A high-throughput approach to developing and optimizing mixed-mode membrane chromatography for protein purification.

Membrane chromatography has been established as a viable alternative to packed-bed column chromatography for the purification of therapeutic proteins. Purification via membrane chromatography offers key advantages, including higher productivity and reduced buffer usage. Unlike column chromatography purification, the utilization of high-throughput screening in order to reduce development times and material requirements has been a challenge for membrane chromatography. This research focused on the development of a new, high-throughput screening technique for use in screening membrane chromatography conditions for monoclonal antibody purification. The developed screen utilizes a 96-well plate format, thereby allowing for the screening of multiple different membrane conditions at once. For this study, four mixed-mode cation exchange membranes and one cation exchange membrane were evaluated on the plate. The screen is performed in a similar manner to that of a resin slurry plate screen, however, instead of a single loading step, the antibody feed was loaded in 50 mg/ml increments up to a maximum loading of 450 mg/ml. Performing a similar, incremental loading on a resin plate would be impractical, as mixing times are substantially longer due to pore diffusion limitations. However, due to the significantly faster rate of mass transfer for membranes relative to resin, mixing times could be reduced by up to a factor of sixty on the membrane plate. Additional optimization showed that higher hydrophobicity can potentially lead to slower kinetics and mixing times that may need to be adjusted accordingly. The end result is a screen that has been proven to provide results comparable to those obtained on larger-scale membrane purification runs while also enabling exploration of a much greater operating space and significantly reducing the feed materials required.

Considerations for Implementation of a New Drug Substance Container for Subzero Storage.

Therapeutic manufacturing has become globalized in recent decades, necessitating transportation of drug substance across the world. The outcome of this expansion is significant costs for shipment and added risk of damage to the drug substance containers. There are multiple container options with various materials of construction for storage of Biologics drug substance (DS). This study evaluates a newly designed CryoVault™ container and previously characterized CelsiusPak® bag container using a well-represented scale-down model. Consideration of an appropriate storage container includes the risk assessment of the design and material of construction, which can potentially impact product quality attributes, stability and container leachables. An extensive data package, including product stability over time and temperature with respect to impact of extractables and leachables from different containers undergoing a typical one freeze/thaw cycle process was evaluated. This drove to the decision for implementation of a container into the drug substance manufacturing process.

Accurate and Rapid Protein Concentration Measurement of In-Process, High Concentration Protein Pools.

Protein concentration is a critical product quality attribute and required for any therapeutic protein. Many commercial and investigational new biologics are now formulated at high concentrations (>100 mg/ml) to achieve successful subcutaneous administration. Assaying protein concentration in high concentration formulations poses a challenge, as traditional absorption spectroscopy and UPLC/HPLC (ultra/high performance liquid chromatography) assays cannot accurately measure such high concentrations without further solution manipulation. However, recent advances in UV/vis technology have led to the creation of instruments that measure samples at relatively short (<1 cm) path lengths, which would allow them to accurately measure high concentration protein samples in accordance with Beer Lambert Law principles. In this research, samples of five different proteins at concentrations ranging from 0.15 to 242 mg/ml (corresponding to OD280 vales of 0.15-315 AU) were measured on two different instruments employing different techniques of low path length UV/vis measurements. In order for the techniques to meet MSD's acceptance criteria for release assays, measurements were required to be accurate to within 10% of a reference measurement (performed on a traditional UV/vis spectrophotometer) and to be precise within 5% CV. The results show that using a technique known as slope spectroscopy, it is possible to measure OD280 from 0.5 to 315 AU with <7% error relative to the reference measurement. If instead measurements are taken using an instrument utilizing a single, small path length, it is possible to measure absorbances from 0.2 to ~75 AU with <7% error. This article concludes that the slope spectroscopy technique performed within the acceptance criteria across the full range of measured absorbances and that the single, short path length measurement performed within the acceptance criteria up to 75 AU.