Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 49
Filter
Add more filters










Publication year range
1.
Biotechnol Lett ; 40(1): 85-92, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28993910

ABSTRACT

OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.


Subject(s)
Cell Culture Techniques/methods , HLA-DR1 Antigen/biosynthesis , Recombinant Proteins/biosynthesis , Animals , Bioreactors , Biotechnology/methods , Cell Line , Cell Proliferation , Drosophila , HLA-DR1 Antigen/genetics , Recombinant Proteins/genetics
2.
Biotechnol Prog ; 32(5): 1174-1180, 2016 09.
Article in English | MEDLINE | ID: mdl-27453130

ABSTRACT

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred-tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH-controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process-dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension-adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174-1180, 2016.


Subject(s)
Bioreactors , Animals , CHO Cells , Cell Proliferation , Cells, Cultured , Cricetulus , Glycosylation , Hydrogen-Ion Concentration , Polysaccharides/analysis , Polysaccharides/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
3.
Anal Biochem ; 505: 26-8, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27130502

ABSTRACT

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.


Subject(s)
Bioreactors , Sf9 Cells/cytology , Animals , Cells, Cultured , Suspensions
4.
Biotechnol Bioeng ; 113(6): 1234-43, 2016 06.
Article in English | MEDLINE | ID: mdl-26616356

ABSTRACT

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. © 2015 Wiley Periodicals, Inc.


Subject(s)
Batch Cell Culture Techniques/methods , DNA Transposable Elements/genetics , Genetic Enhancement/methods , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Animals , CHO Cells , Cricetulus/genetics , Cricetulus/metabolism , Escherichia coli Proteins/genetics , Nerve Tissue Proteins/genetics , Transposases/genetics
5.
J Biotechnol ; 216: 67-75, 2015 Dec 20.
Article in English | MEDLINE | ID: mdl-26476358

ABSTRACT

The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc). After screening several promoter and enhancer combinations for high levels of TNFR:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2×10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNFR-Fc yields over 150µg/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells.


Subject(s)
Gene Expression , Lepidoptera/cytology , Plasmids/metabolism , Transfection/methods , Animals , Cell Count , Cell Line , DNA/metabolism , Humans , Polyethyleneimine/chemistry , Promoter Regions, Genetic , Reproducibility of Results , Suspensions , Transgenes
6.
Biotechnol Prog ; 31(6): 1571-8, 2015.
Article in English | MEDLINE | ID: mdl-26260195

ABSTRACT

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/10(6) 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.


Subject(s)
DNA/chemistry , DNA/isolation & purification , Plasmids/chemistry , Plasmids/isolation & purification , Transfection/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Electrophoresis, Polyacrylamide Gel , Hydrophobic and Hydrophilic Interactions , Polyethyleneimine/chemistry , Solvents/chemistry
7.
J Biotechnol ; 200: 61-9, 2015 Apr 20.
Article in English | MEDLINE | ID: mdl-25758242

ABSTRACT

Heterogeneous populations of stably transfected cells (cell pools) can serve for the rapid production of moderate amounts of recombinant proteins. Here, we propose the use of the piggyBac (PB) transposon system to improve the productivity and long-term stability of cell pools derived from Chinese hamster ovary (CHO) cells. PB is a naturally occurring genetic element that has been engineered to facilitate the integration of a transgene into the genome of the host cell. In this report PB-derived cell pools were generated after 10 days of selection with puromycin. The resulting cell pools had volumetric productivities that were 3-4 times higher than those achieved with cell pools generated by conventional plasmid transfection even though the number of integrated transgene copies per cell was similar in the two populations. In 14-day batch cultures, protein levels up to 600 and 800 mg/L were obtained for an Fc-fusion protein and a monoclonal antibody, respectively, at volumetric scales up to 1L. In general, the volumetric protein yield from cell pools remained constant for up to 3 months in the absence of selection. In conclusion, transfection of CHO cells with the PB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools for the rapid production of recombinant proteins.


Subject(s)
DNA Transposable Elements/genetics , Recombinant Proteins/metabolism , Animals , Antibodies, Monoclonal/metabolism , CHO Cells , Cricetinae , Cricetulus , Etanercept/metabolism , Plasmids , Recombinant Proteins/genetics , Transfection
8.
Biotechnol Prog ; 31(2): 541-9, 2015.
Article in English | MEDLINE | ID: mdl-25683738

ABSTRACT

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 10(3) 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.


