Accelerated CMC workflows to enable speed to clinic in the COVID-19 era: A multi-company view from the biopharmaceutical industry.

The COVID-19 pandemic has placed unprecedented pressure on biopharmaceutical companies to develop efficacious preventative and therapeutic treatments, which is unlikely to abate in the coming years. The importance of fast progress to clinical evaluation for treatments, which tackle unmet medical needs puts strain on traditional product development timelines, which can take years from start to finish. Although previous work has been successful in reducing phase 1 timelines for recombinant antibodies, through utilization of the latest technological advances and acceptance of greater business risk or costs, substantially faster development is likely achievable without increased risk to patients during initial clinical evaluation. To optimize lessons learned from the pandemic and maximize multi-stakeholder (i.e., patients, clinicians, companies, regulatory agencies) benefit, we conducted an industry wide benchmarking survey in September/October 2021. The aims of this survey were to: (i) benchmark current technical practices of key process and product development activities related to manufacturing of therapeutic proteins, (ii) understand the impact of changes implemented in COVID-19 accelerated Ab programs, and whether any such changes can be retained as part of sustainable long-term business practices and (iii) understand whether any accelerative action(s) taken have (negatively) impacted the wider development process. This article provides an in-depth analysis of this data, ultimately highlighting an industry perspective of how biopharmaceutical companies can sustainably adopt new approaches to therapeutic protein development and production.

Spheciosterol sulfates, PKCzeta inhibitors from a philippine sponge Spheciospongia sp.

Three new sterol sulfates, spheciosterol sulfates A-C (1-3), and the known sterol sulfate topsentiasterol sulfate E (4) have been isolated from the sponge Spheciospongia sp., collected in the Philippines. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1-4 inhibited PKCzeta with IC50 values of 1.59, 0.53, 0.11, and 1.21 microM, respectively. In a cell-based assay, 1-4 also inhibited NF-kappaB activation with EC50 values of 12-64 microM.

Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies.

The culture of Chinese Hamster Ovary (CHO) cells for modern industrial applications, such as expression of recombinant proteins, requires media that support growth and production. Such media must support high viable cell densities while also stimulating the synthesis and extracellular transport of biologic products. Early media development efforts in this area yielded basic formulations to sustain growth, viability, and cellular function, albeit comprising animal sourced components, and complex constituents used in batch culture mode. Subsequent improvements included the development of serum-free and chemically defined (CD) media, the identification of critical nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the use of fed-batch and perfusion culture techniques to optimize nutrient delivery while minimizing accumulation of unwanted waste products. This review is comprised of sections covering milestones in the evolution of mammalian cell culture media, nutrient composition and formulation requirements, optimization strategies, consistency and scalability of powder and liquid media preparation for industrial applications, and key recent advances driving progress in CHO cell culture medium design and development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1407-1426, 2018.

Novel sulfur-containing rapamycin analogs prepared by precursor-directed biosynthesis.

[reaction: see text] Two novel sulfur-containing analogs of the immunosuppressive natural product rapamycin (1) were obtained by feeding cultures of Streptomyces hygroscopicus with l-nipecotic acid (4) and either (S)-1,3-thiazane-4-carboxylic acid (5) or (S)-1,4-thiazane-3-carboxylic acid (6). The structures of the two new compounds, 20-thiarapamycin (2) and 15-deoxo-19-sulfoxylrapamycin (3), were determined by spectroscopic methods.

Molecular and phenotypic comparison of phaeochromycin-producing strains of Streptomyces phaeochromogenes and Streptomyces ederensis.

Streptomyces strain LL-P018 produces the phaeochromycins, novel anti-inflammatory polyketides. This organism was identified as a strain of Streptomyces phaeochromogenes by physiological and genetic taxonomic analysis. In order to gain greater taxonomic perspective, LL-P018 was compared to related strains from major culture collections by 16S rRNA gene sequence, ribotype, HPLC-MS metabolite profile, and rpoB sequence. Using BioNumerics software, genetic and chemical fingerprint data were integrated via multivariate cluster analysis into a single, robust comparison. Based upon this analysis, strain LL-P018 is very closely related to the type strains of both S. phaeochromogenes and Streptomyces ederensis, indicating that these two types may in fact represent a single species. This novel comparative multi-cluster analysis is most useful for clarifying relationships between closely related species.

Phaeochromycins A-E, anti-inflammatory polyketides isolated from the soil actinomycete Streptomyces phaeochromogenes LL-P018.

Five new polyketide metabolites, phaeochromycins A-E (1-5), were isolated from an actinomycete designated Streptomyces phaeochromogenes LL-P018, cultured from a soil sample collected from a riverbank in Westevenger, Germany. Phaeochromycins A and C were found to be weak inhibitors of MAPKAP kinase-2 (IC50 = 39 and 130 microM, respectively). The structures of the compounds were determined by spectroscopic analysis, primarily two-dimensional NMR, and revealed that phaeochromycins A, B, C, and E were octaketides, elaborated from a C4 starter unit, related to shunt products of the actinorhodin pathway, namely, mutactin, dehydromutactin, SEK34b, and BSM1. Phaeochromycin D (4) is an unusual partially cyclized degraded octaketide intermediate.

Production of novel rapamycin analogs by precursor-directed biosynthesis.

The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.

Further evidence that a cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc] serves as an acceptor in a sorting reaction.

Previous studies suggested that a Gly-containing branch of cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.