Commercial Production Of Recombinant Proteins Research Articles

Gene targeting in livestock

The development of nuclear transfer from tissue culture cells in livestock made it possible in principle to produce animals with subtle, directed genetic changes by in vitro modification of nuclear donor cells. In the short period since nuclear transfer was first performed, gene targeting in livestock has become a reality. Although gene targeting has immediate potential in biotechnology, it is unclear whether there are practical agricultural applications, at present. The first livestock targeting experiments have been directed at engineering animals either to render their organs immunologically compatible for human transplantation, or for improving the commercial production of recombinant proteins in the transgenic mammary gland. All successful examples of targeting have involved target loci that are expressed in the nuclear donor cell line. Two important barriers to the further development of this technology are adapting protocols for non-expressed genes and modifying procedures to enhance the lifespan of targeted cells in vitro. This review provides data that illustrate the difficulty in targeting non-expressed genes and discusses some of the practical issues associated with providing targeted nuclear donor cells that are competent for nuclear transfer.

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m−3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.

Virus-Based Vectors: A New Approach for Production of Recombinant Proteins

Recombinant proteins have become one of the basic human needs today for pharmaceutical, industrial and research purposes. The conventional methods of producing recombinant proteins, including bacterial systems, yeast, and human cells, have led to rising prices and biosafety problems for these products. Research on commercial production of recombinant proteins prompted a new method based on plant RNA viruses and plant hosts, which is known as Transient Expression System. Accordingly, a viral vector based on the plant RNA virus genome was designed in which the target gene is placed underneath a viral subgenomic promoter. In these vectors, despite the genetic manipulation, the ability to infect the entire plant that was present in the virus was preserved and, several days after plant infestation, it became bioreactor for the production of recombinant protein. The advances made in this field led to the creation of hybrid vectors based on plant viruses and Agrobacterium T-DNA (Transfer DNA). The amount of recombinant protein produced by these vectors was up to 5 grams per kilogram of fresh weight, which is considered a reliable record. The abundant benefits of this method have attracted the attention of researchers in this way, including the rapid and high production rate, reduced production costs and bio-safety of manufactured products. Several recombinant drugs are currently produced by the transient expression system, either delivered to the consumer or by clinical trials. This review presents how to discover, creation and development of viral vectors in transient expression systems in order to produce recombinant proteins.

Efficient production of human interferon beta in the white of eggs from ovalbumin gene\u2013targeted hens

Transgenic chickens could potentially serve as bioreactors for commercial production of recombinant proteins in egg white. Many transgenic chickens have been generated by randomly integrating viral vectors into their genomes, but transgene expression has proved insufficient and/or limited to the initial cohort. Herein, we demonstrate the feasibility of integrating human interferon beta (hIFN-β) into the chicken ovalbumin locus and producing hIFN-β in egg white. We knocked in hIFN-β into primordial germ cells using a CRISPR/Cas9 protocol and then generated germline chimeric roosters by cell transplantation into recipient embryos. Two generation-zero founder roosters produced hIFN-β knock-in offspring, and all knock-in female offspring produced abundant egg-white hIFN-β (~3.5 mg/ml). Although female offspring of the first generation were sterile, their male counterparts were fertile and produced a second generation of knock-in hens, for which egg-white hIFN-β production was comparable with that of the first generation. The hIFN-β bioactivity represented only ~5% of total egg-white hIFN-β, but unfolding and refolding of hIFN-β in the egg white fully recovered the bioactivity. These results suggest that transgene insertion at the chicken ovalbumin locus can result in abundant and stable expression of an exogenous protein deposited into egg white and should be amenable to industrial applications.

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture.

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications.

Media development for large scale Agrobacterium tumefaciens culture.

A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH4 )2 SO4 ), magnesium sulfate heptahydrate (MgSO4 *7H2 O), calcium chloride dihydrate (CaCl2 *2H2 O), iron (II) sulfate heptahydrate (FeSO4 *7H2 O), manganese (II) sulfate monohydrate (MnSO4 *H2 O), zinc sulfate heptahydrate (ZnSO4 *7H2 O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na2 HPO4 /KH2 PO4 ). Calcium and zinc were found to have no detectable impact on biomass concentration or transient expression level, and concentrations of the other components that maximized final biomass concentration were determined. The maximum specific growth rate of Agrobacterium strain C58C1 pTFS40 in this media was 0.33 ± 0.01 h-1 and the final biomass concentration after 26 h of batch growth in shake flasks was 2.6 g dry cell weight/L. Transient expression levels of the reporter protein GUS following infiltration of a recombinant Agrobacterium strain C58C1 into N. benthamiana were comparable when the strain was grown in the defined media, Lysogeny Broth (LB) media, or yeast extract-peptone (YEP) media. In LB and YEP media, free amino acid concentration was measured at three points over the course of batch growth of Agrobacterium strain C58C1 pTFS40; results indicated that l-serine and l-asparagine were depleted from the media first, followed by l-alanine and l-glutamic acid. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1218-1225, 2017.

