Pranhitha Reddy

Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed‐batch CHO cell culture

Cell metabolism can vary considerably over the course of a typical fed‐batch antibody production process. However, the intracellular pathway alterations associated with various phases of growth and antibody production have yet to be fully elucidated using industrially relevant production hosts. Therefore, we performed 13C labeling experiments and metabolic flux analysis (MFA) to characterize CHO cell metabolism during four separate phases of a fed‐batch culture designed to closely represent industrial process conditions. First, we found that peak specific growth rate was associated with high lactate production and minimal TCA cycling. Conversely, we found that lactate metabolism switched from net production to net consumption as the culture transitioned from peak growth to peak antibody production. During the peak antibody production phase, energy was primarily generated through oxidative phosphorylation, which was also associated with elevated oxidative pentose phosphate pathway (oxPPP) activity. Interestingly, as TCA cycling and antibody production reached their peaks, specific growth rate continued to diminish as the culture entered stationary phase. However, TCA cycling and oxPPP activity remained high even as viable cell density began to decline. Overall, we found that a highly oxidative state of metabolism corresponded with peak antibody production, whereas peak cell growth was characterized by a highly glycolytic metabolic state. Biotechnol. Bioeng. 2013; 110: 2013–2024.

Effect of PDI overexpression on recombinant protein secretion in CHO cells.

In eukaryotic cells, protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl‐4‐hydroxylase and the triacylglycerol transfer proteins. Increasing PDI activity in bacterial, yeast, and insect cell expression systems can lead to increased secretion of heterologous proteins containing disulfide bridges. Since Chinese hamster ovary (CHO) cells are widely used for the expression of recombinant proteins, we expressed recombinant human PDI (rhu PDI) in CHO cells to increase cellular PDI levels and examined its effect on the secretion of two different recombinant proteins: interleukin 15 (IL‐15) and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). Secretion of TNFR:Fc (a disulfide‐rich protein) is decreased in cells overexpressing PDI; the TNFR:Fc protein is retained inside these cells and colocalizes with the overexpressed rhu PDI protein in the endoplasmic reticulum. PDI overexpression did not result in intracellular retention of IL15. The nature of the interaction between PDI and TNFR:Fc was further investigated by expressing a disulfide isomerase mutant PDI in CHO cells to determine if the functional activity of PDI is involved in the cellular retention of TNFR:Fc protein.

Metabolic analysis of antibody producing CHO cells in fed‐batch production

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed‐batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed‐batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth. Biotechnol. Bioeng. 2013; 110: 1735–1747.

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1‐checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures. Biotechnol. Bioeng. 2015;112: 141–155.

Identification of novel small molecule enhancers of protein production by cultured mammalian cells.

Small molecule additives to cell culture media (e.g., sodium butyrate) that are capable of enhancing the expression of recombinant proteins have significant utility in the production and manufacture of therapeutic polypeptides. To identify novel small molecule enhancers (SMEs) of recombinant protein expression in Chinese Hamster Ovary (CHO) cells, we screened two separate small molecule libraries for compounds capable of enhancing the expression of either a fluorescent reporter protein or a monoclonal antibody. Several compounds that increased recombinant protein expression were identified, and these compounds fell into three broad classes: (1) aromatic carboxylic acids, (2) hydroxamic acids, and (3) acetamides. We examined the impact of SME addition to CHO cell cultures expressing different classes of recombinant proteins including monoclonal antibodies (MAbs). For CHO cell pools or clones grown in production shake‐flasks or bioreactors, recombinant protein titers up to 60% higher than control cultures were observed. Analysis of mRNA levels suggest that transcriptional activation plays a role in the expression enhancement seen for some SMEs, but other mechanisms may be involved for at least one compound. Finally, we tested many of the identified SMEs for their ability to increase MAb production by a hybridoma cell line. Hexanohydroxamic acid increased shake‐flask MAb production by 40% relative to a control. Taken together, these data demonstrate the potential utility of the compounds in the production of therapeutically relevant proteins from diverse cell‐based production systems. Biotechnol. Bioeng. 2008;100: 1193–1204.

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the CH1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the CH2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN162SGAL in the CH1 domain of the antibody. This highly atypical modification is present at levels of 0.5–2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the CH1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the −1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.

Non-invasive UPR monitoring system and its applications in CHO production cultures.

Unfolded protein response (UPR) is the primary signaling network activated in response to the accumulation of unfolded and/or misfolded protein in the endoplasmic reticulum (ER). The expression of high levels of recombinant proteins in mammalian cell cultures has been linked to the increased UPR. However, the dynamics of different UPR‐mediated events and their impact on cell performance and recombinant protein secretion during production remain poorly defined. Here, we have created a non‐invasive UPR‐responsive, fluorescence‐based reporter system to detect and quantify specific UPR‐mediated transcriptional activation of different intracellular signaling pathways. We have generated stable antibody‐expressing CHO clones containing this UPR responsive system and established FACS‐based methods for real‐time, continuous monitoring of the endogenous UPR activation in live cultures. The results showed that the UPR activation is dynamically regulated during production culture. The clones differed in their UPR patterns; both the timing and the degree of UPR‐induced transcriptional activation were linked to cell performance, such as growth, and viability. In addition, the cell culture environment, such as media composition and osmolarity, significantly impacted endogenous UPR activation. Taken together, these data demonstrate a utility of this UPR monitoring system in recombinant protein production processes and the observations increase our understanding of the critical role of UPR in regulating diverse phenotypes of the cells including growth, survival and recombinant protein secretion under different culture environments and processing conditions. Biotechnol. Bioeng. 2013; 110: 2184–2194.

Analysis of heterogeneity and instability of stable mAb-expressing CHO cells

Screening and isolation of high expression mammalian cell lines for production of recombinant proteins for the clinic is a resource-intensive and time-consuming procedure due to the substantial variation in expression levels of recombinant protein expression amongst transfected cells. Several investigators have reported instability in expression titers early in cell line development and in cell banks. However, in most cases the exact molecular mechanisms of instability remain unknown. In this study we used a fluorescence-activated cell sorting (FACS) based mAb staining method to enable the detection and selective gating of cells with vastly different recombinant expression levels present in transfected pools. Expression diversity and changes within transfected populations were detected and isolated in real time during cell line development. Molecular genetic analysis on the isolated clones revealed an unsuspected rearrangement of the heavy chain in the non-expressing clones. Implications of the genetic rearrangements as well as the use of the FACS method as a tool to improve cell line development to detect expression heterogeneity in pools and to investigate root cause for the molecular genetics of expression instability will be discussed.

Circumventing the “Pay Now or Pay Later” Dilemma: Strategies for Achieving Process Development with Speed and Long-Term Potential

One dilemma faced by process development organizations is balancing the competing demands of speed to clinic with commercial readiness. Highly successful platform approaches, which are common to larger companies, have significantly increased the speed of development leading to testing in humans. The consequences of shortening the timeline for initiation of early phase clinical trials, however, include a limited scope of process and product understanding, increased risk of failure upon scale-up during the Phase I/II clinical campaign(s), and a resulting process that often requires at least some re-development prior to pivotal and commercial manufacturing. At Amgen, we have developed an approach to process development that allows us to satisfy the demands for both early and late-phase processes with efficient use of human and capital resources.