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Host protein detection and quality control

Host protein detection and quality control. This article introduces the definition of HCP, the risks brought by HCP, regulatory issues, commonly used ELISA methods for HCP measurement and their limitations, and orthogonal methods that can be used for HCP characterization.

Host protein (HCP) constitutes the main part of the process-related impurities in the production of biological agents. The amount of residual HCP in a drug is usually considered a critical quality attribute (CQA) because it may affect product safety and efficacy. Therefore, the regulatory requirement is to monitor the removal of HCP in biopharmaceuticals during the development of bioprocesses.

HCP is a protein produced or encoded by a host cell for the production of recombinant therapeutic proteins. Recombinant therapeutic proteins are usually produced by genetically modified prokaryotic or eukaryotic host cells using cell culture or fermentation techniques. Genetic engineering enables host cells to be transformed to selectively express the target protein. In the process of recombinant protein production, host cells also jointly produce proteins related to normal cell functions, such as cell growth, proliferation, survival, gene transcription, protein synthesis, etc. Due to cell apoptosis, death, and lysis, other non-essential proteins can also be released into the cell culture medium.

Risks associated with HCP

Generally, in addition to the target protein, HCP constitutes the main process-related impurities in pharmaceuticals. The risk associated with HCP is mainly immunogenicity. HCP is a complex mixture with various physiochemical and immunological properties. Due to the possibility of triggering an immune response in the human body, almost all HCPs are treated as foreign proteins and carry clinical safety risks. In addition, certain HCPs can also act as adjuvants to enhance the immune response of drugs. If some HCP with proteolytic activity is not fully removed or inactivated, it will also affect the stability and efficacy of the drug. HCP may affect the safety and efficacy of a given drug.

The risks associated with HCP are usually assessed through a combination of downstream purification, HCP residual levels, maximum dose, route of administration, frequency of administration, toxicology data, and clinical data. Although it is generally believed that HCP can potentially trigger an immune response and constitute a clinical safety risk, it is difficult to prove which HCP and its concentration may cause immunogenicity problems. Theoretically, preclinical pharmacological and toxicological evaluations can be performed in the presence of different amounts of HCP impurities.

However, since the size and nature of the immune response depend on the homology of amino acid sequence, the amount of residual HCP and the product dosing regimen, the evaluation result is almost irrelevant. For this reason, it is our main strategy to control risk by developing a powerful purification process, removing HCP to the lowest possible level or removing “undetectable” levels in drug substances or products. However, the detectability of residual HCP also depends on the sensitivity of detection. Pharmaceutical companies solve this risk by carefully developing drug delivery methods and using multiple technologies to evaluate all potential HCPs that may be co-produced or co-purified with drug products during biological drug delivery and drug drug development.

Regulatory requirements for HCP testing and control

According to the International Conference on Harmonization (ICH) Guide Q6B, “For host cell proteins, sensitive assays (such as immunoassays that can detect multiple protein impurities) are usually used. In the case of immunoassays, the polyclonal antibody (pAb) used in the test Produced by immunization with a preparation of production cells (minus product coding genes, fusion partners or other suitable cell lines).

May include laboratory-scale spike experiments to prove that impurities derived from the cell substrate have been removed For example, nucleic acids and host cell proteins can sometimes be used to eliminate the need to establish acceptance criteria for these impurities.” Regulators hope that “whenever possible, using highly sensitive analytical methods, the contaminants introduced in the recovery and purification process should be below the detectable level.”

The European Medicines Agency (EMA) guideline CPMP/BWP/382/97 points out that for HCP, regardless of the product and production system, the residual HCP must be tested regularly. Therefore, it is currently required to conduct routine monitoring of HCP and use appropriate The level of purification and removal of analytical determination methods, and the results between batches should be consistent and meet the drug specification limits.

Regulators from other countries and emerging markets may have their own wording for HCP control, but it is generally believed that sensitive and validated effective methods are needed to monitor residual HCP in accordance with ICH guidelines, usually 1 to 100 ng/mg.

Detection and monitoring of residual HCP

So far, due to its high sensitivity and high throughput, immunoassays usually carried out in the form of sandwich enzyme-linked immunosorbent assay (ELISA) (see Figure 1 below) are still the industry gold standard for HCP measurement. The composition and abundance of HCPs are unique to their respective hosts and manufacturing processes used for biologics production. At the same time, different host cells and manufacturing processes may produce similar amounts of certain HCPs.

he amount of protein derived from a host cell varies from host to host. For example, E. coli has ~4300 genes, while Chinese hamster ovary (CHO) cells have ~30,000 genes. Although not every host gene is transcribed and translated into protein, the complexity of the host genome and post-translational modifications that exist in mammalian cells make it almost impossible to understand the complete HCP composition in a given production process. Because these HCPs are potentially immunogenic, the generally accepted method of assessing the presence of HCP is through immunoassays.

