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GS Bibliography (January 2016) Revision 32 1 Lonza

BIBLIOGRAPHY(REVISION 32)

Lonza has compiled a list of scientific papers relating to the use of the GS GeneExpression System™. For your convenience, we have grouped these papers into thefollowing categories:

1. SECTION 1: GS Expression Methodology and Cell Line Selection Strategies

2. SECTION 2: Cell Line Stability

3. SECTION 3: Antibody Production

4. SECTION 4: Recombinant Proteins (non-antibody) from NS0 Cells

5. SECTION 5: Recombinant Proteins (non-antibody) from CHO Cells

6. SECTION 6: Product Characteristics and Critical Quality Attributes

7. SECTION 7: Process Conditions for GS Cell Lines, including Media and

Feeding Strategies

8. SECTION 8: Metabolism of GS Cell Lines

9. SECTION 9: Virology of NS0 Cell Lines

10. SECTION 10: ‘Omic studies of GS Cell Lines and Systems Biology

11. SECTION 11: Cell Line Engineering

Some references will be found in more than one category.

The file number to the right hand side of the reference is for Lonza’s internal useonly.

To help scan through the literature, there is a summary (if applicable) after eachreference which has been written by Lonza. The summary aims to provide a briefinterpretation of the information within the publication. We do recommend, however,that you read the particular reference for yourself, in order to draw your ownconclusions about the information provided in the publication of interest.


FILENUMBER

SECTION 1: GS Expression Methodology and Cell LineSelection Strategies

Astley K, Naciri M, Racher AJ, Al-Rubeai M. (2007) The role of p21cip1 inadaptation of CHO cells to suspension and protein-free culture. J Biotechnol130:282-290

158

Astley K, Al-Rubeai M (2008) The role of Bcl-2 and its combined effect with p21CIP1

in adaptation of CHO cells to suspension and protein-free culture. Appl. Microbiol.Biotechnol. 78:391-399.

169.

Barnes LM, Bentley CM, Dickson AJ (2000) Advances in animal cell recombinantprotein production: GS-NS0 expression system. Cytotechnol. 32:109-123.

General review of the GS expression system including history of the NS0 cellline.

100.

Barnes LM, Bentley CM, Moy N, Dickson AJ (2007) Molecular analysis ofsuccessful cell line selection in transfected GS-NS0 myeloma cells. Biotechnol.Bioeng. 96:337-348.

The paper describes a study, by Northern, Southern and copy number analysis,of the molecular features of a panel of 17 randomly chosen GS-NS0 cell linesengineered to produce a recombinant antibody. This article discusses thesefindings in relation to vector design.

141.

Bebbington CR (1991) Expression of antibody genes in non-lymphoid mammaliancells. Methods 2:136-145.

Describes GS vector for use in CHO cells and antibody production in excess of200 mg/L.

Plasmid maps for pEE6.1 and pEE14.1

1.

Bebbington CR, Hentschel CCG (1987) The use of vectors based on geneamplification for the expression of cloned genes in mammalian cells. DNA cloning,Glover D (ed.) Acad. Press N.Y. 3:163-180.

Practical methods for use of GS expression systems in CHO.

2.

Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992)High-level expression of a recombinant antibody from myeloma cells using aglutamine synthetase gene as an amplifiable selectable marker. Biotechnol.10:169-175.

Describes use of GS selection system in NS0 for production of recombinantantibody (cB72.3) to yields of 560 mg/L in fed batch airlift.

3.

Bebbington CR (1995) Expression of antibody genes in mammalian cells. In:"Monoclonal antibodies: the next generation" Zola H (ed.). Bios Scientific (Oxford)pp165-181.

Review of expression systems including GS. Also discusses glycosylation ofantibodies.

71.


Bebbington CR (1995) Use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells. In: DNA cloning (3 ed.) Hames Dand Glover D (2nd ed.) pp85-111.

Methods descriptions including GS.

73.

Bloom JW, Madanat MS, Marriott D, Wong T, Chan SY. (1997) Intrachain disulfidebond in the core hinge region of human IgG4. Protein Sci. 6:407-415.

217.

Brand HN, Froud SJ, Metcalfe HK, Onadipe AO, Shaw A, Westlake AJ (1994)Selection strategies for highly productive recombinant cell lines. In: Animal CellTechnology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, GriffithsJB and Belthold W, Butterworth-Heinemann pp55-60.

Compares productivity of attached and suspended GS-CHO. Reviews criteriafor selecting highly productive GS-NS0 and GS-CHO cell lines.

7.

Cockett MI, Bebbington CR, Yarranton GT (1991) The use of engineered EIAgenes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucl.Acids. Res. 19:319-325.

TIMP expressed in GS-CHO with EIA. In the presence of EIA non amplifiedlines gave levels comparable with gene amplified GS-CHO lines (110 g/106

cells/24h). EIA also enhanced production of procollagenase (13 g/106cells/24h).

14.

Davies SL, O'Callaghan PM, McLeod J, Pybus LP, Sung YH, Rance J, WilkinsonSJ, Racher AJ, Young RJ, James DC (2011) Impact of gene vector design on thecontrol of recombinant monoclonal antibody production by Chinese hamster ovarycells. Biotechnol. Prog. 27:1689-1699.

203.

Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC. (2013)Functional heterogeneity and heritability in CHO cell populations. Biotechnol.Bioeng. 110:260-274.

218.

de la Cruz Edmonds MC, Tellers M, Chan C, Salmon P, Robinson DK, MarkusenJF. (2006) Development of transfection and high-producer screening protocols forthe CHOK1SV cell system. Mol. Biotechnol. 34:179-190.

175.

Fan L; Kadura I; Krebs LE; Larson JL; Bowden DM; Frye CC. (2013) Developmentof a highly-efficient CHO cell line generation system with engineered SV40Epromoter. J. Biotechnol. 168:652-658.

http://www.sciencedirect.com/science/article/pii/S0168165613003660

236.

Fan L; Frye CC; Racher AJ. (2013) The use of glutamine synthetase as a selectionmarker: recent advances in Chinese hamster ovary cell line generation processes.Pharm. Bioprocess. 1:487-502.

http://www.future-science.com/doi/abs/10.4155/pbp.13.56

239.

Froud SJ (1994) Selection strategies for highly productive recombinant lines.

Paper read at 'Commercializing human monoclonal antibodies' conference. SanDiego, Feb 7-8 1994. Describes strategies for isolating cell lines making up to

18.


1300 mg of recombinant antibody/litre.

Galbraith DJ, Tait AS, Racher AJ, Birch JR, James DC (2006) Control of cultureenvironment for improved polyethylenimine-mediated transient production ofrecombinant monoclonal antibodies by CHO cells. Biotechnol. Prog. 22:753-762.

Describes optimisation of polyethyleneimine-mediated transfection as a methodfor the very rapid production of milligramme quantities of antibody.

134.

Hayward BE, Hussain A, Wilson RH, Lyons A, Woodcock V, McIntosh B, HarrisTJR (1986) The cloning and nucleotide sequence of cDNA for an amplifiedglutamine synthetase gene from the chinese hamster. Nucl. Acids. Res. 14:999-1008.

79.

Hovey A, Bebbington CR, Jenkins N (1994) Simultaneous control of growth andproductivity using a mutant CHO cell line. In: Animal Cell Technology: Products oftoday, prospects for tomorrow. Eds: Spier RE, Griffiths JB, Berthold W. Butterworth– Heinemann, pp422-424.

Heat shock induction used to produce recombinant TIMP from a mutant CHOcell line. TIMP was expressed in a GS vector.

70.

Hovey A, Bebbington CR, Jenkins N (1994) Control of growth and recombinantprotein synthesis by heat-shock in a mutant mammalian cell line. Biotechnol.Letters 16:215-220.

Describes use of heat shock to induce recombinant protein (TIMP) synthesis ina temperature sensitive CHO cell line. TIMP expressed in a GS vector.

75.

Jenkins N, Hovey A (1993) Generation of CHO cell mutants for growth control. In:Animal Cell Technology: Basic and Applied Aspects 5. Eds: Kaminogawa S et alpp267-272.

Created temperature sensitive mutant of CHO expressing TIMP from GS vector.Temperature switching arrested cell growth and increased product yield.

76.

Jenkins N, Hovey A (1993) Temperature control of growth and productivity inmutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotech.Bioeng. 42:1029-1036.

Temperature sensitive mutants of CHO cells were transfected with the gene forTIMP using GS amplification. Alternating incubation between permissive (34oC)and non permissive (39oC) temperatures arrested cell growth whilst maintaininghigh cell viability for up to 170 hours. TIMP production rate was increased 3 to 4fold over the period of arrest compared with controls at 34oC resulting in a 35%increase in yield.

69.

Kalwy S, Rance J, Young R (2006) Toward more efficient protein expression: keepthe message simple. Mol. Biotechnol. 34:151-156.

Discusses benefits of optimising gene coding sequence, eg by removal ofcryptic splice sites or mRNA-destabilising motifs, for improving productivity.

140.

Kennard ML, Goosney DL, Monteith D, Roe S, Fischer D, Mott JE (2009)Auditioning of CHO host cell lines using the artificial chromosome expression (ACE)technology. Biotechnol. Bioeng. 104:526-539.

180.


Describes head-to-head comparison of CHOK1SV with CHO DG44 and CHO-Sas hosts for antibody expression using an artificial chromosome vector.Antibody concentrations in excess of 1.2 g/L for CHOK1SV-derived cell lines,whereas concentrations for the CHO-S and DG44 cell lines were in the range0.1 to 0.48 g/L.

Kennard ML, Goosney DL, Monteith D, Zhang L, Moffat M, Fischer D, Mott JE(2009) The generation of stable, high MAb expressing CHO cell lines based on theartificial chromosome expression (ACE) technology. Biotechnol. Bioeng. 104:540-553.

181.

Kingston RE, Kaufman RJ, Bebbington CR, Rolfe MR (1992) Amplification usingCHO cell expression vectors. In: Current Protocols in Molecular BiologySupplement 18 Unit 16.14.

Protocol for use of GS (and other expression vectors) in CHO cells.

33.

Knox R.; Nettleship JE; Chang VT; Hui ZK; Santos AM; Rahman N; Ho LP; OwensRJ; Davis SJ. (2013) A streamlined implementation of the glutamine synthetase-based protein expression system. BMC Biotechnol. 13:74.

245.

Lindgren K, Salmén A, Lundgren M, Bylund L, Ebler Å, Fäldt E, Sörvik L, Fenge C,Skoging-Nyberg U. (2009) Automation of cell line development. Cytotechnology59:1-10.

179.

Marchant RJ, Al-Fageeh MB, Underhill MF, Racher AJ, Smales CM (2008)Metabolic rates, growth phase, and mRNA levels influence cell-specific antibodyproduction levels from in vitro-cultured mammalian cells at sub-physiologicaltemperatures. Mol. Biotechnol. 39:69-77.

162

Nakamura T, Omasa T (2015) Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selectionsystem. J. Biosci. Bioeng. 120:323-329.

254.

Onadipe AO, Metcalfe HK, Freeman PR, James C (2001) Capillary-aided cellcloning. In: Animal Cell Technology: from Target to Market. Eds: Lindner-Olsson Eet al. Kluwer Academic Publishers pp72-74.

A method for one-step cloning with a high probability of monoclonality isdescribed.

112.

Peakman TC, Worden J, Harris RH, Cooper H, Tite J, Page MJ, Gewert DR,Bartholemew M, Crowe JS, Brett S (1994) Comparison of expression of ahumanized monoclonal antibody in mouse NS0 myeloma cells and Chinesehamster ovary cells. Hum. Antibod. Hybrid. 5:65-74.

Compared DHFR-CHO and GS-NS0 for expression of an IgG1. Monitored copynumber and steady state mRNA levels during selection and amplification forboth systems. In NS0 and CHO cells making equivalent amounts of antibody,the copy number was lower in NS0 although heavy chain mRNA levels werevirtually identical. Antibody purified from both systems behaved identically in anumber of functional assays.

56.

Porter AJ, Dickson AJ, Racher AJ (2010) Strategies for selecting recombinantCHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors.

189.


Biotechnol. Prog. 26:1446-1454.

Porter AJ, Racher A, Preziosi R, Dickson AJ (2010) Strategies for selectingrecombinant CHO cell lines for cGMP manufacturing: Improving the efficiency ofcell line generation. Biotechnol. Bioeng. 26:1455-1464.

190.

Povey JF; O'Malley CJ; Root TS; Martin EB; Montague GA; Feary M; Trim CM;Lang DA; Alldread R; Racher AJ, Smales CM. (2014) Rapid high-throughputcharacterisation, classification and selection of recombinant mammalian cell linephenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling. J. Biotechnol. 184:84-93.

http://www.sciencedirect.com/science/article/pii/S016816561400234X

246.

Stephens PE, Cockett MI (1989) The construction of a highly efficient and versatileset of mammalian expression vectors. Nucleic Acids Res 17:1710

Contains plasmid map for pEE6.1 vector. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC318444/

249.

Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC (2004)Transient production of recombinant proteins by Chinese hamster ovary cells usingpolyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol. Bioeng. 88:707-721.

Describes use of polyethyleneimine-mediated transfection as a method for thevery rapid production of milligramme quantities of antibody.

143.

Underhill MF, Smales CM, Naylor LH, Birch JR, James DC (2007) Transient geneexpression levels from multigene expression vectors. Biotechnol. Prog. 23:435-443.

Authors evaluated use of IRES elements in GS expression vectors forexpression of multimeric proteins. Overall, it appeared that relative proteinexpression levels expected from heterologous gene products in a multigenevector could not be predicted on copy number alone and it is important tocharacterize multigene or oligocistronic systems prior to use.

137.

Underhill MF, Birch JR, Smales CM, Naylor LH (2005) eIF2 phosphorylation,stress perception, and the shutdown of global protein synthesis in cultured CHOcells. Biotechnol. Bioeng. 89:805-814.

Used GS-CHO expressing TIMP-1 as a model system to investigate relationshipbetween cellular protein synthesis, cellular perception of stress, translationattenuation and specific antibody production rate.

