Temperature dependent viral tropism: understanding viral seasonality and pathogenicity as applied to the avoidance and treatment of endemic viral respiratory illnesses.

This review seeks to explain three features of viral respiratory illnesses that have perplexed generations of virologists: (1) the seasonal timing of respiratory illness and the rapid response of outbreaks to weather, specifically temperature; (2) the common viruses causing respiratory illness worldwide, including year-round disease in the Tropics; (3) the rapid arrival and termination of epidemics caused by influenza and other viruses. The inadequacy of the popular explanations of seasonality is discussed, and a simple hypothesis is proposed, called temperature dependent viral tropism (TDVT), that is compatible with the above features of respiratory illness. TDVT notes that viruses can spread more effectively if they moderate their pathogenicity (thereby maintaining host mobility) and suggests that endemic respiratory viruses accomplish this by developing thermal sensitivity within a range that supports organ-specific viral tropism within the human body, whereby they replicate most rapidly at temperatures below body temperature. This can confine them to the upper respiratory tract and allow them to avoid infecting the lungs, heart, gut etc. Biochemical and tissue-culture studies show that 'wild' respiratory viruses show such natural thermal sensitivity. The typical early autumn surge of colds and the occurrence of respiratory illness in the Tropics year-round at intermediate levels are explained by the tendency for strains to adapt their thermal sensitivity to their local climate and season. TDVT has important practical implications for preventing and treating respiratory illness including Covid-19. It is testable with many options for experiments to increase our understanding of viral seasonality and pathogenicity.

PrecISE: Precision Medicine in Severe Asthma: An adaptive platform trial with biomarker ascertainment.

Severe asthma accounts for almost half the cost associated with asthma. Severe asthma is driven by heterogeneous molecular mechanisms. Conventional clinical trial design often lacks the power and efficiency to target subgroups with specific pathobiological mechanisms. Furthermore, the validation and approval of new asthma therapies is a lengthy process. A large proportion of that time is taken by clinical trials to validate asthma interventions. The National Institutes of Health Precision Medicine in Severe and/or Exacerbation Prone Asthma (PrecISE) program was established with the goal of designing and executing a trial that uses adaptive design techniques to rapidly evaluate novel interventions in biomarker-defined subgroups of severe asthma, while seeking to refine these biomarker subgroups, and to identify early markers of response to therapy. The novel trial design is an adaptive platform trial conducted under a single master protocol that incorporates precision medicine components. Furthermore, it includes innovative applications of futility analysis, cross-over design with use of shared placebo groups, and early futility analysis to permit more rapid identification of effective interventions. The development and rationale behind the study design are described. The interventions chosen for the initial investigation and the criteria used to identify these interventions are enumerated. The biomarker-based adaptive design and analytic scheme are detailed as well as special considerations involved in the final trial design.

Clearance of the rodent retrovirus, XMuLV, by protein A chromatography.

Protein A chromatography is the most common unit operation used in the manufacture of therapeutic monoclonal antibodies (mAbs) due to its high affinity and specificity for the IgG Fc domain. However, protein A chromatography is often not effective for viral clearance. Typical log reduction values (LRV) for the model retrovirus XMuLV range between 1 and 4 logs, while effective steps such as viral filtration can achieve 5-7 logs of clearance. XMuLV LRVs obtained on protein A are reproducible for a given mAb, but can vary widely for different mAbs, even with the same operating conditions. In order to understand the mechanism of XMuLV clearance on protein A, we have investigated its partitioning on Mabselect SuRe protein A resin and explored how the virus interacts with resin, product, and impurities. The results show that XMuLV has some interaction with the resin backbone and ligand, but also appears to bind to and coelute with the mAb. The interaction with product was further examined by evaluating the effect of feed conditions, loading, and different washes on XMuLV partitioning on the column. Understanding the mechanism of XMuLV removal on a protein A, resin provides insight into the variability and low viral clearance of this step and suggests ways in which the removal of virus by this step can be improved.

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes.

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO 3⁻, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO 3⁻ with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO⁻ and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV.

Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding.

Hydroxocobalamin association during cell culture results in pink therapeutic proteins.

Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing.