Host Cell Protein impurity testing FAQs

One of the major challenges of producing a generic anti-HCP antibody is achieving sufficient coverage in a broad range of sample types. Almost every bioprocess is unique in some way, which means that the subpopulation of HCP present can also be unique. For this reason, process-specific and platform assays are considered a less risky choice, as they have a higher chance of achieving better coverage.

This depends on the harvest method and conditions. We found in the case of monoclonal antibody (mAb) production, cell viability is high and the drug substance, mAb, is secreted to the external culture media. In this case, supernatant (suspension) proteins would be better for preparing HCP antigens to generate the HCP ELISA antibody.

Almost every bioprocess is unique in some way, which means that the expression of HCP can also be unique. This is also true for mock versus expressing cell lines. Subtle changes in the culture conditions can alter the expression of HCP, as could the presence of a large amount of drug substance. This is one of the reasons that coverage assays are qualitative and not quantitative. Only the presence or absence of a protein spot is considered, and not relative expression or signal. However, there is a small risk that some HCPs are only expressed in the production cell line and would therefore not be accounted for in a coverage assay using a mock cell line.

For HCPs that bind to a product, there is a risk that the bound drug blocks a specific epitope. This is more problematic for low molecular weight HCPs, where there might only be a single epitope. In this case, you can run the drug substance on an IEF or a native PAGE gel, and then probe the resulting bands with anti-HCP antibodies to see if they react with any bound HCPs.

Generic ELISA kits are developed and validated using a diluent (sample buffer) that is designed to minimize matrix effects from samples and process buffers. The sample buffer also contains blocking reagents to prevent non-specific interactions and reduce background signal. It is entirely possible to use a process specific sample buffer in place of the sample buffer provided in the kit, but this could affect the kit performance and would have to be validated by the end user.

We performed spiked control experiments using high concentrations of monoclonal antibody as the sample buffer (up to 5 mg/mL) and did not see any additional signal or inhibition of signal that could be attributed to the mAb. The antibodies used in generic HCP ELISAs are generated against mock cell lines, so the ELISA should not recognize mAb or any other drug product.

We have limited data at this moment. There are several commercial kits available for some platforms, but all of these assays are antibody-based, so you need to have appropriate orthogonal assay(s). It is important to note that different antibody-based technologies can give different results (i.e., higher or lower ppm of HCP), even when using an identical antibody.

For generic, platform and process-specific ELISA antibodies, the validation criteria are the same. Critical criteria are demonstrating good dilutional linearity, good sensitivity, dynamic range, and ELISA and coverage antibody specificity.

The different HCP ELISA kits might generate different HCP values, so it is possible to get significantly different HCP levels between kits. We have experienced this quite often. We have also seen that different ELISA kits can generate the same level of HCP measurements.

OD values lower than the LOQ of a kit cannot be considered a valid result for dilutional linearity, even if the results appear linear, and should be reported as less than LOQ (<LOQ). If dilution linearity is not achieved within the range of a kit, it may be an indication that this kit does not have optimal compatibility with the sample. In this case, another ELISA kit could be tested.

The most reliable criterion for dilutional linearity assessment is a CV% below 20% for all dilutions. If you only use the values from the highest reported value, there is a risk that the CV is calculated from an invalid data point (e.g., in the event of dilutional linearity failure due to a high concentration (ppm) of a single problematic HCP.)

The CV% should be calculated across all dilutions.

A good standard curve should have at least seven points. A wide range, e.g., 0 - 200 ng/mL, enables more sample dilutions to fall within the range of the standard curve and gives more robust data.

We have tested HCP ELISAs with several downstream purification buffers commonly used in protein A purification of monoclonal antibodies. In most cases, using the sample buffer include in the kit to dilute the sample by a factor of 10 completely resolved low spike recovery caused by matrix effects.

The most relevant buffer compositions to test are the process specific buffers used in purification. One should start by spiking a known concentration of HCP into process specific buffers, not in process samples, to determine if the process buffers can affect recovery.

If it can be demonstrated that there is no matrix effect from the process buffers, then you should test the kit with in-process samples. You can spike HCP into these samples — however, because there is already HCP in the sample, it might be difficult to judge dilutional linearity.

