Sanchayita Ghose

Use and Optimization of a Dual-Flowrate Loading Strategy To Maximize Throughput in Protein-A Affinity Chromatography

The effect of an alternate strategy employing two different flowrates during loading was explored as a means of increasing system productivity in Protein‐A chromatography. The effect of such a loading strategy was evaluated using a chromatographic model that was able to accurately predict experimental breakthrough curves for this Protein‐A system. A gradient‐based optimization routine is carried out to establish the optimal loading conditions (initial and final flowrates and switching time). The two‐step loading strategy (using a higher flowrate during the initial stages followed by a lower flowrate) was evaluated for an Fc‐fusion protein and was found to result in significant improvements in process throughput. In an extension of this optimization routine, dynamic loading capacity and productivity were simultaneously optimized using a weighted objective function, and this result was compared to that obtained with the single flowrate. Again, the dual‐flowrate strategy was found to be superior.

pH Transitions in Ion-Exchange Systems: Role in the Development of a Cation-Exchange Process for a Recombinant Protein

Unexpected transient changes in effluent pH can occur during ion‐exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation‐exchange resin. Incomplete column equilibration was eliminated as a possible cause of these effects. Various buffering species and various salt ions were studied at different solution concentrations to investigate pH transitions on strong cation‐exchange resins. A further comparison was made between cation‐exchange resins with different backbone chemistries. On the basis of these studies, a mechanism is proposed for these phenomena based on competitive equilibria between ions from the buffer salts and H+/OH‐ ions. In addition to the equilibria between these ions and the functional groups on the resins, charged groups on the resin backbone were also found to contribute to transient pH changes. The results from this study were applied to the cation‐exchange step for a recombinant protein that was sensitive to pH excursions to help maintain activity of the protein during the purification process.

Vitamin B12 association with mAbs: Mechanism and potential mitigation strategies

Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in‐process pools and drug substances in the antibody manufacturing process. Vitamin B12 is covalently bound to mAbs via a cobalt‐sulfur coordinate bond via the cysteine residues. The vitamin B12 was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys‐214), and heavy chain (Cys‐134, Cys‐321, Cys‐367, and Cys‐425). Prior to attachment to mAbs, the vitamin B12 needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B12 added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11–14 days at room temperature and with room light intensity about 500–1,000 lux) and by short‐wavelength visible light (400–550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B12. Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light‐induced color variations in final products.

Comparison of the chromatographic properties of IgG1 and IgG2 antibody subclasses

All therapeutic antibodies that are currently approved or under development are IgGs, with IgG1 and IgG2 being the most prevalent subclasses. The present study investigates the effect of antibody subclass on its chromatographic properties. To this end, two sets of antibodies with identical variable domains and differing subclasses (IgG1 and IgG2) were compared by using purification methods that are most commonly used in industrial antibody manufacturing processes. Each antibody was examined using Protein A chromatography, cation‐exchange chromatography, hydrophobic interaction chromatography and salt‐induced precipitation studies. The results of these experiments show that the variable region of an antibody has a greater influence on its chromatographic behaviour than antibody subclass.

Ultrafiltration behavior of partially retained proteins and completely retained proteins using equally-staged single pass tangential flow filtration membranes

This work examines the ultrafiltration behavior of partially retained proteins like lysozyme and completely retained proteins like monoclonal antibodies using single pass tangential flow filtration (SPTFF) modules with different screen channels and molecular weight cut‐offs. When the staging of the SPTFF used the same membrane area in each stage, there was no impact of the module screened channel or the buffer matrix on the final concentration achieved for completely retained monoclonal antibodies. A hybrid configuration containing 30 kDa membranes and 50 kDa membranes increased the maximum achievable concentration for both the monoclonal antibodies used in this work, at the same time, allowing a two‐fold to four‐fold increase in normalized feed flow‐rate through the system compared to only the 30 kDa or only the 50 kDa membranes. The sieving coefficient of lysozyme measured and calculated using SPTFF was lower than those measured during conventional recirculation TFF indicating a more complicated concentration polarization effect than conventional recirculation TFF. Moreover, the sieving coefficients of lysozyme were the same for the 10 kDa regenerated cellulose and 50 kDa PES membranes while it was higher for the 30 kDa regenerated cellulose membrane. The difference in TFF and SPTFF behavior is important when the product of interest is desired to be permeated. This work presents the first body of data for partially and completely retained solutes together in the SPTFF mode and provides a strategy to increase protein concentration at higher feed flow rates.