What do We Know about HCPs – Learning from Literature (1)

Host cell proteins (HCPs) refer to a broad spectrum of proteins produced by the cells in which the product of interest is made. HCPs are process related impurities and are one of many critical quality attributes. Both the FDA and EMA require residual HCP to be reduced to an acceptable level.

• Downstream purification is a critical part of the HCP risk mitigation strategy¹,²;

• Typical assays for HCP analysis include immunoassays (e.g. ELISA), CE-Western, AlphaScreen, Ella, and Octet;

• HCPs can be enriched to improve resolution. There are services available to conduct HCP enrichment;

• Although the general acceptance level for residual HCPs is lower than 100 ppm, individual residual HCP limits should be case dependent ¹,²;

• Different HCPs will have different isoelectric points and hydrophobicities ³;

• “High risk” HCPs are those that are highly immunogenic, reduce product stability, or are especially difficult to remove. The types of “high-risk” HCPs are host cell dependent¹,²;

• Different processes for the same antibody can have different HCPs;

• Protein A can reduce HCP levels but may not reduce the number of types of HCPs ⁴;

• Protein A chromatography columns can also potentially concentrate HCPs

• HCP impurities can have direct or indirect negative effects on drug product stability ¹⁻⁶;

Examples of product stability problems caused by HCPs ⁵,⁶:

• Lipoprotein lipase can degrade polysorbate, which is used to stabilize mAb drug ;

• Cathepsin can cause fragmentation of mAbs

Examples of hard to remove HCPs ⁵,⁶

• Actin

• Annexin A2

• Biglycan

• Cathepsin D

• Clusterin

• Endoplasmic reticulum resident protein 29

• 78 kDa glycose regulated protein

• Galectin-3-binding protein

• Glutathione S-transferase P

• Glyceraldehyde 3 phosphate dehydrogenase

• G-protein coupled receptor 56

• Legumain

• Nidogen-1

• N(4)-(Beta-N-acetylglucosaminyl)-L-asparaginase

• Peroxiredoxin 1

• Pyruvate kinase

• Tubulointerstitial nephritis antigen-like

How can Purilogics help you conquer your HCP removal challenge?

- For cell line development, Purilogics offers high capacity and high-speed Protein A membrane adsorbers (Purexa™ PrA) for high-throughput mAb purification to accelerate analysis;

- For downstream purification, Purilogics offers tentacle-based weak anion-exchange (Purexa™ NAEX Plus) and multimodal strong anion-exchange membrane adsorbers (Purexa™ MQ) with improved impurity removal capabilities.

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References

1. Wang, F., Richardson, D., Mueller, H., Gaza-Bulseco, G., Liu, X., Liu, X., Zhao, Y., Sharma, S., Haindl, M., and Co, C. (2018). Host-cell protein risk management and control during bioprocess development: A consolidated biotech industry review, part 1. Bioprocess International

2. Wang, F., Richardson, D., Mueller, H., Gaza-Bulseco, G., Liu, X., Liu, X., Zhao, Y., Sharma, S., Haindl, M., and Co, C. (2018). Host-cell protein risk management and control during bioprocess development: A consolidated biotech industry review, part 2. Bioprocess International

3. Kornecki, M., Mestmäcker, F., Zobel-Roos, S., Heikaus de Figueiredo, L., Schlüter, H., & Strube, J. (2017). Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly. Antibodies, 6(3), 13.

4. Lenhoff A. Patterns and Determinants of Persistence of CHO Host-Cell Proteins in mAb Bioprocessing, Bioprocessing Summit Virtual, April 24-28, 2020

5. Valente, K. N., Levy, N. E., Lee, K. H., & Lenhoff, A. M. (2018). Applications of proteomic methods for CHO host cell protein characterization in biopharmaceutical manufacturing. Current opinion in biotechnology, 53, 144-150.

6. Kol, S., Ley, D., Wulff, T., Decker, M., Arnsdorf, J., Schoffelen, S., ... & Masson, H. O. (2020). Multiplex secretome engineering enhances recombinant protein production and purity. Nature communications, 11(1), 1-10.