Human \u03b2-defensin-2 production from S. cerevisiae using the repressible MET17 promoter

BackgroundBaker’s yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems.ResultsHere we show improved secretion of the antimicrobial protein, human β-defensin-2, (hBD2), using the S. cerevisiae MET17 promoter by repressing expression during the growth phase. In shake flask culture, a higher final concentration of human β-defensin-2 was obtained using the repressible MET17 promoter system than when using the strong constitutive promoter from proteinase B (PRB1) in a yeast strain developed for high-level commercial production of recombinant proteins. Furthermore, this was achieved in under half the time using the MET17 promoter compared to the PRB1 promoter. Cell density, plasmid copy-number, transcript level and protein concentration in the culture supernatant were used to study the effects of different initial methionine concentrations in the culture media for the production of human β-defensin-2 secreted from S. cerevisiae.ConclusionsThe repressible S. cerevisiae MET17 promoter was more efficient than a strong constitutive promoter for the production of human β-defensin-2 from S. cerevisiae in small-scale culture and offers advantages for the commercial production of this and other heterologous proteins which are deleterious to the host organism. Furthermore, the MET17 promoter activity can be modulated by methionine alone, which has a safety profile applicable to biopharmaceutical manufacturing.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging.

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low‐density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 31:1172–1178, 2015

유전자재조합 CHO 세포에서 Histone Deacetylase Inhibitor를 이용한Albumin-erythropoietin 생산성 증진

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for the commercial production of recombinant proteins. However, they show relatively low yields of recombinant proteins in comparison with microbial cells. Various strategies have been tried to overcome this drawback. The acetyl moieties are attached to the N-terminus of histone by histone acetyltransferase (HAT) while histone deacetylase (HDAC) removes histone-bound acetyl groups. HDAC inhibitor (HDACi), such as sodium butyrate, sodium propionate and valproic acid, can enhance specific productivity of CHO cells. Human albumin-erythropoietin (Alb-EPO) is a novel 105 kDa protein comprising recombinant human EPO fused to human albumin. In this study, we examined the effects of HDACi on the production of Alb-EPO in CHO cells with various concentrations in the range of 0-1 mM. The results showed that sodium butyrate was found to be the best HDACi for enhancing productivity. It enhanced not only the production of Alb-EPO but also the apoptosis of recombinant CHO cells.

Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

Evaluating nuclear transgene expression systems in Chlamydomonas reinhardtii

Chlamydomonas offers several potential advantages as a single-celled autotrophic recombinant protein production system including: facile transformation systems for all three genomes, low production costs, and the ability to secrete proteins. However, transgene expression levels from the nuclear genome of Chlamydomonas are often inadequate for industrial applications. With the objective of optimizing nuclear transgene expression, we surveyed transgene (luciferase) expression driven by seven different nuclear gene promoters and three different transcription terminators. The results demonstrate that in addition to the choice of nuclear gene promoters used, transcriptional terminators can have strong influence on transgene expression. We show that the psaD terminator improved transgene expression when paired with a variety of different gene promoters. Among those tested, the psaD–psaD promoter–terminator expression cassette gave the highest expression levels. This expression cassette was then used to express a human protein of pharmaceutical value, human butyrylcholinesterase (huBuChE). Chlamydomonas cells were able to accumulate the luciferase–huBuChE fusion protein to 0.4% of total soluble protein levels, which is comparable to the expression levels of the same protein obtained in plants. In addition, this study found that the form (linear or supercoiled) of the transforming DNA used for Chlamydomonas transformation had significant impact on the transformation efficiency and the level of transgene expression and stability. These results demonstrate that transgene expression in Chlamydomonas can potentially be scaled for commercial production of recombinant proteins.

New Generation Expression Host System- Aiming high for commercial production of recombinant protein

The global requirements of recombinant protein have increased exponentially in last one decade in various aspects. The commercial production of recombinant proteins requires an ideal host expression system which effectively meets the demand. Numerous host expression systems have been used to achieve level of expression. Escherichia coli BL21 (DE3) have been used ideally for expression of numbers of recombinant proteins from many decades. Though, Escherichia coli Bl21 is quite enough to express recombinant protein in higher fold but often get fails to achieve over expression of protein as essential requirement for today demand. Recently many engineered strains have been implemented in order to bring expression level as per requirement. The complications which arise in the expression of recombinant protein are mainly due to codon bias or inability of vector promoter to utilize host polymerase; while for over-expression of recombinant proteins often inhibit itself due to protein toxicity in conventional expression host systems. Escherichia coli C41 (DE3), Escherichia coli C43 (DE3) and Escherichia coli Rosetta are the refined and engineered strains which have shown over-expression of recombinant protein in recent time. These strains are designed to overcome the complication often limits conventional expression system.

