Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer.

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.

Cored anion-exchange chromatography media for antibody flow-through purification.

Agarose-based anion-exchangers (e.g. quaternary amine, Q) have been widely used in monoclonal antibody flow-through purification to remove trace levels of impurities. Such media are often packed in a large column and the operation is usually robust but with limited throughput due to the compressibility of agarose and consequentially low bed permeability. In order to address this limitation, cored Q beads consisting of a rigid core and a thin agarose gel coating were developed and evaluated for protein flow-through chromatography. Using laboratory-scale columns it was found that, the cored beads indeed provide significantly enhanced rigidity and flow permeability relative to conventional homogeneous agarose resins. Depending on the structure and size of the cored beads, the permeability was 2-4-fold higher than that of a commonly used commercial agarose resin. Good virus and host cell protein clearance was achieved with the cored Q beads even at increased flow velocities. In addition, the impermeable core allows for more efficient use of buffers without loss of useful capacity in polishing applications. Process analyses based upon the experimental data demonstrated that the enhanced permeability achieved with the cored beads can significantly improve process throughput and economics.