Precipitation of Process-Derived Impurities in Non-Protein A Purification Schemes for Antibodies - Precipitation prior to capture chromatography offers a simple, robust, and economical method to

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Precipitation of Process-Derived Impurities in Non-Protein A Purification Schemes for Antibodies
Precipitation prior to capture chromatography offers a simple, robust, and economical method to remove CHO host cell proteins and DNA.


BioPharm International Supplements


ABSTRACT

Precipitation of process-derived impurities prior to capture chromatography in antibody purification offers a simple, robust, and economical method to efficiently remove Chinese hamster ovary (CHO) host cell proteins and DNA. By optimizing the major process parameters—pH, caprylic acid concentration, and mixing time—and understanding their interdependency, one can develop a cost-effective process step. When precipitation is applied directly in CHO cell culture, it combines the clarification and precipitation unit operations. The direct precipitation of contaminants results in seamless transition from upstream to downstream purification processes, particularly in high cell density and high titer cell culture. As a result, demand on the purification process is significantly lowered, and a simple two-step ion exchange process is sufficient to achieve therapeutic purity.


Sartorius Stedim Biotech GmbH
Cation exchange (CEX) capture chromatography in non-affinity processes generally requires a feed conditioning step to lower the pH and conductivity in order to attain high binding capacity (~100 mg/mL). Typically, this is achieved by including a concentration and diafiltration step for primary recovery or, less preferably, through dilution and pH titration of the clarified cell culture bulk, resulting in large processing volumes.1

Primary recovery TFF offers several advantages, including batch volume reduction, partial purification of process-derived DNA, and a cleaner feed stream for better column lifetime and performance.1 On the other hand, it is a lengthy unit operation, with high costs in terms of buffer consumption and TFF cassettes. Diafiltration also adds further constraints in the case of high titers by requiring even higher buffer and tank volumes at production scale.

For those reasons, alternative technologies such as precipitation have been explored, particularly to achieve higher throughput for cell culture processes with high titers. Precipitation of the protein of interest has proven successful in food, blood, and enzyme manufacturing. For example, antibodies have been precipitated successfully at large scale by adding polymers of ethylene glycol (PEG)2 or salts3 and pH titration,4 and research-scale precipitation of contaminants by charged polymers,5 cationic detergents, or short-chain fatty acids6,7 has shown promising application at the cell culture clarified bulk (CB) stage.

In this study, we extend the application of precipitation that combines pH control and caprylic acid (a short-chain fatty acid) to remove process-derived impurities directly from CHO cell culture in the production of human monoclonal antibodies (HuMAbs).


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