(T1430-01-06) Multiple Root Causes Impact Methionine Oxidation in a Coformulated Drug Product

Purpose: Oxidation of therapeutic proteins is known to be a problem in the pharmaceutical industry that can impact the quality, efficacy, and potential shelf-life of a drug product. Fundamentally, understanding the mechanisms involved would be vital in the development of robust formulations as oxidation propensity could be mitigated, leading to more efficacious products with longer shelf-life. As a case study, we have used a coformulated mAb drug product (CFDP) where one of the antibodies contains a methionine residue in the CDR susceptible to oxidation under long-term stability conditions. We have studied multiple potential root causes ranging from metal catalyzed reactive oxygen species (ROS) formation resulting from stainless-steel (SS) exposure in the process train, assessing if the metal catalyzed ROS formation is local near the oxidation target, variability in the container closure system, degradation of surfactant as sources of ROS and the batch-to-batch variability of the drug substance inputs. The results show that the levels of metals are not the sole culprit driving oxidation, but SS exposure in combination with other process parameters and the DS batch-to-batch variability are part of the root cause. Additionally, linoleic acid degradation from the surfactant PS80 appears to be a precursor to methionine oxidation and an unknown variability in the container closure system impacts oxidation performance on stability. Together the results from these studies show that multiple mechanisms are intertwined and contribute to methionine oxidation, making it difficult to draw mechanistic conclusions.

Methods: HPLC, ICPMS, ICPMS-SEC, LC-MS

Results: The levels of metals are comparable across multiple batches tested (high and low oxidation), but stainless-steel exposure seems to drive oxidation on stability. Evaluated the metal speciation (bound vs unbound) by ICP-MS-SEC and comparable levels of bound metal (Cu and Fe) are detected for high and low oxidation batches. Additionally, PS80 purity analysis of high and low oxidation batches depicts faster linoleic acid degradation relative to methionine oxidation for high oxidation batches suggesting that PS80 more specifically the low levels of linoleic acid may lead to the formation of ROS leading to methionine oxidation. The DS contribution was also interrogated coupled with SS exposure by generating lab scale prototype DP batches made from multiple DS combinations.  Results show that the high oxidation is isolated to a subset of DS batches for one of the mAbs in the coformulated drug product. Lastly, unusual stability is observed for stability vials from high oxidation batches.  Some of the vials are depicting different levels of oxidation on stability where in some cases no oxidation is observed, this shows that a component in the container closure system may impact oxidation making it difficult to assess the contributions from the process and other parameters evaluated.

Conclusion: The results from these studies show that multiple mechanisms are intertwined and contribute to methionine oxidation, making it difficult to draw mechanistic conclusions.  Studies are still ongoing to decipher the relative contributions and identify the dominant pathway or assess if all pathways are active and how are they interconnected.