Florian M. Wurm

Production of recombinant protein therapeutics in cultivated mammalian cells

Cultivated mammalian cells have become the dominant system for the production of recombinant proteins for clinical applications because of their capacity for proper protein folding, assembly and post-translational modification. Thus, the quality and efficacy of a protein can be superior when expressed in mammalian cells versus other hosts such as bacteria, plants and yeast. Recently, the productivity of mammalian cells cultivated in bioreactors has reached the gram per liter range in a number of cases, a more than 100-fold yield improvement over titers seen for similar processes in the mid-1980s. This increase in volumetric productivity has resulted mainly from improvements in media composition and process control. Opportunities still exist for improving mammalian cell systems through further advancements in production systems as well as through vector and host cell engineering.

Large-scale transient expression in mammalian cells for recombinant protein production

Large-scale transient expression from mammalian cells is a new technology. Breakthroughs have been achieved for non-viral delivery methods: transfections can now be done at the 1-10 L scale with mammalian cells grown in suspension. Production of 1-20 mg/L of recombinant protein have been obtained in stirred bioreactors. Modified alphaviruses have provided a fast and efficient expression technology based on viral vectors.

Development of stable cell lines for production or regulated expression using matrix attachment regions

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.

GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode

This study correlates the fluorescent signal from stable recombinant CHO cell lines expressing the green fluorescent protein (GFP) at high levels with biomass or cell number, extending the use of fluorescent proteins to applications and assays where cell growth rates are important. Using a standard fluorometer, growth of these cells can be quantified noninvasively in multiwell plates, and because signals are obtained without preparation, the same culture samples can be measured repeatedly. Even with a small relative change in biomass, the specific growth rate can be determined in a few hours. The dynamics of cell populations can now be studied with high sensitivity, low error rate, and minimum sample preparation.

Acrylic acid grafting and collagen immobilization on poly(ethylene terephthalate) surfaces for adherence and growth of human bladder smooth muscle cells.

In tissue engineering, degradable or non-degradable polymer matrices can act as cell-carrier-scaffolds. Cell adhesion and growth on these scaffolds can be promoted by immobilizing extracellular matrix proteins. Therefore, in this study, polymer poly(ethylene terephthalate) (PET) films were surface modified by graft polymerization of acrylic acid, to subsequently allow collagen (types I and III) immobilization and human smooth muscle cell expansion. The surfaces of PET were activated by plasma, followed by acrylic acid graft polymerization, resulting in covalently bound brushes, containing an average of either 0.22+/-0.1 or 5.93+/-0.87 microg/cm2 of poly(acrylic acid) (PAA). Subsequent electrostatic adsorption of collagen gave a surface concentration of 4.96 and 17.2 microg/cm2, respectively, as determined using radiolabelled 125I collagen. Both PET films grafted with 0.22 microg/cm2 of PAA with or without adsorbed collagen were apt for smooth muscle cell adhesion and proliferation. However, films grafted with 5.93 microg/cm2 were not. PAA-grafted PET films, onto which serum proteins of the culture medium adsorbed spontaneously, proved to be better matrices than films on which collagen has been immobilized. It, therefore, can be speculated that other serum proteins are more important than collagen for the human smooth muscle cell adhesion and growth on surface-modified polymer matrices.

25 years of recombinant proteins from reactor-grown cells — Where do we go from here?

The purpose of this review is to describe the current status and to highlight several emerging trends in the manufacture of recombinant therapeutic proteins in cultivated mammalian cells, focusing on Chinese hamster ovary cells as the major production host. Over the past 25 years, specific and volumetric productivities for recombinant cell lines have increased about 20-fold as the result of improvements in media and bioprocess design. Future yield increases are expected to come from further developments in gene delivery and genetic selection for more efficient recovery of high-producing cell lines and in high-throughput cultivation systems to simplify medium design and bioprocess development. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight current research in our own laboratories.

Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection.

Using flow cytometry, single cell sorting, confocal microscopy and fluorescent plasmids, a thorough study of DNA uptake, DNA fate and DNA expression in mammalian cells transfected with the widely used calcium-phosphate precipitation method was executed. We show for the first time that up to 100,000 plasmid molecules can be delivered into individual cells, but also that DNA transfer into cells is a dynamic process that follows a defined kinetics of uptake and intracellular processing. Analyses by flow cytometry and confocal microscopy have also supported results suggesting endocytosis during Ca-Pi transfection. We also demonstrate that expression-enhancing treatment with glycerol during transfection did not result in increased DNA uptake. While cells with maximal DNA load appear to express the highest level of the transgene, these cells are negatively impacted in terms of growth and survival.

Transient Gene Expression in Suspension HEK-293 Cells: Application to Large-Scale Protein Production

Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r‐protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r‐proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension‐adapted HEK‐293 and CHO cells: (1) a calcium phosphate based method (Ca‐Pi), (2) a calcium‐mediated method called Calfection, and (3) a polyethylenimine‐based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK‐293 cells in bioreactors. The third method, entirely serum‐free, has been successfully applied to both suspension‐adapted CHO and HEK‐293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK‐293 EBNA cells of some out of more than 20 secreted r‐proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5–10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12‐well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch‐to‐batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.

100-liter transient transfection

This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2–4% of DNA in transfection cocktail)were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.