This talk focuses on an issue encountered during scale-up of a monoclonal antibody (mAb) commercial manufacturing process. Low molecular weight (LMW) species were observed in some pilot plant scale (500 L) batches. Specifically, these LMW species were determined to be light chain (LC), heavy chain (HC), and heavy/light chain (HL) by chip-based electrophoresis, suggesting that disulfide reduction occurred. Small scale models were constructed, which revealed that the LMW formation was dependent on upstream conditions, harvest conditions, as well as the storage conditions of the harvest cell culture fluid (HCCF). By using statistical software, a correlation was established between the LMW level and the operation parameters. A kinetic study by monitoring the level of LMW showed different disulfide reduction susceptibility for different mAbs.<br/><br/>Furthermore, we found there was a linkage between the presence of LMW and pink color of the product stream, which was determined to be related to the high level of vitamin B12 (VB12). The pink color was carried to the final drug substance, indicating that the VB12 was tightly bound to the product. Mass spectrum data revealed that VB12 was specifically associated to LC or HC, suggesting there may be an association between the free thiol and the cobalt in B12.<br/><br/>For those batches that we observed LMW, elevated levels of Thioredoxin (Trx) and Thioredoxin reductase (TrxR) were detected. Conversely, by addition of various Trx/TrxR inhibitors, the LMW formation was controlled, suggesting that the Trx system may play an important role in the disulfide reduction. A mitigation strategy was developed to prevent LMW formation by controlling the reducing power of the HCCF. For this purpose, we tried to minimize cell lysis to reduce soluble cellular components by maintaining high cell viability, using mild harvest conditions, and storing HCCF under chilled temperature. The strategy has demonstrated to be effective in controlling LMW and led to successful manufacturing campaigns.

Investigation of low molecular weight species and implementation of mitigation strategies into large scale biologics manufacturing

251st National Meeting (2016)

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