138

Wilson RH (1993) Glutamine synthetase gene amplification in Chinese hamsterovary cells. In: "Gene Amplification in Mammalian Cells" (ed.) Kellens RE, MarcelDekker Inc (New York) pp 301-311.

Review of GS properties, its amplification in CHO cells using methioninesulphoximine and its cloning and use as a selectable marker.

50.

Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF (2009) High-level proteinexpression in scalable CHO transient transfection. Biotechnol. Bioeng. 103:542-551.

176.


Describes development of transient expression system using polyethyleneiminefor GS-CHO. Achieved antibody concentrations up-to 80 mg/L. Saw similarproduct characteristics for the same antibody expressed in both transient andstably transfected cells. Differences were seen between antibody expressedtransiently in HEK293 cells compared to stable GS-CHO cell lines.

Ye J, Alvin K, Latif H, Hsu A, Parikh V, Whitmer T, Tellers M, de la Cruz EdmondsMC, Ly J, Salmon P, Markusen JF (2010) Rapid protein production using CHOstable transfection pools. Biotechnol. Prog. 1431-1437.

194.

SECTION 2: Cell line Stability

Bailey LA, Hatton D, Field RP, Dickson AJ (2012) Determination of Chinesehamster ovary cell line stability and recombinant antibody expression during long-term culture. Biotechnol. Bioeng. 109:2093-2103.

210.

Barnes LM, Bentley CM, Dickson AJ (2004) Molecular definition of predictiveindicators of stable protein expression in recombinant NS0 myeloma cells.Biotechnol. Bioeng. 85:115 – 121.

Following their 2003 paper Barnes et al have focused in on a number of NS0cell lines, both stable and unstable for recombinant gene expression in longterm culture. No change in transfected gene copy number is associated withdeclining productivity in unstable cell lines. However, levels of mRNA oncebelow a critical “threshold point” appears to indicate a cell line whoseproductivity will decline. The authors hypothesise that genomic responses todisturbances in the genomic environment (possibly the result of vectorintegration) may be responsible. A number of solutions are proposed.

115.

Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production fromrecombinant mammalian cells. Biotechnol. Bioeng. 81:631 – 639.

This paper provided a comprehensive review of stability issues related torecombinant protein expression. Instability in hybrodomas, DHFR-CHOs andGS-NS0s is discussed. On the whole GS-NS0 cell lines used for manufacturingare stable. Any unstable cell lines created are usually discarded early on, forexample, during the selection stage of cell line creation. Prospective molecularmechanisms for instability are discussed and approaches to improve expressionlevels and ensuring stability of production are also presented.

116.

Barnes LM, Bentley CM, Dickson AJ (2003) Stability of recombinant proteinproduction in the GS-NS0 expression system is unaffected by cryopreservation.Biotechnol. Prog. 19:233 – 237.

Study shows that cryopreservation and revival procedures do not alter thestability characteristics of GS-NS0 cell lines.

117.

Barnes LM, Bentley CM, Dickson AJ (2001) Characterisation of the stability ofrecombinant protein production in the GS-NS0 expression system. Biotechnol.Bioeng. 73:261-270.

Selection of production clones on the basis of growth and productivity alone willnot predict stability during long-term culture. Our research indicates that stablehigh-producing clones can readily be obtained from use of the GS-NS0 system

142.


in the absence of amplification but there may be molecular features of theoriginal transfectants that could serve as very important predictive indicators ofthe stability of recombinant protein production.

Bird P, Bolam E, Castell L., Obeid O, Darton N, Hale G (1998) Glutaminesynthetase transfected cells may avoid selection by releasing glutamine. In: NewDevelopments and New Applications in Animal Cell Technology. Eds: Merten OW,Perrin P, Griffiths B Kluwer Acad. Publishers, pp43-49.

Noted that in some circumstances, particularly hollow fibre systems, GStranfected cell lines may release sufficient glutamine to overcome the selectionpressure imposed by glutamine free media.

87.

Cosgrove L, Lovrecz GO, Verkuylen A, Cavaleri L, Black LA, Bentley JD, HowlettGJ, Gray PP, Ward CW, McKern NM (1995) Purification and properties of insulinreceptor ectodomain from large scale mammalian cell culture. Protein Expressionand Purification. 6:789-798.

Ectodomain expressed in GS-CHO and cells grown in a perfused 40 litre airliftfermenter on microcarrier beads. MSX required to maintain stable production(amplified cell line) but was omitted from production fermenter.

78.

Dorai H, Corisdeo S, Ellis D, Kinney C, Chomo M, Hawley-Nelson P, Moore G,Betenbaugh MJ, Ganguly S (2012) Early prediction of instability of Chinese hamsterovary cell lines expressing recombinant antibodies and antibody-fusion proteins.Biotechnol. Bioeng. 109:1016-1030.

208.

de la Cruz Edmonds MC, Tellers M, Chan C, Salmon P, Robinson DK, MarkusenJF. (2006) Development of transfection and high-producer screening protocols forthe CHOK1SV cell system. Mol. Biotechnol. 34:179-190.

175.

Guerini D, Schroder S, Foletti D, Carafoli E (1995) Isolation and characterization ofa stable Chinese hamster ovary cell line overexpressing the plasma membraneCa2+ ATPase. J. Biol. Chem. 270:14643-14650.

GS–CHO cell lines created. Initial transfectants were subjected to additionalrounds of selections with MSX resulting in clones with 4 to 10 gene copies percell. The stability of expression of transfected cells was examined in thepresence and absence of selective drug (MSX). In the absence of MSXproductivity was retained over short culture periods but was substantiallydecreased after 30 to 40 passages. In contrast stable production wasmaintained for 6 months in the presence of MSX.

67.

Hassell T, Brand H, Renner G, Westlake A, Field RP (1992) Stability of productionof recombinant antibodies from glutamine synthetase amplified CHO and NS0 celllines. In: Animal Cell Technology. Developments Processes and Products,Butterworths, pp42-47.

Examines stability of amplified and non amplified cell lines. Yield of 895 mg/lachieved for a GS-NS0 cell line in serum-free fed-batch antibody culture.

30.

He L, Winterrowd C, Kadura I, Frye C (2012) Transgene copy number distributionprofiles in recombinant CHO cell lines revealed by single cell analyses. Biotechnol.Bioeng. 109:1713-1722.

211.


Kearns B, Lindsay D, Manahan M, McDowall J, Rendeiro D (2003) NS0 batch cellculture process characterisation: a case study. BioProcessing Journal Jan/Feb 52-57.

Investigation into decline in productivity as cell culture generation numberincreased. No genetic instability seen but a phenotypic instability, related to themetabolic state of the culture, is reported.

110.

Kim M, O'Callaghan PM, Droms KA, James DC (2011) A mechanisticunderstanding of production instability in CHO cell lines expressing recombinantmonoclonal antibodies. Biotechnol. Bioeng. 108:2434-2446.

204.

Laubach VE, Garvey EP, Sherman PA (1996) High-level expression of humaninducible nitric oxide synthase in Chinese hamster ovary cells and characterisationof the purified enzyme. Biochem. Biophys. Res. Comm. 218:802-807.

iNOS expressed in CHO cells using GS system. Amplification in the presenceof 400m MSX increased product levels 3 to 4 fold. Expression was enhancedby sodium butyrate (2mM). Expression levels were at least 20-fold higher thanthose reported for a baculovirus expression system. Also noted that sodiumbutyrate could restore high level expression in some cell lines in which iNOSexpression declined with time in culture.

88.

Porter AJ, Dickson AJ, Barnes LM, Racher AJ (2007) Antibody production by GS-CHO cell lines over extended culture periods. In: Cell technology for Cell Products(Ed: Smith R) Springer, Dordrecht, pp137-140.

157.

Racher AJ (2005) Stability and Suitability of GS-NS0 Cell Lines for ManufacturingAntibodies. BioProcessing Journal.4:61-65.

124.

SECTION 3: Antibody Production

Alete DE, Racher AJ, Birch JR, James DC, Smales CM (2005) The functionalcompetence of animal cells: analysis of the secretory pathway. In: Animal CellTechnology meets Genomics (Eds: Gòdia F, Fussenegger,M), Springer, Dordrecht,pp71-74.

129.

Alete DE, Racher AJ, Birch JR, Stansfield SH, James DC, Smales CM (2005)Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myelomacells with varying secreted recombinant monoclonal antibody productivities.Proteomics 5:4689-4704.

122.

Barnard GC, Hougland MD, Rajendra Y (2015) High-throughput mAb expressionand purification platform based on transient CHO. Biotechnol Prog 31:239-247.

http://dx.doi.org/10.1002/btpr.2012

250.

Bebbington CR (1991) Expression of antibody genes in non lymphoid mammaliancells. Methods in Enzymol. 2:136-145.

Describes GS vector for use in CHO cells and antibody production in excess of200mg/L.

1.

Bebbington CR, Renner G, Thomson S, King D, Abrams D, Yarranton GT (1992)High-level expression of a recombinant antibody from myeloma cells using a

3.


glutamine synthetase gene as an amplifiable selectable marker. Biotechnol.10:169-175.

Describes use of GS selection system in NS0 for production of recombinantantibody (cB72.3) to yields of 560 mg/L in fed batch airlift.

Bebbington CR (1995) Expression of antibody genes in mammalian cells. In:"Monoclonal antibodies: the next generation", Zola, H. (ed.) Bios Scientific (Oxford)pp165-181.


71.

Bell SL, Bebbington CR, Bushell ME, Sanders PG, Scott MF, Spier RE, Wardell JN(1991) Genetic engineering of cellular physiology. In: Production of Biologicalsfrom Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp304-306.

GS gene used to confer glutamine independence on a hybridoma.

4.

Bell SL, Bushell ME, Scott MF, Wardell JN, Spier RE, Sanders PG (1992) Geneticmodification of hybridoma glutamine metabolism: physiological consequences. In:Animal Cell Technology: Developments, Processes and Products. Eds: Spier RE,Griffiths JB, MacDonald C. Butterworth-Heinemann, pp 180-182.

GS gene transfected into hybridoma cell to confer glutamine independence.

5.

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushell ME, Sanders PG(1995) Genetic engineering of hybridoma glutamine metabolism. Enzyme andMicrobial Technol. 17:98-106.


65.

Bentley KJ, Gewert R, Harris WJ (1998) Differential efficiency of expression ofhumanized antibodies in transient transfected mammalian cells. Hybridoma 17:559-567.

Used GS vectors to examine factors influencing level of transient expression ofantibodies in CHO cells. Expression levels greatly influenced by amino acidsequence of variable regions and by different combinations of antibody heavyand light chains. The authors note that it was possible to obtain stabletransfectants that overcame the limitations observed in transient expression.

106.

Beyer T. Lohse S, Berger S, Peipp M, Valerius T, Dechant M (2009) Serum-freeproduction and purification of chimeric IgA antibodies. J Immunol Meth 346:26-37.

Describes use of GS-CHO cell lines to express an IgA molecule.

181.

Bi J-X, Shuttleworth J, Al-Rubeai M (2004) Uncoupling of cell growth andproliferation results in enhancement of productivity in p21CIP1-arrested CHO cells.Biotechnol. Bioeng. 85:741-749.

150.

Bibila TA, Ranucci CS, Glazomitsky K, Buckland BC, Aunins JG (1994)Monoclonal antibody process development using medium concentrates. Biotechnol.Prog. 10:87-96.

54.


Describes fed-batch process using concentrated medium with GS-NS0 cell linesmaking recombinant antibodies. Up to 7-fold increases in antibody titre wereachieved compared with batch culture.

Bibila TA, Robinson DK (1995) In pursuit of the optimal fed-batch process formonoclonal antibody production. Biotechnol. Prog. 11:1-13.

A review of approaches taken to optimise fed-batch processes for antibodyproduction.

60.

Birch JR, Bebbington CR, Field R, Renner G, Brand H, Finney H (1993) Theproduction of recombinant antibodies using the glutamine synthetase (GS) system.In: Animal Cell Technology: Basic and Applied Aspects 5:573-577.

Review of GS system with examples of fermenter productivity and processimprovement.

6.

Birch JR, Froud S (1994) Mammalian cell culture systems for recombinant proteinproduction. Biologicals 22:127-133.

Gives example of Mab production (200 mg/L) using GS-NS0 cells in protein-freemedium.

53.

Birch JR, Mainwaring DO, Racher AJ (2005) Use of the Glutamine Synthetase(GS) Expression System for the rapid development of highly productive mammaliancell processes. In: Modern Biopharmaceuticals (Knäblein, J. ed), WILEY-VCHVerlag GmbH & Co KGaA, pp809-832.

Status of GS Gene Expression System, Summer of 2004.

131.

Birch JR, Racher AJ (2006) Antibody production. Advanced Drug Delivery Reviews58:671-685.

General review of antibody production systems, including the GS GeneExpression System.

130.

Bird P, Bolam E, Castell L, Obeid O, Darton N, Hale G (1998) Glutaminesynthetase transfected cells may avoid selection by releasing glutamine. In: NewDevelopments and New Application in Animal Cell Technology. Eds: Merten OW,Perrin P, Griffiths B. Kluwer Acad. Publishers, pp43-49.

Noted that in some circumstances, particularly hollow fibre systems, GStranfected cell lines may release sufficient glutamine to overcome the selectionpressure imposed by glutamine free media.

87.

Bloom JW, Madanat MS, Marriott D, Wong T, Chan SY. (1997) Intrachain disulfidebond in the core hinge region of human IgG4. Protein Sci. 6:407-415.

217.

Brand HN, Froud SJ, Metcalfe HK, Onadipe AO, Shaw A, Westlake AJ (1994)Selection strategies for highly productive recombinant cell lines. In: Animal CellTechnology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, GriffithsJB, Belthold W, Butterworth-Heinemann, 55-60.

Compares productivity of attached and suspended GS-CHO. Reviews criteriafor selecting highly productive GS-NS0 and GS-CHO cell lines.

7.


