Leveraging continuous manufacturing for efficient mAb production

Enzene Biosciences was approached by a customer working on a monoclonal antibody (mAb) for the treatment of osteoporosis in women who have an increased risk of fractures. Using a fed-batch approach, the process suffered from low productivity.

Turning to Enzene, the customer aimed to increase productivity by transitioning from a fed-batch to an intensified perfusion method.

Moving from the original fed-batch system to the incorporation of Enzene’s intensified perfusion process, productivity increased from 15g in a 5L fed-batch to 48g in a 2L perfusion and 730g in a 30L perfusion.

However, despite this increase in productivity of the target mAb, several issues arose when developing the perfusion process. Additional peaks were observed in the tox batch (55L) when analyzed using capillary electrophoresis sodium dodecyl sulfate (CE-SDS), which were not observed in the satellite batch (5L), indicating that either impurities or degradation products were present in the final tox batch drug substance. To identify these products and determine the root cause, an extensive analysis was performed.

Starting with a comparison of the 5L and 55L batches, analytical tests were completed to measure the differences between the two batches, measuring glutamine, ammonia, glucose, lactate, osmolality, pH, and productivity profiles. With this data on hand, the upstream processes were examined, with the following differences scrutinized for possible causality — harvest method, harvesting time, container used, harvest storage temperature, and transfer to downstream processing (DSP).

The probable root causes were identified as:

• The addition of sodium bicarbonate in upstream processing, leading to an increase in osmolality.
• The shear stress when using depth filtration, as opposed to centrifugation and filtration as is used for the satellite batch.
• Longer hold of the clarified harvest in media (hold for harvest at 2-8 and hold and loading at room temperature (RT) for DSP).

CE-SDS analysis of the 55L tox batch at day 14 (before depth filtration) showed no significant difference from the reference spectrum, with no impurities present, eliminating the addition of sodium bicarbonate as a potential cause. After depth filtration and holding at 2-8°, CE-SDS was repeated, with no low molecular weight (LMW) peaks observed. Therefore, it was identified that the final step, a longer hold of the clarified harvest media at RT, was leading to the presence of these peaks. Additional analytical testing determined that these impurities were in fact fragments of the mAb product.

With almost double the free thiol content present in the 55L tox batch (1.683 mole of free thiol/ mole of protein) compared to the 5L satellite batch (0.8 mole of free thiol/mole of protein), the mAb was undergoing partial reduction leading to an increase in free thiol. However, despite this reduction, the molecule remained intact. Instead, the presence of free thiol generated peaks during sample preparation for CE-SDS resulting in binding and co-purifcation of B12 with the molecule.

To overcome these challenges, both corrective and preventative methods were put in place. Firstly, as reverse-phase high-performance liquid chromatography (RP-HPLC) indicated that the molecule remained intact, regulatory services were used to identify if it was acceptable to go ahead with the 55L tox batch. In addition, the redevelopment of the CE-SDS method to consider the presence of additional free thiol was performed. A third purification step using Capto(TM) MMC or hydrophobic interaction chromatography (HIC) was also incorporated, to ensure the product was of the highest purity.

The preventative measures included the lowering of the pH to 6.0 at harvest, oxygen sparging in harvest, and the addition of EDTA before depth filtration to inhibit any thioredoxin and thioredoxin reductases that may have been leading to the appearance of excess thiol in the final 55L tox batch.

With these measures in place, Enzene Biosciences was able to assure product quality and safety when transitioning the mAb for the treatment of osteoporosis to a perfusion process. The final product, therefore, met the same standards as the fed-batch method with an increase in productivity driven by the efficiency improvements.