The first step is to establish a null cell that does not express the product gene, either by using non-transfected (i.e., parental) cells with a common origin to those used to make product alone, or to transfect them with the vector used to create the production cell line without the product coding gene (historically described as a “mock” transfection). The main advantage to the latter is that selective markers (e.g., dihydrofolate reductase or glutamine synthetase) are expressed. Alternatively, antibodies can be raised to selective markers independently. Another advantage is that mock-transfected null cells express selection markers and may be grown in cell culture conditions more closely resembling the manufacturing process. Using pools of mock transfected null lines which have not been cloned allows for the maximum potential genetic diversity and therefore gives the highest potential of generating a broad host cell protein population. However, these cultures, which are seeded with cell pools (not cloned), may demonstrate more variability from run to run. Both approaches [non-transfected (parental) and mock transfected] are commonly used. Both are also subject to the following issues: the cell culture process optimized for the production clone may not lead to similar viability and cell density of the null cells. In addition, in the absence of the stresses experienced by cells producing large quantities of product protein, the null cell lines may exhibit a different HCP profile. These changes in viability, cellular metabolism, and cell density may alter the HCP profile of the antigen, making it less representative of the manufacturing process. More about choosing appropriate HCP antigen preparation conditions is discussed below.

Once the null cells have been established, the HCP antigen is prepared in a bioreactor, cell culture flask, or cell culture bag reflective of the product or process cell culture conditions. A platform cell culture process may be applied, which represents a common manufacturing process for several products (e.g., CHO-derived monoclonal antibodies). Typically, the resulting HCP antigen is minimally processed to ensure a broad HCP spectrum, making the antigen suitable for HCP monitoring of multiple products generated from the platform process. A platform HCP antigen also may be produced by combining several null cell runs with slightly different cell culture conditions (e.g., allowing the culture to remain in the bioreactor for longer periods of time to vary the cell viability). This approach is used to obtain a broader HCP spectrum, and antisera raised against this immunogen may be suitable for HCP detection in products generated from a variety of slightly different processes. These reagents also may increase the robustness of the HCP immunoassay toward process changes.

Alternatively, the HCP antigen may be prepared using an upstream, process-specific approach in which the cell culture process is tailored to mimic the production process for a unique product or specific process. The advantage of this approach may be the high relevance of the resulting HCP antigen to a particular upstream process. As with platform reagents, because no additional processing purification steps are included, the antigen contains a broad range of HCPs, and the antigen will often remain suitable for HCP monitoring after downstream production-process changes. The disadvantage of this approach is that the use of the reagents typically will be limited to a single (or few) product(s) or process(es).

The HCP antigen can be produced from a representative small scale, pilot scale, or production scale run. There are pros and cons for each approach. Pilot or production scale may mimic the actual process best; however, normal variation between cell culture runs may not be reflected if only one run at large scale is applied. Conversely, several small-scale preparations can be pooled and may better reflect the run-to-run variability of the cell culture process. Regardless of the scale, the presence of product should be aggressively avoided, because its presence in the HCP immunogen will compromise the quality of the resulting immunoassay antisera.

After performing the null cell run, the HCP antigen can be prepared from the cell lysate, the concentrated CCF, or as a mixture of both as shown in Figure 2. If the antigen is prepared from CCF, the cells may be harvested at a time when production line harvest is performed or some days later, to allow for some of the cells to lyse and release HCPs into CCF and thereby broaden the HCP spectrum.

To prepare immunogen from cell lysate, the cells are harvested by centrifugation, and the cell pellets are washed and lysed (e.g., by using repeated cycles of freeze/thaw, or high pressure homogenization). Conditions are typically selected to mimic the production process (i.e., to prepare immunogen from CCF, cells and debris are removed by centrifugation, and the CCF is then treated by ultrafiltration/diafiltration). The buffer is exchanged (e.g., into PBS or HEPES), and the CCF is concentrated. The cut-off for filtration should be chosen to minimize loss of HCP, e.g., 10 kDa or less.