Subject(s)
Polyethyleneimine/chemistry , Recombinant Proteins/metabolism , Transfection/methods , Animals , CHO Cells , Cricetinae , Cricetulus , DNA/genetics , DNA/pharmacokinetics , HEK293 Cells , Humans , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Plasmids/genetics , Plasmids/pharmacokinetics , Recombinant Proteins/genetics
10.
Int J Mol Cell Med ; 3(3): 146-56, 2014.
Article in English | MEDLINE | ID: mdl-25317401

ABSTRACT

The optimization of cell culture conditions for growth and productivity of recombinant Chinese hamster ovary (CHO) cells is a critical step in biopharmaceutical manufacturing. In the present study, the effects of the timing and amount of peptone feeding of a recombinant CHO cell line grown in a basal medium in serum-free suspension culture were determined for eight peptones of different origin (plant and casein). The amino acid content and the average molecular weight of the peptones chosen were available. In optimized feeding strategies with single peptones, increase 100 % volumetric productivity and 40 % in cell number were achieved. In feeding strategies with two peptones, several combinations stimulated protein productivity more than either peptone alone, depending on the peptone concentration and time of feeding. Some peptones, which did not stimulate productivity when added alone proved to be effective when used in combination. The combined peptones feeding strategies were more effective with peptones of different origin. Our data support the notion that the origin of peptones provides some guidance in identifying the most effective feeding strategies for recombinant CHO cells.

11.
Biomacromolecules ; 15(8): 2839-48, 2014 Aug 11.
Article in English | MEDLINE | ID: mdl-24963693

ABSTRACT

The success of gene therapy depends on safe and effective gene carriers. Despite being widely used, synthetic vectors based on poly(ethylenimine) (PEI), poly(l-lysine) (PLL), or poly(l-arginine) (poly-Arg) are not yet fully satisfactory. Thus, both improvement of established carriers and creation of new synthetic vectors are necessary. A series of biodegradable arginine-based ether-ester polycations was developed, which consists of three main classes: amides, urethanes, and ureas. Compared to that of PEI, PLL, and poly-Arg, much lower cytotoxicity was achieved for the new cationic arginine-based ether-ester polymers. Even at polycation concentrations up to 2 mg/mL, no significant negative effect on cell viability was observed upon exposure of several cell lines (murine mammary carcinoma, human cervical adenocarcinoma, murine melanoma, and mouse fibroblast) to the new polymers. Interaction with plasmid DNA yielded compact and stable complexes. The results demonstrate the potential of arginine-based ether-ester polycations as nonviral carriers for gene therapy applications.


Subject(s)
Biodegradable Plastics , Gene Transfer Techniques , Genetic Therapy/methods , Peptides , Plasmids , Animals , Biodegradable Plastics/chemistry , Biodegradable Plastics/pharmacology , HeLa Cells , Humans , Mice , NIH 3T3 Cells , Peptides/chemistry , Peptides/pharmacology , Plasmids/chemistry , Plasmids/pharmacology , Swine
12.
Adv Exp Med Biol ; 807: 59-73, 2014.
Article in English | MEDLINE | ID: mdl-24619618

ABSTRACT

Synthetic cationic polymers are of interest as both nonviral vectors for intracellular gene delivery and antimicrobial agents. For both applications synthetic polymers containing guanidine groups are of special interest since such kind of organic compounds/polymers show a high transfection potential along with antibacterial activity. It is important that the delocalization of the positive charge of the cationic group in guanidine significantly decreases the toxicity compared to the ammonium functionality. One of the most convenient ways for incorporating guanidine groups is the synthesis of polymers composed of the amino acid arginine (Arg) via either application of Arg-based monomers or chemical modification of polymers with derivatives of Arg. It is also important to have biodegradable cationic polymers that will be cleared from the body after their function as transfection or antimicrobial agent is fulfilled. This chapter deals with a two-step/one-pot synthesis of a new biodegradable cationic polymer-poly(ethylene malamide) containing L-arginine methyl ester covalently attached to the macrochains in ß-position of the malamide residue via the α-amino group. The goal cationic polymer was synthesized by in situ interaction of arginine methyl ester dihydrochloride with intermediary poly(ethylene epoxy succinimide) formed by polycondensation of di-p-nitrophenyl-trans-epoxy succinate with ethylenediamine. The cell compatibility study with Chinese hamster ovary (CHO) and insect Schneider 2 cells (S2) within the concentration range of 0.02-500 mg/mL revealed that the new polymer is not cytotoxic. It formed nanocomplexes with pDNA (120-180 nm in size) at low polymer/DNA weight ratios (WR = 5-10). A preliminarily transfection efficiency of the Arg-containing new cationic polymer was assessed using CHO, S2, H5, and Sf9 cells.


