Arvia E. Morris

Incorporation of an Isoleucine Zipper Motif Enhances the Biological Activity of Soluble CD40L (CD154)

Recent progress in the understanding of immune function indicates that the interaction of CD40L with its receptor, CD40, plays a pivotal role in both humoral immunity and cell-mediated defense against pathogens. Functional studies of this interaction on both dendritic cells and malignant cells have demonstrated that CD40L also plays an important role in immune surveillance and anti-tumor immunity. CD40L exists in nature predominantly as a membrane-anchored molecule. To develop CD40L as a potential therapeutic, it is important to optimize soluble forms of this molecule that could be used in a clinical setting. Several reports have shown that soluble forms of CD40L, like CD40 antibodies, are biologically active. In the present report we demonstrate that the incorporation of an isoleucine zipper trimerization motif significantly enhances the biological activity of soluble CD40L.

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.

Effects of Insulin and LongR3 on Serum-Free Chinese Hamster Ovary Cell Cultures Expressing Two Recombinant Proteins

Insulin is the most commonly used growth factor for sustaining cell growth and viability in serum‐free Chinese hamster ovary (CHO) cell cultures. In the present study insulin and IGF‐1 analogue (LongR3) were compared for their ability to support growth, viability, and production of two serum‐free CHO cell lines expressing recombinant protein. The first cell line, VA12, expresses protein B, and the second cell line, CL23, expresses protein C. Both molecules are recombinant cytokine receptors. VA12 will grow in serum‐free media lacking growth factor, while CL23 requires either insulin or LongR3 for cell growth. Both cell lines, however, require a growth factor for optimal performance under production conditions. In this study, LongR3 was better able to sustain the viability of both cell lines under production conditions than insulin. These data indicate that while insulin and LongR3 can both serve as growth and viability factors for CHO cells, LongR3 is the preferred growth factor for cell lines VA12 and CL23.

EASE Vectors for Rapid Stable Expression of Recombinant Antibodies

Over the past 10 years, monoclonal antibodies and antibody fragments have become an increasingly important source of therapeutic molecules in the biotechnology industry. Drug development strategies rely on screening large numbers of candidate molecules in search of an optimized drug candidate. This strategy requires efficient production of ten to a few hundred milligrams of candidate molecules for screening in bioassays and animal models. Typically, this amount of recombinant protein expression involves large numbers of transient transfections or cloning of a recombinant cell line. Both of these approaches are time‐consuming and labor‐intensive. In this report, we describe the application of an EASE vector system that is capable of generating stable pools of transfected Chinese hamster ovary cells. These pooled populations of cells produce high quantities of antibody candidates without labor‐intensive cloning in a 3–5 week time frame. When an optimal drug candidate has been selected, pools generated with EASE‐containing vectors can also be used in subsequent cloning steps to make cell lines with improved expression levels. We demonstrate that EASE increases expression in nonamplified pools in addition to increasing amplification and viability of clonal cell lines generated with the EASE‐containing vectors compared with pools and cell lines generated without EASE.

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. Biotechnol. Biotechnol. Bioeng. 2014;111: 965–979.

Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels.

Pressures for cost‐effective new therapies and an increased emphasis on emerging markets require technological advancements and a flexible future manufacturing network for the production of biologic medicines. The safety and efficacy of a product is crucial, and consistent product quality is an essential feature of any therapeutic manufacturing process. The active control of product quality in a typical biologic process is challenging because of measurement lags and nonlinearities present in the system. The current study uses nonlinear model predictive control to maintain a critical product quality attribute at a predetermined value during pilot scale manufacturing operations. This approach to product quality control ensures a more consistent product for patients, enables greater manufacturing efficiency, and eliminates the need for extensive process characterization by providing direct measures of critical product quality attributes for real time release of drug product.

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.