The session provided an update on the application and mechanistic understanding of intensified unit operations (e.g., mixed mode depth filters, mixed mode AEX) since the last conference in 2019. One of the key gaps identified in the 2019 Viral Clearance Symposium session on the topic was for more investigation required to achieve a clear understanding of the molecular mechanisms of virus removal and the relevance of different moleculés interactions including resin, virus, and product. Further investigation into worst-case conditions for these unit operations is also warranted. One of the key outcomes from that 2019 discussion was also that multimodal anion exchangers can have robust and effective virus removal, depending on process and impurities-an observation that was recapitulated with more specific case studies and evidenced by broader application of these chromatographic resins in late-stage regulatory filings.

Visible light (400-800 nm) can lead to photooxidation of protein formulations, which might impair protein integrity. However, the relevant mechanism of photooxidation upon visible light exposure is still unclear for therapeutic proteins, since proteinogenic structures do not absorb light in the visible range. Here, we show that exposure of monoclonal antibody formulations to visible light, lead to the formation of reactive oxygen species (ROS), which subsequently induce specific protein degradations. The formation of ROS and singlet oxygen upon visible light exposure is investigated using electron paramagnetic resonance (EPR) spectroscopy. We describe the initial formation of ROS, most likely after direct reaction of molecular oxygen with a triplet state photosensitizer, generated from intersystem crossing of the excited singlet state. Since these radicals affect the oxygen content in the headspace of the vial, we monitored photooxidation of these mAb formulations. With increasing protein concentrations, we found (i) a decreasing headspace oxygen content in the sample, (ii) a higher relative number of radicals in solution and (iii) a higher protein degradation. Thus, the protein concentration dependence indicates the presence of higher concentration of a currently unknown photosensitizer.

The presence of subvisible or visible particles in mAb formulations can pose significant challenges to pharmaceutical development as it can lead to reduced shelf life, batch rejection, and recalls. Among all type of particles, proteinaceous particles are the most concerning due to their potential role in immunogenicity. Nevertheless, the underlying mechanism for protein particle formation remains poorly understood. Past research highlighted the importance of interfaces and mechanical agitation in causing protein particle formation. Current research suggests that fatty acids, as impurities present in excipients or as a result of polysorbate degradation, can also induce protein assembly and promote particle formation. In this work, we assessed oleic and lauric acid for their impact on particle formation as each represents the main hydrolysis product of PS80 or PS20, respectively. It was found that co-existence of either fatty acids with 10 mg/mL mAb A can cause protein particles, with a similar morphology to those observed previously in mAb formulations. FTIR spectra showed that the particles are proteinaceous, heterogeneous in its composition, but contain corresponding fatty acids. Interestingly, it was found that oleic acid is significantly more effective in causing protein particles than lauric acid in these experiments. This suggests that PS20 containing formulations might have a lower likelihood to have protein particles compared to PS80 containing mAb formulations if hydrolysis of polysorbate were to occur. Lastly, the presence of 0.01% polysorbate in the mAb A formulation was able to fully mitigate the effect of fatty acids and reduce the protein particles significantly, suggesting a potential mechanism where interfacial action is involved. The present study can help to understand the root cause for protein particles in a mAb formulation where fatty acids are introduced because of polysorbate hydrolysis. With further work, it will help to shed light into product control strategy as well as design approaches for robust mAb products.

The WFDC1 gene is frequently down-regulated or lost in prostate cancer, and the encoded protein, ps20, has been implicated in epithelial cell behaviour and angiogenesis. However, ps20 remains largely uncharacterised with respect to its structure and interacting partners. This study characterised the evolution, functionality and structural characteristics of WFDC1/ps20 using phylogenetic reconstruction and other computational approaches. Bayesian phylogenetic analyses suggested that ps20 appeared in a common ancestor of deuterostomes-protostomes. The rate of evolutionary change within the coding regions of vertebrate WFDC1 genes and the synteny conservation in mammals differed from that of other vertebrate clades, indicating a possible functional diversity of ps20 homologues. A gene set enrichment analysis of the genes around WFDC1 (conserved synteny) showed functional relationships between the WFDC1, CDH13, CRISPLD2, IRF8 and TFPI2 genes. The molecular evolution of ps20 has been driven by purifying selection, particularly in the segments corresponding to exons 3 and 4, which encode the most conserved regions of the protein. A co-evolution analysis showed that residues within these regions co-vary with each other during the evolution of ps20. These results show that the regions corresponding to exons 3 and 4 are ps20-specific structure-function modules. Homology modelling of the exon 2-encoded polypeptide and subsequent dynamics calculus using a Gaussian network model showed that residues with high conformational flexibility are part of a loop region involved in protein-protein recognition, given the similarity with other serine protease inhibitors. Residues C96, R94, L105, and C66 are critical for the integrity and functionality of this ps20 region.

OBJECTIVE: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations.
METHODS: A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations.
RESULTS: Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients.
CONCLUSIONS: An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

Whey-acidic-protein (WAP) four-disulphide core (WFDC) proteins have important roles in the regulation of innate immunity, anti-microbial function, and the inhibition of inflammatory proteases at mucosal surfaces. It was recently demonstrated that the WFDC protein, prostate stromal 20 (ps20), encoded by the WFDC1 gene, is a potent growth inhibitory factor, and shares with other WFDC proteins the ability to modulate wound healing processes and immune responses to viral infections. However, ps20 remains relatively uncharacterised at the protein level. Using a panel of ps20 antibodies for western-blotting (WB), ELISA and immunoaffinity purification, we isolated, biochemically characterised and tested ps20 preparations for three biological properties: (i) interactions with glycosaminoglycans (GAG) (ii) inhibition of cell proliferation, and (iii) transglutaminase2 (TG2) mediated crosslinking of ps20 to fibronectin, a process implicated in wound healing. We show herein that ps20 preparations contain multiple molecular forms including full-length ps20 (resolving at ≈27 kDa), an exon 3 truncated form (≈22 kDa) that lacks aa113-140, and variable amounts of a putatively cleaved lower MW (≈15-17 kDa) species. Untagged purified ps20 preparations containing a mixture of these forms are biologically active in significantly suppressing prostate cell proliferation. We show that one mechanism by which lower LMW forms of ps20 arise is through cathepsin L (CL) cleavage, and confirm that CL cleaves ps20 at the C-terminus, but this does not inhibit its growth inhibitory function. However, CL cleavage abrogated the interaction between ps20 and solid-phase fibronectin. Therefore, we demonstrate for the first time that LMW forms of ps20 that lack a C-terminal immunogenic epitope can arise through CL cleavage and this cleavage impairs multimerisation and potential capacity to cross-link to ECM, but not the capacity of ps20 to inhibit cell proliferation. We propose that ps20 like other WFDC proteins can become associated with GAGs and the ECM. Furthermore, we suggest post-translational processing and cleavage of ps20 is required to generate functional protein species, and TG2 mediated crosslinking and CL cleavage form components of a ps20 regulatory apparatus.