Theoretically, injection of HCP mixture into animals (such as rabbits, goats or chickens) will trigger an immune response, and the animals will produce anti-HCP antibodies against these foreign proteins. Although the main components of all HCPs are not yet known, polyclonal antibodies produced in animal cells should be able to recognize the protein content of most (if not all) HCP mixtures. Using these polyclonal antibodies, a multi-analyte sandwich ELISA can be developed (see Figure 1 below). In Figure 1, the captured antibody enriches the HCP in the sample and fixes it in a 96-well plate.

Then, the detection antibody coupled directly to the enzyme or through the biotin-avidin amplifier binds to the captured HCP. Using an enzyme, usually horseradish peroxidase (HRP), can catalyze the substrate to produce colorimetric, chemiluminescent, or fluorescent signals, which are related to the amount of HCP in the test sample. The detection antibody that binds directly to the enzyme or through biotin-avidin amplification binds to the captured HCP.

Host protein detection and quality control — Figure 1: Schematic diagram of sandwich enzyme-linked immunosorbent assay (ELISA) for host protein (HCP) detection.

The universal HCP ELISA developed using polyclonal antibodies produced against parental cell lysates or cell culture supernatants can detect most HCP species, but may not detect protein subsets specific to a particular manufacturing process. The universal ELISA kit is commercially available, and its advantage is that it eliminates the lengthy measurement development time shown in Figure 2 below, and only needs to follow the steps indicated in yellow before being used for process development. This makes the kit very suitable for early development.

In the later stages (clinical phase III or commercial production phase), cell line-specific platform assays or upstream process-specific ELISA assays are usually required to reduce the risks associated with more general commercial assays. Following the green arrow process, Figure 2 below shows the development cycle of internal HCP analysis. Generally, the HCP of a specific platform or process is generated by a similar upstream process in an empty cell culture (mimetic) without upstream product encoding to represent the HCP in the platform production culture. PAb produced by immunizing animals with these HCPs will be used for ELISA development after coverage evaluation.

Polyclonal antibodies should recognize most HCPs that are co-produced with drugs. When undesirable coverage is observed, different strategies (shown by the orange arrow) can be used to increase coverage or qualify the ELISA at risk, and use orthogonal methods to supplement the ELISA to characterize the HCP process clearance (see Figure 2 below). After identification and verification, HCP ELISA can be used as the QC release test of the stock solution.

In the classic purification process of therapeutic monoclonal antibodies, the highest concentration of HCP is detected in the harvested cell culture fluid (HCCF), and then it is removed by other downstream purification steps, usually at a very low concentration (1-100ng /mg or ppm) HCPs levels were detected in the final stock solution.

Limitations of HCP ELISA

Due to the heterogeneity of HCP, high-abundance/immunogenic proteins usually dominate the multi-analyte ELISA signal, while low-abundance/immunogenic proteins do not have enough antibodies to recognize them. In addition, co-purification of drug-rich HCPs may also have limited antibodies to detect them, resulting in dilution nonlinearity and potential underestimation of the possibility of residual HCP.

In addition, because not every HCP is immunogenic in animals, even the best process-specific HCP ELISA cannot detect 100% HCP co-produced with recombinant protein. As a supplement to HCP ELISA, traditional HCP separation and visualization methods (such as 1D and 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) are still useful tools for HCP characterization.

Genentech’s Zhu-Shimoni et al. published a comprehensive review of the limitations of ELISA for HCP detection, and cited some examples of its process development. One example is HCP named GST-α. GST-α does not cause or has a low immune response in immunized animals, and therefore does not produce antibodies in polyclonal antibodies used in HCP ELISA. However, the protein detected on capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is an impurity peak, and the relative concentration in the bulk drug is ~2000ng/mg.

In addition, some HCP may be co-purified with the recombinant protein, thereby enriching in the final volume. However, the polyclonal antiserum against the mimic HCP library may have a limited number of antibodies against these HCPs, and it is impossible to accurately detect them. In addition, steric hindrance and lack of multiple epitopes (capture and detection) that bind the same antibody may cause inaccurate HCP measurements. The lack of appropriate calibration standards also limits the accuracy of HCP quantification. The aforementioned limitations of HCP ELISA can be overcome using orthogonal methods.

