Removal of B. cereus cereulide toxin from monoclonal antibody bioprocess feed via two-step Protein A affinity and multimodal chromatography

摘要

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Baci his cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (similar to 1 fmol injected) and limit of quantification 0.16 ng/mL (similar to 3 fmol injected) over a standard curve with > 3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chro