Broad D, Boraston R, Rhodes M (1991) Production of recombinant proteins inserum-free media. Cytotechnol. 5:47-55.

Compares productivity of three amplified GS-CHO lines making recombinantproteins in the presence and absence of serum (no significant differences seen).

8.

Brown ME, Renner G, Field RP, Hassell T (1992) Process development for theproduction of recombinant antibodies using the glutamine synthetase (GS) system.Cytotechnol. 9:231-236.

Describes improvements in batch process leading to antibody yields ranging upto 1 g/L. Serum-free medium used.

9.

Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PWHI, Sawyer LSW,Hendry RM, Dunlop N, Nara PL., Lanacchia M, Garratty E, Stiehm ER, Bryson YJ,Cao Y, Moore JP, Ho DD, Barbas CF (1994) Efficient neutralization of primaryisolates of HIV-1 by a recombinant human monoclonal antibody. Science266:1024-1027.

A recombinant human antibody to virus envelope protein gp120 was expressedin GS-CHO cells using methionine sulphoximine amplification. The wholeantibody was constructed from a FAb fragment generated from a combinatorialphage display library.

55.

Charaniya S, Karypis G, Hu W-S (2009) Mining transcriptome data for function-traitrelationship of hyper productivity of recombinant antibody. Biotechnol, Bioeng.102:1654-1669.

172.

Corti D, Zhao J, Pedotti M, Simonelli L, Agnihothram S, Fett C, Fernandez-Rodriguez B, Foglierini M, Agatic G, Vanzetta F, Gopal R, Langrish CJ, Barrett NA,Sallusto F, Baric RS, Varani L, Zambon M, Perlman S, Lanzavecchia A (2015)Prophylactic and postexposure efficacy of a potent human monoclonal antibodyagainst MERS coronavirus. Proc. Natl. Acad. Sci., USA. 112:10473-10478.

Use of GS Xceed™ System described in Supplementary Materials.

256.

Davies SL, O'Callaghan PM, McLeod J, Pybus LP, Sung YH, Rance J, WilkinsonSJ, Racher AJ, Young RJ, James DC (2011) Impact of gene vector design on thecontrol of recombinant monoclonal antibody production by Chinese hamster ovarycells. Biotechnol. Prog. 27:1689-1699.

203.

Davies SL, Lovelady CS, Grainger RK, Racher AJ, Young RJ, James DC. (2013)Functional heterogeneity and heritability in CHO cell populations. Biotechnol.Bioeng. 110:260-274.

218.

Dempsey J, Ruddock S, Osborne M, Ridley A. Sturt S, Field R (2003) Improvedfermentation processes for NS0 cell lines expressing human antibodies andglutamine synthetase. Biotechnol. Prog. 19:175-178.

Repeated nutrient analysis and re-supplementation of serum-free antibodyproducing NS0 cultures performed. As a result media and feeds weredeveloped which gave 10-fold increases in antibody harvest titres up to 600mg/l.

109.

Dinnis DM, Stansfield SH, Schlatter S; Smales CM; Alete D; Birch JR; Racher AJ;Marshall CT; Nielsen LK.; James DC (2006) Functional proteomic analysis of GS-

133.


NS0 murine myeloma cell lines with varying recombinant monoclonal antibodyproduction rate. Biotechnol. Bioeng. 94:830-841.

Dorai,H, Sauerwald,TM, Campbell A, Kyung Y-S, Goldstein J, Magill A, Lewis MJ,Tang QM, Jan D, Ganguly S, Moore G (2007) Investigation of proteinmicroheterogeneity. A case study in rapid detection of mutation in mammalianproduction cell lines. BioProcess Intl 5(8):66-72.

Describes usage of a variety of analytical techniques to characterise anantibody-peptide fusion protein and identify a mutation that occurred post-transfection. Paper also includes data on GS vector copy number in two celllines derived from CHOK1SV.

161

Downham MR, Farrell WE, Jenkins HA (1996) Endoplasmic reticulum proteinexpression in recombinant NS0 myelomas grown in batch culture. Biotechnol. andBioengineer. 51:691–696.

Production of ER proteins (GRP78/BiP, GRP94 and Erp72) increased duringdecline phase of batch culture of GS-NS0 coincident with an increase inproduction rate of recombinant antibody and reduction in uptake of glucose andglutamate.

83.

Fan L; Kadura I; Krebs LE; Larson JL; Bowden DM; Frye CC. (2013) Developmentof a highly-efficient CHO cell line generation system with engineered SV40Epromoter. J. Biotechnol. 168:652-658.


236.

Field RP, Brand H, Renner GL, Robertson HA, Boraston R (1991) Production of achimeric antibody for tumour imaging and therapy from Chinese hamster ovary(CHO) and myeloma cells. In: Production of Biologicals from Animal Cells inCulture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp742-744.

Early work on expression of chimeric antibody (cB72.3) in GS-NS0. Achieved240mg/l in unfed, serum-free fed-batch culture. An alternative expressionsystem using neo and gpt genes as selectable markers in CHO cells gave90mg/l.

16.

Fries S, Glazomitsky K, Woods A, Forrest G, Hsu A, Olewinski R, Robinson DK,Chartrain M (2005) Evaluation of disposable bioreactors. Rapid production ofrecombinant proteins by several animal cells. BioProcess International 3( Supp6):36-44.

Describes growth of antibody-producing GS-NS0 cell line in cell culture-bagdisposable bioreactor system.

155.

Froud SJ (1994) Selection strategies for highly productive recombinant lines.

Paper read at 'Commercializing human monoclonal antibodies' conference. SanDiego, Feb 7-8 1994. Describes strategies for isolating cell lines making up to1300mg of recombinant antibody/litre.

18.

Galbraith DJ, Tait AS; Racher AJ, Birch JR, James DC (2006) Control of cultureenvironment for improved polyethylenimine-mediated transient production of

134.


recombinant monoclonal antibodies by CHO cells. Biotechnol. Prog. 22:753-762.

Describes optimisation of polyethyleneimine-mediated transfection as a methodfor the very rapid production of milligramme quantities of antibody.

Grainger RK, James DC. (2013) CHO cell line specific prediction and control ofrecombinant monoclonal antibody N-glycosylation. Biotechnol. Bioeng. 110:2970-2983.

http://dx.doi.org/10.1002/bit.24959

234.

Hassell T, Brand H, Renner G, Westlake A, Field RP (1992) Stability of productionof recombinant antibodies from glutamine synthetase amplified CHO and NS0 celllines. In: Animal Cell Technology. Developments Processes and Products,Butterworths, pp42-47.

Examines stability of amplified and non amplified cell lines. Yield of 895 mg/lachieved for a GS-NS0 cell line in serum-free fed-batch antibody culture.

30.

Hayes NVL, Smales CM, Klappa P (2010) Protein disulfide isomerase does notcontrol recombinant IgG4 productivity in mammalian cell lines. Biotechnol. Bioeng.105:770-779.

186.

Hermes PA, Castro CD (2010) A fully defined, fed-batch, recombinant NS0 cultureprocess for monoclonal antibody production. Biotechnol. Prog. 26:1411-1416.

193.

Ho Y, Varley J, Mantalaris A (2006) Development and analysis of a mathematicalmodel for antibody-producing GS-NS0 cells under normal and hyperosmotic cultureconditions. Biotechnol. Prog. 22:1560-1569.

Describes a hybrid model, consisting of both unstructured and structuredelements, has been developed to describe cell growth and death, metabolism,and antibody production in the GS-NS0 system under normal culture conditions.The specific transcription and translation rates of heavy and lightimmunoglobulin chains were identified as parameters with the largest impact onthe antibody production process.

127.

Ho Y, Kiparissides A, Pistikopoulos EN, Mantalaris A. (2012) Computationalapproach for understanding and improving GS-NS0 antibody production underhyperosmotic conditions. J. Bioscience Bioeng. 113:88-98.

222.

Ibarra N, Watanabe S, Bi JX, Shuttleworth J, Al-Rubeai M (2003)Modulation of cell cycle for enhancement of antibody production in perfusion cultureof NS0 cells. Biotechnol. Prog. 19:224-228.

NS0 cells metabolically engineered to express cytostatic and anti-apoptoticgenes. Resulting cell line exhibited a 4-fold increase in productivity in thearrested phase compared to the proliferative phase.

111.

Kadarusman J, Bhatia R, McLaughlin J, Lin WR (2005) Growing cholesterol-dependent NS0 myeloma cell line in the Wave bioreactor system: overcomingcholesterol-polymer interaction by using pretreated polymer or inert fluorinatedethylene propylene. Biotechnol. Prog. 21:1341-1346.

Discusses choice of material for manufacture of disposable bioreactor systems

168.


Keen MJ, Hale C (1996) The use of serum-free medium for the production offunctionally active humanised monoclonal antibody from NS0 mouse myeloma cellsengineered using glutamine synthetase as a selectable marker. Cytotechnol.18:207-217.

Describe protein-free medium for GS-NS0 supplemented with cholesterol,phosphatidylcholine and -cyclodextrin. Adapted cells to become cholesterolindependent.

80.

Khoo SHG, Al-Rubeai M (2009) Detailed understanding of enhanced specificantibody productivity in NS0 myeloma cells. Biotechnol. Bioeng. 102:188-189.

171.

Kilgore BR, Lucka A, Patel R, Andrien B, Dhume ST (2005) Cell line Switch fromNS0 to CHO Causes Several Changes in Glycosylation of Recombinant IgG. ACS33rd Northeast Regional Meeting (Abstract)

http://acs.confex.com/acs/nerm05/techprogram/P21476.HTM

214.

King DJ, Byron OD, Mountain A, Weir N, Harvey A, Lawson ADG, Proudfoot KA,Baldock D, Harding SE, Yarranton GT, Owens RJ (1993) Expression, purificationand characterization of B72.3 Fv fragments. Biochem. J. 290:723-729.

Fv expressed in GS-CHO and in E. coli. Yields of 4 mg/L achieved in CHO rollerbottles and 450 mg/L in E. coli fermentations.

32.

Kiparissides A; Pistikopoulos EN; Mantalaris A. (2015) On the model-basedoptimization of secreting mammalian cell (GS-NS0) cultures. Biotechnol, Bioeng.112:536-548.


224.

Lifely MR, Hale C, Boyce S, Keen MJ, Phillips J (1995) Glycosylation andbiological activity of Campath - IH expressed in different cell lines and grown underdifferent culture conditions. Glycobiol. 5:813-822.

Antibody expressed in CHO (DHFR), rat YO myeloma and NS0 (GS) cells.Glycosylation varied significantly between cell lines and to a minor extentdepending on culture conditions. YO antibody was unusual in having abisecting Glc NAc on the core oligoscaccharide and NS0 antibody appeared tobe under glycosylated. YO antibody had enhanced activitity in ADCC assayscompared with CHO or NS0.

74.

Lewis AP, Parry N, Peakman TC, Scott-Crowe J (1992) Rescue and expression ofhuman immunoglobulin genes to generate functional human monoclonalantibodies. Hum. Antibod. Hybrid. 3:146-152.

Human Ig genes rescued from human hybridoma cells and expressed in rat YOcells using GS vector.

34.

Lohse S, Derer S, Beyer T, Klausz K, Peipp M, Leusen JH, van de Winkel JG,Dechant M, Valerius T (2011) Recombinant dimeric IgA antibodies against theepidermal growth factor receptor mediate effective tumour cell killing. J. Immunol.186:3770-3778.

205.

Mason M, Sweeney,B. Cain K, Stephens P, Sharfstein ST (2012) Identifyingbottlenecks in transient and stable production of recombinant monoclonal-antibody

213.


sequence variants in chinese hamster ovary cells. Biotechnol. Prog. 28:846-855.

McKenna SL, Cotter TG (2000) Inhibition of caspase activity delays apoptosis in atransfected NS0 myeloma cell line. Biotech. Bioeng. 67:165-176.

Z-VAD-fmk, a specific inhibitor of caspases reduced apoptosis in GS-NS0 cellsbut did not increase productivity. The inhibitor did not prevent mitochondrialdysfunction.

102.

McLeod J, O'Callaghan PM, Pybus LP, Wilkinson SJ, Root T, Racher AJ, JamesDC (2011) An empirical modeling platform to evaluate the relative control discreteCHO cell synthetic processes exert over recombinant monoclonal antibodyproduction process titer. Biotechnol. Bioeng. 108:2193-2204.

197.

Mead EJ, Chiverton LM, Spurgeon SK, Martin EB, Montague GA, Smales CM, vonder Haar T. (2012) Experimental and in silico modelling analyses of the geneexpression pathway for recombinant antibody and by-product production in NS0cell lines. PLOS One 7(10):e47422.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468484/

221.

Moran EB, McGowan ST, McGuire JM, Frankland JE, Oyebade IA, Waller W,Archer LC, Morris LO, Pandya J, Nathan SR, Smith L., Cadette ML, Michalowski JT(2000) A systematic approach to the validation of process control parameters formonoclonal antibody production in fed-batch culture of a murine myeloma.Biotechnol. Bioeng. 69:242-255.

Describes an approach to validating control parameters for a GS-NS0 cell line.

105.

O'Callaghan PM, McLeod J, Pybus LP, Lovelady CS, Wilkinson SJ, Racher AJ,Porter AJ, James DC (2010) Cell line-specific control of recombinant monoclonalantibody production by CHO cells. Biotechnol. Bioeng. 106:938-951.

191.

O'Connor KC, Muhitch JW, Lacks DJ, Al-Rubeai M (2006) Modelling suppressionof cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells. Biotechnol. Lett.28:1919-1924.

154.

Paterson T, Innes J, McMillan L, Downing I, McCann Carter MC (1998) Variation inIgG1 heavy chain allotype does not contribute to differences in biological activity oftwo human anti-Rhesus (D) monoclonal antibodies. Immunotechnol. 4:37-47.

Two anti-Rhesus (D) antibodies ‘rescued’ from heterohybrid cell lines and re-expressed in NS0 cells using GS selection. Secretion levels were not improvedby amplification. The two recombinant antibodies exhibited identical in vitroproperties to the parent bodies.

97.

