Yves Durocher

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2-6 months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This direct transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.

Therapeutic glycoprotein production in mammalian cells

Over the last years, the biopharmaceutical industry has significantly turned its biologics production towards mammalian cell expression systems. The presence of glycosylation machineries within these systems, and the fact that monoclonal antibodies represent today the vast majority of new therapeutic candidates, has largely influenced this new direction. Recombinant glycoproteins, including monoclonal antibodies, have shown different biological properties based on their glycan profiles. Thus, the industry has developed cell engineering strategies not only to improve cells specific productivity, but also to adapt their glycosylation profiles for increased therapeutic activity. Additionally, the advance of omics technologies has recently given rise to new possibilities in improving these expression platforms and will significantly help developing new strategies, in particular for CHO (Chinese Hamster Ovary) cells.

Metabolic engineering of CHO cells to alter lactate metabolism during fed-batch cultures.

Recombinant yeast pyruvate carboxylase (PYC2) expression was previously shown to be an effective metabolic engineering strategy for reducing lactate formation in a number of relevant mammalian cell lines, but, in the case of CHO cells, did not consistently lead to significant improvement in terms of cell growth, product titer and energy metabolism efficiency. In the present study, we report on the establishment of a PYC2-expressing CHO cell line producing a monoclonal antibody and displaying a significantly altered lactate metabolism compared to its parental line. All clones exhibiting strong PYC2 expression were shown to experience a significant and systematic metabolic shift toward lactate consumption, as well as a prolonged exponential growth phase leading to an increased maximum cell concentration and volumetric product titer. Of salient interest, PYC2-expressing CHO cells were shown to maintain a highly efficient metabolism in fed-batch cultures, even when exposed to high glucose levels, thereby alleviating the need of controlling nutrient at low levels and the potential negative impact of such strategy on product glycosylation. In bioreactor operated in fed-batch mode, the higher maximum cell density achieved with the PYC2 clone led to a net gain (20%) in final volumetric productivity.

Protein tyrosine kinases in human breast cancer: kinetic properties and evidence for the presence of two forms of native enzyme.

SummaryThe protein tyrosine kinase (PTK) of human breast tumors classified as positive (TM +) or negative (TM -) according to their estrogen and progestin receptor levels was partially characterized with regard to its distribution, kinetic parameters, molecular size, and ability to phosphorylate endogenous mammary proteins. For both types of tumors, PTK activity depended upon the presence of Mn++ (2–5 mM) and/or Mg++ (10–20 mM). The activities, total (per g of tissue) and specific (per mg of protein), were similar for both types of tumors, and an average of 60% of activity was located in cytosolic fractions. The autoradiographic detection of alkali-resistant phosphoproteins after SDS-PAGE showed very similar patterns between corresponding fractions from both types of tumors. Upon gel filtration, two peaks of activity of apparent Mr 245 kDa (peak I) and 47 kDa (peak II) were observed. Peak II was found in both cytosols and extracts from particulate fractions, while peak I was present only in the latter fraction for both TM + and TM — tumors. The apparent Kms for ATP ranged from 4.1 to 6.6 µM, and from 11 to 34 µg/ml for the synthetic substrate poly [Glu80, Tyr20], at an optimal pH of 6.5–7.5. When endogenous alkali-resistant phosphorylation of peaks I and II was determined by autoradiography after SDS-PAGE, two major mammary proteins of Mr 60 and 45 kDa were phosphorylated by peak II and three, Mr 145, 74, and 62 kDa, by peak I. Thus, in TM + and TM — human breast cancer tissues, PTKs possess similar enzymatic and molecular properties. The Mr 47 kDa is present in both soluble and particulate fractions, while the Mr 245 kDa is only observed in the latter fraction.

Production of Highly Sialylated Monoclonal Antibodies

The first monoclonal antibody (Mab), developed against kidney transplant rejection, was accepted by the FDA in 1986 [1]. Today, Mabs are leading the biotherapeutics market as 28 have been approved in Europe and the USA, and hundreds are in clinical trials [2-4]. Most of them are of IgG1 subtype, developed for cancer and immune disease treatments. Mabs clinical efficacy not only relies on specific target binding provided by their variable region, but also on their ability to trigger defense mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). These effector functions are mediated by the interaction between the antibody Fc fragment and the Fcγ-receptors expressed on immune cell surfaces or the molecules of the complement involved in ADCC and CDC respectively. In the last decade, these interactions were found to be highly dependent on on the presence and structure of the N-glycan linked to the Fc fragment [5, 6].

The major form of protein tyrosine kinase in the dog prostate is expressed by a 50 kDa polypeptide

We have already reported that the protein tyrosine kinase (PTK) activity in the dog prostate is distributed in cytosolic (75%) and particulate (Triton X‐100‐solubilized) fractions and that upon gel filtration, both PTKs migrate as entities of M r 44 000 [(1991) Biochem. Cell. Biol. 69, 146–153]. Herein we demonstrate by immunoprecipitation with anti‐phosphotyrosine antibodies that the soluble PTK has the ability to undergo self‐phosphorylation. In addition, the polypeptide responsible for that enzymatic activity has been identified by 2 approaches; (1) a two‐dimensional electrophoresis, in which the first dimension performed in non‐denaturing conditions allowed the localization of the native enzyme, while the second dimension (SDS‐PAGE) permitted the analysis of alkali‐resistant phosphoproteins corresponding to the activity; (2) protein renaturation after SDS‐PAGE followed by in situ phosphorylation (with [γ‐32P]ATP) of polyGT electrophoresed together with the enzyme preparation; the exclusive presence of the radiolabeled phosphotyrosine in the renatured protein confirmed its enzymatic nature. Using these methods, the major form of PTK in the dog prostate was shown to be expressed by a 50 kDa polypeptide which possesses autophosphorylation sites and which is present in the cytosol as an active monomer.

Transient Gene Expression in Suspension HEK293-EBNA1 Cells

Transient gene expression in human embryo kidney 293 (HEK293) cells is an established approach for the rapid production of large amounts of recombinant proteins (r-proteins). Milligram to gram quantities of r-proteins can be typically obtained within less than 10xa0days following transfection. In this chapter, we describe a simple and robust transfection process of suspension-growing human embryo kidney 293 cells using two commercially available serum-free media and polyethylenimine as the transfection reagent. This chapter provides examples for the production and purification of a his-tagged recombinant protein and two monoclonal antibodies.