Evaluation of product antibody (mAb) heterogeneity in non-clonal cell pools for early pre-clinical development

During early stage of pre-clinical biologics development, grams of product antibody is needed for process development, formulation development, and analytical assay development. To accelerate the pre-clinical timeline, it\u27s a common practice to use master wells (non-clonal stable cell pools) to generate development material. The advantage of using master wells is that the product antibody is generated from the same host cell line and expression vector in an earlier and shorter time. Thus, the purified product antibody can be representative to the final therapeutic product. However, the non-clonal nature of the cell pools can give rise to potential risk of heterogeneity in product quality exhibited in charge variants, glycan profile, etc. Herein, we present a case study on the evaluation of charge variant heterogeneity, its root cause, and impact. The increase fraction of acidic variants was first discovered in the in-process analytics by isoelectric focusing (iCIEF). Tryptic peptide mapping LC-MS analysis of purified drug substance further indicated an exchange of Lysine to Asparagine in the Fc region. Subsequent cDNA analysis of the single-cell clones from a master well that produced the purified product revealed a single substitution mutation that results in the amino acid substitution. Although the material used for development was a mixture of antibody product, process development, formulation development, and analytical development were not impacted. The risk of using mutated, potential non-representative variant was further mitigated by bridging studies, confirming product produced by single-cell clones. This case is a demonstration of a worst case scenario, in which a larger percentage (about 40%) heterogeneity was introduced via a point mutation at the DNA level. Nevertheless, overall time line for this program was not affected; thus the time saving benefits of this strategy outweigh the disadvantages and supports the use of non-clonal cell pool in the fast paced early stage development space

Use of the Ambr 250 to enable rapid clone selection and process development for large scale manufacturing processes

Currently, widely used bench scale bioreactor systems require much user manipulation, a large amount of raw materials, and have a long turnaround time for reactor cleaning and rebuilding. New technologies such as robotic disposable bioreactor systems provide a solution that is miniaturized, high throughput, and substantially automated. The Ambr® 250 offers such a solution, with 24x250mL bioreactors controlled independently. Although this new technology is rapidly being adopted by several groups as a way to increase efficiency and speed within upstream development, it remains to be proven that these systems are complete models for process characterization. We have shown that Ambr 250 is a good scale down model for multiple cell line systems. The aim of this work is to further characterize the engineering environment of the Ambr® 250 with a view of defining its role in industrial cell culture process development and scale-up. CFD modeling of the Ambr 250 mammalian vessel with validation via Particle Image Velocimetry (PIV) was conducted to simulate the hydrodynamic environment in the vessel. These findings were evaluated against current benchtop models and manufacturing scales. Cultures were run utilizing different engineering parameters (vvm, P/V, kLa) to assess the scalability of the current system. Cell growth, production, and product quality were compared across to recommend operating conditions for the Ambr® 250 that best match manufacturing scale reactors. Multiple CHO host cell lines were examined in order to find optimal operating conditions for the Ambr® 250 system

Enabling pandemic speed to clinic for SARS-CoV2 neutralizing antibodies

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Upstream control strategy development for afucosylated species in mAb biomanufacturing

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Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process