Peakman TC, Worden J, Harris RH, Cooper H, Tite J, Page MJ, Gewert DR,Bartholemew M, Crowe JS, Brett S (1994) Comparison of expression of ahumanized monoclonal antibody in mouse NS0 myeloma cells and Chinesehamster ovary cells. Hum. Antibod. Hybrid. 5:65-74.

Compared CHO DHFR and GS NS0 for expression of an IgG1. Monitored copynumber and steady state mRNA levels during selection and amplification forboth systems. In NS0 and CHO making equivalent amounts of antibody, thecopy number was lower in NS0 although heavy chain mRNA levels werevirtually identical. Antibody purified from both systems behaved identically in a

56.


number of functional assays.

Porter AJ, Mohindra A, Porter JM, Racher AJ (2011) Does earlier use ofproductivity enhancers during cell line selection lead to the identification of moreproductive cell lines? BMC Proceedings 5(Suppl8):9.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3284934/?tool=pubmed

209.

Porter AJ, Dickson AJ, Racher AJ (2010) Strategies for selecting recombinantCHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors.Biotechnol. Prog. 26:1446-1454.

189.

Porter AJ, Racher A, Preziosi R, Dickson AJ (2010) Strategies for selectingrecombinant CHO cell lines for cGMP manufacturing: Improving the efficiency ofcell line generation. Biotechnol. Bioeng. 26:1455-1464.

190.

Povey JF; O'Malley CJ; Root TS; Martin EB; Montague GA; Feary M; Trim CM;Lang DA; Alldread R; Racher AJ, Smales CM. (2014) Rapid high-throughputcharacterisation, classification and selection of recombinant mammalian cell linephenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling. J. Biotechnol. 184:84-93.

http://www.sciencedirect.com/science/article/pii/S016816561400234X

246.

Rajendra Y, Hougland MD, Alam R, Morehead TA, Barnard GC (2015) A high celldensity transient transfection system for therapeutic protein expression based on aCHO GS-knockout cell line: Process development and product quality assessment.Biotechnol Bioeng 112:977-986.

252.

Reid CQ, Tait AS, Baldascini H, Mohindra A, Racher AJ, Bilsborough S, SmalesCM, Hoare M (2010) Rapid whole monoclonal antibody analysis by massspectrometry: An ultra scale-down study of the effect of harvesting by centrifugationon the post-translational modification profile. Biotechnol. Bioeng. 107:85-95.

188.

Rendall MH, Maxwell A, Tatham D, Khan P, Gay RD, Kallmeier RC, Wayte JRT,Racher AJ (2005) Transfection to manufacturing: reducing timelines for highyielding GS-CHO processes. In: Animal Cell Technology meets Genomics (Eds:Gòdia F, Fussenegger M), Springer, Dordrecht, pp701-704.

135.

Robinson DK, Chan CP, Yu Ip CC, Seamans TC, Lee DK, Lenny AB, Tung JS,DiStefano DJ, Munshi S, Gould SL, Tsai PK, Irwin J, Mark GE, Silberklang M.(1994) Product consistency during long-term fed-batch culture. In: Animal CellTechnology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, GriffithsJB, Berthold W. Butterworth-Heinemann, pp763-767.

Expressing antibody genes in GS-NS0. Yields up to 1.8g/l in three to four weekduration fed-batch. Glycoform pattern shifts towards incomplete mannose-terminated glycans as culture progresses.

46.

Robinson DK, Chan C., Yu Ip C, Tsai PK, Tung J, Seamans TC, Lenny AB, LeeDK, Irwin J, Silberklang M (1994) Characterization of a recombinant antibodyproduced in the course of a high yield fed-batch process. Biotechnol. Bioeng.44:727-735.

Anti-HIV gp120 MAb expressed in non-amplified GS-NS0 system. Fed-batchprocess developed which increased yields ten-fold compared with batch culture.

52.


The clone produced 0.86g MAb/l over 22 days. Antigen binding and MWtremained constant throughout culture. Four major IEF bands were observedwith a minor fifth more acidic band appearing later in culture. Pattern ofglycoforms secreted by cells changes as culture progresses.

Robinson DK, Seamans TC, Gould SL, DiStefano DJ, Chan CP, Lee DK, Bibila T,Glazomitsky K, Munshi S, Daugherty B, O'Neill Palladino L, Stafford-Hollis J, HollisGF, Silberklang M (1994) Optimization of a fed-batch process for production of arecombinant antibody. Reprinted from Biochem. Engineering VIII, Ann. (NY) Acad.Sci. 745:285-296.

Describes the development of an optimized fed-batch process for a GS-NS0 cellline secreting a monoclonal antibody. Includes data on amino acid consumptionrates.

62.

Robinson DK, DiStefano D, Gould SL, Cuca G, Seamans TC, Benincasa D,Munshi S, Chan CP, Stafford-Hollis J, Hollis GF, Jain D, Ramasubramanyan K,Mark GE, Silberklang M (1995) Production of engineered antibodies in myelomaand hybridoma cells. In: Antibod. Engineering. Eds: Wang H, Imanaka T pp1 - 14,Worthington: ACS.

Detailed description of production of recombinant antibody in a GS-NS0 systemfor production of a recombinant antibody.

84.

Schenerman MA, Hope JN, Kletke C, Singh JK, Kimura R, Tsao EI, Folena-Wasserman G (1999) Comparability Testing of a Humanized Monoclonal Antibody(Synagis®) to Support Cell Line Stability, Process Validation and Scale-Up forManufacturing. Biologicals. 27:203-215.

126.

Schlatter S, Stansfield SH, Dinnis DM, Racher Andrew J, Birch John R, David CJames (2005) On the Optimal Ratio of Heavy to Light Chain Genes for EfficientRecombinant Antibody Production by CHO Cells. Biotechnol. Prog. 21:122-133.

121.

Seamans TC, Gould SL, DiStefano DJ, Silberklang M, Robinson DK (1994) Use oflipid emulsions as nutritional supplements in mammalian cell culture. Ann. (NY)Acad. Sci. 745:240-243.

Describes preparation of stable lipid emulsions to substitute for lipoproteins inthe culture of NS0 cells secreting a recombinant antibody.

61.

Seamans TC, Fries S, Beck A, Wurch T., Chenu S, Chan C, Ushio M, Bailey J,Kejariwal A, Ranucci C, Villani N, Ozuna S, Goetsch L, Corvaia N, Salmon P,Robinson DK, Chartrain M (2008) Cell cultivation process transfer and scale-up insupport of production of early clinical supplies of an anti IGF-1R antibody, Part 1.BioProcess International 6(3):26-36.

163.


164.

Sellick CA, Croxford AS, Maqsood AR, Stephens G, Westerhoff HV, Dickson(2011) Metabolite profiling of recombinant CHO cells: Designing tailored feedingregimes that enhance recombinant antibody production. Biotechnol. Bioeng.

212.


108:3025-3031.

Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ,Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murinemyeloma cell lines with varying recombinant monoclonal antibody production rate.Biotechnol. Bioeng. 88:474–488.

Proteomic analysis of four different NS0 cell lines, with different levels ofantibody production, identified a number of proteins with altered abundances.Amongst these proteins a number of molecular chaperones involved in foldingand assembly of immunoglobulins were identified. The authors also report anabundance of light chain protein over heavy chain protein a likely prerequisitefor efficient MAb production.

118.

Stephens S, Emtage S, Vetterlein O, Chaplin L., Bebbington CR, Nesbitt A,Sopwith M, Athwal D, Novak C, Bodmer M (1995) Comprehensivepharmacokinetics of a humanised antibody and analysis of residual anti-idiotypicresponses. Immunol. 85:668-674.

Describes pharmacokinetics in monkeys and human volunteers of anti TNFantibody (CDP571) produced in GS-NS0. In humans the antibody was welltolerated with a half life of approx. 13 days and anti CDP571 antibodies werelow or undetectable at higher doses. At lower doses there was a transient IgMresponse which was anti-idiotypic.

72.

Tait AS, Hogwood CEM, Smales CM, Bracewell DG (2012) Host cell proteindynamics in the supernatant of a mAb producing CHO cell line. Biotechnol. Bioeng.109:971-982.

206.

Teixeira AP, Duarte TM, Carrondo MJT, Alves PM (2011) Synchronousfluorescence spectroscopy as a novel tool to enable PAT applications inbioprocesses. Biotechnol. Bioeng. 108:1852-1861.

198.

Teixeira AP; Duarte TM; Oliveira R; Carrondo MJT; Alves PM (2011) High-throughput analysis of animal cell cultures using two-dimensional fluorometry.J.Biotechnol. 151:255-260.

229.

Tey BT, Singh RP, Al-Rubeai M (1999) Influence of bcl-2 over-expression on NS0and CHO culture viability and chimeric antibody productivity. In: Animal CellTechnology: Products from Cells, Cells as Products. Eds: Bernard A et al. pp 59-61Kluwer.

Over-expression of bcl-2 significantly reduced the rate of cell death in a GS-CHO and in GS-NS0 cell line and resulted in a 19% and 25% increase,respectively in antibody production.

98.

Tey BT, Singh RP, Piredda L., Piacentini M, Al-Rubeai M (2000) Influence of bcl-2on cell death during the cultivation of a Chinese hamster ovary cell line expressinga chimeric antibody. Biotech. Bioeng. 68:31-43.

Transfection with the control expression vector (Neo marker) used in this studyand exposure to the selective drug G418 led to upregulation of endogenousbcl-2. This led to an increase in cell viability in cell cultures and prolongedsurvival. There was no influence on antibody titre.

99.

Tey BT, Singh RP, Piredda L, Piacentini MP, Al-Rubeai M (2000) Bcl-2 mediated 147.


suppression of apoptosis in myeloma NS0 cultures. J. Biotechnol. 79:147-159.

In batch culture, no difference was seen in product concentration between cellline over-expressing Bcl-2 and control cell line. However, in fed-batch cultureusing a concentrated amino acid feed, antibody concentration was increased60% in the Bcl-2 over-expressing cell line..

Tey BT, Al-Rubeai M. (2004) Suppression of apoptosis in perfusion culture ofMyeloma NS0 cells enhances cell growth but reduces antibody productivity.Apoptosis 9:843-852.

160

Tey BT, Al-Rubeai M (2005) Effect of Bcl-2 overexpression on cell cycle andantibody productivity in chemostat cultures of myeloma NS0 cells. J Biosci Bioeng100:303-310.

159

van Berkel PHC, Gerritsen J, Perdok G, Valbjørn J, Vink T, van de Winkel JGJ,Parren PWHI. (2009) N-linked glycosylation is an important parameter for optimalselection of cell lines producing biopharmaceutical human IgG. Biotechnol. Prog.25:244-251.

174.

Wayte J, Boraston R, Bland H, Varley J, Brown M (1997) pH: effects on growth andproductivity of cell lines producing monoclonal antibodies: control in large-scalefermenters. The Genetic Engineer and Biotechnol. 17:125-132.

Describes effect of pH on growth and productivity of a GS-NS0 cell line makinga humanised monoclonal antibody. Productivity increased at pH7.1 comparedwith 7.4.

94.

Yoon S, Konstantinov KB (1994) Continuous, real-time monitoring of the oxygenuptake rate (OUR) in animal cell bioreactors. Biotech. Bioeng. 44:983-990.

Describes oxygen uptake monitoring for cultures of GS-NS0 cells makinghumanised anti TNF antibody. Used a perfused stirred reactor with cellretention device. OUR was in the range 7 to 13 pg/cell per hour depending oncell density.

59.

Watanabe S, Shuttleworth J, Al-Rubeai M (2002) Regulation of cell cycle andproductivity in NS0 cells by the over-expression of p21CIP1. Biotechnol. Bioeng.77:1-7.

148.

Wu MH, Dimopoulos G, Mantalaris A, Varley J. (2004) The effect of hyperosmoticpressure on antibody production and gene expression in the GS-NS0 cell line.Biotechnol. Appl. Biochem. 40:41-46.

Describes growth and productivity kinetics as well as gene expression, usingmicroarray technology, at various osmolalities..

128.

Zhou W, Chen C-C, Buckland B, Aunins J (1997) Fed-batch culture of recombinantNS0 myeloma cells with high monoclonal antibody production. Biotechnol. Bioeng.55:783-792.

Describes feeding strategy leading to final antibody concentration in excess of2.7g/L. Describes transitions in metabolism caused by nutrient depletion. Thecell line used in the study had three vector copies.

90.


SECTION 4: Recombinant Proteins (non-antibody) fromNS0 Cells

Cannon-Carlson S, Varnerin J, Tsarbopoulos A, Jenh C-H, Cox MA, Chou C-C,Connelly N, Zavody P, Tang JC-T (1998) Expression Purification andCharacterisation of Recombinant Human Interleukin-13 from NS0 Cells. ProteinExpression and Purification. 12:239-248.

IL-13 expressed in NS0 cells using GS expression system. Cells grown in 15litre fed batch culture. Details given of the medium and feed used. The proteinis unglycosylated.

92.

Chen P, Cataldo S, Dennis P, Popoloski J, Dabora R (1994) A comparison ofChinese hamster ovary (CHO) and mouse myeloma (NS0) cell lines producing arecombinant protein. American Chem. Soc. Abs. of 207th National Meeting SanDiego.

Rec protein expressed at level of 120 mg/l with CHO DHFR in suspension andat 450 mg/l in GS-NS0 in batch culture (following medium optimisation).Substantial differences seen in the carbohydrate structure and pharmacokineticproperties of the protein produced in the two cell lines.

11.

Flesher AR, Marzowski J, Wang W-C, Raff HV (1995) Fluorophore-labeledcarbohydrate analysis of immunoglobulin fusion protein: correlations ofoligosaccharide content with in-vivo clearance profile. Biotech. and Bioeng.46:399-407.

Compared glycosylation and in-vivo clearance of a CTLA4/immunoglobulinfusion protein made in GS-NS0 cells with that made in CHO cells. NS0 derivedprotein had no detectable N-acetylneuraminic acid and had concomitantaccelerated clearance.

66.