Because there is no obvious approach for overcoming these limitations, in practice the bacterial HCP antigen is usually generated from the lysates of washed cells, using null cell fermentations, that are grown and induced under conditions representing the upstream manufacturing process as closely as possible. As for any HCP antigen, it is necessary to demonstrate that the HCP profile is representative of the manufacturing process before use in assay development. This approach yields the full complement of HCPs expressed and hence is very broad.

Because most animals have been exposed to bacterial antigens and may have pre-existing antibodies to many bacterial cell proteins, it is important to characterize the pre-existing antibody responses, and if appropriate, consider the use of specific pathogen-free (SPF) animals. The presence of significant levels of pre-existing anti-HCP antibodies may confound the analysis of the antibody responses; therefore, it is important to screen preimmune sera from animals for pre-existing antibodies prior to immunization.

Null bacterial cells transfected with the expression vector contain chaperone genes that may be expressed at high levels (e.g., 5% of the total HCPs). Such overexpression of particular HCP impurities might necessitate the development of single-analyte assays for these particular impurities (see 6.1 Assays for Individual HCPs).

Appropriate controls within the assay range may be established to monitor assay performance. Controls may be prepared from independent dilutions of the HCP standard, product samples or intermediate pools, or spiked product samples. However, there is a slight risk that control samples prepared as a dilution from the same HCP standard material can degrade at the same rate as undiluted HCP standard, and degradation of the standard will not be detected. To mitigate the risk, data from several standard curve parameters (e.g., signal, background, slope, coefficient of determination) are often assessed for each assay and used to support HCP standard stability. Using material different from the reference to prepare controls will help ensure that the degradation rate will be independent, and HCP standard degradation can be detected easily.

Assay control charts can be established and used to record reference curve parameters, control sample values, and lot-specific information for the critical reagents, such as labeled HCP antibodies. Acceptable ranges for the controls should be established on the basis of multiple runs (usually 20–30) and may be used as part of routine assay acceptance criteria. In addition, the values for % coefficient of variation (CV) of standard and control replicates are often a part of the assay acceptance criteria. Differences over time in HCP standard curve performance or HCP control levels, or an increase in assay variability, may indicate a lack of HCP standard stability.

A portion of the prepared HCP antigen is mixed with an appropriate adjuvant (most commonly, Freund’s adjuvant or in combination with incomplete adjuvant) and used for the immunization of animals. Each animal receives an initial priming immunization, followed by multiple (often 4 to 8) booster immunizations, to mature the immune response and generate high-titer, high-affinity antibody responses. Antiserum is collected from each animal before the initial priming immunization (time 0) and at appropriate intervals (usually 10–14 days) after booster doses. Four to six bleeds are usually collected from each animal. However, the duration of the immunization protocol may be extended by increasing the number of booster immunizations, making it possible to collect additional high-titer antisera. Other immunization strategies may also be helpful (e.g., cascade immunization or size fractionation of the HCP immunogen).

Investigators should perform titer or Western blot analyses of the individual test bleeds before antiserum pooling to screen for and remove sera that 1) have low titer, 2) display an immunodominant immune response to a small group of HCPs, or 3) have nonspecific binding characteristics or anti-product reactivity. This evaluation should also be performed on the final antibody pools to demonstrate broad coverage of HCPs and a lack of binding to the therapeutic product. In addition, some laboratories have found it useful to evaluate antibodies for aggregates using size exclusion chromatography as described below. Acid elution conditions may cause some antibodies to aggregate, and multimers of this type, particularly when labeled with either biotin or horseradish peroxidase, may lead to increased assay background. In addition, some laboratories include a final process-scale size-exclusion chromatography step to reduce aggregate levels to low levels (e.g., <5%).

