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Isomerization of aspartic acid (Asp) in therapeutic proteins could lead to safety and efficacy concerns. Thus, accurate quantitation of various Asp isomerization along with kinetic understanding of the variant formations is needed to ensure optimal process development and sufficient product quality control. In this study, we first observed Asp-succinimide conversion in complementarity-determining regions (CDRs) Asp-Gly motif of a recombinant mAb through ion exchange chromatography, intact protein analysis by mass spectrometry, and LC-MS/MS. Then, we developed a specific peptide mapping method, with optimized sample digestion conditions, to accurately quantitate Asp-succinimide-isoAsp variants at peptide level without method-induced isomerization. Various kinetics of Asp-succinimide-isoAsp isomerization pathways were elucidated using 18O labeling followed by LC-MS analysis. Molecular modeling and molecular dynamic simulation provide additional insight on the kinetics of Asp-succinimide formation and stability of succinimide intermediate. Findings of this work shed light on the molecular construct and the kinetics of the formation of isoAsp and succinimide in peptides and proteins, which facilitates analytical method development, protein engineering, and late phase development for commercialization of therapeutic proteins.

Fed-batch process intensification with a significantly shorter culture duration or higher titer for monoclonal antibody (mAb) production by Chinese hamster ovary (CHO) cells can be achieved by implementing perfusion operation at the N-1 stage for biomanufacturing. N-1 perfusion seed with much higher final viable cell density (VCD) than a conventional N-1 batch seed can be used to significantly increase the inoculation VCD for the subsequent fed-batch production (referred as N stage), which results in a shorter cell growth phase, higher peak VCD, or higher titer. In this report, we incorporated a process analytical technology (PAT) tool into our N-1 perfusion platform, using an in-line capacitance probe to automatically adjust the perfusion rate based on real-time VCD measurements. The capacitance measurements correlated linearly with the offline VCD at all cell densities tested (i.e., up to 130 × 106 cells/mL). Online control of the perfusion rate via the cell-specific perfusion rate (CSPR) decreased media usage by approximately 25% when compared with a platform volume-specific perfusion rate approach and did not lead to any detrimental effects on cell growth. This PAT tool was applied to six mAbs, and a platform CSPR of 0.04 nL/cell/day was selected, which enabled rapid growth and maintenance of high viabilities for four of six cell lines. In addition, small-scale capacitance data were used in the scaling-up of N-1 perfusion processes in the pilot plant and in the GMP manufacturing suite. Implementing a platform approach based on capacitance measurements to control perfusion rates led to efficient process development of perfusion N-1 for supporting high-density CHO cell cultures for the fed-batch process intensification.

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

The R2R3-Myb proteins comprise a large family of plant transcription factors that regulate the expression of multiple drought-responsive and cold-responsive genes by binding special cis-elements at the promoter. There have been previous studies in transgenic plant over-expression of the myb protein. In this article, we analyzed the function of MdMYB6 , an R2R3-type Myb transcription factor from apples (Malus × domestica), in transgenic Arabidopsis plants. In contrast to wild type plants, these transgenic lines accumulated less anthocyanin. Equimolar concentrations of sorbitol did not alter the anthocyanin accumulation. Real-time polymerase chain reaction (PCR) indicated that MdMYB6 over-expression suppressed enzymes involved in anthocyanin biosynthesis and some Arabidopsis basic helix-loop-helix (bHLH) genes. The MdMYB6 transcription faction may be an important repressor of anthocyanin biosynthesis in plants.

Therapeutic monoclonal antibodies (mAbs), primarily immunoglobin G1 (IgG1) and IgG4 with an engineered CPPC motif in its hinge region, are predominant biologics. Inter-chain disulfide bonds of IgG mAbs are crucial to maintaining IgG integrity. Inter-chain disulfide bond–reduced low molecular weight (LMW) is considered as one of quality attributes of IgG drug substance and is observed in drug substance manufacturing. In this study, we demonstrate that IgG1 and IgG4 are susceptible to the reducing agent TCEP differently and they follow different pathways to form LMWs. Our study shows that IgG1 is more sensitive to TCEP than IgG4. Both therapeutic IgG1 and human blood plasma IgG1 follow a heavy-heavy-light chain (HHL) pathway, featured with HHL and HH as intermediate species. Human blood plasma IgG4 with a CPSC motif in its hinge region follows heavy-light chain (HL) pathway, featured with HL as the intermediate species. However, therapeutic IgG4 follows a hybrid pathway with the HL pathway as the primary and the HHL pathway as the secondary. These experimental observations are further explained using solvent accessibility of inter-chain disulfide bonds obtained from computational modeling and molecular dynamics simulations. Findings from this study provide mechanistic insights of LMW formation of IgG1 and IgG4, which suggest selection of IgG1 or IgG4 for bispecific antibodies and cysteine-based antibody–drug conjugates. Key points • Experimentally discovered preferable disulfide bond reduction pathways between IgG1 and IgG4 antibodies, driven by the different solvent accessibilities of these disulfide bonds. • Computationally explained the solvent accessibility aided by molecular dynamics simulations. • Provided insights in developing robust biologics process and designing bispecific antibodies and cysteine-based antibody–drug conjugates.

