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1.
Biotechnol Bioeng ; 114(3): 576-588, 2017 03.
Article in English | MEDLINE | ID: mdl-27642072

ABSTRACT

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.


Subject(s)
CHO Cells , Disease Resistance/genetics , Genetic Engineering/methods , Host-Pathogen Interactions/genetics , Minute Virus of Mice/immunology , N-Acetylneuraminic Acid/genetics , Animals , Cricetinae , Cricetulus , Host-Pathogen Interactions/immunology , Models, Biological , N-Acetylneuraminic Acid/immunology , N-Acetylneuraminic Acid/metabolism
2.
Biotechnol Prog ; 31(2): 334-46, 2015.
Article in English | MEDLINE | ID: mdl-25641927

ABSTRACT

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.


Subject(s)
Cell Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sialyltransferases/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Metabolic Engineering , N-Acetylneuraminic Acid/analysis , N-Acetylneuraminic Acid/metabolism , Polysaccharides/metabolism
3.
J Biotechnol ; 193: 91-9, 2015 Jan 10.
Article in English | MEDLINE | ID: mdl-25444873

ABSTRACT

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.


Subject(s)
Immunoglobulin G/metabolism , Recombinant Proteins/metabolism , Serpins/metabolism , Animals , Biotechnology , CHO Cells , Cricetinae , Cricetulus , Immunoglobulin G/analysis , Immunoglobulin G/genetics , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Serpins/chemistry , Serpins/genetics
4.
J Biotechnol ; 167(1): 24-32, 2013 Aug 10.
Article in English | MEDLINE | ID: mdl-23777858

ABSTRACT

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.


Subject(s)
Endoribonucleases/genetics , N-Acetylglucosaminyltransferases/genetics , Transcription Factors/genetics , Animals , CHO Cells , Cricetinae , Cricetulus , Gene Knockout Techniques , Glutamate-Ammonia Ligase/genetics , Immunoglobulin G/metabolism , Recombinant Proteins
5.
Protein Expr Purif ; 24(2): 274-81, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11858723

ABSTRACT

Human fibroblast activation protein (FAP), an integral membrane serine protease, was produced in insect cells as a hexa-His-tagged protein using a recombinant baculovirus expression system. Two isoforms of FAP, glycosylated and nonglycosylated, were identified by Western blotting using an anti-His-tag antibody and separated by lectin chromatography. The glycosylated FAP was purified to near homogeneity using immobilized metal affinity chromatography and was shown to have both postprolyl dipeptidyl peptidase and postgelatinase activities. In contrast, the nonglycosylated isoform demonstrated no detectable gelatinase activity by either zymography or a fluorescence-based gelatinase activity assay. The kinetic parameters of the dipeptidyl peptidase activity for glycosylated FAP were determined using dipeptide Ala-Pro-7-amino-trifluoromethyl-coumarin as the substrate. The k(cat) is 2.0 s(-1) and k(cat)/K(m) is 1.0 x 10(4) M(-1) s(-1) at pH 8.5. The pH dependence of k(cat) reveals two ionization groups with pK(a1) of 7.0 and pK(a2) of 11.0. The pH profile of k(cat)/K(m) yields similar results with pK(a1) 6.2 and pK(a2) 11.0. The neutral pK(a1) is associated with His at the active site. The basic pK(a2) might be contributed from an ionization group that is not involved directly in catalysis, instead associated with the stability of the active site structure.


Subject(s)
Antigens, Neoplasm , Biomarkers, Tumor , Growth Substances/genetics , Serine Endopeptidases/genetics , Animals , Baculoviridae , Cell Line , Chromatography, Affinity , Cloning, Molecular , Endopeptidases , Gelatinases , Glycosylation , Growth Substances/biosynthesis , Growth Substances/isolation & purification , Humans , Hydrogen-Ion Concentration , Kinetics , Membrane Proteins , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/isolation & purification
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