Recombinant DNA (rDNA) technologies provide a wide range of tools for producing a broad array of recombinant proteins. Since the early 1970s, the biotechnology industry has harnessed those tools — together with genetic engineering and genomics — for developing new classes of innovative and effective therapeutic molecules. The therapeutic recombinant protein market segment now represents the core of the medical biotechnology industry, with hundreds of companies involved in discovery, development, and marketing.

Although recombinant technologies are extremely powerful tools, significant limitations in expression, upstream, and downstream processes still exist for many proteins that have therapeutic potential. Failure to obtain specific product features such as posttranslational modifications and high yields jeopardize the probability of developing relevant proteins into approved therapeutic drugs. The need for technologies that allow reliable and effective therapeutic protein production capabilities is growing.

PRODUCT FOCUS: RECOMBINANT PROTEINS

PROCESS FOCUS: UPSTREAM PROCESSING

WHO SHOULD READ: PROCESS ENGINEERS

KEYWORDS: CHO, EXPRESSION SYSTEMS, PLATFORM SCREENING

LEVEL: BASIC

Because of their capacity to provide proper protein folding, posttranslational modifications, and secretion of complex proteins in an active form, mammalian cells have become the major system for recombinant therapeutic protein production. Significant efforts have been made to develop mammalian cell expression platforms. However, along the early drug discovery process, other production systems (e.g., prokaryotic, insect cell, and cell-free) remain widely used for convenience. Easy to handle and fast, those systems allow the production of small amounts of products required for validation and screening. Transient expression in mammalian cells is also routinely used for difficult-to-express products and provide research suitable yields (1).

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Drug discovery includes a variety of successive systems for candidate screening until the identification of a lead candidate (2). Typically, the initial screening is carried out in a prokaryotic or insect-cell system until a panel of candidates are identified. That process is followed by transient transfections in mammalian cells, but not necessarily in cell lines appropriate for manufacturing. Finally, a manufacturing cell line is developed that expresses the lead clinical candidate, to fulfill the quantitative needs of clinical trials. This approach relies on system switches and is time consuming and not cost-effective. In addition, it may influence product quality and efficacy, and the final production system will differ from the screening system, possibly delivering dissimilar products.

Selexis has developed solutions for more robust and efficient identification of individual lead protein candidates that rely on a single stable expression system throughout the drug development process. We have built an effective, stably transfected mammalian-cell platform based on a combination of DNA vectors harboring proprietary epigenetic regulatory elements such as the Selexis Genetic Elements (SGEs) technology with optimized gene transfer methods (3). Those elements promote an elevation of plasmid integration into a host-cell genome by homologous recombination associated with transfection (4).

Increased recombination may result from the unwinding properties of the SGE region in a plasmid, which could favor strand exchange between plasmids and at specific sites in the recipient cell genome (3). The SGE-containing plasmids are likely preferentially targeted to endogenous SGE sites in a cell genome because thousands of SGEs have been identified in mammalian-cell genomes (3,5). Independent transformants contain transgenes in different chromosomes. Furthermore, multiple copies of the recombinant gene are not scattered throughout the host genome but are integrated as a concatemer at one site in the host-cell chromosome (3). Thus, the increased number of copies integrated into a host-cell genome lead to a proportional increase in recombinant protein expression. That differs from the high copy number found in DHFR-mediated gene-amplified cells, which are often associated with repeat-induced transcriptional silencing (6,7).

By allowing for unwinding of the genomic environment of a transgene, SGEs may also prevent transgene silencing, thereby providing high transcription rates of all copies. This distinctive feature of high-level stable protein expression within days of transfection enables accelerated screening of SGE-generated CHO transformants. So timelines for cell line development are reduced. This feature, when applied to a screening campaign, also allows identification of relevant therapeutic protein variants and high-expressing clones earlier in a screening process. That saves considerable labor by screening top lead candidates in a production-ready platform and represents an integrated and reliable solution to advance biopharmaceuticals toward human clinical trials.

Here, we describe a platform for expression of a combinatorial library of human antibodies expressing 250 different MAbs. It was based on mRNA sequences isolated from B-cell selected clones and phage display directed toward a viral antigen. Our purpose was to identify an antibody lead compound by screening full-length and appropriately folded MAbs.