What is Host Cell Protein?
Host Cell Proteins, or HCPs, are endogenous to the cell line which has been used to express your protein or biopharmaceutical. They are removed during the manufacturing process using a variety of purification techniques. However, it is likely that a low-level of HCP material will remain, and this is where host cell protein analysis comes into play. It is important to continuously monitor the level of host cell protein throughout your process and show a reduction following each purification step. This is because HCPs can cause immunogenicity in the patient and therefore pose a significant safety risk.
Acceptable limits of host cell protein in the final drug product have been set by the FDA and EMA as 1-100ppm, but unknown HCPs at a level lower than this could still cause an immunogenic effect. This is why it is crucial to fully characterize the HCP profile in your drug using various orthogonal tests.
Host Cell Protein Analysis- Orthogonal Testing
HCP analysis has historically been performed using ELISA (enzyme-linked immunosorbent assay) tests. The level of coverage achieved using off-the-shelf kits can be improved with the use of product-specific methods, but a significant number of HCPs are still likely to be missed, especially very weakly immunogenic or non-immunogenic species.
In contrast to HCP-ELISA, which relies on the presence of polyclonal antibodies for positive Host Cell Protein detection, Mass Spectrometric (MS) approaches have the advantage of detecting a wider population of HCPs in a de novo manner, without specialized polyclonal antibody reagents or product and process-specific method development.
Due to these technical benefits, HCP-MS has emerged as an orthogonal technique that serves to expand the identification of Host Cell Proteins, with regulatory advice stating that HCP assessments should include MS as part of the normal process and product development.
The BioPharmaSpec Approach to Host Cell Protein Analysis
BioPharmaSpec has developed a service for MS based HCP detection and quantitation as an orthogonal support to HCP-ELISA. The service is set up as a phased approach:
Phase 1 – Qualitative analysis to identify protein impurities present in each sample
A cell line extract (used for the biopharmaceutical synthesis) should be analyzed in this phase to build an HCP library and provide a complete overview of the cell line HCPs that could potentially be in the sample of interest.
Direct on line LC/ES-MS-MS analysis
- Digestion of the HCP protein mixture from a cell lysate followed by on-line LC/ES-MS/MS and/or MSe analysis of the products (2-Dimensional LC methods can be developed as required).
- Creation of a protein library, based on the proteins observed in the HCP mixture.
- Digestion of the biopharmaceutical product of interest and other samples of interest followed by on-line LC/ES-MS/MS and/or MSe analysis of the products (2-Dimensional LC methods can be developed as required).
- HCPs present in the biopharmaceutical product are identified based on an assessment of the data obtained against the expected sequence for the product, the reference HCP protein library and relevant database searches.
The on-line LC/ES-MS/MS work involves:
- Proteolytic digestion of the samples of interest.
- Analysis of the products of digestion using UPLC-MS/MS (and/or MSe) on a Q-TOF instrument. Various analyses are undertaken to optimize the BioPharmaSpec in-house method for analysis of HCPs specifically for the samples.
- An assessment of the data obtained against the expected protein sequence for the product, the HCP protein library and the relevant protein database.
Gel Based Analysis
Preparation of 1D or 2D-gels using a Mass Spectrometric-compatible stain followed by identification of the proteins using in-gel digestion and MS/MS analysis (MALDI-TOF/TOF) with protein database searches to identify the proteins.
The gel-based work involves:
- Analysis of each of the supplied samples using 1-Dimensional or 2-Dimensional Gel Electrophoresis (including supplied HCP mixtures).
- A comparison of the gel images obtained from analysis of the product samples and the HCP mixtures from a cell lysate.
- Excision of the gel bands/spots consistent with protein impurities (HCPs) in the gels obtained from the product samples and the equivalent spots on the gels obtained from analysis of HCP mixtures.
- In-gel digestion of the excised spots.
- Extraction and clean-up of the released peptides.
- MALDI-MS/MS of the released peptides.
- Assessment of the data against a relevant protein database.
- Identification of the proteins from the gel spots of interest.
Phase 2 – Quantitation against the response from a known protein spiked into the sample
Following assessment of the results from Phase 1, digestion of the protein mixture followed by on-line LC/ES-MS/MS and/or MSe analysis of the products will be repeated with the addition of a suitable mix of protein standards, to estimate the levels of a sample’s protein impurities in relative terms.
Quantitation against a protein standard will therefore provide a quantitative estimate relative to the protein standard or mixture. This estimate will be compromised to an extent by the differences in ionization efficiency between the various peptides in the sample and reference protein mixture, something that cannot be taken into account in the quantitation calculation. For this reason, BioPharmaSpec also recommends using a more accurate quantitation method, such as that described in Phase 3 below, to quantify the HCPs of concern or of particular interest.
Phase 3 – Quantitation against heavy-labeled marker peptides
The data obtained from Phases 1 and 2 will define marker peptides for the host cell protein impurities of interest. In order to provide an accurate quantitation of these impurities BioPharmaSpec recommends synthesis of carefully selected heavy-labeled peptide analogues, which are then spiked into samples for analysis. Protein amounts are then calculated based on a comparison of the response from the native and heavy-labeled peptide analogues as outlined below:
- Multiple Reaction Monitoring (MRM) transitions are developed for each native and heavy-labeled peptide.
- Samples are spiked with the heavy-labeled peptides.
- Samples are then analyzed using the developed MRM based method on a triple-quadrupole mass spectrometer and the amounts of the components of interest are quantified relative to the relevant heavy-labeled peptide markers and, if required, the known amount of the purified protein in the sample.
The analysis method within Phases 2 and 3 above can be qualified and transferred to a GMP compliant facility for routine testing if required.
- HCP-MS is an orthogonal approach to Host Cell Protein analysis, to be used to enhance ELISA data.
- BioPharmaSpec can provide a full service for MS analysis of HCPs including accurate detection of HCPs of interest i.e. those present in amounts >100ng per mg of product or those that are immunogenic.
- HCPs should be assessed from early in the product development. It can then be shown that improvements in the downstream purification through process-development are having a positive impact on the levels of HCPs present.