Gofton CM, Roberts G, Bergin S, Owens RJ (1992) The rapid production ofrecombinant rabbit metalloproteinases in myeloma cells. In: Animal CellTechnology: Developments, processes and products. Butterworths, pp48-50.

Procollagenase, prostromelysin and TIMP expressed rapidly in NS0 using GSgiving non-optimised fermenter yields of 46, 48.5 and 59.2 mg/l respectively.

23.

Hornick JL; Khawli LA; Hu PS; Lynch M; Anderson PM; Epstein AL. (1997)Chimeric CLL-1 antibody fusion proteins containing granulocyte-macrophagecolony-stimulating factor or interleukin-2 with specificity for B-cell malignanciesexhibit enhanced effector functions while retaining tumour targeting properties.Blood 89:4437-4447.

225.

Li SL, Liang SJ, Guo N, Wu AM, Fujita-Yamaguchi Y (2000) Single-chainantibodies against human insulin-like growth factor I receptor: expression,purification and effect on tumor growth.

Recloning of anti-IGF-IR monoclonal antibody as a single-chain Fv withsuccessful expression obtained in a NS0 cell line.

113.

Murphy G, Houbrechts A., Cockett MI, Williamson RA, O'Shea M, Docherty AJP(1991a) The N-terminal domain of the tissue inhibitor of metalloproteinases (TIMP)is inhibitory. Biochem. 30:8097-8102.

41.


TIMP 1 and a truncated version expressed in GS-NS0.

Murphy G, Cockett MI, Ward RV, Docherty AJ (1991b) Matrix metalloproteinasedegradation of elastin, type IV collagen and proteoglycan. A quantitativecomparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and–2 and punctuated metalloproteinase (PUMP). Biochem. J. 277:277-279.

Punctuated metalloproteinase (PUMP) and stromelysin 2 expressed in GS-NS0cells.

40.

Murphy G, Allan JA, Willenbrock F, Cockett MI, O'Connell JP, Docherty AJ(1992a) The role of the C-terminal domain in collagenase and stromelysinspecificity. J. Biol. Chem. 267:9612-9618.

Collagenase, stromelysin and truncated versions of these enzymes expressedin GS-NS0 cells.

39.

Murphy G, Willenbrock F, Ward RV, Cockett MI, Eaton D, Docherty AJP (1992b)The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but isessential for membrane activation and modulates interactions with tissue inhibitorsof metalloproteinases. Biochem. J. 283:637-641.

Gelatinase and a variant expressed in GS-NS0.

42.

O Shea M, Willenbrock F, Williamson RA, Cockett MI, Freedman RB, Reynolds JJ,Docherty AJP, Murphy G (1992) Site-directed mutations that alter the inhibitoryactivity of the tissue inhibitor of metalloproteinases-1: Importance of the N-terminalregion between cysteine 3 and cysteine 13. Biochem. 31:10146-10152.

Wild type and mutated TIMPS in GS-NS0 cells. Most produced at 30mg/l but forsome variants productivity very low.

43.

Robinson MK, Andrew D, Rosen H, Brown D, Ortlepp S, Stephens P, Butcher EC(1992) Antibody against the leu-CAM b-chain (CD18) promotes both LFA-1 andCR3-dependent adhesion events. J. Immunol. 148:1080-1085.

CD18 expressed in GS-NS0 cells.

45.

Rossman C, Sharp N, Allen G, Gewert D (1996) Expression and purification ofrecombinant, glycosylated human interferon alpha 2b in murine Myeloma NS0 cells.Protein Expression and Purification. 7:335–342.

Expressed Interferon alpha in GS-NS0 cells and isolated a cell line expressing20 g/106 cells/24h and accumulating 120 g/mL in culture. Glycosylation wassimilar to that of non recombinant interferon purified from human Namalwa cells.This is the highest reported level of glycosylated, recombinant IFN expression ina stable mammalian system.

85.

Young RJ, Owens RJ, Mackay GA, Chan CMW, Shi J, Hide M, Francis DM, HenryAJ, Sutton BJ, Gould HJ (1995) Secretion of recombinant human IgE-Fc bymammalian cells and biological activity of glycosylation site mutants. Protein Eng.8:193-199.

Established permanent GS-CHO and GS-NS0 cell lines making Fc. The NS0cell line accumulated up to 100 mg product per litre in contrast to ~2 mg/L in

107


CHO.

Zhou W, Bibila T, Glazomitsky K, Montalvo J, Chan C, DiStefano D, Munshi S,Robinson D, Buckland B, Aunins J. (1996) Large scale production of recombinantmouse and rat growth hormone by fed-batch GS-NS0 cell cultures. Cytotechnol.22:239-250.

Transfected NS0 cells with GS vector containing growth hormone genes andsequence for targeting integration by homologous recombination (for optimalexpression). Operated fed-batch cultures at 250l scale and information onoperating conditions is given. Yields of mouse and rat growth hormone were580 and 240 mg/l respectively. Data presented on cell metabolism. Glucoseconsumption rate decreased during transition to stationary phase and lactatewas consumed during stationary phase.

77.

SECTION 5: Recombinant Proteins (non-antibody) fromCHO Cells

Blochberger TC, Cooper C, Peretz D, Tatzelt J, Griffith OH, Baldwin MA, PrusinerSB (1997) Prion protein expression in Chinese hamster ovary cells using aglutamine synthetase selection and amplification system. Protein Engineering10:1465-1473

Describes expression of hamster prion proteins in CHO cells using the GSsystem.

91.

Brady RL, Dodson EJ, Dodson GG, Lange G, Davis SJ, Williams AF, Barclay AN(1993) Crystal structure of domains 3 and 4 of rat CD4: Relation to the NH2-terminal domains, Science 260:979-983.

Recombinant rat CD4 D3 and D4 expressed using GS.

96.

Brown MH, Barclay AN (1994) Expression of immunoglubulin andscavenger receptor superfamily domains as chimeric proteins with domains 3 and 4of CD4 for ligand analysis. Protein Engineering 7:515-521.

Describes the use of GS-CHO cells to express proteins in which immunoglobulinsuperfamily domains ( CD4d 3 + 4 ) are used as expression vehicles for theproduction of chimeric proteins containing other superfamily domains.

95.

Castro MG, Tomasec P, Morrison E, Murray CA, Hodge P, Blanning P, Linton E,Lowry PJ, Lowenstein PR (1995) Mitogenic effects and nuclear localisation ofprocorticotrophin-releasing hormone expressed within stably transfected fibroblastcells (CHO-K1). Molecular and Cellular Endocrinol. 107:17-27.

Hormone expressed in GS-CHO cells. Cells expressing hormone had anincreased proliferation rate.

63.

Clarke S, Dillon J, Smith A, Sotheran E (2004) Strategies for producing commercialcell lines. BioProcess Interntl. 2(4) April 2004, pp 48 – 52.

The cell culture experiences of Lorantis Ltd from Cambridge in the UK. Theychose the GS expression system and CHOK1 cells to construct a stable cell lineexpressing recombinant protein (Delta-1 derived from Notch binding protein)fused to an antibody constant region. Details of their project along methods for

114.


selection and cloning are presented.

Classon BJ, Brown MH, Garnett D, Somoza C, Barclay AN, Willis AC, Williams AF(1992) The hinge regions of the CD8 alpha chain: structure, antigenicity and utilityin expression of immunoglobulin superfamily domains. Int. Immunol. 4:(2), 215-225.

Expression of SCD8 in GS-CHO. Yields of ca. 20mg/l in roller bottle culture.Used 2mM sodium butyrate to enhance production.

12.

Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissueinhibitor of metalloproteinases in Chinese hamster ovary cells using glutaminesynthetase gene amplification. BioTechnol. 8:662-667.

Yields of 180mg/L achieved in shake flask culture. Sodium butyrate enhancedproduction.

13.

Cockett MI, Bebbington CR, Yarranton GT (1991) The use of engineered EIAgenes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. Nucl.Acids. Res. 19:319-325.

TIMP expressed in GS-CHO with EIA. In the presence of EIA non amplifiedlines gave levels comparable with gene amplified GS-CHO lines (110 g/106

cells/24h). EIA also enhanced production of procollagenase (13 g/106cells/24h).

14.

Cosgrove L, Lovrecz GO, Verkuylen A, Cavaleri L., Black LA, Bentley JD, HowlettGJ, Gray PP, Ward CW, McKern NM (1995) Purification and properties of insulinreceptor ectodomain from large scale mammalian cell culture. Protein Expressionand Purification 6:789-798.

Ectodomain expressed in GS-CHO and cells grown in a perfused 40 litre airliftfermenter on microcarrier beads. MSX required to maintain stable production(amplified cell line) but was omitted from production fermenter.

78.

Crouch E, Chang D, Rust K, Pesson A, Heuser J (1994) Recombinant pulmonarysurfactant protein D. J. Biol. Chem. 269:15808-15813

GS-CHO used to express rat SP-D.

64.

Davis SJ, Ward HA, Puklaver MJ, Willis AC, Williams AF, Barclay AN (1990) Highlevel expression in Chinese hamster ovary cells of soluble forms of CD4 Tlymphocyte glycoprotein including glycosylation variants. J. Biol. Chem. 265:10410-10418.

S CD4 and S CD4 (half) expressed at levels of 80 and 25 g/L from GS-CHOcells. Used GMEM-S + 5% FCS and MSX (15 or 100 M) in roller bottles.Sodium butyrate added to enhance production. Also used switch to serum freemedium (GMEM-S + 2 mM butyrate) at final stage, to aid purification.

15.

Dorai H, Nemeth JF, Cammaart E Wang Y, Tang QM, Magill A, Lewis MJ, Raju TS,Picha K, O'Neil K, Ganguly S, Moore G. (2009) Development of mammalianproduction cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODYTM: Factors that influence productivity and product quality. Biotechnol.Bioeng. 103:162-176.

173.


Fahnestock M, Johnson JL., Feldman RMR, Tsomides TJ, Mayer J, Narhi LO,Bjorkman PJ (1994) Effects of peptide length and composition on binding to anempty class 1 MHC heterodimer. Biochem. 33:8149-8158.

Heterodimers expressed in GS CHO.

57.

Fahnestock ML, Johnson JL, Renny Feldman RM, Neveu JM, Lane WS, BjorkmanPJ (1995) The MHC class I homolog encoded by human cytomegalovirus bindsendogenous peptides. Immunity 3:583-590.

A secreted form of the MHC class I heavy chain homolog was expressedtogether with human 2m using the GS expression system in CHO cells.

68.

Field R, Cockett M, Froud SJ (1988) Glutamine synthetase amplification of TIMPexpression in CHO cells. In: Advances in Cell Biology and Technology forBioprocesses, Butterworths, pp195-197.

80mg/L TIMP made in fermenters.

17.

Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Preneta A,Stephens PE, Thompson S, Yarranton GT (1989) The development of a process forthe production of HIV1 GP120 from recombinant cell lines. In: Production ofBiologicals from Animal Cells in Culture, Butterworths, pp110-116.

GP120 expressed in amplified GS-CHO. Production levels of 1-3mg/l Cleavageof GP120 coincides with depletion of asparagine, glutamate, aspartate andserine from medium.

19.

Gastinel LN, Simister NE, Bjorkman PH (1992) Expression and crystallisation of asoluble and functional form of an Fc receptor related to class 1 histocompatibilitymolecules. Proc. Natl. Acad. Sci. 89(2): 638-642.

Expression of neonatal Fc receptor (cell surface and soluble forms) in amplifiedGS-CHO. Soluble form gave ca. 40mg/l. Cells also grown in hollow fibrereactor.

20.

Gjorloff A, Hedlund G, Kallard T, Sarson D, Fischer H, Trowsdale J, Sjogren HO,Dohlsten M (1992) The LFA-3 adhesion pathway is differently utilised by super-antigen-activated human CD4+ T-cell subsets. Scand. J. Immunol. 36(2):243-250.

HLA – DR4 and LFA3 expressed in amplified GS-CHO. Protein expressed oncell membrane.

21.

Gloor S, Nasse K, Essen LO, Appel F (1992) Production and secretion in CHOcells of the extracellular domain of AMOG.beta 2, a type-11 membrane protein.Gene 120(2):307-312.

Recombinant AMOG.beta 2 membrane protein secreted by amplified GS-CHOcells and concentrations of 15mg/L achieved.

22.

Guerini D, Schroder S, Foletti D, Carafoli E (1995) Isolation and characterization ofa stable Chinese hamster ovary cell line overexpressing the plasma membraneCa2+ ATPase. J. Biol. Chem. 270:14643-14650.

GS – CHO cell lines created. Initial transfectants were subjected to additionalrounds of selections with MSX resulting in clones with 4 to 10 gene copies per

67.


cell. The stability of expression of transfected cells was examined in thepresence and absence of selective drug (MSX). In the absence of MSXproductivity was retained over short culture periods but was substantiallydecreased after 30 to 40 passages. In contrast stable production wasmaintained for 6 months in the presence of MSX.

Gutman O, Danieli T, White JM, Henis YI (1993) Effects of exposure to low pH onthe lateral mobility of influenza haemagglutinin expressed at the cell surface.Correlation between mobility inhibition and inactivation. Biochem. 32:101-106.

HA expressed on surface of amplified GS-CHO cells.

25.

Harfst E, Johnstone AP (1992) Characterisation of the glutamine synthesaseamplifiable eukaryotic expression system applied to an integral membrane protein –the human thyrotropin receptor. Anal. Biochem. 207:80-84.

Thyrotropin expressed on surface of CHO cells using GS selection vector atlevels at least 10 x that achieved in other systems. Receptor is efficientlycoupled to adenylate cyclase.

26.

Harfst E, Johnstone AP, Gout I, Taylor AH, Waterfield MD, Nussey SS (1992a) Theuse of the amplifiable high-expression vector pEE14 to study the interactions ofautoantibodies with recombinant human thyrotrophin receptor. Mol. Cell.Endocrinol. 83(2-3):117-123.

Functional Thyrotrophin receptor expressed at high level on surface of GS-CHO.In comparison Baculovirus system gave no demonstrable protein production.

