Stress Temperature Studies in Small Scale Hastelloy® Drug Substance Containers Lead to Increased Extent of and Increased Variability in Antibody-Drug Conjugate and Monoclonal Antibody Aggregation: Evidence for Novel Oxidation-Induced Crosslinking in Monoclonal Antibodies.

Health authorities require that suitable stability of the drug substance be shown in relevant materials of construction. ICH Q1A(R2) explicitly states that "stability studies should be conducted on drug substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution". Stainless steel containers are commonly used for the long-term storage of frozen bulk drug substances (DSs). Hastelloy®-based metal containers are sometimes used due to their higher corrosion resistance and significantly lower iron content to mitigate the potential corrosion-related risks associated with high salt formulations. Despite their benefits, we have found that elevated temperature stability studies in small scale Hastelloy® containers can lead to degradation that is not representative of degradation under typical storage conditions relevant to the manufacturing process. We provide evidence for an oxidation-induced aggregation mechanism that is based on Fenton chemistry with peroxide being supplied by the autoxidation of polysorbate at stress temperatures. Further, variation in the rates of iron leaching between individual small scale containers is shown to be the cause of the variable rates of degradation through strong correlations between leached iron levels and the extents of oxidation and aggregation. The addition of a metal chelator or the removal of polysorbate from the formulation mitigates the oxidation and the non-representative behavior. Extended characterization by LC-MS and 18O labeled peptide mapping shows that a significant portion of the aggregate formed under these conditions is covalently crosslinked and that the predominant covalent species is either a dityrosine or tyrosine-tryptophan crosslink between an Fc peptide and a Fab peptide. This report is the first time either of these two crosslinks have been reported for antibodies with detailed analytical characterization. Because the behavior observed in these studies is not representative of degradation under typical storage conditions relevant to the manufacturing process, this study demonstrates that small scale stress studies in metal containers should be performed with caution and that extended incubation times can lead to non-representative degradation mechanisms.

A Small-scale Model to Assess the Risk of Leachables from Single-use Bioprocess Containers through Protein Quality Characterization.

Leachables from single-use bioprocess containers (BPCs) are a source of process-related impurities that have the potential to alter product quality of biotherapeutics and affect patient health. Leachables often exist at very low concentrations, making it difficult to detect their presence and challenging to assess their impact on protein quality. A small-scale stress model based on assessing protein stability was developed to evaluate the potential risks associated with storing biotherapeutics in disposable bags caused by the presence of leachables. Small-scale BPCs were filled with protein solution at high surface area-to-volume ratios (≥3× the surface area-to-volume ratio of manufacturing-scale BPCs) and incubated at stress temperatures (e.g., 25 °C or 30 °C for up to 12 weeks) along with an appropriate storage vessel (e.g., glass vial or stainless steel) as a control for side-by-side comparison. Changes in protein size variants measured by size exclusion chromatography, capillary electrophoresis, and particle formation for two monoclonal antibodies using both the small-scale stress model and a control revealed a detrimental effect of gamma-irradiated BPCs on protein aggregation and significant BPC difference between earlier and later batches. It was found that preincubation of the empty BPCs prior to protein storage improved protein stability, suggesting the presence of volatile or heat-sensitive leachables (heat-labile or thermally degraded). In addition, increasing the polysorbate 20 concentration lowered, but did not completely mitigate, the leachable-protein interactions, indicating the presence of a hydrophobic leachable. Overall, this model can inform the risk of BPC leachables on biotherapeutics during routine manufacturing and assist in making decisions on the selection of a suitable BPC for the manufacturing process by assessing changes in product quality. LAY ABSTRACT: Leachables from single-use systems often exist in small quantities and are difficult to detect with existing analytical methods. The presence of relevant detrimental leachables from single-use bioprocess containers (BPCs) can be indirectly detected by studying the stability of monoclonal antibodies via changes by size exclusion chromatography, capillary electrophoresis sodium dodecyl sulfate, and visible/sub-visible particles using a small-scale stress model containing high surface area-to-volume ratio at elevated temperature alongside with an appropriate control (e.g., glass vials or stainless steel containers). These changes in protein quality attributes allowed the evaluation of potential risks associated with adopting single-use bioprocess containers for storage as well as bag quality and bag differences between earlier and later batches. These leachables appear to be generated during the bag sterilization process by gamma irradiation. Improvements in protein stability after storage in "preheated" bags indicated that these leachables may be thermally unstable or volatile. The effect of surfactant levels, storage temperatures, surface area-to-volume ratios, filtration, and buffer exchange on leachables and protein stability were also assessed.

Leachables from saline-containing IV bags can alter therapeutic protein properties.

PURPOSE: To investigate the cause of the observed instability of dulanermin in 100 ml polyolefin (PO) infusion bags containing saline. METHODS: Diluted dulanermin in IV bags was collected and frozen prior to analysis by size exclusion chromatography. The UV absorption profiles of the IV bag solutions were characterized by using spectrophotometry. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) measured the metal content. Leachables from IV bags were identified by LC-UV-high resolution MS/MS analysis. RESULTS: An elevated loss of dulanermin monomers was observed only in 100 ml PO bags. These IV bag solutions have a compound that contains zinc and has absorbance at 320 nm. This compound was identified to be 2-Mercaptobenzothiazole, and its zinc salt and was found to come from the stopper used in the 100 ml PO bags. The manufacturer has subsequently corrected this problem by using non-latex components in the 100 ml PO IV bag. CONCLUSIONS: End-users need to be aware that IV bags made from a particular polymer by the same manufacturer may contain components or use a manufacturing process that results in a different product. Analysis of samples after freezing and thawing proved to be useful in identifying potential incompatibility of dulanermin in the IV bags.