Expression systems used in the generation of biopharmaceuticals (often called biologics or biologicals) contain many proteins. These proteins, termed host cell proteins (HCPs), have to be removed during product purification. Any residual HCP, even low levels, can potentially compromise the safety and efficacy of biopharmaceuticals.
The rapid growth in production of biologics has generated an expanding requirement for identification and evaluation of residual HCP. Antibodies are the most commonly used tool for detecting and assessing HCP, since they can be used for identification, detection, and quantification. Evaluation and validation of anti-HCP antibodies is a crucial step in effective residual host cell protein monitoring.
Biopharmaceuticals are therapeutic agents synthesized, at least in part, using biological processes. The main categories of biologics are organisms (for example, attenuated viruses for vaccination), products derived from organisms, tissues or cultured cells (for example, blood products, stem cells, and monoclonal antibodies), and recombinant proteins.
The two categories of biologicals with the most rapid growth are recombinant proteins and monoclonal antibodies. The many types of expression hosts used for generation of recombinant proteins for therapeutics include mammalian cells, such as Chinese hamster ovary (CHO) cell lines, insect cells, yeast, and bacteria. CHO cells are the most commonly used expression host for recombinant proteins and are also increasingly being used for generation of monoclonal antibodies.
One challenge for the production of biopharmaceuticals is the large number of proteins, present in the expression system or substrate, that have to be removed during the purification process. Even after multiple purification steps, there will often be HCP that copurifies with the biologic. This HCP remaining after product purification is termed residual host cell protein. Due to the inherent variability in biological systems, the amount of residual HCP will change from production run to run, as well as between products.
The use of eukaryotic hosts, particularly mammalian ones, provides the advantage of similar or identical posttranslational processing (for example, glycosylation patterns) and codon bias to that of humans. Though bacterial vectors are used for generation of many biologics and have been developed with mammalian codon bias and altered glycosylation patterns, these bacterial hosts are likely to continue to be less common as hosts. Therefore, mammalian residual HCPs, particularly CHO, are increasing in importance. The CHO protein predicted immunity (CHOPPI) database has been established to identify the immunogenicity of CHO proteins.
The presence of HCP in biologics can potentially affect the efficacy and toxicity of the final product. The presence of residual HCP could change the therapeutic window through a number of different mechanisms such as receptor antagonism or agonism, or albumin binding changing the apparent volume of distribution.
The main concern for residual HCP is immunogenicity in humans being treated with biologics. One advantage of mammalian host systems, in addition to correct posttranslational processing, is that most host cell proteins will be homologous, or similar, to endogenous human proteins. This reduces the potential for triggering immune responses.
However, clinical trials have been suspended due to detection of anti-HCP antibodies. For instance, 26% of the participants developed anti-CHO antibodies in the trial of IB1001 (IXinity, recombinant factor IX). As some proteins can be immunogenic at low concentrations, current regulations state that there should be no detectable HCP. Consequently, there is no concentration threshold for HCP in final products.
Polyclonal antibodies are the main tool for detecting and monitoring HCPs. The broad heterogeneity in the types of residual HCPs are due to differences in the properties of biologics, types of host cells, and both production and purification protocols. This heterogeneity necessitates the use of complex mixtures of polyclonal antibodies. Ideally, in addition to detection of a broad array of proteins, there should also be antibodies against multiple epitopes within proteins.
Different strategies are used to increase HCP coverage. Frequently, a number of different animal species are immunized, since immunogenicity of proteins can vary between species. Additionally, different adjuvants and different antigen fractionation/treatment protocols can increase the complexity of the antibody pool. One method for increasing coverage is cascade immunization (Thalhamer and Freund 1984). In this method a mixture of proteins is used as the immunogen. After development of an immune response, serum from the host is used to precipitate out the immunogenic proteins. The remaining proteins, which were poorly immunogenic or nonimmunogenic, are used to immunize a new host animal. By removing the most immunogenic proteins for the second set of immunizations, this method can increase antibody coverage.