27.

Harfst E, Johnstone AP, Nussey SS (1992b) Characterisation of the extracellularregion of the human thyrotropin receptor expressed as a recombinant protein. J.Mol. Endocrinol. 9:227-236.

Describes isolation and partial characterisation of recombinant thyrotropinreceptor from GS-CHO cells.

28.

Harfst E, Johnstone AP, Nussey SS (1992c) Interaction of thyrotropin and thyroid-stimulating antibodies with recombinanct extracellular region of human TSHreceptor. Lancet 339:193 - 194.

CHO cells expressing recombinant thyrotropin receptor from a GS-vector usedto study interaction of receptor with autoantibodies from sera of patients withautoimmune disease.

29.

Hovey A, Bebbington CR, Jenkins N (1994) Simultaneous control of growth andproductivity using a mutant CHO cell line. In Animal Cell Technology: Products oftoday, prospects for tomorrow. eds R.E. Spier, J.B. Griffiths and W.Berthold.Butterworth–Heinemann, pp422-424.

Heat shock induction used to produce recombinant TIMP from a mutant CHOcell line. TIMP was expressed in a GS vector.

70.

Hovey A, Bebbington CR, Jenkins N (1994) Control of growth and recombinantprotein synthesis by heat-shock in a mutant mammalian cell line. Biotechnol.Letters. 16:215-220.

Describes use of heat shock to induce recombinant protein (TIMP) synthesis in

75.


a temperature sensitive CHO cell line. TIMP expressed in a GS vector.

Jenkins N, Hovey A (1993) Generation of CHO cell mutants for growth control. InKaminogawa, S. et al. (eds) Animal Cell Technology: Basic and Applied Aspects 5,pp267-272.

Created temperature sensitive mutant of CHO expressing TIMP from GSvector. Temperature switching arrested cell growth and increased productyield.

76.

Jenkins N, Hovey A (1993) Temperature control of growth and productivity inmutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotech.Bioeng. 42:1029-1036.

Temperature sensitive mutants of CHO cells were transfected with the gene forTIMP using GS amplification. Alternating incubation between permissive (34oC)and non permissive (39°C) temperatures arrested cell growth whilst maintaininghigh cell viability for up to 170 hours. TIMP production rate was increased 3 to 4fold over the period of arrest compared with controls at 34° resulting in a 35%increase in yield.

69.

Kemble GW, Henis YI, White JM (1993) GPI- and transmembrane anchoredinfluenza haemagglutinin differ in structure and receptor binding activity. J. CellBiol. 122:1253-1265.

Expressed haemagglutinin including engineered variants in amplified GS-CHOcells.

31.

King DJ, Byron OD, Mountain A, Weir N, Harvey A, Lawson ADG, Proudfoot KA,Baldock D, Harding SE, Yarranton GT, Owens RJ (1993) Expression, purificationand characterization of B72.3 Fv fragments. Biochem. J. 290:723-729.

Fv expressed in GS-CHO and in E.coli. Yields of 4mg/L achieved in CHO rollerbottles and 450 mg/Lin E. coli fermentations.

32.

Kuwae S, Ohda T, Tamashima H, Miki H. Kobayashi K (2005) Development of afed-batch culture process for enhanced production of recombinant humanantithrombin by Chinese hamster ovary cells. J. Biosci. Bioeng. 100:502-510.

Expressed human antithrombin at 1 g/L using GS-CHO cells..

145.

Laubach VE, Garvey EP, Sherman PA (1996) High-level expression of humaninducible nitric oxide synthase in Chinese hamster ovary cells and characterisationof the purified enzyme. Biochem. Biophys. Res. Comm. 218:802-807.

iNOS expressed in CHO cells using GS system. Amplification in the presenceof 400 M MSX increased product levels 3 to 4 fold. Expression was enhancedby sodium butyrate (2 mM). Expression levels were at least 20-fold higher thanthose reported for a baculovirus expression system. Also noted that sodiumbutyrate could restore high level expression in some cell lines in which iNOSexpression declined with time in culture.

88.

McCall MN, Shotton DM, Barclay AN (1992) Expression of soluble isoforms of ratCD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45Rmonoclonal antibodies. Immunol. 76:310-317.

35.


Four soluble isoforms of CD45 expressed in GS-CHO at 5mg/L of spent culturemedium.

McKnight AJ, Classon BJ (1992) Biochemical and immunological properties of ratrecombinant interleukin 2 and interleukin 4. Immunol. 75:286-292.

IL-2 and IL-4 expressed in GS-CHO.

36.

Moore JP, McKeating JA, Jones IM, Stephens PE, Clements G, Thomson S, WeissRA (1989) Characterisation of recombinant gp120 and gp160 from HIV-1: bindingto monoclonal antibodies and soluble CD4. AIDS 4:307-315.

Compares properties of GP120 made in GS-CHO cells with that made in insectcells. The CHO derived protein bound SCD4 with affinity similar to viral GP120.Product from insect cells had lower affinity.

38.

Quilliam AL, Osman N, McKenzie IFC, Hogarth PM (1993) Biochemicalcharacterization of murine FcRI. Immunol. 78:358-363.

Fc receptor expressed on surface of GS-CHO cells.

44.

Sano H, Chiba H, Iwaki D, Sohma H, Voelker DR, Kuroki Y (2000) Surfactantproteins A and D bind CD14 by different mechanisms. J. Biol. Chem. 275:22442 -22451.

Used GS expression system with CHOK1 and pEE14.4 to express CD14 from agenerated stable cell line. An insect cell line was also used for comparison.Mammalian expressed CD14 consistent with insect expression. Lung surfactantproteins SP-A and D are important in the innate immunity of the lung and wereinvestigated in to their mechanism for binding CD14.

120.

Springer S, Döring K, Skipper JCA, Townsend ARM, Cerundolo V (1998) Fastassociation rates suggest a conformational change in the MHC class 1 molecule H-2Db upon peptide binding. Biochem. 37:3001-3012.

Soluble H-2Db class 1 molecules expressed in CHO cells using GS systems.Several rounds of amplification used.

89.

Stennicke HR; Kjalke M; Karppf DM; Balling KW; Johansen PB.; Elm T; Øvlisen K;Möller F; Holmberg HL; Gudme CN; Persson E; Hilden I; Pelzer H; Rahbek-NielsenH; Jespersgaard C; Bogsnes A; Pedersen AA; Kristensen AK; Peschke B; KappersW; Rode F; Thim L; Tranholm M; Ezban M; Olsen EH; Bjørn SE. (2013) A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolongedeffect in hemophilic mice models. Blood 121:2106-2108.

http://www.bloodjournal.org/content/121/11/2108.full-text.pdf+html

243.

Trowbridge IS, Johnson P, Ostergaard H, Hole N (1992) Structure and function ofCD45: a leukocyte-specific protein tyrosine phosphatase. Adv. Exp. Med. Biol.323:29-37.

CD45 external domain expressed at 20 mg/L in GS CHO cells.

48.

Williams AF, Davis SJ, He Q, Barclay AN (1989). Structural diversity in domains ofthe immunoglobulin superfamily. Cold Spring Harb. Symp. Quart. Biol. 54:Pt 2, 49.


637-647.mead Expression of soluble rat CD4 in amplified GS-CHO.

SECTION 6: Product Characteristics and Critical QualityAttributes

Baker KN, Rendal, MH, Hills AE, Hoare M, Freedman RB, James DC (2001)Metabolic control of recombinant protein N – glycan processing in NS0 and CHOcells. Biotech. Bioeng. 73:188-202.

The authors compare glycosylation of TIMP (tissue inhibitor ofmetalloproteinases 1) made in GS-NS0 and GS-CHO. Significant differenceswere found. In NS0 derived material, 30% of N-glycan antennae terminated inalpha 1, 3 linked galactose (none found in CHO). The level of sialylation wasslightly higher in NS0 than CHO. Sialic acid from CHO was predominantly N-acetylneuraminic acid whilst NS0 – derived glycan contained equal proportionsof N-glycolyl and N-acetyl variants. It was demonstrated that manipulation ofnucleotide - sugar metabolism (by addition of precursors) could be used tomodify glycan processing.

104.

Ball C, Fox B, Hufton S; Sharp G; Poole S; Stebbings R; Eastwood D; Findlay L;Parren PWHI; Thorpe R; Bristow A; Thorpe SJ. (2012) Antibody C region influencesTGN1412-like functional activity in vitro. J. Immunol. 189:5831-5840.

http://www.jimmunol.org/content/189/12/5831.abstract

244.

Bebbington CR (1995) Expression of antibody genes in mammalian cells. In Zola,H. (ed). "Monoclonal antibodies: the next generation" Bios Scientific. (Oxford)pp165-181.


71.

Chen P, Cataldo S, Dennis P, Popoloski J, Dabora R (1994) A comparison ofChinese hamster ovary (CHO) and mouse myeloma (NS0) cell lines producing arecombinant protein . American Chem. Soc. Abs. of 207th National Meeting SanDiego.

Rec protein expressed at level of 120 mg/L with DHFR-CHO in suspension andat 450 mg/L in GS-NS0 in batch culture (following medium optimisation).Substantial differences seen in the carbohydrate structure and pharmacokineticproperties of the protein produced in the two cell lines.

11.

Dorai H, Nemeth JF, Cammaart E Wang Y, Tang QM, Magill A, Lewis MJ, Raju TS,Picha K, O'Neil K, Ganguly S, Moore G. (2009) Development of mammalianproduction cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODYTM: Factors that influence productivity and product quality. Biotechnol.Bioeng. 103:162-176.

173.

Dorai H; Santiago A; Campbell M; Tang QM; Lewis MJ; Wang Y; Lu QZ; Wu SL;Hancock W. (2011) Characterization of the proteases involved in the N-terminalclipping of glucagon-like-peptide-1-antibody fusion proteins. Biotechnol. Prog.

228.


27:220-231.

Flesher AR, Marzowski J, Wang W-C, Raff HV (1995) Fluorophore-labeledcarbohydrate analysis of immunoglobulin fusion protein: correlations ofoligosaccharide content with in-vivo clearance profile. Biotech. Bioeng. 46:399-407.

Compared glycosylation and in vivo clearance of a CTLA4/immunoglobulinfusion protein made in GS-NS0 cells with that made in CHO cells. NS0 derivedprotein had no detectable N-acetylneuraminic acid and had concomitantaccelerated clearance.

66.

Fu J, Bongers J, Tao L, Huang D, Ludwig R, Huang Y, Qian Y, Basch , Goldstein J,Krishnan R, You L, Li ZJ, Russell RJ. (2012) Characterization and identification ofalanine to serine sequence variants in an IgG4 monoclonal antibody produced inmammalian cell lines. J. Chromat. B 908:1-8.

216.

Grainger RK, James DC. (2013) CHO cell line specific prediction and control ofrecombinant monoclonal antibody N-glycosylation. Biotechnol. Bioeng. 110:2970-2983.


234.

Gramer MJ, Goochee CF (1994) Glycosidase activities of the 293 and NS0 celllines, and of an antibody producing hybridoma cell line. Biotechnol. Bioeng.43:423-428.

Several glycosidases detected in NS0 cell lysates. Sialidase present and stableat pH 4.5 but unstable at 7.5. CHO sialidase stable at 7.5.

24.

Gramer MJ, Eckblad JJ, Donahue R, Brown J, Shultz C, Vickerman K, Priem P,van den Bremer ETJ, Gerritsen J, van Berkel PHC (2011) Modulation of antibodygalactosylation through feeding of uridine, manganese chloride, and galactose.Biotechnol. Bioeng. 108:1591-1602.

200.

Hansen R, Dickson AJ, Goodacre R, Stephens GM, Sellick CA (2010) Rapidcharacterization of N-linked glycans from secreted and gel-purified monoclonalantibodies using MALDI-ToF mass spectrometry. Biotechnol. Bioeng. 107:902-908.

195.

Hills AE, Patel A, Boyd P, James DC (2001) Metabolic control of recombinantmonoclonal antibody N-glycosylation in GS-NS0 cells. Biotechnol. Bioeng. 75:239-251.

Describes impact of manipulating intracellular pools of UDP-sugars uponglycosylation of an IgG4.

144.

Kilgore BR, Lucka A, Patel R, Andrien B, Dhume ST (2005) Cell line Switch fromNS0 to CHO Causes Several Changes in Glycosylation of Recombinant IgG. ACS33rd Northeast Regional Meeting (Abstract)

http://acs.confex.com/acs/nerm05/techprogram/P21476.HTM

214.

Kilgore BR; Lucka AW; Patel R; Andrien Jr BA; Dhume ST. (2008) Comparabilityand monitoring immunogenic N-linked oligosaccharides from recombinantmonoclonal antibodies from two different cell lines using HPLC with fluorescencedetection and mass spectrometry. Meth. Mol. Biol. 446:333-346.

232.


Lifely MR, Hale C, Boyce S, Keen MJ, Phillips J (1995) Glycosylation andbiological activity of Campath - IH expressed in different cell lines and grown underdifferent culture conditions. Glycobiol. 5:813-822.

Antibody expressed in CHO (DHFR) rat YO myeloma and NS0 (GS) cells.Glycosylation varied significantly between cell lines and to a minor extentdepending on culture conditions. YO antibody was unusual in having abisecting GlcNAc on the core oligoscaccharide and NS0 antibody appeared tobe under glycosylated. YO antibody had enhanced activitity in ADCC assayscompared with CHO or NS0.

74.

McCracken NA; Kowle R; Ouyang A. (2014) Control of galactosylated glycoformsdistribution in cell culture system. Biotechnol. Prog. 30:547-553.


241.

Obrezanova O, Arnell A, Gómez de la Cuesta R, Berthelot ME, Gallagher TRA,Zurdo J, Stallwood Y (2015) Aggregation risk prediction for antibodies and itsapplication to biotherapeutic development. mAbs 7:352-363

http://www.tandfonline.com/doi/full/10.1080/19420862.2015.1007828#abstract

251.