Through size exclusion (SEC), ion exchange (IEX) or reverse phase (RP)-high performance liquid chromatography (HPLC), SDS-PAGE can detect a large amount of residual HCP (>0.1%) in the final drug, CE-SDS , Isoelectric focusing capillary electrophoresis (iCE) and other analytical methods, and confirmed by LC-MS. However, the content of most HCP in the final stock solution is very low, and a more sensitive detection method is required.

The aforementioned analytical methods usually do not have the sensitivity and resolution to separate and detect a single HCP. Methods based on immunoassays, such as slot blot analysis and western blot analysis, are sensitive, but usually only semi-quantitative. Protein separation and visualization methods, such as 2D-SDS-PAGE, supplement HCP ELISA by providing information about the characteristics of individual HCPs. 2D-SDS-PAGE with silver staining or other sensitive staining methods, such as Sypro Ruby (Life Technologies, Grand Island, New York), is traditionally available due to its ability to separate proteins by two isoelectric points (pI) It has been used to analyze complex protein mixtures and molecular weight (MW).

Silver staining has a detection sensitivity of ~0.2-0.5ng/protein spot, so very low abundance proteins can be detected, but the staining process is lengthy and tedious, and there are large differences between gels. To overcome the differences between gels, DIGE can be used to run two or three samples on the same gel, each sample is pre-labeled with a different fluorescent dye. Using 2D-DIGE to analyze APIs or in-process libraries can provide laboratory scientists with a snapshot of HCP profiles and easily compare HCP composition differences between two samples (see Figure 3 below).

Combining spot selection and LC-MS, 2D-DIGE can provide direct information about HCP properties (pI, MW, abundance and identification), which can help improve HCP removal strategies in downstream process development.

In the last five years, two-dimensional LC-MS has become a new orthogonal method for HCP characterization. Its advantages are sensitivity, specificity, gel-free, and automation. By using high pH reverse phase, ion exchange or size exclusion to achieve additional separation size before LC-MS/MS identification, the two-dimensional liquid chromatography-mass spectrometry system can reduce the interference of dominant peptides digested from recombinant proteins. And to maximize the detection of low-abundance HCP-related peptides. In addition, by identifying potential residual HCPs through 2D-LC-MS, a target-based multiple reaction monitoring (MRM)-LC-MS method can be developed to quantify multiple HCPs in the stock solution.

Sum up:

Proper HCP removal is an important indicator of robust and well-controlled biological treatment. However, due to the low content of HCP in the stock solution, it is quite difficult to detect and measure HCP in a matrix based on recombinant proteins. HCP ELISA has the sensitivity to detect the level of ng/mg remaining in the drug, but it is limited by the pAb used in the assay.

Generally, pAbs are produced against HCP populations present in the host cell proteome upstream of biological processes or more commonly. These antibodies have insufficient specificity for downstream processes, therefore, the amount of available antibody present is not related to the amount of HCP in the stock solution, which leads to antigen excess and lack of dilution linearity.

On the other hand, certain HCPs may be missed by the downstream specific process ELISA. These HCPs may leak during the specific downstream purification process and/or when the downstream process changes. Given the process-specific reagents, antibodies, and the time required to develop and validate the analysis, developing a process-specific HCP ELISA for each biological agent is also expensive and time-consuming. Therefore, if the same cell line or cell line with similar HCP profile is used, the multi-product platform assay will be more feasible and cost-effective.

The data shows that different upstream processes only change the expression/secretion of a very small HCP subgroup among thousands of proteins. Using the platform HCP ELISA or the process-specific HCP ELISA for HCP testing and control is a strategic decision to balance the benefits and risks of both. Regardless of the method chosen, the antibody reagents used in each procedure in the ELISA must be carefully identified to prove that they cover the vast majority of HCPs that may be co-produced with therapeutic proteins.

In addition, orthogonal methods such as 2D-DIGE and 2D-LC-MS can be used to provide additional evidence about the robustness of the HCP removal process. These methods can also characterize HCP co-purified with recombinant proteins, which do not cause immune responses in animals.

Orthogonal methods (such as 2D-DIGE and 2D-LC-MS) can be used to provide additional evidence about the robustness of the HCP removal process. These methods can also characterize HCP co-purified with recombinant proteins, which do not cause immune responses in animals.

When combined with HCP ELISA, the above-mentioned characterization method can reduce the risk of HCP supervision and provide valuable information about the characteristics and characteristics of HCP to guide the development of downstream processes.

In addition, single or multiple analyte ELISAs can be developed to target co-purified HCPs after LC-MS/MS identification and understanding of the platform or process specific HCP ELISA for antigen excess.