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We have investigated the independent effects of selective gene amplification (using the dhfr amplifiable selection marker) and culture operating strategy (batch vs repeated fed-batch vs semicontinuous perfusion) on the glycosylation of a recombinant reporter protein (secreted alkaline phosphatase, SEAP) produced in transfected Chinese hamster ovary (CHO) cells. HPLC analyses coupled with susceptibility to various exoglycosidases were used to determine the N-glycosylation profile of SEAP samples. The dhfr amplified cell line yielded an almost 10-fold increase in specific productivity as compared to that of the unamplified cell line. The glycosylation pattern of the reporter protein produced in batch bioreactor cultures of the amplified cell line showed only slight differences as compared to the glycosylation pattern of the protein from batch bioreactor cultures of the unamplified cell line. In contrast, analysis of SEAP glycosylation structures from the protein isolated from semicontinuous perfusion cultures indicated that both relative glycan content and extent of sialylation were increased as compared to samples isolated from repeated fed-batch cultures. These results suggest that the slow growing perfusion cultures produce more completely glycosylated proteins than the faster growing repeated fed-batch cultures.

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.

We report the identification of a novel small open reading frame in Escherichia coli. The sORF (called iroK) encodes a 21 amino cid peptide, which when translated confers a 133% (ca. 20 g/L) increase in resistance to 3-hydroxypropionic acid. We show that iroK conferred tolerance is additive to previously identified tolerance mechanisms involving relief of inhibited metabolism, yet does not involve altered 3-HP transport. This result demonstrates the continued surprises that microbial genomes hold and emphasize the importance of comprehensive discovery methods in future strain and metabolic engineering efforts.

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