RESUMO
Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN® GS-/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-ß-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc.
Assuntos
Células CHO , Resistência à Doença/genética , Engenharia Genética/métodos , Interações Hospedeiro-Patógeno/genética , Vírus Miúdo do Camundongo/imunologia , Ácido N-Acetilneuramínico/genética , Animais , Cricetinae , Cricetulus , Interações Hospedeiro-Patógeno/imunologia , Modelos Biológicos , Ácido N-Acetilneuramínico/imunologia , Ácido N-Acetilneuramínico/metabolismoRESUMO
N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production.
Assuntos
Engenharia Celular/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Engenharia Metabólica , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismoRESUMO
We report the discovery and validation of a novel CHO cell engineering target for improving IgG expression, serpin peptidase inhibitor, clade B, member 1 (Serpinb1). Transcriptomic studies using microarrays revealed that Serpinb1 was up-regulated in cultures with IgG heavy and light chain transcription transiently repressed compared with cultures treated with non-targeting siRNA. As proof of concept, a lentiviral vector was employed to overexpress the Chinese Hamster Serpinb1 in a CHOZN(®) Glutamine Synthetase (-/-) recombinant IgG producing CHO line. The lentiviral stable pool demonstrated 4.2-fold SERPINB1 overexpression compared with the non-transduced control. The peak viable cell density (VCD) and peak IgG volumetric productivity of the lentiviral stable pool increased 1.3 and 2.0 fold, respectively, compared with the non-transduced control. For host cell engineering, a plasmid encoding SERPINB1 was transfected into the CHOZN(®) GS (-/-) host cell line to create several stable pools. Single-cell clones isolated from the pools were characterized for their SERPINB1 expression levels and growth. The clone (SERPINB1_OE_27) with the highest SERPINB1 expression had decreased peak viable cell density and exponential phase growth rate. Selected SERPINB1 OE clones were subsequently evaluated for their IgG expression capabilities using GS selection. Clone SERPINB1_OE_42 with moderate SERPINB1 overexpression demonstrated increased IgG productivity in "bulk" selection. We conclude that manipulating Serpinb1 expression can lead to increased recombinant IgG productivity, but the effect in host cell lines may vary by clone and by overexpression level. This work represents the ongoing effort in applying "-omics" findings to novel CHO host cell line engineering.
Assuntos
Imunoglobulina G/metabolismo , Proteínas Recombinantes/metabolismo , Serpinas/metabolismo , Animais , Biotecnologia , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/análise , Imunoglobulina G/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Serpinas/química , Serpinas/genéticaRESUMO
The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).
Assuntos
Acetilgalactosamina/metabolismo , Eritropoetina/biossíntese , Glicoproteínas/química , N-Acetilgalactosaminiltransferases/metabolismo , Proteoma/química , Proteínas Recombinantes/biossíntese , Acetilgalactosamina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Engenharia Celular , Linhagem Celular , Cricetulus , Eritropoetina/genética , Expressão Gênica , Glicômica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/genética , Proteoma/genética , Proteoma/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Alinhamento de Sequência , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production.
Assuntos
Endorribonucleases/genética , N-Acetilglucosaminiltransferases/genética , Fatores de Transcrição/genética , Animais , Células CHO , Cricetinae , Cricetulus , Técnicas de Inativação de Genes , Glutamato-Amônia Ligase/genética , Imunoglobulina G/metabolismo , Proteínas RecombinantesRESUMO
Recombinant glycoproteins produced by mammalian cells represent an important category of therapeutic pharmaceuticals used in human health care. Of the numerous sugars moieties found in glycoproteins, the terminal sialic acid is considered particularly important. Sialic acid has been found to influence the solubility, thermal stability, resistance to protease attack, antigenicity, and specific activity of various glycoproteins. In mammalian cells, it is often desirable to maximize the final sialic acid content of a glycoprotein to ensure its quality and consistency as an effective pharmaceutical. In this study, CHO cells overexpressing recombinant human interferon gamma (hIFNgamma) were treated using short interfering RNA (siRNA) and short-hairpin RNA (shRNA) to reduce expression of two newly identified sialidase genes, Neu1 and Neu3. By knocking down expression of Neu3 we achieved a 98% reduction in sialidase function in CHO cells. The recombinant hIFNgamma was examined for sialic acid content that was found to be increased 33% and 26% respectively with samples from cell stationary phase and death phase as compared to control. Here, we demonstrate an effective targeted gene silencing strategy to enhance protein sialylation using RNA interference (RNAi) technology.
Assuntos
Inativação Gênica , Glicoproteínas/biossíntese , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/genética , RNA Interferente Pequeno/genética , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Clonagem Molecular , Cricetinae , Cricetulus , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Humanos , Interferon gama/biossíntese , Interferon gama/química , Interferon gama/genética , Dados de Sequência Molecular , Neuraminidase/biossíntese , Proteínas Recombinantes , Alinhamento de SequênciaRESUMO
In the present study, we have identified species-specific housekeeping genes (HKGs) for Chinese Hamster Ovary (CHO) cells using data from microarray gene expression profiling. HKGs suitable for quantitative RT-PCR normalization should display relatively stable expression levels across experimental conditions. We analyzed transcription profiles of several IgG-producing recombinant CHO cell lines under numerous culture conditions using a custom CHO DNA microarray platform and calculated relative expression variability from 124 microarrays. We selected a novel panel of candidate HKGs based on their low variability in expression from the microarray data. Compared to three traditional HKGs (Gapdh, Actb, and B2m), the majority of genes on this panel demonstrated lower or equal variability. Each candidate HKG was then validated using qRT-PCR. Final selection of CHO-specific HKGs include Actr5, Eif3i, Hirip3, Pabpn1, Vezt, Cog1, and Yaf2. The results reported here provide a useful tool for gene expression analyses in CHO cells, a critical expression platform used in biotherapeutics.
