Most biotechnology unit operations are complex in nature with numerous process variables, feed material attributes, and raw material attributes that can have significant impact on the performance of the process. Design of experiments (DOE)-based approach offers a solution to this conundrum and allows for an efficient estimation of the main effects and the interactions with minimal number of experiments. Numerous publications illustrate application of DOE towards development of different bioprocessing unit operations. However, a systematic approach for evaluation of the different DOE designs and for choosing the optimal design for a given application has not been published yet. Through this work we have compared the I-optimal and D-optimal designs to the commonly used central composite and Box-Behnken designs for bioprocess applications. A systematic methodology is proposed for construction of the model and for precise prediction of the responses for the three case studies involving some of the commonly used unit operations in downstream processing. Use of Akaike information criterion for model selection has been examined and found to be suitable for the applications under consideration.

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Aggregation of biotech products used therapeutically, such as antibodies, can contribute to potential immunogenicity of the product. Charge-based heterogeneities may also impact the safety and/or efficacy of a therapeutic. In this study, an approach based on empirical modeling and least squares regression is suggested for establishing hold times for process intermediates during production of monoclonal antibody (Mab) therapeutics. Two immunoglobulins were analyzed with respect to aggregation and charge heterogeneity in buffer conditions that are typically used during downstream processing of Mab products. Size exclusion chromatography, ion exchange chromatography (IEC), and circular dichroism were used. We found that aggregation primarily occurs at pH 3 (buffers used in affinity chromatography) and is higher in citrate buffer compared to acetate and glycine buffers. Aggregation is minimal in buffers used in anion exchange chromatography (Tris-HCl buffer at pH 7.2 and 8) and in cation exchange chromatography (citrate buffer at pH 6, acetate buffer at pH 6, and phosphate buffer at pH 6.5 and 7.5). The behavior is opposite in the case of charged heterogeneities (basic and acidic variants) as measured by IEC. The product is more susceptible to degradation at high pH than at low pH. The data presented here demonstrate that product stability can be a significant issue within the routinely used manufacturing conditions. We suggest that the approach presented needs to be adopted by all manufacturers to ensure product stability during processing.

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