The most common method for screening biopharmaceutical products and testing for HCP is enzyme-linked immunosorbent assay (ELISA), a sensitive assay with a low detection limit, high level of reproducibility, and compatibility with high-throughput screening.
Since the ELISA technique does not permit identification of antigens when using mixtures of antibodies, but only provides titers, the accuracy and utility of ELISA relies on a thorough prior assessment of the antibodies used. Therefore, accurate evaluation and validation of antibodies reactive against HCP is crucial for detecting and monitoring HCP both during the product development cycle and during manufacture of biologics. Lack of detection of residual HCP can have major business impacts including delays in development and introduction, longer cycle times, and even discontinuation of the development of promising therapies.
The predominant method for assessing anti-HCP antibodies involves 1-D or 2-D electrophoresis followed by western blotting. In 1-D electrophoresis, polypeptides with similar molecular masses will not be fully resolved from complex mixtures of proteins in some regions of the gel. 2-D electrophoresis gives much better resolution, due to separation of proteins in the first dimension by isoelectric point (pI) followed by molecular mass in the second dimension. Additionally, the combination of pI and mass can assist in identifying proteins.
Bio-Rad's streamlined HCP workflow provides fast, precise evaluation of antibodies, and is particularly well suited for complex mixtures such as anti-HCP. The components of the workflow have been optimized to provide consistent protein separations and high-sensitivity, reliable detection on western blots.
The fast run times of the novel Laemmli-like Criterion™ TGX™ Precast Gels and use of the rapid Trans-Blot® Turbo™ Transfer System significantly decrease the time needed for antibody evaluation when compared to traditional systems. The complete HCP analysis workflow from protein extraction to analysis can be completed in less than 2 days.
The host cell protein detection workflow uses the ReadyPrep 2-D Cleanup Kit for protein extraction and removes ionic contaminants, such as detergents and lipids, which can lower the resolution of 2-D electrophoresis. This kit can also be used to concentrate dilute samples.
After protein extraction, the next step is isoelectric focusing (IEF) to separate proteins using immobilized pH gradient (IPG) strips. During IEF, proteins move through a pH gradient until they reach their pI, the pH where they have no net charge. The PROTEAN i12 IEF System provides the flexibility required for different screening parameters with the capacity to run up to 12 channels under multiple conditions in a single run, while running different samples, protocols, and pH gradients simultaneously.
Long-life Criterion TGX Precast Gels, with combs to fit IPG strips, provide fast separation by molecular mass in the second dimension in as little as 20 minutes using the Criterion Dodeca Cell. TGX Precast Gels have a novel chemistry that provides consistent polypeptide separation with fast run times and a shelf life of one year. After electrophoresis, the quality of the run can be evaluated using either the noncovalent, reversible SYPRO Ruby Protein Gel Stain or the one-step Oriole Fluorescent Gel Stain.
The Trans-Blot Turbo System can transfer proteins in a second-dimension gel using the PVDF or low fluorescence PVDF transfer packs in less than 10 minutes. The host cell protein assay workflow has been optimized for the Clarity™ Western ECL Substrate, which has femtogram-level sensitivity.
The ChemiDoc MP or the ChemiDoc Imaging System, when combined with PDQuest 2-D Analysis Software, provides rapid imaging and analysis, including automated spot detection and matching, and quantification.
European Medicines Agency (2012). Withdrawal Assessment Report IXinity (formerly IB1001). Accessed June 4, 2015.
Thalhamer J and Freund J (1984). Cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens. J Immunol Methods 10, 245–251. PMID 6361152
Berkelman T et al. (2013). Enhanced 2-D electrophoresis and western blotting workflow for reliable evaluations of anti-HCP antibodies. Bioprocess Int 11, 50–61.
CHOPPI (CHO protein predicted immunity) database
Posch A and Berkelman T (2014). Improving HCP impurity assessment. Genet Eng News 34, Tutorial
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