Profiling of N-glycosylation gene expression in CHO cell fed-batch cultures.

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N-glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N-glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed-batch culture of a CHO cell line producing recombinant human interferon-gamma (IFN-gamma). Of the 24 N-glycosylation genes examined, 21 showed significant up- or down-regulation of gene expression as the fed-batch culture progressed from exponential, stationary and death phase. As the fed-batch culture progressed, there was also an increase in less sialylated IFN-gamma glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-gamma. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed-batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N-glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible "bottlenecks" or "compromised" pathways in N-glycosylation and subsequently allow for the development of strategies to improve glycosylation quality.

Targeting early apoptotic genes in batch and fed-batch CHO cell cultures.

Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, were identified and CHO GT (Gene Targeted) cell lines were developed by targeting these four genes. Two were CHO GT(O) cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GT(KD) cell lines involving knockdown of Alg-2 and Requiem which are pro-apoptotic genes using small interfering RNA (siRNA) technology. Comparisons of these CHO GT cell lines with the parental cell line in batch culture (BC) and fed-batch culture (FBC) were performed. Compared to parental cells, the CHO GT cell lines showed apoptosis resistance as they significantly delayed and/or suppressed initiator caspase-8 and -9 and executioner caspase-3 activities during culture. FBC of CHO GT cell lines reached significantly higher maximum viable cell densities (up to 9 x 10(6) cells/mL) compared with the parental cell line (5 x 10(6) cells/mL). The recombinant interferon gamma (IFN-gamma) yields were increased by up to 2.5-fold. Furthermore, it was observed that the IFN-gamma was more highly sialylated.

Transcriptional profiling of apoptotic pathways in batch and fed-batch CHO cell cultures.

Chinese Hamster ovary (CHO) cells are regarded as one of the "work-horses" for complex biotherapeutics production. In these processes, loss in culture viability occurs primarily via apoptosis, a genetically controlled form of cellular suicide. Using our "in-house" developed CHO cDNA array and a mouse oligonucleotide array for time profile expression analysis of batch and fed-batch CHO cell cultures, the genetic circuitry that regulates and executes apoptosis induction were examined. During periods of high viability, most pro-apoptotic genes were down-regulated but upon loss in viability, several early pro-apoptotic signaling genes were up-regulated. At later stages of viability loss, we detected late pro-apoptotic effector genes such as caspases and DNases being up-regulated. This sequential regulation of apoptotic genes showed that DNA microarrays could be used as a tool to study apoptosis. We found that in batch and fed-batch cultures, apoptosis signaling occurred primarily via death receptor- and mitochondria-mediated signaling pathways rather than endoplasmic reticulum-mediated signaling. These insights provide a greater understanding of the regulatory circuitry of apoptosis during cell culture and allow for subsequent targeting of relevant apoptosis signaling genes to prolong cell culture.