Purifying Monoclonal Antibodies

What is Critical to Monoclonal Antibody Purification

Purification of monoclonal antibodies (mAbs) involves isolation of antibodies from ascites fluid or cell culture supernatant of a hybridoma cell line. When purifying monoclonal antibodies, monomer purity and endotoxin level are two important factors.

Monomer purity

Monomer purity is a critical parameter for monoclonal antibody therapeutic candidate in pre-clinical in vivo studies, because aggregate can affect antibody's bioactivity and immunogenicity as well as pharmacokinetics. Therefore, for in vivo studies, antibody's monomer purity typically needs to be controlled above 95%, and in some high-dose studies, one would need to increase the monomer purity to as high as 99%.

Monomer purity can be increased by removing antibody aggregates and fragments through polishing purification steps such as ion exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. For some antibodies that are prone to forming new aggregates in certain types of buffer, polishing steps are not sufficient to increase monomer purity because new aggregates are formed in the polishing process. In such a case, one would need to optimize the formulation buffer that can prevent new aggregate formation.

Sino Biological has successfully produced several thousands of high monomer purity antibodies for customers worldwide. We are highly experienced in handling many challenging antibodies.

Our expertise and experience in antibody purification can be of great value to our customers worldwide, especially for small biotech companies without a large in-house process development team.

SEC-HPLC of recombiant antibody
Antibody purity>99.7% (by SEC-HPLC)

Endotoxin level

Endotoxin is a class of large molecules consisting of a lipid and a polysaccharide. The polysaccharide is composed of O-antigen, outer core and inner core joined by a covalent bond. Endotoxin is derived from the outer membrane of Gram-negative bacteria. Endotoxin can elicit strong immune responses in animals and human, and hence it is important to limit the amount of endotoxin contaminant in injectable protein and antibody products.

In production of monoclonal antibodies for pre-clinical animal studies, one would usually need to control the endotoxin level in the final product to avoid any size effects caused by endotoxin. Endotoxin is usually introduced into the culture systems through raw material, culture medium, container, and plasmids used for transfection. Because the DNA plasmid is produced from E. coli. fermentation, it usually contains very high level of endotoxin. Therefore, it is important to control the quality of raw materials and cell culture medium, and equally important to eliminate endotoxin from the DNA plasmid for transfection. After protein A affinity purification, the endotoxin level is usually still above the acceptable level, and hence typically one would need to use polishing chromatography steps to separate and remove endotoxin from the antibody product.

Sino Biological has produced hundreds of endotoxin controlled monoclonal antibodies in various animal studies for customers worldwide. Our endotoxin controlled antibody production services can meet antibody quality suitable for human use. Specifically, if required, the endotoxin level can be reduced to as low as 0.01-0.05 EU/mg.

Monoclonal Antibody Purification from Various Samples

Centrifugation and filtration are basic laboratory techniques for sample clarification. These steps removes lipids and particle matter, such as cell debris. In order to ensure the quality of monoclonal antibodies, it should be performed before chromatographic purification.

Major contaminants in different sources of antibodies

Source Quantity Major contaminants
Hybridoma Up to 1 mg/ml Serum proteins, phenol red, water, albumin, transferrin, α2-macroglobulin, haptoglobulin, ceruloplasmin, bovine IgG, viruses
Ascitic fluid 1–15 mg/ml Lipids, albumin, transferrin, lipoproteins, endogenous IgG, other host proteins
Recombinant antibodies Depends upon expression system Proteins from the host, e.g., E. coli.

Hybridoma antibody purification

Hybridoma supernatants are easy to produce, especially for large numbers of different monoclonal antibodies, but are relatively low in monoclonal antibody concentration.

Hybridoma supernatants are advantageous because small amounts (<100 ml) can be easily obtained in a few days (<1 week) and from multiple hybridomas with relatively little effort. The first step for this mAb purification, is usually by affinity chromatography (protein A or L column). Further polishing methods can also be performed.

Ascites fluid antibody purification

The mouse ascites fluid contains a higher concentration of the monoclonal antibody than hybridoma supernatants. However, the fluid has a lot of nonspecific immunoglobulins from the host. Immobilized Protein A and Protein G can be used to purify monoclonal antibodies from ascites fluid.

Recombinant antibody purification

Purification of recombinant antibody formats can be achieved using single-step purification protocols, employing ligands specific for the affinity tags co-expressed with recombinant antibodies. Affinity tags are short polypeptide sequences or whole proteins, fused at the gene level with the rAbs. Different types of affinity tags can be used; however, polyhistidine tags are widely preferred. Immobilized metal affinity chromatography (IMAC) is employed to purify recombinant antibodies expressed with polyhistidine tags.

References

1. Hober, S., Nord, K., & Linhult, M. (2007). Protein A chromatography for antibody purification. Journal of Chromatography B, 848(1), 40-47.
2. Low, D., O'Leary, R., & Pujar, N. S. (2007). Future of antibody purification. Journal of Chromatography B, 848(1), 48-63.
3. Fishman, J. B., & Berg, E. A. (2019). Antibody Purification and Storage. Cold Spring Harbor Protocols, 2019(5), pdb-top099101.
4. Arora, S., Ayyar, B. V., & O'Kennedy, R. (2014). Affinity chromatography for antibody purification. In Protein Downstream Processing (pp. 497-516). Humana Press, Totowa, NJ.
5. Yokoyama, W. M. (2008). Production of monoclonal antibody supernatant and ascites fluid. Current protocols in molecular biology, 83(1), 11-10.

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