Unexpected mucin-type O-glycosylation and host-specific N-glycosylation of human recombinant interleukin-17A expressed in a human kidney cell line

The T helper cell-derived cytokine interleukin-17A (IL-17A) is a variably glycosylated disulfide-linked homodimer of 34–38 kDa. Its polypeptide monomer contains one canonical N-glycosylation site at Asn68, and human recombinant IL-17A was partly N-glycosylated when expressed in human kidney (HEK293) cells as a fusion protein with a melittin signal sequence and an N-terminal hexahistidine tag. Orbitrap mass analyses of the tryptic N-glycopeptide 63–69 indicated that the N-glycosylation was of the GalNAc-terminated type characteristic of cultured kidney cells. The mass spectrum of IL-17A monomer also included peaks shifted by +948 Da from the respective masses of unglycosylated and N-glycosylated polypeptides. These were caused by unpredicted partial O-glycosylation of Thr26 with the mucin-like structure -GalNAc(-NeuNAc)-Gal-NeuNAc. Identical O-glycosylation occurred in commercially sourced recombinant IL-17A also expressed in HEK293 cells but with a different N-terminal sequence. Therefore, the kidney host cell line not only imposed its characteristic pattern of N-glycosylation on recombinant IL-17A but additionally created an O-glycosylation not known to be present in the T cell-derived cytokine. Mammalian host cell lines for recombinant protein expression generally impose their characteristic patterns of N-glycosylation on the product, but this work exemplifies how a host may also unpredictably O-glycosylate a protein that is probably not normally O-glycosylated.     

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCA^r and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCA^r mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCA^r mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway.     

CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins

Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. (c) 1996 John Wiley & Sons, Inc.     

In vivo deglycosylation of recombinant glycoproteins in tobacco BY-2 cells

Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines.     

Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells

Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

     

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation,alter glycosylation processing,and produce altered glycoforms in CHO cell cultures

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d -glucose (2DG) and 5-thio- d -glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%–45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%–33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.     

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4‐O‐Bu₃ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4‐O‐Bu₃ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ～20% decrease in maximal viable cell density and ～30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4‐O‐Bu₃ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4‐O‐Bu₃ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up‐regulated more in sodium butyrate treated wild‐type, erythropoietin, or IgG expressing CHO‐K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4‐O‐Bu₃ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4‐O‐Bu₃ManNAc‐treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4‐O‐Bu₃ManNAc‐treatment. These findings demonstrate that 1,3,4‐O‐Bu₃ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4‐O‐Bu₃ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.

     

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.     

Combinatorial engineering of <Emphasis Type="Italic">ldh-a</Emphasis> and <Emphasis Type="Italic">bcl-2</Emphasis> for reducing lactate production and improving cell growth in dihydrofolate reductase-deficient Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate dehydrogenase-A (LDH-A), the enzyme converting pyruvate into lactate. However, despite the reduced lactate accumulation, such cell cultures are eventually terminated in the late period of the culture, mainly due to apoptosis. Therefore, we developed an apoptosis-resistant, less lactate-producing dhfr ⁻ CHO cell line (CHO-Bcl2-LDHAsi) by overexpressing Bcl-2, one of the most well-known anti-apoptotic proteins, and by downregulating LDH-A in a dhfr ⁻ CHO cell line. When the dhfr ⁻ CHO-Bcl2-LDHAsi cell line was used as a host cell line for the development of recombinant CHO (rCHO) cells producing an Fc-fusion protein, the culture longevity of the rCHO cells was extended without any detrimental effect of genetic engineering on specific protein productivity. Simultaneously, the specific lactate production rate and apparent yield of lactate from glucose were reduced to 21–65% and 37–78% of the control cells, respectively. Taken together, these results show that the use of an apoptosis-resistant, less lactate-producing dhfr ⁻ CHO cell line as a host cell line saves the time and the effort of establishing an apoptosis-resistant, less lactate-producing rCHO cells for producing therapeutic proteins.     

Beneficial effect of 30Kc6 gene expression on production of recombinant interferon-β in serum-free suspension culture of CHO cells

The Bombyx mori 30Kc gene is known to have anti-apoptotic activity and can enhance the cell growth and expression of recombinant proteins in anchorage-dependent CHO cell cultures. In this study, an interferon-β (IFN-β)-producing CHO cell line, which expresses the recombinant 30Kc6 gene, was constructed to investigate the effect of 30Kc6 expression on the production of IFN-β in serum-free suspension culture. The 30Kc6 expressing cell line showed lower apoptotic activity and prolonged cell viability under apoptotic conditions induced by the addition of sodium butyrate, staurosporine, or the removal of serum. The 30Kc6 expressing cell line also suppressed the loss of mitochondrial membrane potential induced under these conditions. It was observed that viability, and production of IFN-β were also enhanced by 30Kc6 expression in serum-free suspension cultures. These results indicate that the 30Kc6 gene can positively affect the viability and production of recombinant therapeutic proteins in serum-free suspension cultures of CHO cell lines.     

