A HCP Enzyme-linked Immunosorbent Assay is directed against all proteins of the cell line used to manufacture the biopharmaceutical. HCP ELISAs are routinely used to quantify and monitor HCPs. They combine sensitivity with high sample throughput – necessary requirement for industrial scaling.
Generic HCP ELISA Kits
For this assay the biggest challenge is development of the polyclonal antibody.1 In order to match regulations and to avoid product contamination a high coverage ratio is mandatory.2 This antibody differs between species (the host chosen for the experimental set-up) and even single cell lines. Although application range of this ELISA is narrow, it is referred to as “Generic HCP ELISA Kit” or “Commercial HCP assay”. Generic HCP ELISAs are useful in both process development and process validation (Figure 1).
HCP ELISA for Process Monitoring
During manufacturing process development, a new version of ELISA is required to account for process changes which influence the proteome and therefore the HCP composition. In-House HCP assays or process specific HCP ELISAs take more effort to develop (Figure 1) but support with higher HCP coverage.
Orthogonal Methods for HCP detection
However, generic ELISAs do not offer complete coverage for all process-specific HCPs and process-specific ELISAs might be not qualified to evaluate the HCP content after process changes.3 During assay qualification and validation and release process validation and quality control (Figure 1) it is crucial to utilize additional methods beside ELISAs that provide very different selectivity to the primary method, that is, is orthogonal(Table 1). The orthogonal method can be used to evaluate the primary method on an ongoing basis, to assure that the primary method remains specific if new synthetic impurities or degradation products are formed in subsequent batches of drug substance or drug product4.
|Quantify HCP content||LC-MS/MS: identify individual HCPs|
|Analyze sample linearity||Electrophoresis (2D-DIGE, 2D WB|
|Monitor in-process steps||Capillary electrophoresis to account for prevalent HCPs|
|Monitor process consistency and product purity||Complement ELISA in process consistency and product purity, especially in after process changes|
A comparative protein analysis by two dimensional difference gel electrophoresis (2D-DIGE) combined with fluorescent staining is applied to verify the representative character of null cell culture fermentation HCP proteins towards HCP in manufacturing process. This approach enables simultaneous separation of HCPs in a single gel and yields at least the detection sensitivity of silver staining procedures. The technique is semi-quantitative, has a limited dynamic range, and needs mass spectrometry for HCP identification5.
Similar to 2D-DIGE, the fundamental deficiency of 2D WB is that it does not predict how the ELISA or similar sandwich assay format using that Ab will quantitatively react to the reduced array of HCP in final drug substance. The determination of anti-HCP reactivity to individual HCP curcial for development of process-specific HCP.
Antibody Affinity Extraction
Antibody Affinity Extraction (AAE) is another alternative, especially for upstream samples such as cell lysates or conditioned media. The anti-HCP antibody is covalently bound to a chromatography support. The starting HCP sample is re-extracted and eluted until no significant HCP can be recovered. The eluted HCPs from all extractions are combined and prepared for 2D gel fractionation. Both, the starting, unextracted sample and the AAE sample are fractioned in separate 2D gels and can be compared afterwards.
2D HPLC-ELISA aims to fractionate HCPs into two dimensions. First dimension is chromatofocusing step able to process milligram quantities of HCP. The enormous loading capacity leads to improved detection sensitivity in comparison to 2D WB8. Second dimension is created by reverse phase gradient chromatography. 1000 to 2000 single fractions emerge which can be tested in ELISA for reactivity. ELISA provides a much higher sensitivity than WB, enough to identify and assess individual reactivity to downstream HCPs and therefore a higher specifity as well. The denaturation is reversible in this case, too. It is a more quantitative approach than 2D WB – comparison of total mass instead of spot matching.9
LC / MS MS
Approaches involving liquid chromatography coupled to mass spectrometry (LC-MS) provide alternative solutions for product characterization within the biopharmaceutical industry and have enabled the analysis of low-abundance analytes in complex protein mixtures. LC-MS is able to provide additional details about the individual HCPs and their quantity without the long development and immunization process of a specific ELISA.10
SWATH Acquisition has recently become one of the premier mass spec acquisition strategies for identification and quantitation of analytes complex samples. SWATH provides an unbiased data-independent method for detecting low-level HCPs – even those that were previously unknown or did not generate an immunogenic response in animal studies. SWATH is the only data independent acquisition (DIA) technique enabling detection and quantitation of virtually every detectable substance in the sample. The obtained extensive values of total HCP content in ng/mg drug substance, pI, MW, and quantity in ppm are robust and reproducible. SWATH LC-MS provides a list of proteins and quantities in each process step for HCP clearance analysis in virtual 2D plot.11
The technique is comprehensive, sensitive, fast, and reproducible, enabling the profiling and identification of the HCP complement of 1000s of proteins at a sub-ppm level in a single 1-hour run. Still, rather than replacing ELISA, SWATH Analysis can be seen as a complement for fully characterizing and monitoring HCPs.12
No complicated and extensive LC fractionation is required, together with the speed, sensitivity, and dynamic range of the TripleTOF System the full HCP complement can be detected at sub-ppm levels in 1 hour.
- Sarah Gilgunn (2018). “Challenges to industrial mAb bioprocessing—removal of host cell proteins in CHO cell bioprocesses” in ScienceDirect. 22: 98 – 106. DOI
- FDA, (2018). “Establishing Impurity Acceptance Criteria As Part of Specifications for NDAs,
ANDAs, and BLAs Based on Clinical Relevance” in MANUAL OF POLICIES AND PROCEDURES, Link
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- Kesh Prakash, (2015). “Analytical Methods for the Measurement of Host Cell Proteins and Other Process-Related Impurities” in ACS Symposium Series, Chapter 9, pp 387-404 DOI
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- Weibin Chen, (2015). “Improved Identification and Quantification of Host Cell Proteins (HCPs) in Biotherapeutics Using Liquid Chromatography-Mass Spectrometry” in ACS Symposium Series, Vol. 1202, Chapter 13, pp 357–393 DOI
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