Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis, gating pyruvate generation, and increasing pyruvate flux into the TCA cycle

Abstract

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM‐KO) and pyruvate kinase muscle isoform‐1 knockout (PKM1‐KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild‐type (WT), PKM‐KO, and PKM1‐KO stable cell lines, expressing two different antibodies, were analyzed in 14‐day fed‐batch production assays using different vessels. PKM‐KO and PKM1‐KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM‐KO and PKM1‐KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1‐KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.

CRISPR‐interceded CHO cell line development approaches

“…Lactate and ammonia are known to be major byproducts that limit cell growth and productivity and can affect glycosylation patterns at certain quantities in supernatant (Mulukutla et al, 2019). Alternative culture methodologies, media design, and metabolic engineering have all been employed in the past to lower lactate production (Tang et al, 2021; Wilkens et al, 2019).…”

Chemical inhibitors of hexokinase‐2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures

“…Metabolic engineering has also been employed to reduce lactate accumulation by either targeting enzymes in the glycolysis pathway or by improving the poor connectivity between the mitochondrial and cytosolic metabolic systems. These efforts include reducing the expression of enzymes such as pyruvate kinase muscle (PKM), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinases (PDHks) as well as overexpressing enzymes such as recombinant yeast pyruvate carboxylase (PYC2) enzyme and malate dehydrogenase‐2 (MDH‐2) (Chong et al, 2010; Gupta et al, 2017; Tang et al, 2021; Toussaint et al, 2016; Zhou et al, 2011). In these studies, reduction in lactate levels were observed along with a redirection of more pyruvate into the TCA cycle for oxidation in the mitochondria, higher specific productivities, and in some cases improved product quality (Chong et al, 2010; Gupta et al, 2017; Tang et al, 2021; Toussaint et al, 2016; Zhou et al, 2011).…”