Host cell proteins (HCPs) are process-related impurities derived from a host cell expression system that may be present in trace amounts in a final drug substance. During biologics development, it is important to demonstrate that a bioprocess is efficient in removing HCPs and that it provides consistent control of HCP levels. Several techniques are typically used for detection, quantitation, and risk evaluation of HCPs in biologics. The most common are enzyme-linked immunosorbent assays (ELISAs), Western blotting, sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and chromatographic separation methods (1). Genomics, proteomics, and bioinformatics also have been used recently in analysis and evaluation of host cell proteins (2).

PRODUCT FOCUS:MONOCLONAL ANTIBODIES

PROCESS FOCUS:PRODUCTION AND DOWNSTREAM PROCESSING

WHO SHOULD READ:MANUFACTURING, PROCESS DEVELOPMENT, QA/QC

KEYWORDS: ELISA, HOST-CELL PROTEINS, WESTERN BLOTTING, CROSS-REACTIVITY, RISK MANAGEMENT

LEVEL:INTERMEDIATE

From our recent understanding, thousands of potential HCP species could be present in a bioprocess (3,4,5). In a typical two-dimensional gel electrophoresis of HCPs from mammalian cells or Escherichia coli, hundreds of relatively abundant HCPs were detected. This is different from residual DNA, another process-related impurity, which is negatively charged in most bioprocesses and relatively easy to separate from the product stream. HCPs present a wide spectrum of variability, from different molecular sizes to charge heterogeneities. It is safe to say that any given biologic will always have a subset of HCPs with similar properties, which makes separating them from such products more challenging.

During the development of one monoclonal antibody (MAb), our group found that drug substance containing the antibody had a higher than normal level of HCPs. Initially, the process development team tested a variety of separation approaches trying to lower the HCP value, but with limited success. Further analytical studies led to a hypothesis that cross-reactivity of anti-HCP antibodies to this particular MAb might be the problem. The same preparation of polyclonal anti-HCP antibodies was used to both capture and report antibody for HCP quantitation in the HCP ELISA (Figure 1), so we hypothesized that even a small fraction of cross-reactivity could cause a measurable HCP signal (because of the assay's nanogram sensitivity).

Systematic analysis indicated that ~30% of internally generated MAbs have such nonspecific cross-reactivity. We implemented a modified HCP ELISA to minimize this cross-reactivity and improve the accuracy of our HCP analysis. Other biologics, especially MAbs under development in the biotechnology industry, may have similar properties and should benefit from our findings. Improvement of this HCP assay will increase the accuracy of HCP quantitation to prevent the commitment of costly and unnecessary resources to improve our bioprocess when the actual cause for high HCP readings was antibody cross-reactivity.

Materials and Methods

All the MAbs we tested were from our own company. Precast gels for SDS-PAGE came from Bio-Rad Laboratories (catalog #345-0043, www.bio-rad.com). Antihuman IgG polyclonal antibodies came from Thermo Scientific (catalog #31143, www.thermo.com). And Streptavidin–IRDye-800 fluorescence conjugate came from Rockland Immunochemicals (catalog #S000-32, www.rockland-inc.com).

ELISA: We developed a process-specific Chinese hamster ovary (CHO) host cell protein ELISA and used it to support our group's MAb development. A typical quantitation limit (QL) of 5 ng/mg (5 ppm) was demonstrated according to the ICH method validation guidelines (6). The ELISA is in a sandwich format, with HCP binding antibodies first coated onto a 96-well plate and incubated overnight, followed by an incubation with 1% BSA, then testing samples added and incubated for 90 minutes at room temperature. After multiple washes, the HCP reporting antibodies (biotinylated) were added and a colorimetric reaction determined the sample's HCP level against a predetermined standard curve.

For cases in which a cross-reactivity occurred, we added an additional step of antihuman IgG antibody incubation before the HCP reporting antibody incubation. This step blocks nonspecific interactions among anti-HCP antibodies and MAbs under development.

Gel Electrophoresis and Western Blot: We used 10–20% gradient SDS-PAGE gels for separation of all MAbs, treating samples with SDS and reducing reagent. for total protein detection, the gel was fixed in a solution with 10% each of acetic acid and ethanol. Sypro Ruby protein stain was used for over-night fluorescence staining. After performing two washes in Milli-Q water (from Millipore, www.millipore.com) for five minutes each, we used a VersaDoc image system from Bio-Rad to acquire the gel images.

For Western blotting, the proteins separated in SDS-PAGE were transferred to a nitrocellulose membrane over night with NuPAGE transfer buffer (Invitrogen catalog #NP0006-1, www.invitrogen.com). After blocking with 1% BSA in PBS-T (0.1% Tween-20 in PBS), we used a biotin-labeled polyclonal antibody against CHO HCP to test for cross-reactivity between the internal MAbs and the anti-HCP antibody. A streptavidin-IR Dye800 was used to detect the interaction, and the image was acquired using an Odyssey system from LI-COR Biosciences (www.licor.com).