Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

As we pursue the means to improve yields to meet growing therapy demands, it is important to examine the impact of process control on glycosylation patterns to ensure product efficacy and consistency. In this study, we describe a dynamic on-line fed-batch strategy based on low glutamine/glucose concentrations and its impact on cellular metabolism and, more importantly, the productivity and N-glycosylation quality of a model recombinant glycoprotein, interferon gamma (IFN-gamma). We found that low glutamine fed-batch strategy enabled up to 10-fold improvement in IFN-gamma yields, which can be attributed to reduced specific productivity of ammonia and lactate. Furthermore, the low glutamine concentration (0.3 mM) used in this fed-batch strategy could maintain both the N-glycosylation macro- and microheterogeneity of IFN-gamma. However, very low glutamine (<0.1 mM) or glucose (<0.70 mM) concentrations can lead to decreased sialylation and increased presence of minor glycan species consisting of hybrid and high-mannose types. This shows that glycan chain extension and sialylation can be affected by nutrient limitation. In addition to nutrient limitation, we also found that N-glycosylation quality can be detrimentally affected by low culture viability. IFN-gamma purified at low culture viability had both lower sialylation as well as glycans of lower molecular masses, which can be attributed to extensive degradation by intracellular glycosidases released by cytolysis. Therefore, in order to maintain good N-glycosylation quality, there is a need to consider both culture viability and nutrient control setpoint in a nutrient-limiting fed-batch culture strategy. A greater understanding of these major factors that affect N-glycosylation quality would surely facilitate future development of effective process controls.