Perani A; Gloria B; Wang D; Murphy R; Singh HK; Smyth FE; Scott AM. (2011)Impact on product quality of high productive GS-CHO cell lines. BMC Proceedings5(S8):17.


233.

Reid CQ, Tait AS, Baldascini H, Mohindra A, Racher AJ, Bilsborough S, SmalesCM, Hoare M (2010) Rapid whole monoclonal antibody analysis by massspectrometry: An ultra scale-down study of the effect of harvesting by centrifugationon the post-translational modification profile. Biotechnol. Bioeng. 107:85-95.

188.

Robinson DK, Chan CP, Yu Ip, CC, Seamans TC, Lee DK, Lenny AB, Tung J-S,DiStefano DJ, Munshi S, Gould SL, Tsai PK, Irwin J, Mark GE, Silberklang M(1994) Product consistency during long-term fed-batch culture. In:Animal CellTechnology: Products of Today, Prospects for Tomorrow. Eds: Spier RE, GriffithsJB, Berthold W, Butterworths-Heinemann, pp763-767.

Expressing antibody genes in GS-NS0. Yields up to 1.8g/L in three to four weekduration fed batch. Glycoform pattern shifts towards incomplete mannose-terminated glycans as culture progresses.

46.

Robinson DK, Chan CP, Yu Ip, C, Tsai PK, Tung J, Seamans TC, Lenny AB, LeeDK, Irwin J, Silberklang M (1994) Characterization of a recombinant antibodyproduced in the course of a high yield fed-batch process. Biotech. Bioeng. 44:727-735.

Anti-HIV gp120 Mab expressed in non-amplified GS-NS0 system. Fed-batchprocess developed which increased yields ten-fold compared with batch culture.The clone produced 0.86g mAb/L over 22 days. Antigen binding and MWtremained constant throughout culture. Four major IEF bands were observedwith a minor fifth more acidic band appearing later in culture. Pattern ofglycoforms secreted by cells changes as culture progresses.

52.


Rossman C, Sharp N, Allen G, Gewert D (1996) Expression and purification ofrecombinant, glycosylated human interferon alpha 2b in murine myeloma NS0 cells.Protein Expression and Purification 7:335 – 342.

Expressed Interferon Alpha in GS-NS0 cells and isolated a cell line expressing20 g/106 cells/24h and accumulating 120 g/mLl in culture. Glycosylation wassimilar to that of non recombinant interferon purified from human Namalwa cells.This is the highest reported level of glycosylated, recombinant IFN expression ina stable mammalian system.

85.

van Berkel PHC, Gerritsen J, Perdok G, Valbjørn J, Vink T, van de Winkel JGJ,Parren PWHI. (2009) N-linked glycosylation is an important parameter for optimalselection of cell lines producing biopharmaceutical human IgG. Biotechnol. Prog.25:244-251.

174.

Yu Ip CC, Miller WJ, Silberklang M, Mark GE, Ellis RW, Huang L, Glushka J, VanHalbeek H, Zhu J, Alhadeff JA (1994) Structural characterisation of the N-Glycansof a humanised Anti-CD18 murine immunoglobulin G. Arch Biochem. Biophys.308:87 – 399.

Identifies glycan structures of humanised IgG4 expressed in GS-NS0.

51.

SECTION 7: Process Conditions for GS Cell Linesincluding Media and Feeding Strategies

Bibila TA, Ranucci CS, Glazomitsky K, Buckland BC, Aunins JG (1994)Monoclonal antibody process development using medium concentrates. Biotechnol.Prog. 10:87-96.

Describes fed-batch process using concentrated medium with GS-NS0 cell linesmaking recombinant antibodies. Up to 7-fold increases in antibody titre wereachieved compared with batch culture.

54.

Bibila TA, Robinson DK (1995) In pursuit of the optimal fed-batch process formonoclonal antibody production. Biotechnol. Prog. 11:1-13.

A review of approaches taken to optimise fed-batch processes for antibodyproduction.

60.

Birch JR, Boraston RC, Metcalfe H, Bebbington CR, Field RP (1994) Selecting anddesigning cell lines for improved physiological characteristics. Cytotechnol. 15:11-16.

Describes transfection of GS into hybridoma to give glutamine independence.Productivity was increased in glutamine free medium. Also describes theisolation of a cholesterol independent variant of the NS0 myeloma cell line.

58.

Birch JR, Froud S (1994) Mammalian cell culture systems for recombinant proteinproduction. Biologicals 22:127-133.

Gives example of MAb production (200 mg/L) using GS-NS0 cells in protein-freemedium.

53.

Broad D, Boraston R, Rhodes M (1991) Production of recombinant proteins inserum-free media. Cytotechnol. 5:47-55.

8.


Compares productivity of three amplified GS-CHO lines making recombinantproteins in the presence and absence of serum (no significant differences seen).

Brown ME, Renner G, Field RP, Hassell T (1992) Process development for theproduction of recombinant antibodies using the glutamine synthetase (GS) system.Cytotechnol. 9:231-236.

Describes improvements in batch process leading to antibody yields ranging upto 1g/L. Serum-free medium used.

9.

Buser CW, Beaudet R, Soohoo N, Pugh GG (1994) Development of serum-freemedia for engineered NS0 cell lines. In Animal Cell Technology: Products ofToday, Prospects for Tomorrow. Eds: Spier RE, Griffiths JB, Berthold W.Butterworth-Heinemann, pp121-139.

Describes development of serum-free medium for a GS-NS0 cell line, based onWilliams medium E. Lipid supplements added to the medium.

10.

Cannon-Carlson S, Varnerin J, Tsarbopoulos A, Jenh C-H, Cox MA, Chou C-C,Connelly N, Zavody P, Tang J C-T (1998) Expression purification andcharacterisation of recombinant human Interleukin-13 from NS0 Cells. ProteinExpression and Purification 12:239-248.

IL-13 expressed in NS0 cells using GS expression system. Cells grown in 15litre fed batch culture. Details given of the medium and feed used. The proteinis unglycosylated.

92.

Cockett MI, Bebbington CR, Yarranton GT (1990) High level expression of tissueinhibitor of metalloproteinases in Chinese hamster ovary cells using glutaminesynthetase gene amplification. BioTechnol. 8:662-667.

Yields of 180 mg/L achieved in shake flask culture. Sodium butyrate enhancedproduction.

13.

Davis SJ, Ward HA, Puklaver MJ, Willis AC, Williams AF, Barclay AN (1990) Highlevel expression in Chinese hamster ovary cells of soluble forms of CD4 Tlymphocyte glycoprotein including glycosylation variants. J. Biol. Chem.265:10410-10418.

S CD4 and S CD4 (half) expressed at levels of 80 and 25 g/L from GS-CHOcells. Used GMEM-S + 5% FCS and MSX (15 or 100M) in roller bottles.Sodium butyrate added to enhance production. Also used switch to serum freemedium (GMEM-S + 2 mM butyrate) at final stage, to aid purification.

15.

deZengotita VM; Miller WM; Aunins JG; Zhou W. (2000) Phosphate feedingimproves high-cell-concentration NS0 myeloma culture performance for monoclonalantibody production. Biotechnol. Bioeng. 69:566-576.

226.

DiStefano DJ, Mark GE, Robinson DK (1996) Feeding of nutrients delays apoptoticdeath in fed-batch cultures of recombinant NS0 myeloma cells. Biotechnol. Letters18:1067 – 1072.

Nutrient solutions added to fed-batch cultures of recombinant GS-NS0 cellsdelayed onset of apoptosis and decreased rate of apoptotic death. This seemsto be responsible for increase in culture longevity and five to ten fold increase in

82.


product concentration.

Duncan PJ, Jenkins HA, Hobbs G (1997) The effect of hyperosmotic conditions ongrowth and recombinant protein expression by NS0 myeloma cells in culture. TheGenetic Engineer and Biotechnologist 17:75-78.

Hyperosmotic conditions (400 mOsm) led to a 1.8 fold increase in specificproductivity compared with control conditions (275 mOsm). The conditions alsoled to large increases in glucose and glutamate uptake rates and in lactateproduction rates.

93.

Fries S, Glazomitsky K, Woods A, Forrest G, Hsu A, Olewinski R, Robinson DK,Chartrain M (2005) Evaluation of disposable bioreactors. Rapid production ofrecombinant proteins by several animal cells. BioProcess International 3( Supp6):36-44.

Describes growth of antibody-producing GS-NS0 cell line in cell culture-bagdisposable bioreactor system.

155.

Frahm B, Lane P, Märkl H, Pörtner R (2003) Improvement of a mammalian cellculture process by adaptive, model-based dialysis fed-batch cultivation andsuppression of apoptosis. Bioprocess Biosyst. Eng. 26:1-10.

149.

Froud SJ, Clements GJ, Doyle ME, Harris ELV, Lloyd C, Murray P, Preneta A,Stephens PE, Thompson S, Yarranton GT (1989) The development of a process forthe production of HIV1 GP120 from recombinant cell lines. In:Production ofBiologicals from Animal Cells in Culture, Butterworths, pp110-116.

GP120 expressed in amplified GS-CHO. Production levels of 1-3 mg/LCleavage of GP120 coincides with depletion of asparagine, glutamate, aspartateand serine from medium.

19.

Hermes PA, Castro CD (2010) A fully defined, fed-batch, recombinant NS0 cultureprocess for monoclonal antibody production. Biotechnol. Prog. 26:1411-1416.

193.

Hogwood CEM, Tait AS, Koloteva-Levine N, Bracewell DG, Smales CM. (2013)The dynamics of the CHO host cell protein profile during clarification and protein Acapture in a platform antibody purification process. Biotechnol. Bioeng. 110:240-251.

219.

Hutchinson N; Bingham N; Murrell N; Farid SS; Hoare M. (2006) Shear stressanalysis of mammalian cell suspensions for prediction of industrial centrifugationand its verification. Biotechnol. Bioeng. 95:483-491.

230.

Jiang Z, Droms KA, Geng Z, Casnocha SA, Xiao Z, Gorfien SF, Jacobia SJ (2012)Fed-batch cell culture process optimisation. BioProcess Intl. 10:40-45

215.

Kadarusman J, Bhatia R, McLaughlin J, Lin WR (2005) Growing cholesterol-dependent NS0 myeloma cell line in the Wave bioreactor system: overcomingcholesterol-polymer interaction by using pretreated polymer or inert fluorinatedethylene propylene. Biotechnol. Prog. 21:1341-1346.

Discusses choice of material for manufacture of disposable bioreactor systems

168.

Keen MJ, Hale C (1996) The use of serum-free medium for the production offunctionally active humanised monoclonal antibody from NS0 mouse myeloma cells

80.


engineered using glutamine synthetase as a selectable marker. Cytotechnol.18:207-217.

Describes protein free medium for GS-NS0 supplemented with cholesterol,phosphatidylcholine and -cyclodextrin. Adapted cells to become cholesterolindependent.

Kuwae S, Ohda T, Tamashima H, Miki H, Kobayashi K (2005) Development of afed-batch culture process for enhanced production of recombinant humanantithrombin by Chinese hamster ovary cells. J. Biosci. Bioeng. 100:502-510.

Expressed human antithrombin at 1 g/L using GS-CHO cells. Developed fed-batch process and evaluated the process at different pH values.

145.

Legmann R, Schreyer HB, Combs RG, McCormick EL, Russo AP, Rodgers ST(2009) A predictive high-throughput scale-down model of monoclonal antibodyproduction in CHO cells. Biotechnol. Bioeng. 104:1107-1120.

185.

Ma N, Ellet J, Okediadi C, Hermes P, McCormick E, Casnocha SA (2009) A singlenutrient feed supports both chemically defined NS0 and CHO fed-batch processes:Improved productivity and lactate metabolism. Biotechnol. Prog. 25:1353-1363.

182.

Metcalfe H, Field RP, Froud SJ (1994) The use of 2-hydroxy-2, 4, 6-cycloheptarin-1-one (tropolone) as a replacement for transferrin. In:Animal Cell Technology:Products of Today, Prospects for Tomorrow. Eds: Spier RG, Griffiths JB, MoignierB. Butterworth-Heinemann. pp 88-90.

Tropolone used as a transferrin replacement for culture of GS-NS0 cells.

37.

Naciri M, Kuystermans D, Al-Rubeai M (2008) Monitoring pH and dissolved oxygenin mammalian cell culture using optical sensors. Cytotechnol. 57:245-250.

170.

Osman JJ, Birch JR, Varley J (2001) The response of GS-NS0 myeloma cells topH shifts and pH perturbations. Biotechnol. Bioeng. 75:63-73.

Describe the effects of pH shifts and perturbations on growth, productivity andglucose metabolism for a GS-NS0 cell line.

108.

Osman JJ, Birch JR, Varley J (2002) The response of GS-NS0 myeloma cells tosingle and multiple pH perturbations. Biotech. Bioeng. 79:398–407.

Used GS-NS0 mouse myeloma cells expressing cB72.3 antibody in stirred tankbatch fermentations. Analysed the effects of single and multiple pHperturbations at pH 8 and 9 from pH7.3. For multiple pH perturbations a 2component system was used. Increasing the number of perturbations orexposure time to changed pH increases cell death.

119.

Popova D; Stonier A; Pain D; Titchener-Hooker NJ; Farid SS. (2015)Representative mammalian cell culture test materials for assessment of primaryrecovery technologies: A rapid method with industrial applicability. Biotechnol. J.10:162-170.

http://dx.doi.org/10.1002/biot.201400294

248.

Ray NG, Rivera R, Gupta R, Mueller D (1997) Large scale production of humanisedmonoclonal antibody expressed in a GS-NS0 cell line. In: Animal Cell Technology,

81.


Ed: Carrondo MJT. Kluwer Academic Publishers pp235-241.

Describes production of a humanised antibody from GS-NS0 cell line in a 2000litre stirred tank reactor run in fed-batch mode. Achieved 370-470 mgantibody/L after approx. 10 days. Deals with scale-up issues. Demonstrateseffect of sparge rate on lactate accumulation which is attributed to levels ofcarbon dioxide in the culture. Accumulation of CO2 at low sparge rates led toincreased lactate accumulation.

