Regulators require testing of drug products for process-related impurities throughout development to monitor product safety, purity, and efficacy. Low levels of most impurities can be inconsequential, but patient safety demands that host-cell proteins (HCPs) be eliminated or reduced to the lowest levels practical. Enzyme-linked immunosorbent assays (ELISAs) represent a key tool in that endeavor. Antibody-coverage analysis is one part of assessing a platform kit or custom HCP ELISA. In a 15 June 2021 webinar, Jared Isaac (senior scientist at Cygnus Technologies) discussed orthogonal antibody affinity extraction (AAE) and mass spectrometry (MS) methods for that purpose. He included case studies from several cell-based processes.
Viral-vector and vaccine manufacturing requires rigorous analytics for process-related impurities such as HCPs and DNA, growth-media components, enzymes, and purification process additives. HCPs can remain after multiple purification steps and must be reduced or eliminated to prevent stability problems, immune reactions, and off-target biological effects. Identifying HCPs early can lower process-development costs while ensuring product efficacy and safety.
The AAE method was developed to address specificity and sensitivity limitations of two-dimensional (2D) Western blotting. Antibodies are mobilized on a solid-phase support in a column, HCP-containing samples pass through, and immunoreactive proteins are captured while others wash away. HCPs that elute from an AAE column can be detected by 2D polyacrylamide gel electrophoresis (PAGE) or liquid chromatography (LC) with MS.
To analyze cell lysate or harvest material and determine antibody coverage, an aliquot is saved and the remainder of the sample is passed over an AAE column. Once the immunoreactive HCPs are captured by immobilized polyclonal ELISA antibodies, they can be analyzed with 2D-PAGE or LC-MS. Antibody coverage is calculated from gel images. LC-MS provides a â€śvirtualâ€ť 2D-PAGE image with percent coverage and a list of all HCPs present in the sample with their molecular weights and isoelectric points. Drug-substance material can be analyzed for copurified HCPs in a similar AAE process. Pre- and post-AAE samples are analyzed by LC-MS, then results are reported as percent antibody coverage and as a list of all identified proteins, including their molecular weights and isolectric points.
ELISA polyclonal antibody coverage depends on the detection method. 2D Western blotting typically has a coverage range of 50â€“60%; the AAE method combined with 2D-PAGE typically shows a coverage range of 60â€“85% depending on the staining methodology. Combining the AAE method with MS expands that range to 70â€“90%. ELISA polyclonal antibody coverage can be determined by calculating a lower boundary (i.e., dividing the post-AAE spots by the number of unique spots) and an upper boundary (dividing the post-AAE spots by the pre-AAE spots). With gel-based detection, visual spots are qualitative representations of proteins; with MS, the mass spectra represent quantitative ions.
We analyzed the same sample with Cygnus Technologies antibodies and used 2D Western blotting, AAE with 2D-PAGE, and AAE-MS detection. Both visual methods showed the same number of pre-AAE spots; but Western blotting detected fewer spots than were seen in the post-AAE 2D PAGE gel. AAE-MS gave the highest coverage range, identifying more pre-AAE and post-AAE proteins alike. It is a highly sensitive detection method for determining ELISA antibody coverage. My group at Cygnus Technologies has used it to curate proteomic databases for many cell lines, including HEK293, HeLa, Vero, NS/O, PG13, Sf9, and MDCK cells.
We developed a custom ELISA for a clientâ€™s HEK293 process based on a sample with 1,454 spots (1,356 in the AAE elution fraction). We used both 2D-PAGE and MS orthogonally to determine ELISA antibody coverage for this gene-therapy bioprocess. Because final drug-substance sample volumes are limited â€” with proteins at concentrations too low for 2D-PAGE detection â€” MS is ideal for gene therapies. Using AAE-MS, we determined the ELISA antibody coverage to be 80â€“89%, with 235 identified proteins. LC-MS and ELISA are confirmatory orthogonal methods that correlate well for quantification and qualification of HCPs throughout downstream processing.
Questions and Answers
What is the sample volume requirement for coverage analysis by AAE-MS? I ask for â‰Ą20-mL samples with 2â€“5 mg/mL of total protein.
Do you provide top-40 problematic HCPs for each cell line? Weâ€™ve developed extensive lists for many different cell lines.
What about proteins that are not recognized by anti-HCP antibodies? We document all proteins present and identify those that are not antibody reactive. You can use risk assessment to determine whether a process-specific ELISA is necessary.