Cross-Reactivity Among Anti-HCP Antibodies and Therapeutic Monoclonal Antibodies: In typical biologics development efforts, a sandwich ELISA is used for quantitation of HCPs (Figure 1). The main reason to use this format is that HCPs present in the final drug substance must be reduced to ppm levels to minimize potential health risks, so an enrichment step (use of binding antibody) is necessary for their accurate measurement at such low levels. Because of the diversity of HCPs that may be present in the early bioprocess steps (3,5,7,8), for HCP quantitation we used a polyclonal antibody prepared against a null cell of the same production cell line for HCP analysis, as described in a recent review (2). Anti-HCP antibodies were raised as usual in either rabbits or goats, and the IgGs were purified using either a protein A or protein G column for use in the ELISA assay. As Figure 1 indicates, a portion of the anti-HCP IgGs was labeled with biotin and used as the reporting antibody for HCP detection. Using this format, a minimum of two antibody-generating epitopes is required for HCP detection.

During the development of one particular internal MAb candidate, HCP testing showed that the drug substance appeared to have higher levels of HCP than other MAbs under development even though they all use a very similar purification platform. Because typical HCP levels in biologics are reported at 1–100 ppm (9), we made an effort to further reduce the HCP level for this particular MAb. Initially, our efforts focused on process improvement. However, soon we realized that altering different purification conditions had very limited impact on HCP reduction (data not shown).

A hypothesis was proposed that this MAb might have cross-reactivity with anti-HCP antibodies, so a portion of the signal reported as HCPs actually resulted from binding to a subpopulation of the MAbs with anti-HCP antibodies (Figure 2). Because this particular MAb is a human IgG, we tested our hypothesis using polyclonal antihuman IgG antibodies in a sandwich HCP ELISA.

During the assay, after incubation of the drug substance with the capture antibody, we included a step in which the antihuman IgG antibody was added before the biotinylated anti-HCP antibodies. That additional step lowered the HCP signal (Figure 3), which confirmed that some of the HCP signal was indeed due to cross-reactivity. Further testing showed that the maximum blocking effect from antihuman IgG antibodies was reached at 100 µg/mL (data not shown), so all subsequent tests reported herein included the additional blocking step.

Development of a Modified Sandwich ELISA and Analysis of Additional MAbs: To demonstrate that decreasing HCP values were not the results of cross-reactivity among HCPs and antihuman IgG antibodies, we used HCP standards (in the absence of MAbs) to incubate with polyclonal antihuman IgG antibodies. Figure 4 indicates that incubation with antihuman IgG antibodies does not significantly affect HCP detection. When tested at different MAb concentrations, it can be seen that ~80% of the HCP value results from cross-reactivity (Figure 5).

To further evaluate the cross-reactivity phenomenon, we tested seven other MAbs with and without blocking antihuman IgG antibodies (Table 1). The majority of MAbs in this study do not exhibit cross-reactivity with anti-HCP antibodies. However, two of them (MAb-6 and MAb-8) in addition to original MAb-9 showed a more modest cross-reactivity with the anti-HCP antibodies.

We analyzed nine MAbs (eight used in the ELISA plus one more) by Western blot as an orthogonal technique to further evaluate the cross-reactivity phenomenon. Figure 6 showed that some had cross-reactivity with anti-HCP antibodies.Comparing the Western blot analysis with ELISA results (Table 1) indicated some correlation between the two assays, especially with heavy-chain cross-reactivity. However, Western blot binding is to a denatured sample, so the signal may be due to buried epitopes such as MAb-1, which showed little cross-reactivity in ELISA (Table 1) yet a visible binding by anti-HCP to the light chain (Figure 6).