Jeff Jia Cheng Hou

High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system

Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

Intracellular trafficking pathways for nuclear delivery of plasmid DNA complexed with highly efficient endosome escape polymers.

Understanding the pathways for nuclear entry could see vast improvements in polymer design for the delivery of genetic materials to cells. Here, we use a novel diblock copolymer complexed with plasmid DNA (pDNA) to determine both its cellular entry and nuclear pathways. The diblock copolymer (A-C3) is specifically designed to bind and protect pDNA, release it at a specific time, but more importantly, rapidly escape the endosome. The copolymer was taken up by HEK293 cells preferentially via the clathrin-mediated endocytosis (CME) pathway, and the pDNA entered the nucleus to produce high gene expression levels in all cells after 48 h, a similar observation to the commercially available polymer transfection agent, PEI Max. This demonstrates that the polymers must first escape the endosome and then mediate transport of pDNA to the nucleus for occurrence of gene expression. The amount of pDNA within the nucleus was found to be higher for our A-C3 polymer than PEI Max, with our polymer delivering 7 times more pDNA than PEI Max after 24 h. We further found that entry into the nucleus was primarily through the small nuclear pores and did not occur during mitosis when the nuclear envelope becomes compromised. The observation that the polymers are also found in the nucleus supports the hypothesis that the large pDNA/polymer complex (size ~200 nm) must dissociate prior to nucleus entry and that cationic and hydrophobic monomer units on the polymer may facilitate active transport of the pDNA through the nuclear pore.

Targeting membrane proteins for antibody discovery using phage display.

A critical factor in the successful isolation of new antibodies by phage display is the presentation of a correctly folded antigen. While this is relatively simple for soluble proteins which can be purified and immobilized onto a plastic surface, membrane proteins offer significant challenges for antibody discovery. Whole cell panning allows presentation of the membrane protein in its native conformation, but is complicated by a low target antigen density, high background of irrelevant antigens and non-specific binding of phage particles to cell surfaces. The method described here uses transient transfection of alternating host cell lines and stringent washing steps to address each of these limitations. The successful isolation of antibodies from a naive scFv library is described for three membrane bound proteins; human CD83, canine CD117 and bat CD11b.

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal‐derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum‐free, protein‐free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single‐cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD‐CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

Intracellular trafficking pathways for plasmid DNA complexed with highly efficient endosome escape polymers.

Background Non-viral gene delivery vectors are widely used for the delivery of genetic materials into mammalian cells. Currently, there is a need to develop cheap and efficient transfection agents for use in production of recombinant proteins via transient gene expression. There are several barriers that non-viral vectors must overcome for successful transfection, these include cellular internalisation, endosome escape, protection of DNA and delivery of DNA into the nucleus. The ability to escape the endosome and gain entry to the nucleus are of the two primary barriers to successful transfection. The processes involved in the pathways for cellular uptake, intracellular trafficking, and nuclear entry are still not fully understood. More detailed understanding of the pathways involved in transfection is needed in order to develop highly efficient transfection agents.

Effects of perfusion processes under limiting conditions on different Chinese Hamster Ovary cells

Background The use of perfusion culture to generate biopharmaceuticals is an attractive alternative to fed-batch bioreactor operation. The process allows for generation of high cell densities, stable culture conditions and a short residence time of active ingredients to facilitate the production of sensitive therapeutic proteins. However, challenges remain for efficient perfusion based production at industrial scale, primarily complexity of required equipment and strategies adopted for downstream processing. For perfusion systems to be industrially viable there is a need to increase product yields from a perfusion-based platform. We have shown previously that one effective way to enhance the cell specific productivity is via glucose limitation [1,2]. The mechanisms leading to an increased productivity under these glucose limiting conditions are still under investigation. Preliminary studies using proteomic analysis have indicated changes in histone acetylation [2]. In this work, we investigated the influence of glucose limited conditions on the production of two different recombinant proteins in perfusion processes.

Novel cell engineering of the unfolded protein response to achieve efficient therapeutic protein production cell line

Background The bioproduction of therapeutic proteins such as monoclonal antibodies (mAb) continues to be a fast growing sector of advanced manufacturing. The ever increasing repertoire of therapeutic proteins coupled with the emergence of biosimilars led to increasing global demand for higher, faster, more cost-effective manufacturing process. Central to any good production platform is the capacity of the production cell line. A suitable production cell line exhibit physiological traits such as high specific productivity (qp), rapid doubling time, high peak cell density and efficient metabolism. The emergence of a suitable production cell with all the aforementioned traits is an extremely rare event, as it requires all facets of cellular transcription, translation, secretion and metabolic efficiency are individually optimized and collectively synchronized into a system capable of high level protein expression. One of the major cellular bottlenecks thought to limit protein production in mammalian cells lies in the cellular translation and secretion capacity. The over-expression of complex recombinant proteins such as mAbs driven by strong viral promoters exert considerable burden in the Endoplasmic Reticulum (ER) and Golgi apparatus. The increase in ER stress triggers the Unfolded Protein Response (UPR) which can result in cell apoptosis and possibly eliminating cells with high uptake of the Gene of Interest (GOI). We adopted host cell engineering approach using XBP1 spice ratio to expand cellular translation and secretion capacity to increase qp and improve the probability of isolating suitable high producers. Materials and methods We screened a panel of mAb producing clonal cell lines using qPCR to identify XBP1 mRNA splice ratio as a suitable target to augment host cell translation and secretion machinery. We then employed FACS to isolate cell population with high ratio of spliced over unspliced XBP1 in order to overcome the negative regulatory effect of unspliced XBP1. We created a host cell line with high XBP1 splice ratio, exhibiting increased expression of chaperones, secretary vesicle proteins and quality assurance proteins without increasing UPR associated apoptotic markers as identified using qPCR. This host cell line was then used for in comparative mAb transient bioproduction studies with two different mAbs. Transient studies were performed using PEI-mediated transfection in industrial relevant chemically defined medium and feeding regime.

Analysis of Protein Expression via Alternate 3’ Untranslated Region (UTR) Signals Through the Use of Site Specific Recombination

Chinese Hamster Ovary (CHO) cells remain the workhorse of the biopharmaceutical industry. We used site specific recombination in CHO cells to evaluate the post-translational effects of different poly-A signals. Recombinase (Flp) assisted generation of CHO pools show a significant difference in intra-clonal variation within in the pools. Moreover, the use of FRT/Flp resulted in an isogenic population which has allowed accurate correlation between mRNA levels and recombinant protein yields. We identified a human derived 3’UTR which generated a higher level of mRNA when compared to the more commonly used SV40 poly-A, the corresponding recombinant protein levels was found to be independent of the transcript levels.