(M1430-09-55) Process Change in Cell Culture Significantly Improves Aggregates in Drug Product

Purpose: MabA is a high concentration IgG1 biosimilar monoclonal antibody (mAb). The product quality attributes and stability behavior of a proposed biosimilar are expected to be comparable to those of the reference product (RP) both in amount and degradation rates. However, during development, the aggregate levels of MabA were higher than those of the RP; additionally, they increased over drug product stability, contrary to the behaviour of the RP. Aggregates in MabA first increased soon after the concentration step (i.e.) tangential flow filtration (TFF), stayed stable through the quiescent, frozen storage of the drug substance (DS), but continued to increase over the initial part of drug product (DP) stability (at 2-8°C) after which they plateaued (Figure 1). A series of troubleshooting activities were undertaken to find the root cause. After eliminating common formulation-related contributors to aggregation, focus shifted further upstream in the process to explore alternative sources. Purdie et al (2016) found that certain compounds added in upstream media were implicated in forming modified by-products that led to aggregation. Since the problem description in Purdie et al (2016) were similar to the observations in the MabA product, experiments were performed to understand the impact from such compounds added in Upstream cell culture on achieving aggregate contents commensurate with those in the RP and reduce the propensity to aggregate over DP shelf-life. This was successfully achieved, tested and implemented for at-scale manufacture.

Methods: Multiple pilot scale batches were run, each one modulating the amount of the compounds added to the process. Since the readout for the issue was aggregation over DP stability, the material from the trial runs was purified, formulated, filled into DP containers and stability was followed under accelerated and stress conditions in addition to real time conditions. Results were compared for aggregate content by Size Exclusion High performance liquid Chromatography (SE-HPLC), with UV detection at 220 nm.

Results: Results showed that modulating the contents of redox-related compounds used in Upstream cell culture reduced the aggregates both at the TFF step/DS release and in the DP over stability (Figure 2, Figure 3). The purported mechanism though not confirmed, was understood to be similar to Purdie et al. Presence of the redox compounds in significant quantities in cell culture purportedly led to the formation of reactive species. In MabA, it is likely that the reactive species targeted Cysteine residues in the mAbs and formed mAb sub-populations with altered cysteine residues. It is inferred that cysteine residues were the likely targets of the reactive groups as extensive characterization of isolated aggregates showed that they were covalently linked via multiple, reducible, disulphide bonds. During DS purification where the mAb concentration is low, high rates of aggregate formation were not observed. However, when the protein was concentrated in the TFF step, due to the proximity of multiple mAb molecules and the possible availability of ample levels of altered mAbs formed in cell culture, aggregate formation was observed. Aggregation was arrested when the drug substance was subsequently frozen. However, it re-started when the product became mobile post thawing. In liquid state, (DP) aggregation continued until a new equilibrium was reached (i.e.) possibly corresponding to a scenario where most of the altered molecules being already engaged in aggregates, and no new altered products being formed. This phenomenon was observed over stability of the drug product as a gradual deceleration of the rate of aggregate increase until a steady state (plateau) was obtained. Reducing the redox compound levels in cell culture likely lowered the initial number of altered by-products and thereby stemmed the aggregate increase observed in subsequent stages. A detailed comparability of the modified process data with that of the initial process data indicated a few additional product quality changes due to the modulation of these compounds. The changes were risk assessed to be of low risk and changes were then implemented at-scale.

Conclusion: Modulating redox compound contents in the upstream cell culture stage helped in reducing aggregates in MabA drug product over shelf-life. This exercise is one of the instances where a cell culture step - one of the first steps in the mAb production process - was demonstrated to directly impact quality attributes of the drug product – at the opposite end of the process. Very often in development, it is common to address issues through a siloed, function-specific lens. However, certain challenges demand a cohesive, cross-functional team approach—one that embraces the full scope of the problem and works collaboratively toward integrated solutions.

References: Cell Culture Media Impact on Drug Product Solution Stability, Purdie et al, Biotechnol. Prog., 32: 998 – 1008, 2016.

Acknowledgements: Conflict of interest: The authors declare no conflict of interest.

Figure 1: Trend of aggregate increase in MabA over different stages of manufacturing and storage

Figure 2: Comparison of %HMWP in the pre-change process and post-change process with RP

Figure 3: Stability data for the pre-change process and post-change process at 40°C. Reference Product (RP) data is also shown for comparison. Data shows that except post-change Process 4, others are comparable to the RP profile and rates.