An unexpected high-level of impurity formation (heavy-heavy-light chain (HHL)) was observed in a legacy monoclonal antibody cell culture fed-batch process when transferred from a 5000-L stainless steel facility to a 2000-L single-use bioreactor (SUB). Root cause analysis identified that the formation likely occurred during the production bioreactor or harvest steps. To our surprise, the lab studies mimicking the reducing environment of a single-use harvest operation did not induce HHL formation for a normal batch. Further investigation using a 5-L scale-down model of the production step identified that the root cause of HHL formation was actually the byproduct of changing sparger types and the use of a microsparger in the single-use bioreactor (Figure 1). Additional scale-down model work and material hold studies identified that the HHL formation occurs extracellularly. Overall, the evidence suggests that the microsparger itself is not inducing HHL formation; rather its presence set off a cascade of events that ultimately led to excess free thiols in the culture broth present at the end of the run. Specifically, the presence of microsparging leads to poor growth, resulting in overfeeding the culture and unconsumed free-thiol containing components that induce HHL formation. Using this knowledge we were able to replicate HHL formation using orthogonal methods and ultimately close the process characterization gap. This talk will overview the tech transfer considerations used to design the process in the initial 2000-L SUB and supporting characterization studies to characterize HHL formation. Further, this talk will also review tech transfer considerations to a secondary 2000-L SUB facility with a drilled-hold sparger that did not produce any elevated levels of HHL.

Unexpected high levels of  HHL formation observed during a tech transfer to a 2000-L single-use bioreactor: A case study

ACS Fall 2020 Virtual Meeting

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