Continuous bind-and-elute protein A capture chromatography: Optimization under process scale column constraints and comparison to batch operation.

Recently, continuous downstream processing has become a topic of discussion and analysis at conferences while no industrial applications of continuous downstream processing for biopharmaceutical manufacturing have been reported. There is significant potential to increase the productivity of a Protein A capture step by converting the operation to simulated moving bed (SMB) mode. In this mode, shorter columns are operated at higher process flow and corresponding short residence times. The ability to significantly shorten the product residence time during loading without appreciable capacity loss can dramatically increase productivity of the capture step and consequently reduce the amount of Protein A resin required in the process. Previous studies have not considered the physical limitations of how short columns can be packed and the flow rate limitations due to pressure drop of stacked columns. In this study, we are evaluating the process behavior of a continuous Protein A capture column cycling operation under the known pressure drop constraints of a compressible media. The results are compared to the same resin operated under traditional batch operating conditions. We analyze the optimum system design point for a range of feed concentrations, bed heights, and load residence times and determine achievable productivity for any feed concentration and any column bed height. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:938-948, 2016.

Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system.

Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.

Monitoring ceramic hydroxyapatite media degradation using dynamic image analysis and uniaxial confined bulk compression.

Ceramic hydroxyapatite (CHT) is a multimodal chromatographic medium widely used in the pharmaceutical industry for the purification of biomolecules. CHT is a sintered form of hydroxyapatite crystals with moderate stability at acidic conditions. This moderate stability may lead to underperformance of CHT packed bed lifetime, especially under acidic conditions, which should be monitored by diagnostic tools to design optimal buffer systems for the step. This study presents the application of dynamic image analysis (DIA) and uniaxial confined bulk compression (UCBC) to monitor CHT particle degradation as a function of buffer composition. DIA was used to evaluate changes in solidity and morphology, while UCBC was used to evaluate changes in resistance to uniaxial compression. All properties were studied as a function of bed position and operational parameters. Results show that when CHT is exposed to acidic pH, adding phosphate and/or calcium at concentrations of 1 mM minimizes changes in particle solidity and mechanical strength. Changes in CHT morphological properties (i.e., convexity, aspect ratio) are also affected by the presence of calcium and/or phosphate in the inlet buffers. Furthermore, calcium and phosphate have a positive effect on the mechanical behavior of CHT, which is related to changes in the CHT particle solidity.

Recovery modeling of tangential flow systems.

The demand for increased formulation concentrations for protein therapeutics puts a significant strain on already existing tangential flow filtration (TFF) systems that were constructed with lower protein concentration targets as part of their design criteria. TFF is commonly used to buffer exchange and concentrate the product to the appropriate drug substance concentration. Analyzing the ability of an existing TFF system to process under conditions outside its original design specifications can be challenging. In this analysis, we present a systematic approach to assess the operational limits of a TFF process with consideration of system performance parameters for changing process targets. In two new engineering diagrams, the recovery efficiency diagram and the operating space plot, all relevant operational constraints and parameters are related to allow rapid process fit evaluation. The engineering assessment of TFF systems presented in this article allows a rational review of system limitations during process fit evaluations of existing TFF systems. It also provides a rational basis for targeted system upgrades and setting system design specifications for the design of new systems if existing systems are found inadequate.

Risk-benefit evaluation of on-line high-performance liquid chromatography analysis for pooling decisions in large-scale chromatography.

In the production of a human therapeutic protein from inclusion bodies, product related impurities of very similar size and charge to the product are created as byproducts of the refold process. Their removal is usually challenging even when using chromatography with high performance resins and elution by shallow linear gradients. Additionally, performing this type of separation for commercial production adds increased complexity. To maximize productivity, columns are loaded so high that product elution profiles are not well separated from the impurities and pooling decisions are challenging. In this paper, conventional UV pooling based on fractionation or predefined absorbance based criteria will be compared to pooling based on fast on-line HPLC analytic. The development and implementation in a GMP process will be shown for a specific challenging separation by hydrophobic interaction chromatography. The different approaches have their unique complexities, timelines, uncertainties, and risks during development and implementation as well as during manufacturing. This study presents a probabilistic framework for quantitative comparison of two processes with unequal variability and uncertainty to evaluate the potential benefits of a PAT technology for its routine use in GMP Bioprocess manufacturing.