Subject(s)
Arginine/analogs & derivatives , Polymers/chemical synthesis , Animals , Arginine/chemistry , Cations , Cell Line , Humans , Polymers/pharmacology , Transfection
13.
J Biotechnol ; 171: 61-70, 2014 Feb 10.
Article in English | MEDLINE | ID: mdl-24333123

ABSTRACT

A method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed with the gene of interest being expressed from a plasmid carrying the homologous region 5 enhancer (hr5) and the immediate early 1 (ie1) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Under the optimal conditions described in the study, cells were transfected at a density of 30×106 cells/mL with 0.9 µg DNA and 1.35 µg of linear 25 kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4×106 cells/mL for the protein production phase. The volumetric yield of tumor necrosis factor receptor (ectodomain) fused to an Fc domain (TNFR-Fc) was about 100 µg/mL for cultures at volumes up to 300 mL. As expected, the molecular weight of the dimeric TNFR-Fc produced from Sf9 cells was about 6 kDa less than that produced from a recombinant Chinese hamster ovary (CHO) cell line due to differences in glycosylation between the two hosts. Transient transfection provides an alternative to the baculovirus expression vector system (BEVS) for the rapid production of recombinant proteins from Sf9 cells.


Subject(s)
Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Sf9 Cells/metabolism , Transfection/methods , Animals , CHO Cells , Cell Count , Cell Culture Techniques , Cricetinae , Cricetulus , Genetic Vectors/genetics , Nucleopolyhedroviruses/genetics , Plasmids/genetics , Polyethyleneimine , Promoter Regions, Genetic/genetics , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
14.
Protein Expr Purif ; 92(1): 67-76, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24021764

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.


Subject(s)
Drug Carriers/metabolism , Genetic Vectors/administration & dosage , Polyethyleneimine/metabolism , Transfection/methods , Animals , CHO Cells , Cell Culture Techniques/instrumentation , Cricetinae , Cricetulus , Equipment Design , HEK293 Cells , Humans , Recombinant Proteins/genetics , Viruses/genetics
15.
J Control Release ; 169(3): 276-88, 2013 Aug 10.
Article in English | MEDLINE | ID: mdl-23379996

ABSTRACT

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.


Subject(s)
DNA/administration & dosage , Dendrimers/chemistry , Polylysine/chemistry , Transfection , Animals , CHO Cells , Cricetinae , Cricetulus , DNA/genetics , Dendrimers/analysis , Dendrimers/metabolism , Green Fluorescent Proteins/genetics , Immunoglobulin G/genetics , Polylysine/analysis , Polylysine/metabolism
16.
Anal Biochem ; 430(2): 138-40, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22960013

ABSTRACT

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280nm (A(280)) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A(280) was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension.


Subject(s)
Cell Culture Techniques , Nanotechnology , Spectrophotometry, Ultraviolet , Animals , CHO Cells , Cell Survival/physiology , Cricetinae , Cricetulus , HEK293 Cells , Humans
17.
Biomacromolecules ; 13(10): 3127-37, 2012 Oct 08.
Article in English | MEDLINE | ID: mdl-22931162

ABSTRACT

Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC(50) values for the LPL analogues were ∼5-250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (≥3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain ε-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA.


Subject(s)
Dendrimers/pharmacology , Polylysine/pharmacology , Animals , Apoptosis/drug effects , CHO Cells , Cell Membrane/drug effects , Cell Membrane Permeability/drug effects , Cell Survival/drug effects , Cells, Cultured , Cricetinae , Dendrimers/chemical synthesis , Dendrimers/chemistry , Dose-Response Relationship, Drug , Molecular Structure , Molecular Weight , Polylysine/chemical synthesis , Polylysine/chemistry , Structure-Activity Relationship
18.
Macromol Biosci ; 12(6): 794-804, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22496075

ABSTRACT

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.


Subject(s)
Gene Expression , Immunoglobulin G/biosynthesis , Polylysine/chemistry , Transfection/methods , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin G/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
19.
N Biotechnol ; 29(4): 471-6, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22401955

ABSTRACT

In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 ± 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.


Subject(s)
Immunoglobulin G/chemistry , Polysaccharides/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Glycopeptides/analysis , Glycosylation , HEK293 Cells , Humans , Immunoglobulin G/biosynthesis , Immunoglobulin G/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
20.
Biotechnol Bioeng ; 109(9): 2271-8, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22422519

ABSTRACT

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 cell's transcription machinery, resulting in a higher level of transgene mRNA.


Subject(s)
DNA/genetics , Plasmids/genetics , Recombinant Proteins/biosynthesis , Transfection/methods , Animals , CHO Cells , Cricetinae , Cricetulus , DNA/chemistry , DNA/metabolism , Gene Expression , HEK293 Cells , Humans , Polyethyleneimine , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...