Contained and high‐level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor

Chloroplast transformation is a promising approach for the commercial production of recombinant proteins in plants. However, gene containment still remains an issue for the large-scale cultivation of transplastomic plants in the field. Here, we have evaluated the potential of using tobacco transplastomic cell suspensions for the fully contained production of a modified form of the green fluorescent protein (GFP+) and, a vaccine antigen, fragment C of tetanus toxin (TetC). Expression of these proteins in cell suspension cultures (and calli) was much less than in leaves, reaching 0.5%-1.5% of total soluble protein (TSP), but still produced 2.4-7.2 mg/L of liquid culture. Much better expression levels were achieved with a novel protein production platform in which transgenic cell suspension cultures were placed in a temporary immersion bioreactor in the presence of Thidiazuron to initiate shoot formation. GFP+ yield reached 660 mg/L of bioreactor (33% TSP), and TetC accumulated to about 95 mg/L (8% TSP). This new production platform, combining the rapid generation of transplastomic cell suspension cultures and the use of temporary immersion bioreactors, is a promising route for the fully contained low-cost production of recombinant proteins in chloroplasts.

Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis

Although many different crop species have been used to produce a wide range of vaccines, antibodies, biopharmaceuticals and industrial enzymes, tobacco has the most established history for the production of recombinant proteins. To further improve the heterologous protein yield of tobacco platforms, transient and stable expression of four recombinant proteins (i.e. human erythropoietin and interleukin-10, an antibody against Pseudomonas aeruginosa, and a hyperthermostable α-amylase) was evaluated in numerous species and cultivars of Nicotiana. Whereas the transient level of recombinant protein accumulation varied significantly amongst the different Nicotiana plant hosts, the variety of Nicotiana had little practical impact on the recombinant protein concentration in stable transgenic plants. In addition, this study examined the growth rate, amount of leaf biomass, total soluble protein levels and the alkaloid content of the various Nicotiana varieties to establish the best plant platform for commercial production of recombinant proteins. Of the 52 Nicotiana varieties evaluated, Nicotiana tabacum (cv. I 64) produced the highest transient concentrations of recombinant proteins, in addition to producing a large amount of biomass and a relatively low quantity of alkaloids, probably making it the most effective plant host for recombinant protein production.

Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

The culture of Escherichia coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct retards cell growth, inhibits protein formation, and diverts carbon from biomass to protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP–PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutation caused a severe reduction in growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation, and eliminated acetate accumulation. The mutant strain (TC110) apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2×LB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of significantly reduced acetate accumulation (9.1±6.6 vs. 90.4±1.6 mM in GJT001, P<0.05). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5±0.7 in TC110 vs. 8.0±0.1 in GJT001, P<0.05). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and β-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flasks with 2×LB broth with 2% glucose. The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation=466±35%) than in TC110 (coefficient of variation=55±1%). In corn steep liquor medium with 2% glucose, we observed a 28.5-fold improvement in final volumetric production of GFP in TC110 over GJT001. TC110 had a 7.5-fold improvement in final volumetric productivity of β-galactosidase over GJT001 in 2×LB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2×LB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors.

Application of sodium propionate to the suspension culture of Chinese hamster ovary cells for enhanced production of follicle-stimulating hormone

In this study, we found that adding sodium propionate to batch culture medium resulted in cell growth suppression in a dosedependent manner, while it enhanced follicle-stimulating hormone (FSH) production in Chinese hamster ovary (CHO) cells. In a pseudo-perfusion culture, with the exchange of fresh medium containing 2.5 mM sodium propionate, a final FSH of 532 μg was obtained, which was approximately 1.5-fold higher than the control. Overall, the results demonstrate that the application of sodium proplonate for the commercial production of recombinant proteins in rCHO cells is feasible.

Production of scFv-Fc fusion protein using genetically manipulated quails

The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins.

Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo

Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.

Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry.

Rapid growth in the biotechnology industry has led to a dramatic increase in attention to the protein folding problem. Understanding protein-folding pathways is essential to the production of biopharmaceuticals since commercial production of recombinant proteins often requires a protein-refolding process for recovery of high yields. Protein folding coupled to the formation of disulfide bonds presents one of the simplest approaches to studying folding intermediates. On-line capillary isoelectric focusing-electrospray ionization mass spectrometry (CIEF-ESIMS) is demonstrated for kinetic studies of disulfide bond-induced protein refolding. Refolding intermediates of bovine pancreatic ribonuclease A, a model system for this study, are blocked at different stages by alkylating free thiols with iodoacetate. The alkylation reaction results in the introduction of charge (-1) and mass (59) differences for each alkylation site, providing the means for predictable separation and direct identification of refolding intermediates using CIEF-ESIMS. Besides the observation of refolding intermediates containing different numbers of disulfide bonds and even mixed disulfides, the two-dimensional resolving power of CIEF-ESIMS allows the determination of conformational heterogeneity among groups of refolding intermediates.