Assuntos
Células CHO , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Cricetinae , Cricetulus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse FisiológicoRESUMO
The LEAP (Laser-Enabled Analysis and Processing) platform combines in situ imaging with laser manipulation to efficiently identify, purify, and monitor expansion of high secreting clones. It also allows for rapid analysis of cell population heterogeneity. This unit describes the LEAP instrumentation as well as basic and alternate protocols of the major applications in recombinant human or humanized IgG expression. The protocols include fluorescent cell counting, secreted recombinant IgG capture and detection, and IgG-secreting clone selection by laser processing.
Assuntos
Citometria de Varredura a Laser/métodos , Lasers , Adesão Celular , Contagem de Células , Técnicas de Cultura de Células , Células Cultivadas , Células Clonais , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Expressão Gênica , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Citometria de Varredura a Laser/instrumentação , Proteínas Recombinantes/metabolismoRESUMO
Enzymes from thermophiles are preferred for industrial applications because they generally show improved tolerance to temperature, pressure, solvents, and pH as compared with enzymes from mesophiles. However, nearly all thermostable enzymes used in industrial applications or available commercially are produced as recombinant enzymes in mesophiles, typically Escherichia coli. The development of high-temperature bioprocesses, particularly those involving cofactor-requiring enzymes and/or multi-step enzymatic pathways, requires a thermophilic host. The extreme thermophile most amenable to genetic manipulation is Thermus thermophilus, but the study of expression of heterologous genes in T. thermophilus is in its infancy. While several heterologous genes have previously been expressed in T. thermophilus, the data reported here include the first examples of the functional expression of a gene from an archaeal hyperthermophile ( bglA from Pyrococcus woesei), a cofactor-requiring enzyme ( dszC from Rhodococcus erythropolis IGTS8), and a two-component enzyme ( carBa and carBb from Sphingomonas sp. GTIN11). A thermostable derivative of pnbA from Bacillus subtilis was also expressed, further expanding the list of genes from heterologous hosts that have been expressed in T. thermophilus.
Assuntos
Dioxigenases , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Thermus thermophilus/enzimologia , Thermus thermophilus/genética , beta-Galactosidase/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Hidrolases de Éster Carboxílico/genética , Cloranfenicol O-Acetiltransferase/genética , Regulação da Expressão Gênica em Archaea , Microbiologia Industrial , Oxirredutases/genética , Oxigenases/genética , Plasmídeos , Pyrococcus/enzimologia , Pyrococcus/genética , Rhodococcus/enzimologia , Rhodococcus/genética , Sphingomonas/enzimologia , Sphingomonas/genética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
An alkane-degrading bacterium, designated GTI MVAB Hex1(T), was isolated from chronically crude oil-contaminated soil from an oilfield in southern Illinois. The isolate grew very weakly or not at all in minimal or rich media without hydrocarbons. Straight-chain aliphatic hydrocarbons, such as hexadecane and heptadecane, greatly stimulated growth; shorter-chain (=C(15)) hydrocarbons did not (with decane as the sole exception). Growth was also greatly enhanced by the branched aliphatic hydrocarbons pristane and squalane. The latter of these was most intriguing, as catabolism of squalane has hitherto been reported only for Mycobacterium species. Although unable to utilize mono- or polycyclic aromatic hydrocarbons as sole carbon sources, the isolate did show slight fluorene-mineralizing capability in Luria-Bertani medium, which was partially repressed by hexadecane. In contrast, hexadecane supplementation greatly increased mineralization of (14)C-dodecane, which was not a growth substrate. Further testing emphasized the isolate's extremely narrow substrate range, as only Tween 40 and Tween 80 supported significant growth. Microscopic examination (by scanning and transmission electron microscopy) revealed a slightly polymorphic coccoidal to bacillar morphology, with hydrocarbon-grown cells tending to be more elongated. When grown with hexadecane, GTI MVAB Hex1(T) accumulated a large number of electron-transparent intracytoplasmic inclusion bodies. These were also prevalent during growth in the presence of squalane. Smaller inclusion bodies were observed occasionally with pristane supplementation; they were, however, absent during growth on crude oil. On the basis of 16S rRNA gene sequence data and range of growth substrates, classification of this isolate as the type strain of Alkanindiges illinoisensis gen. nov., sp. nov. is proposed, which is most closely related (approx. 94 % sequence similarity) to Acinetobacter junii.
Assuntos
Moraxellaceae/isolamento & purificação , Moraxellaceae/metabolismo , Esqualeno/metabolismo , Aerobiose , Biodegradação Ambiental , DNA Bacteriano/genética , DNA Ribossômico/genética , Óleos Combustíveis , Hidrocarbonetos/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Moraxellaceae/classificação , Moraxellaceae/genética , Fenótipo , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Microbiologia do SoloRESUMO
A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.