Impurity ELISA automation for accelerated process development

Overview

Cytiva impurity ELISA kits are easily integrated into automated systems such as the Biomek i5 to increase throughput, while maintaining the same robust and sensitive results you’re used to. This accelerates drug development, so lifesaving therapies reach patients sooner.

Throughout development and release of biologics such as monoclonal antibody therapies, regulatory authorities demand precise measurement and control of process impurities like residual ligand leakage and host cell protein (HCP) clearance. These are critical for quality control in biologics because they are a risk to patients if not suitably monitored. Monitoring impurities in biologics is routinely performed using ELISAs, which are specifically recommended in regulatory guidelines. Cytiva’s impurity and host cell protein ELISA kits help drug manufacturers mitigate risk by offering rapid, accurate, and sensitive assays to assist in making the best design and optimization choices for their purification processes.

ELISAs are robust and considered a workhorse for impurity measurements. However, multiple hands-on protocol steps limit the number of assays a scientist can run in parallel. This creates a bottleneck at multiple stages in the development workflow. Automation of impurity ELISAs enables a much higher assay throughput by reducing hands-on time, freeing scientists to complete other tasks.

We automated our Amersham™ HCPQuant™ CHO ELISA kit using a Biomek i5 liquid handling workstation from Beckman Coulter Life Sciences, with an integrated AquaMax 4000 plate washer from Molecular Devices. We demonstrated that automation gives the same robust results as the standard, manual protocol (Figs 1-5, Table 1). Using our setup, it was possible to run three automated HCP assays simultaneously, with potential to increase this by adding additional shaking positions and tip loading positions to the system (Fig 6).

Our robust solution enables higher throughput of impurity analysis with less user interaction, which advances and accelerates biotherapeutic development.

Fig 1. HCP clearance. Amersham™ HCPQuant™ CHO was used to measure residual HCP in four downstream process steps for two unique mAb purifications (downstream process 1 and 2). Each process step was measured using automation and manual handling of the ELISA. Eash assay was performed in triplicate. Concentration is given in ppm (ng HCP per mg IgG). Error bars represent standard deviation. Numbers above the columns represent the relative change in calculated HCP concentration between the methods. A t-test performed on each process step revealed no significant difference between the two methods.

Fig 2. A summary of the mean interplate variation. A robust HCP method must be able to accurately report the same concentration of HCP in samples across multiple experiments. To measure the variation across multiple assays (interplate variation), we determined the coefficient of variation (CV) of the calculated concentration of HCP. We performed this for each of the eight process steps across triplicate assays for each handling method. The mean interplate CV of the automated method was 3.96% compared to 7.89% using the manual method. Both methods had good reproducibility, well within the assay specification of < 20%. We used a t-test to determine that there was no significant difference (ns) between the means of the two groups. Error bars represent standard error of the mean (SEM).

Fig 3. A summary of the intraplate variation. To determine well to well (intraplate) variation, we calculated CV from the OD measurements of 112 replicate sample measurements. The mean CV using the automated method was 4.1%, compared to 3.6% using the manual method, both well under the assay specification of < 20%. Ninety percent of automated replicate measurements had a CV less than 9.2%. Using a t-test, we did not detect any significant difference in the mean variation between the methods.

Fig 4. Standard curve summary. For accurate determination of HCP concentration, an assay’s calibration curve must be reproducible. To measure the reproducibility, three standard curves from each handling method were compared. Standard curves were plotted from three replicate experiments for each handling method and overlaid. Standard curves from the manual handling method had lower overall absorbance in two replicates. This did not affect the assay performance. Error bars represent standard deviation.

Fig 5. Mean standard curve recovery. For a robust HCP method, the standard curve must accurately interpolate the concentration of analyte. Using the standard curve from triplicate experiments for each handling method, we interpolated the concentration of each calibrator to calculate recovery percentage. All standard curves in both groups could be used to interpolate accurate concentrations within the assay specification of 80 to 120%. Error bars represent standard deviation.

Table 1. Mean standard curve recovery and variation. An accurate HCP method demands the standard curve to have accurate recovery and good reproducibility. In the table, the average recovery for each calibrator taken from triplicate experiments is summarized. Recovery of calibrators ranged from 91.3% to 104.4%, which was within the assay specification of 80 to 120%. CV was used to determine the reproducibility of recovery across the three experiments from each group. This was within the assay specification of < 20% for both methods.

Fig 6. A schematic representation of the deck layout of the Biomek i5 used to automate Amersham HCPQuant CHO. It was possible to completely automate three assays simultaneously. The limiting factor was the number of plate shakers used (BS1-3). Capacity and therefore throughput can be increased by adding additional shakers and tip loading (TL) positions. P, Standard Position. TL, Tip Loading. BS, Bioshaker. AM, AquaMax 4000. TR, trash receptacle.

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Beckman and Biomek are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. AquaMax and SpectraMax are registered trademarks of Molecular Devices, LLC. All other trademarks are the property of the respective owners.