Helena Nilshans Product Manager Gyros
The highly competitive market for biotherapeutics now includes many companies that are developing therapies, often for similar targets and clinical indications. This puts great pressure on companies to maintain quality, reduce time to market, and maintain cost effectiveness in the process development of antibody therapeutics.
High productivity demands efficient bioanalytical procedures to measure, for example, product titer and process impurities; however, commonly used methods, such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography, can create bottlenecks in critical workflows. Immunoassays used to monitor bioprocess development should ideally have a number of qualities to maximize efficiency:
- High-quality data quickly delivered to support confident data-driven decision making
- Rapid assay development
- High analytical performance that is consistent between process stages
- Broad dynamic range and high matrix tolerance to cope with a diversity of samples, from expression to production
- Minimal demand on resources (hands-on-time and reagent consumption)
- High throughput when it counts, e.g., in cell-line development and clonal selection
- Robustness for ready validation to meet regulatory demands.
The Value of Automated Nanoliter-Scale Immunoassays
Gyrolab™ systems meet the needs of many biopharmaceutical companies that apply immunoassays to bioanalysis throughout bioprocess development. The Gyrolab automated, nanoliter-scale immunoassay platform addresses bottlenecks and delays by providing rapid assay development time, automation, high throughput, and a broad dynamic range that minimizes dilutions and repeats. In this article, we illustrate how Gyrolab immunoassays meet the critical requirements for immunoassays used in process development of an immunoglobulin G (IgG) therapeutic antibody, from initial cell culture to final purified product, to measure:
- IgG product titer
- Host cell protein (HCP) impurities
Assays to determine monoclonal antibody (mAb) concentration (product titer) during biopharmaceutical development must quantify titers in a broad concentration range. In cell line development and clone selection, titers can vary greatly and sample numbers may be high, whereas in downstream process development, concentrations are high and must be measured with high analytical precision and reproducibility.
High Throughput in Cell Line Development and Clonal Selection
Decisions on the cell line and final clone to be used are typically made early because changes to the cell line are considered major and require comparability studies. Rapid screening and timely selection of highly productive and stable clones are major challenges. Gyrolab immunoassays deliver the robustness, rapid turnaround time, and high throughput needed for efficient screening. Figure 1 shows titer distribution of clones in a screen of low titers during early cell line development. Posttransfection samples were measured for IgG titer using the Gyrolab xP workstation, which processed 332 samples in 4 hours (data kindly supplied by Merck).
IgG Titer in Cell Culture
An IgG assay for use in the development of a human mAb therapeutic must be able to measure the broad range of product concentrations experienced in cell line development and during the optimization of upstream and downstream processes. Gyros’ kits to determine intact human IgG are composed of ready-to-use reagents and are compatible with two Bioaffy CDs that differ in sample volumes to address low- and high-titer samples. Together, the two kits cover a concentration range of 6 logs from concentrations of 1 ng/mL upward.
Figure 2 illustrates how the IgG titer throughout a 12-day cell culture was measured using the Gyrolab huIgG Kit-High Titer on both Gyrolab xPlore™ and Gyrolab xP workstation. The data correlated well with data from ELISA. Gyros wishes to thank POLYMUN Scientific Immunbiologische Forschung GmbH for generously supplying the samples for analysis.
IgG Titer during Purification
The high-titer kit was also used to measure the high IgG titers during the two-step purification of the IgG biologic from harvested cells. Overall, there was good correlation between IgG concentration data from Gyrolab xPlore, ELISA, and OD280 (typically used in later process stages to measure titer) (see Figure 3).
Host Cell Impurities
Ensuring minimal levels of HCP impurities from expression systems is critical during bioprocess development for recombinant therapeutic antibodies. HCPs in biotherapeutics are potentially immunogenic and therefore regulated. Residual HCP is a complex protein mixture that is routinely measured using ELISA with polyclonal antibodies; however, ELISA suffers from a narrow dynamic range and long turnaround time and often requires manual interventions and repeat analysis.
Precise Determination of HCP Impurities
In their search for more robust methods for HCP analysis, MedImmune compared an ELISA with a Gyrolab assay in the analysis of HCP in samples from different stages in the purification of a therapeutic protein. They found that there was excellent correlation of results from the two methods (Figure 4) and that the Gyrolab assay improved dilutional linearity as a result of broader dynamic ranges and good precision, enabling them to make rapid data-driven decisions.
CHO-HCP Assay with Broad Dynamic Range
To address many of the limitations of ELISA, Gyros has developed a range of five ready-to-use Gyrolab kits to measure HCP impurities from Chinese hamster ovary (CHO) cells. This range offers a selection of assays that contain commercially available antibodies developed from slightly different antigen preparations. The menu of kits simplifies the evaluation and selection of a generic CHO-HCP kit that will meet a particular analytical challenge. Gyrolab kits broaden the analytical range typically from 2 orders of magnitude to 4 (Figure 5), thereby simplifying spike recovery and dilution linearity experiments. The automation on Gyrolab systems enables 96 data points to be generated in approximately 75 minutes and without manual intervention.