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.

Alteration of proteins and pigments influence the function of photosystem I under iron deficiency from Chlamydomonas reinhardtii.

BACKGROUND: Iron is an essential micronutrient for all organisms because it is a component of enzyme cofactors that catalyze redox reactions in fundamental metabolic processes. Even though iron is abundant on earth, it is often present in the insoluble ferric [Fe (III)] state, leaving many surface environments Fe-limited. The haploid green alga Chlamydomonas reinhardtii is used as a model organism for studying eukaryotic photosynthesis. This study explores structural and functional changes in PSI-LHCI supercomplexes under Fe deficiency as the eukaryotic photosynthetic apparatus adapts to Fe deficiency. RESULTS: 77K emission spectra and sucrose density gradient data show that PSI and LHCI subunits are affected under iron deficiency conditions. The visible circular dichroism (CD) spectra associated with strongly-coupled chlorophyll dimers increases in intensity. The change in CD signals of pigments originates from the modification of interactions between pigment molecules. Evidence from sucrose gradients and non-denaturing (green) gels indicates that PSI-LHCI levels were reduced after cells were grown for 72 h in Fe-deficient medium. Ultrafast fluorescence spectroscopy suggests that red-shifted pigments in the PSI-LHCI antenna were lost during Fe stress. Further, denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI subunits PsaC and PsaD decreased, while PsaE was completely absent after Fe stress. The light harvesting complexes were also susceptible to iron deficiency, with Lhca1 and Lhca9 showing the most dramatic decreases. These changes in the number and composition of PSI-LHCI supercomplexes may be caused by reactive oxygen species, which increase under Fe deficiency conditions. CONCLUSIONS: Fe deficiency induces rapid reduction of the levels of photosynthetic pigments due to a decrease in chlorophyll synthesis. Chlorophyll is important not only as a light-harvesting pigment, but also has a structural role, particularly in the pigment-rich LHCI subunits. The reduced level of chlorophyll molecules inhibits the formation of large PSI-LHCI supercomplexes, further decreasing the photosynthetic efficiency.

Efficient light harvesting in a dark, hot, acidic environment: the structure and function of PSI-LHCI from Galdieria sulphuraria.

Photosystem I-light harvesting complex I (PSI-LHCI) was isolated from the thermoacidophilic red alga Galdieria sulphuraria, and its structure, composition, and light-harvesting function were characterized by electron microscopy, mass spectrometry, and ultrafast optical spectroscopy. The results show that Galdieria PSI is a monomer with core features similar to those of PSI from green algae, but with significant differences in shape and size. A comparison with the crystal structure of higher plant (pea) PSI-LHCI indicates that Galdieria PSI binds seven to nine light-harvesting proteins. Results from ultrafast optical spectroscopy show that the functional coupling of the LHCI proteins to the PSI core is tighter than in other eukaryotic PSI-LHCI systems reported thus far. This tight coupling helps Galdieria perform efficient light harvesting under the low-light conditions present in its natural endolithic habitat.

Data-directed top-down Fourier-transform mass spectrometry of a large integral membrane protein complex: photosystem II from Galdieria sulphuraria.

High-resolution top-down MS was used to characterize eleven integral and five peripheral subunits of the 750 kDa photosystem II complex from the eukaryotic red alga, Galdieria sulphuraria. The primary separation used LC MS with concomitant fraction collection (LC-MS+), yielding around 40 intact mass tags at 100 ppm mass accuracy on a low-resolution ESI mass spectrometer, whose retention and mass were used to guide subsequent high-resolution top-down nano-electrospray FT ion-cyclotron resonance MS experiments (FT-MS). Both collisionally activated and electron capture dissociation were used to confirm the presence of eleven small subunits to mass accuracy within 5 ppm; PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbT, PsbX and PsbZ. All subunits showed covalent modifications that fall into three classes including retention of initiating formyl-methionine, removal of methionine at the N-terminus with or without acetylation, and removal of a longer N-terminal peptide. Peripheral subunits identified by top-down analysis included oxygen-evolving complex subunits PsbO, PsbU, PsbV, as well as Psb28 (PsbW) and Psb27 ("PsbZ-like"). Top-down high-resolution MS provides the necessary precision, typically less than 5 ppm, for identification and characterization of polypeptide composition of these important membrane protein complexes.

Structural and functional changes of PSI-LHCI supercomplexes of Chlamydomonas reinhardtii cells grown under high salt conditions.

The eVect of high salt concentration (100 mM NaCl) on the organization of photosystem I-light harvesting complex I supercomplexes (PSI-LHCI) of Chlamydomonas reinhardtii was studied. The electron transfer activity was reduced by 39% in isolated PSI-LHCI supercomplexes. The visible circular dichroism (CD) spectra associated with strongly coupled chlorophyll (Chl) dimers were reduced in intensity, indicating that pigment-pigment interactions were disrupted. This data is consistent with results from Xuorescence streak camera spectroscopy, which suggest that red-shifted pigments in the PSI-LHCI antenna had been lost. Denaturing gel electrophoresis and immunoblot analysis reveals that levels of the PSI reaction center proteins PsaD, PsaE and PsaF were reduced due to salt stress. PsaE is almost completely absent under high salt conditions. It is known that the membrane-extrinsic subunits PsaD and E form the ferredoxin-docking site. Our results indicate that the PSI-LHCI supercomplex is damaged by reactive oxygen species at high salt concentration, with particular impact on the ferredoxin-docking site and the PSILHCI interface.