Biomanufacturers face a conflict between low-cost generic host cell protein (HCP) assays and highly sensitive but more costly process-specific HCP assays that are usually not initiated until the proof-of-concept stage. But drug developers cannot expect sufficient sensitivity from most commercially available generic assays. For some companies, multiproduct HCP assays could offer a solution to the dilemma.

Biopharmaceutical manufacture using genetically modified microorganisms and cell lines is typically associated with contamination by process-related impurities. One of the most important contaminations during product development and production of biopharmaceuticals is HCPs originating from the production cell line. HCPs are inherent to all production processes relying on bacterial, yeast, or mammalian cell culture, and they can reduce drug efficacy and even delay or kill promising drug candidates through adverse patient reactions.

PRODUCT FOCUS: PROTEINS (PRODUCTS OF CELL CULTURE AND FERMENTATION)

PROCESS FOCUS: DOWNSTREAM PROCESSING

WHO SHOULD READ: QA/QC, PROCESS DEVELOPMENT, AND ANALYTICAL

KEYWORDS: HOST CELL PROTEINS, CHO CELLS, ELISAS, SDS-PAGE, PLATFORM TECHNOLOGIES

LEVEL: INTERMEDIATE

Biomanufacturing therefore requires high-quality process development to reach a maximum degree of purity and demands suitable analytical methods to ensure accurate determination of residual HCPs. Such proteins in a drug substance should be “below detectable levels using a highly sensitive analytical method” (1). As a rule, they should not exceed ~100 ppm (2).

Host cell protein populations are typically measured using enzyme-linked immunosorbent assays (ELISAs) with polyclonal antibodies. Such assays are widely applied during process development, for example, to demonstrate manufacturing consistency, for routine quality control, and for final product release testing. The first step in developing an HCP assay is generating and preparating an appropriate HCP-containing solution to use as an antigen for generating polyclonal antibodies. Development of a highly sensitive assay that can detect HCPs at levels ≥10–100 ppm is challenging and requires broad experience in antibody development.

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Proteins that are weakly or nonimmunoreactive are especially difficult to detect. The number of immunoreactive HCPs can be increased by chemically modifying an antigen or implementing special immunization protocols. A number of cascade immunization protocols showing improvements in immunoreactivity have been reported (3,4). Several rounds of animal immunizations are often necessary to secure broad antigen recognition. Nonimmunoreactive proteins can be detected by combining one- or two-dimensional sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with sensitive protein silver staining. Protein is subsequently identified with mass spectrometry and/or Western blotting (5). Mass spectrometry (MS) identifies contaminating HCPs, although it is not as sensitive a method as are ELISAs.

The strength of immunoassays is their detection of HCPs at levels that are undetectable using electrophoresis. An ELISA measures the collective sum of immunoreactive proteins; in other words, a signal is obtained from a number of different proteins present at low levels. So even when a recombinant product is so pure that there is not enough HCP species to be detectable with a polyacrylamide gel, an ELISA can measure residual proteins. Overall, the ELISA is more sensitive than are methods that monitor individual HCPs.

Generic and Process-Specific Assays

In early phases of clinical product development, when the risk of drug failure is still high, sponsors often use generic HCP assays to minimize costs. When a drug has passed the proof-of-concept hurdle, then process-specific HCP assays displaying full specificity can be developed.

Commercially available “generic” HCP assays are supposed to measure all host cell proteins that could be present in a given cell line — e.g., Chinese hamster ovary (CHO) cells — independent of the downstream processes used. Such assays are based on polyclonal antibodies developed against a representative HCP preparation, so they broadly react with as many HCPs as possible. Process-specific assays use polyclonal antibodies produced against HCPs sampled from a given mock fermentation and a conditioning or purification step. Such assays are usually highly specific for their corresponding HCP mix, so they are generally expected not to react as broadly as generic assays for an accurate determination of HCPs from different production processes.

Although generic HCP assays are intended to detect nearly all possible HCP contaminations independent of an individual production process, several sponsors have found insufficient sensitivity with such assays and a lack of immunoreactivity with certain HCPs. To investigate this issue, we compared several process-specific HCP ELISAs with a commercially available generic HCP ELISA regarding their ability for quantitative HCP determination in CHO-derived antigen preparations.