Host Cell Protein contaminants in mAb and protein therapy manufacturing

Host cell proteins (HCPs) are process-related impurities produced by the host organism during biotherapeutic manufacturing. During the purification process, a majority of HCPs are removed from the final product. Residual HCPs that copurify with the manufactured antibody therapy, even at a low level, can elicit immune responses in patients. HCPs can also directly effect the quality of the drug product itself. For example, proteolytic HCPs, even in minute quantities, can cleave the desired protein product over time, reducing or eliminating biological potency or altering stability.

The measurement and control of these HCPs are important to achieve the desired efficacy, shelf-life, or other important attributes of the therapy. USP recently released a draft of <1132.1> Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry as a new general chapter to introduce mass spectrometry (MS) techniques for identification and quantitation of HCPs lingering in recombinant therapeutic products. These evolving methods for HCP analysis are orthogonal to ELISA and other analytical methods and enable the user to build an understanding of HCP profiles and clearance patterns throughout the process. To view the chapter visit here.

USP has developed Analytical Reference Materials (ARMs) to help detect and characterize problematic host cell proteins (HCPs) that co-elute with monoclonal antibodies (mAb) and other protein therapeutics manufactured in Chinese Hamster Ovary (CHO) cells.

Benefits of the USP Host Cell Protein ARMs

Multiple SIL peptides per high-risk HCP target
Purity of SIL peptides > 95% (very important for LC-MS sensitivity)
Complements quantitation methods in USP General Chapters:
1. <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals
2. <1132.1> Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry, (in PF 49(3)) (not yet official)

Host Cell Protein Product Types

Recombinant HCP proteins expressed in Chinese Hamster Ovary (CHO) cells

Applications:
- ELISA
  - Confirm if a total HCP ELISA can detect the target HCP
  - Compare performance of anti-target HCP ELISAs
  - Include as control for anti-HCP target ELISA
- Western Blotting
  - Confirm total HCP antibodies can detect the target HCP
  - Compare anti-target HCP antibodies
- Mass Spectrometry
  - Control for development of Multiple Reaction Monitoring (MRM) for target HCP
    - Identification and relative quantitation of target HCP
  - Could be included in LC-MS/MS performance optimization as System Suitability control/standard
- Host Cell Protein targets
  - PLBL2 (Phospholipase B-like 2

Stable isotope labelled (SIL) peptides containing sequences from HCPs of concern (identified in CHO-produced products)

Applications:
- Mass Spectrometry: Identification (presence) of target HCPs
- Mass Spectrometry: Quantitation (relative) of target HCPs
Host Cell Protein Peptide targets
- Clusterin – Coming Soon!
- Lipoprotein Lipase – Coming Soon!
- Contact us at USPBiologics@USP.org to tell us which other HCP targets you need for your work.

Host Cell Protein Targets

Clusterin (CLU)
- Clusterin binds with high affinity to both Fc and Fab domains of IgGs, thereby preventing mAb/protein activities.
Lipoprotein Lipase (LPL)
- Lipoprotein lipase is an enzyme that hydrolyzes triglycerides in lipoproteins, such as chylomicrons and very low-density lipoproteins (VLDL), into free fatty acids and glycerol. One of the primary concerns surrounding LPL is its potential to degrade essential excipients, specifically polysorbate 80 (PS-80) and polysorbate 20 (PS-20), which are commonly found in final product formulations. The structural similarity between polysorbates and triglycerides makes them susceptible to degradation by LPL. This enzymatic activity could have far-reaching implications for the stability of therapeutic molecules, particularly during long-term storage.
Phospholipase B-like 2 (PLBL2)
- Phospholipase B-like 2 protein is a lysosomal enzyme involved in the phospholipid catabolic process. Its presence in the final product can jeopardize patient safety through two mechanisms: inducing immunogenicity and enzymatic activity leading to undesirable drug molecule

When to test for HCPs during mAb/protein manufacturing

While no specific limits exist in regulatory guidance for HCPs, according to ICH Q6B specifications, HCP impurities should be minimized using appropriate, well controlled manufacturing processes.

HCP levels should be measured in:

early development: from clone selection to process development;
preclinical lots used in toxicology assessment;
all lots during clinical development (Phases 1 through 3);
process validation samples from the final commercial manufacturing process;
after approval, HCP monitoring may be required as an element of the control system.

Host Cell Proteins