Spike recovery is a good method to determine if process-specific buffers have a detrimental effect on the assay. Using the assay standard for this is preferred as a control — however, the best results can be obtained by also using HCP from the customer’s specific processes that has been measured by an orthogonal method (e.g., mock cell line HCP quantified by a total protein assay).

Dilutional linearity is the best method to determine the minimum required dilution for the assay. You should use the dilution where the calculated HCP becomes linear with the following dilutions, because more concentrated samples might still be in the hook region of the ELISA.

Yes, we have. Some data is available here in our data file. Different cell strains (e.g., CHO-K1 and CHO-S) can give subtly different 2D patterns and have different coverage with the same anti-HCP antibody. However, it is difficult to draw a clear conclusion about how this affects ELISA because antibody performance in the coverage assay does not always directly relate to the antibody performance in ELISA.

Not necessarily. It depends how much HCP was detected by mass spec. If the amount of HCP detected by mass spec is low, it may still be within the accepted limits for the product. If the amount detected is high relative to the product, it may be an indication that some HCP is not covered by the ELISA and has not been effectively cleared during downstream purification. In this case, the samples should be tested in a different ELISA and the HCP levels compared. If the calculated HCP in the new ELISA is in agreement with the mass spec data, it may be an indication that the new ELISA should be used.

For a generic kit, we consider 60% coverage determined by either 2D DIBE or affinity to be sufficient. However, regulatory guidelines recommend "as broad coverage as possible," so you should always aim for the highest percent coverage possible.

This is a challenging situation. There are a few methods you could use, and each has drawbacks to consider.

• Use an HCP affinity column to capture the HCPs from the sample and then separate by 2D PAGE for DIGE analysis. This method lets you see the number of detected proteins, but the presence of product or serum in the total protein sample could make it difficult to see all of the HCP on a 2D gel as product/serum can obscure them or make it difficult to fully resolve HCPs on the gel.
• Use an affinity column or immunoprecipitation with product/serum-specific antibodies to remove product/serum from the mock sample. This can be pretty effective, but there is a risk that some HCP could stay bound to excess product/serum and get lost from the sample.
• Determine which HCPs are present with mass spectrometry analysis. Keep in mind that a large amount of product/serum in the sample can affect sensitivity and make less abundant HCPs difficult to detect.
• Use mass spectrometry analysis during the capture step of ELISA. This can help identify which HCPs are detected by your ELISA, but you will still need to compare this to the total HCPs present.

Confidence level (CV%) is calculated from the average coverage percentage of triplicate coverage experiments. It is important to measure coverage confidence level in this way, as the coverage analysis can introduce slight variations between replicates.

As the HCP ELISA uses polyclonal antibodies to detect as many HCPs as possible, there will be a good chance you will still be able to detect HCPs cleaved by the enzyme. A good control experiment to determine the effect of enzyme cleavage would be to perform two HCP coverage assays on (1) total HCP and (2) on enzyme-treated HCP. DIGE may also be a useful tool to determine how the HCP 2D pattern changes after enzyme cleavage.

Inclusion bodies are particularly challenging for HCP analysis in general, because it is difficult to predict which proteins from the host cell will be present. In this case, you can use an orthogonal method like mass spectrometry on the inclusion body to determine which proteins are present at harvest.

Cyanine labeling is a well understood and reproducible method of labelling. It is demonstrated to be at least as sensitive as silver staining and has been used for over two decades as an alternative labeling method.

Melanie software has no particular automated functions for this. To reduce background effect, you can take these steps:

• Set a threshold for the spot definition giving uncertain spots.
• Switch to spot-to-spot evaluation using the 3D view function
• Check the surrounding background level against the peak height of the spot.
• Decide if the signal is high enough over the background level.

Yes, we have multiple solutions for you. Please visit our HCP analysis page for more information.

HCP stands for host cell proteins. HCPs are any proteins produced by the host cell (e.g., E. coli and CHO) that are not related to the drug substance or product. The clearance of HCP from a drug substance is considered a critical quality attribute, as the presence of HCP in the final drug can cause adverse effects in patients and affect the efficacy of the product itself. The United States Pharmacopeial Convention (USPC 1132) has more detailed information on why residual HCP must be removed from a drug substance.