Multiple approaches to the purification of antibodies from antiserum pools have successfully generated antibodies suitable for use in HCP immunoassays. Antibody purification is often performed by Protein A, Protein G, or HCP column affinity chromatography. The application of coated magnetic beads can also provide benefits in some scenarios. The choice of Protein A or Protein G is driven by which animal species was used to generate the antibody. Manufacturers provide standard protocols for these routine antibody purifications. When affinity purifying anti-HCP antibodies, materials such as chromatography resins, which may have been used previously for other products, should be avoided, and additional cleaning cycles should be in place to minimize carryover. Ideally, manufacturers should use a resin that has never been exposed to a DP. Before the affinity purification step, some laboratories also include an initial 50% ammonium sulfate precipitation of the crude antiserum to enrich and concentrate the antibody fraction and thereby extend the performance and duration of use of affinity columns. The use of Protein A or Protein G purification strategies generates reagents in which the anti-HCP-specific antibodies compose a smaller proportion of the total antibody. In contrast, HCP affinity purification strategies selectively purify anti-HCP-specific antibodies from the antisera or antibody-enriched preparations. In this case, the HCP antigens are coupled to an activated resin, and the antiserum is purified over the column following established protocols or the resin manufacturer’s instructions. The purified antibodies may be further purified by size-exclusion chromatography to remove aggregates, then dialyzed, concentrated, divided into aliquots, and stored frozen (typically 70 or colder). Neat antisera should also be stored frozen (see 1106 for additional information regarding storage of serum samples).

Affinity purification of specific antibodies using HCP immobilized on a column requires careful management of the column preparation and use so that it can be reproduced reliably for future purifications. In addition, if reused, appropriate resin storage and regeneration procedures should be evaluated and controlled to avoid degradation during storage. Done properly, it is a reliable, reproducible process that has led to consistent anti-HCP antibodies and HCP immunoassays. After loading the column with antisera, an optimized wash procedure is critical. Because the preparation is a mixture of antibodies with differing affinities, extensive washing may remove low-affinity antibodies. Similarly, loading large amounts of anti-HCP antisera onto the column may result in high-affinity antibodies, displacing low-affinity antibodies if they recognize epitopes that are in close proximity. In either case, the low-affinity antibodies may be removed. In theory, higher affinity antibodies may also be more challenging to elute. Whatever load and wash conditions are initially selected for the antigen-specific affinity purification, these conditions need to be maintained in future production lots of the antibodies. A comparison between these two purification strategies is shown in Table 2. Both approaches are commonly used.

The quality of the antibody pair (capture and detector) is often evaluated in two ways:

HCP coverage evaluations help assess the ability of the antibodies to recognize a wide range of HCPs in the calibration standard and those present in in-process and DS samples. Two methods (2-D gels followed by Western blot analysis and immunoaffinity purification followed by 2-D gel analysis) are in fairly common usage, and both have the limitation that they tend to underestimate the true antigen binding in immunoassay methods but may have value in comparing two preparations head-to-head (or a platform to a commercial reagent). Both procedures use reducing 2-D gel electrophoresis to separate HCPs in the HCP standard or in early process sample(s) (e.g., harvest). Numerical coverage comparisons should be used with caution because of the many method variables and the art required to reproduce results, even with the same reagents in the same laboratory. Because of this variability, the results are best evaluated qualitatively. Comparisons of batches (or sources) of antibodies should be done side by side as much as possible to determine if antibody lots are comparable or if one is superior to another. One approach, the SDS-PAGE/Western, is essentially a comparison of the number of spots present in the immunologically stained membrane versus the number in a duplicate gel (or blot) stained for total protein (e.g., by fluorescent dye or silver; see also USP chapter Immunological Test Methods—Immunoblot Analysis 1104). Differential staining of each antibody preparation may power the analysis, because it can be used to analyze the same 2-D gel. The second approach, immunoaffinity binding/SDS-PAGE, involves comparing the flow through and eluate to the load from the HCP calibration standard (or early process sample) passed over a resin to which the capture antibodies have been covalently immobilized. The resin is washed after loading and before elution to remove nonspecifically bound HCPs. For the purpose of sample comparison, difference gel electrophoresis (DIGE) technology could be helpful. Table 3 lists the advantages and disadvantages of these two coverage methods.

More information about the appropriate use of SDS-PAGE/Western blot methods and ways to minimize the disadvantages above can be found in the later section 5.2 Considerations for Western Blot Methods.

An example of a suitable use of this approach is shown in Figure 3.