?

(1) Replacement of asparagine 23 residue with threonine leads to higher stability for 0.5M NaOH. This improvement avoids deamidation, which causes the protein to become unstable at alkaline pH.

(2) The ligands were chemically modified with 5 and 20.7kDa polyethylene glycol (PEG). Static binding affinity was observed after PEgging. The decrease in DBC is due to a decrease in the effective pore diffusion of the pegylated ligand and a decrease in the binding kinetics of the IgG. Despite this, the mass of media-related pollutants was reduced by a factor of ≥2. Finally, a 15% increase in the recovery rate of IgG was observed in the elution after pegylation.

3. What are the improvement directions of protein A purification methods？

Since protein A ligands do not bind to all types of IgG, and new therapeutic approaches such as antibody fragments may lack the Fc region, combinations of different ligands such as protein L or protein A and new affinity ligands may emerge at a commercial level as the next generation of mAb isolation. However, in the case of DBC, alternative ligands such as peptides, FC-binding proteins, and nanobodies need to be improved to be considered a viable option. In addition, further research is needed to develop new or enhanced processes, stationary phases, and ligands that can surpass the simplicity, yield, manufacturability, and cost-effectiveness of protein A-bed chromatography.

4. What are the differences between caprylic acid precipitation and caprylic acid - (NH4) 2SO4 continuous precipitation for antibody purification?

In terms of antibody yield, caprylic acid precipitation is comparable to caprylic acid - (NH4) 2SO4 continuous precipitation, but superior to antibody affinity chromatography and high-performance liquid chromatography. In terms of antibody purity, caprylic acid precipitation is less efficient than the other three methods. It should also be noted that caprylic acid precipitation is associated with reduced affinity of certain antibodies and is not suitable for purifying mouse IgA and IgG3.

5. What are the characteristics of antibody purification by HPLC?

The main problem encountered by this method is the interaction of antibody molecules with the columnar substrates, resulting in a non-ideal chromatography of peak broadening and elution observed at significantly lower molecular sizes. Under such conditions, the decomposition of monomers from other species is greatly affected. The interaction occurs primarily through two mechanisms, including hydrophobic and/or ionic forces. The hydrophobic action can be minimized by adding a small amount of detergent or organic solvent to the mobile phase, while the ionic action is mainly controlled by changing the pH or ionic strength. Due to the weak acidity of many column support materials, alkaline hydrophobic antibodies are a major problem. The addition of charge (basic) ionizing agents can help minimize both types of forces. In this regard, amino acids are the most suitable (for example, lysine and arginine). The separation time is usually more than 30 minutes per sample.

Based on a proven antibody platform system, KMD Bioscience can provide complete upstream and downstream services, from antigen design, synthesis and modification, animal immunization, antibody purification, phage library construction and screening to downstream antibody modification and application identification. We also provide antigen preparation services, including DNA synthesis, plasmid construction, protein expression, protein purification, peptide synthesis and modification, small molecule structure analysis and cross-linking with carrier proteins.