For monoclonal antibodies, protein A-based affinity capture chromatography, followed by anion and cationic exchange chromatography, has been demonstrated to reduce HCP levels to below 50 ppm and remove nucleic acids. Please see more information on this in our data file and application note.

Regulatory authorities do not recommend a specific coverage percentage because this varies by molecule type and treatment schedule. However, an antibody that detects HCPs as evenly as possible in MW and pI, with as high percent coverage as possible is advantageous. Regulatory authorities will also look at historical data from similar drug substances and expect to see equivalent or better results.

Generic assays can certainly be used for early phases and preclinical development. Use of generic kits for late phases can be case by case. As long as you submit enough data to show that the HCP ELISA works correctly and its antibody has sufficient HCP coverage, you can use a generic (commercial) ELISA. Generic ELISAs save time, but they come with an increased risk of undetected HCPs. If time-to-market is considered a greater patient benefit compared to potential risk, there will be a higher chance that regulatory authorities would allow the use of a generic ELISA. In some cases, authorities might ask an applicant to prepare a process-specific ELISA as a "phase 4" or post-approval requirement. Also, security of supply for many years (i.e., lifetime of the drug) could be an issue for a generic ELISA.

For the late clinical phases and commercial releases, the use of generic kits for batch release can be case by case. As long as there is enough data to demonstrate the HCP ELISA is working correctly and its antibody has good coverage, you can use a generic (commercial) ELISA in some instances. The use of generic kits for regulatory approval and batch releasing is also dependent on the molecule and application. You can perform a risk versus benefit assessment to understand the impact to the patient and determine whether a generic kit is suitable for certain treatments.

Many researchers in the field consider 50 ppm a good target. However, no such numbers are available from the regulatory authorities. The level of HCP allowance is dependent on many factors; method of administration, frequency and dose of the drug administrations, drug substance (monoclonal Ab or other recombinant proteins), host cells, historical data from similar molecules (if available), clinical trial results, and so on.

It depends on the phase of development. In early PD phases, all HCP levels in each purification step need to be checked to optimize the purification method. Once the production process is finalized, it might not be necessary or advantageous to measure HCP at all purification steps. However, HCP presence in the final substance is a quality critical attribute and it is best practice to test every production lot to minimize a risk to patient safety.

Mass spectrometry is an excellent orthogonal method, but it is not yet powerful enough to replace ELISA. The main reasons for this are:

• Sensitivity. Although mass spectrometry is very sensitive, the large amount of drug substance makes the relative sensitivity to HCP in the region of 10 ppm, which is around 10 times less sensitive than ELISA (< 1 ppm)
• Speed. ELISA results can be achieved and analyzed in around four hours. Mass spectrometry analysis can take up to two weeks from data collection.
• Volume of data. Mass spectrometry can generate massive amounts of data, which is not practical for regulatory filing as the authorities are not equipped to deal with this amount of information.

Yes, they do. A measurement of percent coverage of the anti-HCP antibody is expected to be filed at all application stages

Yes, coverage percentage of the anti-HCP antibody used in an ELISA is expected at each regulatory filing step.

Not necessarily. You should obtain new versions of the discontinued reagent and test historical samples to determine if the calculated HCP concentration using the new reagents is equivalent, within predefined limits (e.g., <20% variation). Coverage assays should also be performed with the new reagents to ensure equivalent antibody coverage. In the case that HCP levels are inconsistent between the versions of the reagents (e.g., <0.5x or >2x), it may be appropriate to test different available reagents from other sources.

Mass spectrometry is an excellent orthogonal method, but it is not yet powerful enough to replace ELISA. The main reasons for this are:

• Sensitivity. Although mass spectrometry is very sensitive, the large amount of drug substance makes the relative sensitivity to HCP in the region of 10 ppm, which is around 10 times less sensitive than ELISA (< 1 ppm)
• Speed. ELISA results can be achieved and analyzed in around four hours. Mass spectrometry analysis can take up to two weeks from data collection.
• Volume of data. Mass spectrometry can generate massive amounts of data, which is not practical for regulatory filing as the authorities are not equipped to deal with this amount of information.