No abstract

Chinese hamster ovary (CHO) cell lines are used to express a variety of therapeutic proteins. However, lactogenic behavior displayed by some CHO cell lines during manufacturing processes may result in a decline in viability, productivity, and possible alterations in product quality. In cultured cells, lactate is produced during glycolysis through irreversible conversion of phosphoenolpyruvate to pyruvate and then lactate via sequential function of pyruvate kinase and lactate dehydrogenase (LDH) enzymes. In the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules are identified. The lactogenic behaviors of these cell lines can be differentially mitigated through optimization of either nutrient feeds or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway reveal a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform levels and lactogenic behavior. CRISPR mediated knockout of the PKM-1 isoform abolishes lactate accumulation even under lactogenic conditions. Furthermore, a cell line lacking expression of both PKM-1 and PKM-2 enzymes capable of maintaining productivity, viability, and growth without displaying lactogenic behavior is identified. Targeted deletion of PKM in CHO cells may be tolerated due to expression of PKL (liver) and PKR (red blood cell) isoforms of pyruvate kinase. All together, these findings suggest that PKM-1 up-regulation during antibody production could trigger lactogenic behavior and that this enzyme is dispensable for CHO cell survival.

Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

Bispecific antibodies are regarded as the next generation of therapeutic modalities as they can simultaneously bind multiple targets, increasing the efficacy of treatments for several diseases and opening up previously unattainable treatment designs. Linking two half antibodies to form the knob-into-hole bispecific antibody requires an additional in vitro assembly step, starting with reduction of the antibodies and then reoxidization. Analysis of the disulfide bonds (DSBs) is vital to ensuring the correct assembly, stability, and higher-order structures of these important biomolecules because incorrect disulfide bond formation and/or presence of cysteine-related post-translational modifications can cause a loss of biological activity or even elicit an immune response from the host. Despite advancements in analytical methods, characterization of cysteine forms remains technically challenging and time-consuming. Herein, we report the development of an improved nonreduced peptide map method coupled with machine learning to enable rapid identification of disulfide bonds and cysteine-related variants in an IgG1 knob-into-hole bispecific antibody. The enhanced method offers a fast, consistent, and accurate workflow in mapping-out expected disulfide bonds in both half antibodies and bispecific antibodies and identifying cysteine-related modifications. Comparisons between two versions of the bispecific antibody molecule and analysis of stressed samples were also accomplished, indicating this method can be utilized to identify alterations originating from bioprocess changes and to determine the impact of assembly and postassembly stress conditions to product quality.

In this study, the Elementary Metabolite Unit (EMU) algorithm was employed to calculate intracellular fluxes for Chlorella protothecoides using previously generated growth and mass spec data. While the flux through glycolysis remained relatively constant, the pentose phosphate pathway (PPP) flux increased from 3% to 20% of the glucose uptake during nitrogen-limited growth. The TCA cycle flux decreased from 94% to 38% during nitrogen-limited growth while the flux of acetyl-CoA into lipids increased from 58% to 109% of the glucose uptake, increasing total lipid accumulation. Phosphoenolpyruvate carboxylase (PEPCase) activity was higher during nitrogen-sufficient growth. The glyoxylate shunt was found to be partially active in both cases, indicating the nutrient nature has an impact on flux distribution. It was found that the total NADPH supply within the cell remained almost constant under both conditions. In summary, algal cells substantially reorganize their metabolism during the switch from carbon-limited (nitrogen-sufficient) to nitrogen-limited (carbon-sufficient) growth.