Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme‐induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single‐chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv‐fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody‐like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

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A flexible, rigorous laboratory experiment for upper-level biochemistry undergraduates is described that focuses on the Roundup Ready maize line. The work is appropriate for undergraduate laboratory courses that integrate biochemistry, molecular biology, or bioinformatics. In this experiment, DNA is extracted and purified from maize kernel and leaf samples collected from a Roundup Ready maize grower's field. A small segment of DNA (108 base pairs) specific to the Roundup Ready transgene that codes for CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) is amplified with polymerase chain reaction (PCR) to detect the presence of the gene in the maize samples. Students additionally choose a protein closely related to CP4 EPSPS as determined by amino acid sequence alignments. The selected amino acid sequences are submitted to an online protein modeling program where students compare their protein with the herbicide-resistant enzyme found in Roundup Ready crops. This experimental paradigm gives students a physical appreciation for the central dogma of biology, as they are exposed to products derived from the replication, transcription, and translation events belonging to a genetically modified crop. The PCR portion of the laboratory allows students to perform an in vitro replication of a portion of the NK603 transgene and identify the segment via ultraviolet radiation. In direct connection to the PCR portion, the protein structure elucidation gives rise to the central ideas of evolutionthat slight changes in the genetic code of DNA, translated into proteins, produce novel protein structures with significantly different function.

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Nitric oxide signaling is a powerful mechanism for the regulation of blood pressure homeostasis, inducing vasodilation and reducing peripheral resistance. As such, disruption of NO‐inhibitory protein interactions may prove to be a novel therapeutic for the treatment of hypertensive disease states. Focusing on the recent discovery of the alpha globin/eNOS complex in vascular endothelium, we have designed an alpha globin mimetic peptide that binds to eNOS and increases NO signaling and dilation. A key step in the development of therapeutic agents targeting this complex is a high‐resolution structural model of the binding interface of alpha globin and eNOS. Thus, we have used solution NMR to determine the relevant structural details of the alpha globin mimetic peptide known to bind to eNOS. Interestingly, this twenty residue peptide has a stable population of both proline cis and trans isomers, potentially influencing the affinity of the peptide/eNOS interaction. Crosslinking mass spectrometry allows for the mapping of interacting residues between the peptide and eNOS after enzymatic digestion. Using cis and trans proline peptide ensembles, we have generated models of the peptide/eNOS complex through docking simulations which can be verified with full‐length alpha globin. The different conformations adopted by the peptide allows for potential new interaction modes that may not be possible with full alpha globin protein, thereby possibly increasing peptide/protein binding affinity. Determining the binding interface of the alpha globin/eNOS complex allows for targeted searches for molecular inhibitors of this alpha globin/eNOS interaction, in efforts for novel therapeutics to combat vasoconstrictive disease.Support or Funding InformationAmerican Heart Association Grant 16PRE31180040 (TCSK)NIH 088554 (BEI)NIH 087828 (LC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.