Yashas Rajendra

A simple high-yielding process for transient gene expression in CHO cells.

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10⁶ cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE.

DNA delivery with hyperbranched polylysine: A comparative study with linear and dendritic polylysine

PEI and polylysine are among the most investigated synthetic polymeric carriers for DNA delivery. Apart from their practical use, these 2 classes of polymers are also of interest from a fundamental point of view as they both can be prepared in different architectures (linear and branched/dendritic) and in a wide range of molecular weights, which is attractive to establish basic structure-activity relationships. This manuscript reports the results of an extensive study on the influence of molecular weight and architecture of a library of polylysine variants that includes linear, dendritic and hyperbranched polylysine. Hyperbranched polylysine is a new polylysine-based carrier that is structurally related to dendritic polylysine but possesses a randomly branched structure. Hyperbranched polylysine is attractive as it can be prepared in a one-step process on a large scale. The performance of these 3 classes of polylysine analogs was evaluated by assessing eGFP and IgG production in transient gene expression experiments with CHO DG44 cells, which revealed that protein production generally increased with increasing molecular weight and that at comparable molecular weight, the hyperbranched analogs were superior as compared to the dendritic and linear polylysines. To understand the differences between the gene delivery properties of the hyperbranched polylysine analogs on the one hand and the dendritic and linear polylysines on the other hand, the uptake and trafficking of the corresponding polyplexes were investigated. These experiments allowed us to identify (i) polyplex-external cell membrane binding, (ii) free, unbound polylysine coexisting with polyplexes as well as (iii) polymer buffer capacity as three possible factors that may contribute to the superior transfection properties of the hyperbranched polylysines as compared to their linear and dendritic analogs. Altogether, the results of this study indicate that hyperbranched polylysine is an interesting, alternative synthetic gene carrier. Hyperbranched polylysine can be produced at low costs and in large quantities, is partially biodegradable, which may help to prevent cumulative cytotoxicity, and possesses transfection properties that can approach those of PEI.

Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells.

Abstract Transient gene expression (TGE) from mammalian cells is an increasingly important tool for the rapid production of recombinant proteins for research applications in biochemistry, structural biology, and biomedicine. Here we review methods for the transfection of human embryo kidney (HEK-293) and Chinese hamster ovary (CHO) cells in suspension culture using the cationic polymer polyethylenimine (PEI) for gene delivery.

Role of non-specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK-293E cells

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non‐specific (filler) DNA to partially replace the transgene‐bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK‐293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady‐state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA–polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA–PEI complexes, leading to a higher percentage of transfected cells. The co‐assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cells transcription machinery, resulting in a higher level of transgene mRNA. Biotechnol. Bioeng. 2012;109: 2271–2278.

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS‐knockout cell line: Process development and product quality assessment

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS‐KO host cell line. By creating a bi‐allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS‐KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)‐mediated transfections were performed at high cell density (4 × 106 cells/mL) followed by immediate growth arrest at 32°C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre‐complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7‐day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc‐fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7‐day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS‐KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293. Biotechnol. Bioeng. 2015;112: 977–986.

Hyperbranched Polylysine: A Versatile, Biodegradable Transfection Agent for the Production of Recombinant Proteins by Transient Gene Expression and the Transfection of Primary Cells

The feasibility of a new transfection agent, HBPL, for the production of recombinant IgG antibody via TGE as well as for the transfection of primary cells is studied. Under the conditions investigated, transfection of CHO-DG44 cells using HBPL results in IgG yields that are comparable to those obtained with PEI. In experiments with CHO-K1 cells and MEFs the use of HPBL allows to achieve transfection efficiencies comparable to or better than those obtained with PEI or Fugene®. HBPL-mediated transfection does not require complex pre-formation, works well in serum-containing media and is biodegradable, which may prevent cumulative cytotoxicity and facilitates downstream processing.

Comparison of three transposons for the generation of highly productive recombinant CHO cell pools and cell lines

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons—piggyBac, Tol2, and Sleeping Beauty—for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large‐scale production of recombinant proteins using Chinese hamster ovary cells (CHO‐DG44) as the host. Transfection with each of the dual‐vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor‐Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10–12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2‐month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14‐day fed‐batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234–1243.

Transcriptional and post‐transcriptional limitations of high‐yielding, PEI‐mediated transient transfection with CHO and HEK‐293E cells

Transient gene expression (TGE) in human embryonic kidney (HEK‐293) and Chinese hamster ovary (CHO) cells is a well‐established technology for the rapid generation of recombinant proteins. Although the maximum TGE yields have reached 1 g/L or more, the amount of plasmid DNA (pDNA) required for transfection remains high. Although greater than 103 copies of pDNA are present per transfected cell, protein yields are still lower than those achieved in recombinant cell lines with only one or a few copies of the transgene. This indicates a clear limitation to TGE in terms of the maximum level of recombinant protein production. In this study, we investigated the limitations to high‐yielding TGE processes with CHO and HEK‐293E cells using a monoclonal antibody as a model protein. For either cell host, both the intracellular and intranuclear pDNA levels increased linearly with the amount of pDNA added to the culture. In contrast, transgene mRNA accumulation reached a plateau as the intranuclear pDNA amount increased, suggesting a limitation in pDNA transcription. A post‐transcriptional limitation to TGE yields was revealed by calculating the amount of antibody produced per transgene mRNA (mRNA utilization). For both hosts the transgene mRNA utilization decreased dramatically when transfected pDNA amounts increased beyond the level giving the maximum protein yield. The post‐transcriptional limitation did not appear to be due to bottlenecks in antibody assembly or secretion, suggesting that transgene mRNA translation may be limiting. The results show that TGE yields are not limited by pDNA delivery into the nuclei, but in pDNA and transgene mRNA utilization.

Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO–DG44 cells with 0.06 μg pDNA/106 cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7‐day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine‐mediated pDNA delivery into cells or nuclei. The steady‐state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half‐life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc‐fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high‐yielding TGE processes with CHO–DG44 cells by decreasing the amount of pDNA required for transfection.

Transient gene expression with CHO cells in conditioned medium: a study using TubeSpin® bioreactors

Background Transient gene expression (TGE) allows rapid protein production in mammalian cells and has become an important tool in the pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)-mediated, high-density transfection allowed to express recombinant proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly efficient protocols are available, volumetric scale-up of TGE is still a challenge. A major issue is the need to perform the transfection in fresh medium rather than in conditioned (spent) medium. This implies a medium exchange step just before transfection. In CHO-DG44 [3] cells we observed up to a 100-fold decrease in volumetric protein production if transfections were performed in conditioned medium, compared to fresh medium. The reasons for such a negative effect of conditioned medium on transfectability and/or protein production expression are not yet known. To study this problem we transfected CHO cells at small-scale in TubeSpin bioreactor 50 tubes using 41 different commercially available serum-free media formulations in combination with different transfection parameters and culture conditions. By comparing the transient production of a recombinant IgG antibody among the different media, we observed variation of up to 400-fold when transfecting in fresh media and up to 20-fold when using conditioned medium. The optimization of the PEI:DNA ratio allowed a significant improvement in yields of transfection in conditioned medium. Methods Suspension-adapted CHO-DG44 cells [3] were routinely cultivated in TubeSpin bioreactor 50 tubes (TPP, Trasadingen, Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. The 38 media samples for transfection were provided by Excellgene SA. Conditioned medium is defined as a cell culture medium where cells have been growing for more than two days up to a density between 4-5 million cells/mL. Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 μg of pDNA and 76 μg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)