A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification.

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.

Escherichia coli LysU is a potential surrogate for human lysyl tRNA synthetase in interactions with the C-terminal domain of HIV-1 capsid protein.

Human lysyl-tRNA synthetase (hLysRS) is known to interact directly with human immunodeficiency virus type-1 (HIV-1) GagPol polyproteins, and both hLysRS with tRNA(Lys3) are selectively packaged into emerging HIV-1 viral particles. This packaging process appears to be mediated by contact between the motif 1 helix h7 of hLysRS and the C-terminal dimerization domain of the HIV-1 capsid protein (CA) segment of Gag or GagPol. Given similarities between hLysRS and Escherichia coli (E. coli) heat shock protein LysU, we investigate if LysU might be an hLysRS surrogate for interactions with Gag or GagPol proteins. We report on a series of studies involving three CA C-domains: CA(146) (intact domain), CA(151) (truncated domain), and CA(146)-M185A (M185A, CA dimer interface mutant). After confirming that LysU and CA(146) are dimeric whilst CA(151) and M185A remain monomeric, we use glutathione S-transferase (GST) pull-down assays to demonstrate the existence of specific interactions between LysU and all three CA-C domains. By means of (1)H-NMR titration experiments, we estimate K(d) values of 50 µM for the interaction between LysU and CA(146) or >500 µM for interactions between LysU and CA(151) or LysU and M185A. The reason for these binding affinity differences may be that interactions between LysU and CA(146) take place through dimer-dimer interactions resulting in a α(2)ß(2) heterotetramer. LysU/CA-C protein interactions are weaker than those reported between hLysRS and the Gag, CA or CA(146) proteins, and hLysRS/Gag binding interactions have also been suggested to involve only αß heterodimer formation. Nevertheless, we propose that LysU could act as a surrogate for hLysRS with respect to Gag and GagPol polyprotein interactions although arguably not sufficiently for LysU to act as an inhibitor of the HIV-1 life cycle without further adaptation or mutation. Potentially, LysU and/or LysU mutants could represent a new class of anti-HIV-1 therapeutic agent.

Assessing the preferred solution conformation of an interacting sense-antisense (complementary) peptide pair.

Sense peptides and corresponding antisense peptides, are capable of making specific interactions. Such interactions may result from inter-peptide side-chain/side-chain contacts or because peptides adopt mutually complementary three-dimensional shapes. Using a combined (1)H NMR spectroscopy/molecular modeling approach to study the interactions between one sense peptide and its corresponding antisense peptide, data are produced that provide clear support for the former hypothesis.

The discovery and optimisation of pyrido[2,3-d]pyrimidine-2,4-diamines as potent and selective inhibitors of mTOR kinase.

We describe a novel series of potent inhibitors of the kinase activity of mTOR. The compounds display good selectivity relative to other PI3K-related kinase family members and, in cellular assays, inhibit both mTORC1 and mTORC2 complexes and exhibit good antiproliferative activity.

Occupational arsine gas exposure.

Arsine gas exposure is a rare occupational event and can be completely prevented with the use of appropriate protective gear. Exposure often occurs when arsine gas is generated while arsenic-containing crude ores or metals are treated with acid. Cases of toxicity require an index of suspicion and a good history. In particular, it should be in the differential diagnosis in patients who present acutely with red/bronze skin and hemoglobinuria. Treatment is supportive and may include transfusions and dialysis in severe cases. Clinical severity is proportionate to the level of exposure, and severity is directly related to the onset of symptoms.