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.

Cellular responses to individual amino-acid depletion in antibody-expressing and parental CHO cell lines.

Depletion of two nonessential amino acids, asparagine (Asn) and glutamine (Gln), occurred during a fed-batch production process with a CHO cell line expressing a recombinant antibody. This depletion coincided with growth suppression and the onset of the stationary phase. Experimental withdrawal of Asn led to cell cycle arrest of cell line A in G0/G1 phase. On a mechanistic level, withdrawal of either Asn or Gln stimulated the amino-acid response (AAR) pathway, indicating that depletion of nonessential amino acids can induce AAR in this cell line. Compared to withdrawal of an essential amino acid, leucine (Leu), withdrawal of either Asn or Gln induced fewer changes in downstream effectors of mammalian target of rapamycin (mTOR) signaling involved in regulation of global protein synthesis. Global transcriptional analysis followed by pathway analysis revealed that the cultures experienced a down-regulation of cell-cycle progression, DNA replication and nucleotide biosynthesis in an E2F-dependent manner, as well as a down-regulation of lipid metabolism in a SREBP1/2-dependent manner as a result of individual amino-acid withdrawal. Timing and magnitude of observed phenotypic and transcriptional responses to amino-acid withdrawal differed between essential (Leu) and nonessential (Asn and Gln) amino acids examined. Observed responses were similar in parental (CS9 and CHOK-1) and two other antibody-producing CHO cell lines, but the magnitude of the transcriptional response was both cell-line and amino-acid dependent. Overall, these results suggest that depletion of nonessential amino acids in cell culture plays a role in the onset of the stationary phase of production process and offer mechanistic insights into the observed growth attenuation phenotype.

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.

Factors that determine stability of highly concentrated chemically defined production media.

High cell density perfusion processes for the production of therapeutic antibodies require large volumes of media to meet cellular stoichiometric and energy demands. The use of media concentrates provides a way to reduce the cost of manufacturing. Reducing the number and size of liquid media batches reduces the media footprint in the manufacturing plant and cuts costs associated with single-use systems for preparation and storage of liquid media. Concentrates that can be stored at room temperature also reduce costs by eliminating the need for refrigerated storage. To meet these economic and operational objectives, we developed a complete concentrated medium system consisting of a 5X medium concentrate that can be used in conjunction with a concentrated supplement of cystine, tyrosine, and folic acid. The effects of pyruvate, bicarbonate, and glutamine on the stability of the 5X concentrates were studied. Pyruvate and bicarbonate were found to have profound impacts on media stability, including media coloration, precipitate formation and ability to support cell culture. Bicarbonate was found to have detrimental effects in 5X concentrated media, resulting in precipitation of pyruvate-free media and accelerated glutamine degradation. Pyruvate prevented precipitation in bicarbonate-containing concentrates. Moreover, the presence of pyruvate in bicarbonate-free, glutamine-free 5X concentrates resulted in the substantial preservation of the functional activity of the medium for 1 month at room temperature.

Hydroxocobalamin association during cell culture results in pink therapeutic proteins.

Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing.

Psychosocial intervention for rural women with breast cancer: The Sierra-Stanford Partnership.

OBJECTIVE: This study was initiated by breast cancer survivors living in a rural community in California. They formed a partnership with academic researchers to develop and evaluate a low-cost, community-based Workbook-Journal (WBJ) for improving psychosocial functioning in geographically and economically isolated women with primary breast cancer. DESIGN: A randomized controlled trial was used to compare the WBJ intervention plus educational materials to educational materials alone (usual care). SETTING: One rural cancer center and several private medical, surgical, and radiation oncology practices in 7 rural counties in the Sierra Nevada Foothills of California. PARTICIPANTS: One hundred women with primary breast cancer who were either within 3 months of diagnosis or within 3 months of completing treatment. INTERVENTION: A community-initiated, theoretically-based Workbook-Journal, designed by rural breast cancer survivors and providers as a support group alternative. It included compelling personal stories, local rural resources, coping strategies, and messages of hope. RESULTS: Community recruiters enrolled 83% of the women referred to the study. Retention at 3-month follow-up was 98%. There were no main effects for the WBJ. However, 3 significant interactions suggested that women who were treated in rural practices reported decreased fighting spirit and increased emotional venting and posttraumatic stress disorder symptoms if they did not receive the WBJ. Among women who receive the WBJ, 74% felt emotionally supported. CONCLUSIONS: This community-based Workbook-Journal may be an effective psychosocial intervention for rural, isolated, and low-income women with breast cancer. Community involvement was essential to the success of this project.