Rhodospirillum rubrum has the potential for biomass resource recycling combined with sewage purification. However, low biomass production and yield restricts the potential for sewage purification. This research investigated the improvement of biomass production, yield and organics reduction by Mg2+ in R. rubrum wastewater treatment. Results showed that with optimal dosage (120 mg/L), biomass production reached 4,000 mg/L, which was 1.5 times of that of the control group. Biomass yield was improved by 43.3%. Chemical oxygen demand (COD) removal reached over 90%. Hydraulic retention time was shortened by 25%. Mechanism analysis indicated that Mg2+ enhanced the isocitrate dehydrogenase and Ca2+/Mg2+-ATPase activities, bacteriochlorophyll content on respiration and photophosphorylation. These effects then enhanced ATP production, which led to more biomass accumulation and COD removal. With 120 mg/L Mg2+ dosage, the isocitrate dehydrogenase and Ca2+/Mg2+-ATPase activities, bacteriochlorophyll content, ATP production were improved, respectively, by 33.3%, 50%, 67%, 41.3% compared to those of the control group.

The baseline instability for capillary electrophoretic analysis is an intrinsic feature of the technique, which has not been thoroughly examined for its impact on therapeutic protein purity analysis with the capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) applications. For the particular CE-SDS application, this phenomenon was manifested through peak migration time shifts and sliding of the superimposed baseline profile. These dual phenomena are closely associated so that experimental assessment alone may not shed enough light to the underlying drivers. In the current study, both experimental and simulation approaches were employed to assess the systematic drifts. Computer simulation was used to decipher the two underlying factors and test their contributions toward purity and impurity peak determination inaccuracies. The data generated in this study demonstrated that the electrophoretic baseline disturbance had more pronounced impact on the purity data than the migration time shift. In addition, the potential contributing factors to the baseline disturbances were assessed experimentally which indicated that the source is related to thermal disruption during a sample run and the unique baseline patterns came from the background electrolytes. To improve data reproducibility for drug purity testing in the industrial setting and quality control (QC) environment, it is recommended to run shorter injection sequences including fewer samples and closely monitor the baseline drift for accurate integration. Those methods would help reduce the impact of systematic drift and disturbances.

Rubrivivax gelatinosus has the potential of biomass resource recycling combined with sewage purification. However, low biomass production and yield restricts the potential for sewage purification. Thus, this research investigated the improvement of biomass production and yield and organics reduction by Fe3+ in R. gelatinosus wastewater treatment. Results showed that 10–30 mg/L Fe3+ improved biomass yield in wastewater to a level found in culture medium. With optimal dosage (20 mg/L), biomass production reached 4,300 mg/L, which was 1.67 times that of the control group. Biomass yield was improved by 43.3%. Chemical oxygen demand (COD) removal reached above 91%. Hydraulic retention time was shortened by 25%. Mechanism analysis indicated that Fe3+ enhanced the succinate and NADH dehydrogenase activities and, bacteriochlorophyll content in three energy metabolism pathways. These effects then enhanced adenosine triphosphate (ATP) production, which led to more biomass accumulation and COD removal. With 20 mg/L Fe2+ dosage, succinate and NADH dehydrogenase, coproporphyrinogen III oxidase activities, bacteriochlorophyll content and ATP production were improved, respectively, by 48.4, 50.8, 50, 67 and 56% compared to those of the control group.

The goal of cell culture process intensification is to increase volumetric productivity, generally by increasing viable cell density (VCD), cell specific productivity or production bioreactor utilization in manufacturing. In our previous study, process intensification in fed-batch production with higher titer or shorter duration was demonstrated by increasing the inoculation seeding density (SD) from ~ 0.6 (Process A) to 3–6 × 106 cells/mL (Process B) in combination with media enrichment. In this study, we further increased SD to 10–20 × 106 cells/mL (Process C) using perfusion N-1 seed cultures, which increased titers already at industrially relevant levels by 100% in 10–14 day bioreactor durations for four different mAb-expressing CHO cell lines. Redesigned basal and feed media were critical for maintaining higher VCD and cell specific productivity during the entire production duration, while medium enrichment, feeding strategies and temperature shift optimization to accommodate high VCDs were also important. The intensified Process C was successfully scaled up in 500-L bioreactors for 3 of the 4 mAbs, and quality attributes were similar to the corresponding Process A or Process B at 1000-L scale. The fed-batch process intensification strategies developed in this study could be applied for manufacturing of other mAbs using CHO and other host cells.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF.Key points• Bioreactor conditions resulted in reduction susceptible harvest material.• GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction.• Demonstrated role for cell metabolic changes in post-harvest mAb reduction.