Electrophoretic analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian β1,4-galactosyltransferase

Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine -1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT, fluorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.     

Anxa2- and Ctsd-knockout CHO cell lines to diminish the risk of contamination with host cell proteins

Chinese hamster ovary (CHO) cells have been used as host cells in the production of a range of recombinant therapeutic proteins, including monoclonal antibodies and Fc-fusion proteins. Host cell proteins (HCP) represent impurities that must be removed from therapeutic formulations because of their potential risks for immunogenicity. While the majority of HCP impurities are effectively removed in typical downstream purification processes, clearance of a small population of HCP remains challenging. In this study, we knocked out the Anxa2 and Ctsd genes to assess the feasibility of knockout approaches for diminishing the risk of contamination with HCP. Using the CRISPR/Cas9 system, Anxa2-, and Ctsd-knockout CHO cell lines were successfully established, and we confirmed the complete elimination of the corresponding HCP in cell lysates. Importantly, all knockout cell lines showed similar growth and viability to those of the wild-type control during 8 days of cultivation. Thus, knockout of unrequired genes can reduce contamination with HCP in the production of recombinant therapeutic proteins.     

Enhanced Production of Human Recombinant Proteins from CHO cells Grown to High Densities in Macroporous Microcarriers

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q _P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q _P with increasing cell densities up to a threshold of >1 × 10⁸ cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q _P any further. A positive linear correlation (r ² = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 10⁶ to 3 × 10⁸ cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.     

Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre‐engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO‐S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO‐S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO‐S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO‐S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526–539 © 2009 Wiley Periodicals, Inc.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

Gene transfer effects on various cationic amphiphiles in CHO cells

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O′-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.     

Current achievements in the production of complex biopharmaceuticals with moss bioreactors

Transgenic plants are promising alternatives for the low-cost and safe pathogen-free production of complex recombinant pharmaceutical proteins (molecular farming). Plants as higher eukaryotes perform posttranslational modifications similar to those of mammalian cells. However, plant-specific protein N-glycosylation was shown to be immunogenic, a fact that represents a drawback for many plant systems in biopharmaceutical production. The moss Physcomitrella patens offers unique properties as a contained system for protein production. It is grown in the predominant haploid gametophytic stage as tissue suspension cultures in photobioreactors. Efficient secretory signals and a transient transfection system allow the secretion of freshly synthesized proteins to the surrounding medium. The key advantage of Physcomitrella compared to other plant systems is the feasibility of targeted gene replacements. By this means, moss strains with non-immunogenic humanized glycan patterns were created. Here we present an overview of the relevant aspects for establishing moss as a production system for recombinant biopharmaceuticals.     

APRT缺陷型CHO细胞系的建立及其重组蛋白表达能力评价北大核心CSCD

中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是生产复杂重组药物蛋白的首选宿主细胞,腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APRT)催化腺嘌呤与磷酸核糖缩合形成腺苷一磷酸,是嘌呤生物合成步骤中的关键酶。采用基因编辑技术敲除CHO细胞中aprt基因,验证获得的APRT缺陷型CHO细胞系的生物学特性;构建两种真核表达载体:对照载体(含有目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和弱化载体(含有启动子和起始密码子突变的aprt弱化表达盒及EGFP),分别转染APRT缺陷型和野生型CHO细胞并筛选获得稳定转染的细胞池;重组CHO细胞传代培养60代并用流式细胞术检测EGFP表达的平均荧光强度,并比较不同实验组重组蛋白EGFP的表达稳定性。PCR扩增和测序结果表明,CHO细胞aprt基因成功敲除;获得的APRT缺陷型CHO细胞系在细胞形态、生长增殖、倍增时间等生物学特性方面与野生CHO细胞无显著差异。目的蛋白瞬时表达结果表明,与野生型CHO细胞相比,转染对照载体和弱化载体的APRT缺陷型CHO细胞系中EGFP的表达分别提高了42%±6%和56%±9%;特别是长期传代培养时,转染弱化载体的APRT缺陷型细胞中EGFP表达量显著高于野生型CHO细胞(P<0.05);构建的基于APRT缺陷型CHO细胞系能够明显提高重组蛋白的长期表达稳定性。研究结果为建立高效稳定的CHO细胞表达系统提供了一种有效的细胞工程策略。