QED radiative corrections for accelerator neutrinos.

Neutrino oscillation experiments at accelerator energies aim to establish charge-parity violation in the neutrino sector by measuring the energy-dependent rate of νe appearance and νµ disappearance in a νµ beam. These experiments can precisely measure νµ cross sections at near detectors, but νe cross sections are poorly constrained and require theoretical inputs. In particular, quantum electrodynamics radiative corrections are different for electrons and muons. These corrections are proportional to the small quantum electrodynamics coupling α ≈ 1/137; however, the large separation of scales between the neutrino energy and the proton mass (~GeV), and the electron mass and soft-photon detection thresholds (~MeV) introduces large logarithms in the perturbative expansion. The resulting flavor differences exceed the percent-level experimental precision and depend on nonperturbative hadronic structure. We establish a factorization theorem for exclusive charged-current (anti)neutrino scattering cross sections representing them as a product of two factors. The first factor is flavor universal; it depends on hadronic and nuclear structure and can be constrained by high-statistics νµ data. The second factor is non-universal and contains logarithmic enhancements, but can be calculated exactly in perturbation theory. For charged-current elastic scattering, we demonstrate the cancellation of uncertainties in the predicted ratio of νe and νµ cross sections. We point out the potential impact of non-collinear energetic photons and the distortion of the visible lepton spectra, and provide precise predictions for inclusive observables.

Engineering redox sensors into CHO cells enables near-real-time quantification of intracellular redox in bioprocesses.

The production of biologics that treat complex diseases, such as cancer, autoimmune, and infectious disease, requires careful monitoring and control of cell cultures. While bioprocess optimizations have dramatically improved production yields, a lack of analytical tools has made it challenging to identify accompanying intracellular improvements. Intracellular redox can diminish the growth and productivity of biologics-producing cells and adversely impact product quality profiles yet characterizing redox is challenging due to its complex and highly transient nature. In this study, we integrated a fluorescent thiol-based redox biosensor to monitor intracellular redox in one bisAb- and two monoclonal antibody-producing clonal cell lines in a 14-day fed-batch bioreactor. We characterized biosensor functionality using three fluorescence measurement techniques and determined sensor oxidation correlates with the intracellular ratio of reduced (GSH) and oxidized glutathione (GSSG), an important cellular antioxidant. Our fed-batch bioreactor studies showed that sensor expression minimally affected bioprocess outcomes, including growth, productivity, product quality attributes, or intracellular redox attributes, including mitochondrial reactive oxygen species and total cellular GSH levels in all cell lines tested. Biosensor measurements taken throughout the culture revealed that the intracellular environment in these cell lines became more reduced throughout the culture, with the exception of a high pH condition which became more oxidized. Our results demonstrate the potential of using biosensors to monitor intracellular changes in near-real-time with minimal process effects, thus potentially improving future bioprocess optimizations.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.

Redox as a bioprocess parameter: analytical redox quantification in biological therapeutic production.

Engineered Chinese hamster ovary (CHO) cells are the most widely utilized cell line for protein-based therapeutics production at industrial scales. Process development strategies which improve production capacity and quality are often implemented without an understanding of underlying intracellular changes. Intracellular redox conditions drive reactions in pathways critical to biologics production, including bioenergetic and biosynthetic pathways, necessitating methods to quantify redox-related changes. Advances in methods for analytical redox quantification presented here, including bioreactor probes, redox-targeted proteomics, genetically encoded redox-sensitive fluorescent proteins, and biochemical assays, are creating new opportunities to characterize the effects of redox in biologics production. Implementing these methods will lead to enhanced media formulations, improved bioprocess strategies, and new cell line engineering targets and ultimately develop redox into an optimizable bioprocess parameter to improve the yield and quality of these lifesaving medicines.