Robinson DK, Seamans TC, Gould SL, DiStefano DJ, Chan CP, Lee DK, Bibila T,Glazomitsky K, Munshi S, Daugherty B, O'Neill Palladino L., Stafford-Hollis J, HollisGF, Silberklang M (1994) Optimization of a fed-batch process for production of arecombinant antibody. Reprinted from Biochemical Engineering VIII, Ann. N.Y.Acad. Sci. 745:285-296.

Describes the development of an optimized fed-batch process for a GS-NS0 cellline secreting a monoclonal antibody. Includes data on amino acid consumptionrates.

62.

Robinson DK, DiStefano D, Gould SL, Cuca G, Seamans TC, Benincasa D,Munshi S, Chan CP, Stafford-Hollis J, Hollis GF, Jain D, Ramasubramanyan K,Mark GE, Silberklang M (1995) Production of engineered antibodies in myelomaand hybridoma cells. In: Antibody Engineering. Eds: Wang H, Imanaka T.Worthington: ACS, pp1-14.

Detailed description of production of recombinant antibody in a GS-NS0 system.

84.

Sanders PG, Wilson RH (1984) Amplification and cloning of the Chinese hamsterglutamine synthetase gene. The EMBO Journal 3:(1), 65-71.

Descibes amplification of endogenous GS gene in CHO using MSX selection.Describes modified GMEM with glutamate, asparagine, nucleotides andpyruvate.

47.

Seamans TC, Gould SL, DiStefano DJ, Silberklang M, Robinson DK (1994) Use oflipid emulsions as nutritional supplements in mammalian cell culture. Annals N.Y.Acad. Sci. 745:240-243.

Describes preparation of stable lipid emulsions to substitute for lipoproteins inthe culture of NS0 cells secreting a recombinant antibody.

61.


163.

Seamans TC, Fries S, Beck A, Wurch T., Chenu S, Chan C, Ushio M, Bailey J,Kejariwal A, Ranucci C, Villani N, Ozuna S, Goetsch L, Corvaia N, Salmon P,Robinson DK, Chartrain M (2008) Cell cultivation pocess transfer and scale-up insupport of production of early clinical supplies of an anti IGF-1R antibody, Part 2.BioProcess International 6(4):34-42.

164.

Sellick CA, Hansen R, Jarvis RM, Maqsood AR, Stephens GM, Dickson AJ,Goodacre R (2010) Rapid monitoring of recombinant antibody production bymammalian cell cultures using fourier transform infrared spectroscopy and

187.


chemometrics. Biotechnol. Bioeng. 106:432-442.

Sellick CA, Croxford AS, Maqsood AR, Stephens G, Westerhoff HV, Dickson(2011) Metabolite profiling of recombinant CHO cells: Designing tailored feedingregimes that enhance recombinant antibody production. Biotechnol. Bioeng.108:3025-3031.

212.

Shirsat N; Avesh M; English NJ; Glennon B; Al-Rubeai M. (2013) Application ofstatistical techniques for elucidating flow cytometric data of batch and fed-batchcultures. Biotechnol. Appl. Biochem. 60:536-545.

http://dx.doi.org/10.1002/bab.1138

242.

Silk NJ, Denby S, Lewis G, Kuiper M, Hatton D, Field RP, Bagnaz F, Lye GJ(2010) Fed-batch operation of an industrial cell culture process in shakenmicrowells. Biotechnol. Lett. 32:73-78.

Describes a fed-batch culture of a GS-CHO cell line in a shaken 24-well plate.

183.

Tait AS; Tarrant RDR; Velez-Suberbie ML; Spencer DIR; Bracewell DG. (2013)Differential response in downstream processing of CHO cells grown under mildhypothermic conditions. Biotechnol. Prog. 688-696.

231.

Titchener-Hooker NJ, Dunnill P, Hoare M (2008) Micro biochemical engineering toaccelerate the design of industrial-scale downstream processes forbiopharmaceutical proteins. Biotechnol. Bioeng. 100:473-487.

167.

Velez-Suberbie ML, Tarrant RDR, Tait AS, Spencer DIR, Bracewell DG. (2013)Impact of aeration strategy on CHO cell performance during antibody production.Biotechnol. Prog. 29:116-126.

220.

Wayte J, Boraston R, Bland H, Varley J, Brown M (1997) pH: effects on growth andproductivity of cell lines producing monoclonal antibodies: control in large-scalefermenters. The Genetic Engineer and Biotechnol. 17:125-132.

Describes effect of pH on growth and productivity of a GS-NS0 cell line makinga humanised monoclonal antibody. Productivity increased at pH7.1 comparedwith 7.4.

94.

Zhang J, Robinson DK (2005) Development of animal-free, protein-free andchemically-defined media for NS0 culture. Cytotechnol. 48:59-74.

156.

Zhou W, Bibila T, Glazomitsky K, Montalvo J, Chan C, DiStefano D, Munshi S,Robinson D, Buckland B, Aunins J (1996) Large-scale production of recombinantmouse and rat growth hormone by fed-batch GS-NS0 cell cultures. Cytotechnol.22:239-250.

Transfected NS0 cells with GS vector containing growth hormone genes andsequence for targeting integration by homologous recombination (for optimalexpression). Operated fed-batch cultures at 25 litrel scale and information onoperating conditions is given. Yields of mouse and rat growth hormone were580 and 240 mg/L respectively. Data presented on cell metabolism. Glucoseconsumption rate decreased during transition to stationary phase and lactatewas consumed during stationary phase.

77.

Wu MH, Dimopoulos G, Mantalaris A, Varley J (2004) The effect of hyperosmotic 128.


pressure on antibody production and gene expression in the GS-NS0 cell line.Biotechnol. Appl. Biochem. 40:41-46.

Describes growth and productivity kinetics as well as gene expression, usingmicroarray technology, at various osmolalities..

Zhou W, Chen C-C, Buckland B, Aunins J (1997) Fed-batch culture of recombinantNS0 myeloma cells with high monoclonal antibody production. Biotech. Bioeng.55:783-792.

Describes feeding strategy leading to final antibody concentration in excess of2.7g/L. Describes transitions in metabolism caused by nutrient depletion. Thecell line used in the study had three vector copies.

90.

SECTION 8: Metabolism of GS Cell Lines

Bell SL, Bebbington CR, Bushell ME, Sanders PG, Scott MF, Spier RE, Wardell JN(1991) Genetic engineering of cellular physiology. In: Production of Biologicalsfrom Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp 304-306.


4.

Bell SL, Bushell ME, Scott MF, Wardell JN, Spier RE, Sanders PG (1992) Geneticmodification of hybridoma glutamine metabolism: physiological consequences. InAnimal Cell technology: Developments, Processes and Products. Eds: Spier RE,Griffiths JB, MacDonald C. Butterworth-Heinemann, pp180-182.


5.

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushell ME Sanders PG(1995) Genetic engineering of hybridoma glutamine metabolism. Enzyme andmicrobial technol. 17:98-106.


65.

Bibila TA, Ranucci C, Glazomitsky K, Buckland BC, Aunins JG (1994) Investigationof NS0 cell metabolic behaviour in monoclonal antibody producing clones. Ann.N.Y. Acad. Sci. 745:277-284.

146.

Birch JR, Boraston RC, Metcalfe H, Bebbington CR, Field RP (1994) Selecting anddesigning cell lines for improved physiological characteristics. Cytotechnol. 15:11-16.

Describes transfection of GS into hybridoma to give glutamine independence.Productivity was increased in glutamine free medium.

58.

Bond J, Varley J (2005) Use of flow cytometry and SNARF to calibrate andmeasure intracellular pH in NS0 cells. Cyometry Part A 64A:43-50.

Evaluates methods for measurement of intracellular pH.

125.

Cairo JJ, Paredes C, Godia F, Prats E, Azorin F, Cornudella L (1998) Modificationof hybridoma cells metabolism. In: New Developments and New Applications inAnimal Cell Technology. Eds: Merten O-W, Perrin P, Griffiths B. Kluwer AcademicPublishers, pp167-174.

86.


Transfection of hybridoma with GS gene eliminated glutamine requirement andsuppressed ammonia production. Also resulted in halving of glucose uptakerate whilst maintaining similar growth pattern. Discusses metabolic fluxes incells with and without GS.

Carinhas N; Duarte TM; Barreiro LC; Carrondo MJT; Alves PM; Teixeira AP (2013)Metabolic signatures of GS-CHO cell clones associated with butyrate treatment andculture phase transition. Biotechnol. Bioeng. 110:3244-3257.


238.

Dempsey J, Ruddock S, Osborne M, Ridley A, Sturt S, Field R (2003) Improvedfermentation processes for NS0 cell lines expressing human antibodies andglutamine synthetase. Biotechnol. Prog. 19:175-178.

Repeated nutrient analysis and re-supplementation of serum-free antibodyproducing NS0 cultures performed. As a result media and feeds weredeveloped which gave 10-fold increases in antibody harvest titres up to 600mg/L.

109.

DiStefano, DJ, Mark GE, Robinson DK (1996) Feeding of nutrients delaysapoptotic death in fed-batch cultures of recombinant NS0 myeloma cells.Biotechnol. Letters 18:1066 – 1073.

Nutrient solutions added to fed-batch cultures of recombinant GS-NS0 cellsdelayed onset of apoptosis and decreased rate of apoptotic death. This seemsto be responsible for increase in culture longevity and five- to- ten- fold increasein product concentration.

82.

Dorai H. Kyung YS, Ellis D, Kinney C, Lin C, Jan D, Moore G, Betenbaugh MJ(2009) Expression of anti-apoptosis genes alters lactate metabolism of Chinesehamster ovary cells in culture. Biotechnol. Bioeng. 103:592-608.

177.

Downham MR, Farrell WE, Jenkins HA (1996) Endoplasmic reticulum proteinexpression in recombinant NS0 Myelomas grown in batch culture. Biotech. Bioeng.51:691 – 696.

Production of ER proteins (GRP78/BiP, GRP94 and Erp72) increased duringdecline phase of batch culture of GS-NS0 coincident with an increase inproduction rate of recombinant antibody and reduction in uptake of glucose andglutamate.

83.

Duarte TM, Carinhas N, Barreiro LC, Carrondo MJT, Alves PM, Teixeira AP (2014)Metabolic responses of CHO cells to limitation of key amino acids. Biotechnol.Bioeng. 111:2095-2106.

253.

Duncan PJ, Jenkins HA, Hobbs G (1997) The effect of hyperosmotic conditions ongrowth and recombinant protein expression by NS0 myeloma cells in culture. TheGenetic Engineer and Biotechnol. 17:75-78.

Hyperosmotic conditions (400 mOsm) led to a 1.8 fold increase in specificproductivity compared with control conditions (275 mOsm). The conditions alsoled to large increases in glucose and glutamate uptake rates and in lactateproduction rates.

93.


Ibarra N, Watanabe S, Bi JX, Shuttleworth J, Al-Rubeai M (2003) Modulation of cellcycle for enhancement of antibody production in perfusion culture of NS0 cells.Biotechnol. Prog. 19:224-228.


111.

Kearns B, Lindsay D, Manahan M, McDowall J, Rendeiro D (2003) NS0 batch cellculture process characterisation: a case study. BioProcessing Journal 2:52-57.

Investigation into decline in productivity as cell culture generation numberincreased. No genetic instability seen but a phenotypic instability, related to themetabolic state of the culture, is reported.

110.

Kyriakopoulos S,; Polizzi KM; Kontoravdi C. (2013) Comparative analysis ofamino acid metabolism and transport in CHO variants with different levels ofproductivity. J. Biotechnol. 168:543-551.


237.

Khoo SHG, Al-Rubeai M (2009) Metabolic characterization of a hyper-productivestate in an antibody producing NS0 myeloma cell line. Metabol. Eng. 11:199-211.

178.

Ma N, Ellet J, Okediadi C, Hermes P, McCormick E, Casnocha SA (2009) A singlenutrient feed supports both chemically defined NS0 and CHO fed-batch processes:Improved productivity and lactate metabolism. Biotechnol. Prog. 25:1353-1363.

182.

McKenna SL, Cotter TG (2000) Inhibition of caspase activity delays apoptosis in atransfected NS0 myeloma cell line. Biotech. Bioeng. 67:165-176.

Z-VAD-fmk, a specific inhibitor of caspases reduced apoptosis in GS-NS0 cellsbut did not increase productivity. The inhibitor did not prevent mitochondrialdysfunction.

102.

Paredes C, Prats E, Cairo JJ, Azoris F, Cornudella Ll, Godia F (1999) Modificationof glucose and glutamine metabolism in hybridoma cells through metabolicengineering. Cytotechnol. 30:85-93.

Transfected a hybridoma cell line with GS. Transfected cells had a reducedgrowth rate and a lower glucose utilisation rate. Ammonia and alanineproduction was eliminated. Alanine was required for optimal growth.

101.

Ray NG, Rivera R, Gupta R, Mueller D (1997) Large scale production of humanisedmonoclonal antibody expressed in a GS-NS0 cell line. In: Animal Cell Technology.Ed: Carrondo MJT Kluwer Academic Publishers pp235-241.

Describes production of a humanised antibody from GS-NS0 cell line in a 2000lstirred tank reactor run in fed-batch mode. Achieved 370-470mg/antibody/l afterapprox. 10 days. Deals with scale-up issues. Demonstrates effect of spargerate on lactate accumulation which is attributed to levels of carbon dioxide in theculture. Accumulation of CO2 at low sparge rates led to increased lactateaccumulation.

81.

Sellick CA, Hansen R, Maqsood AR, Dunn WB, Stephens GM, Goodacre R, 196.


Dickson AJ (2009) Effective quenching processes for physiologically validmetabolite profiling of suspension cultured mammalian cells. Anal. Chem. 81:174-183.

Sellick CA, Hansen R, Jarvis RM, Maqsood AR, Stephens GM, Dickson AJ,Goodacre R (2010) Rapid monitoring of recombinant antibody production bymammalian cell cultures using fourier transform infrared spectroscopy andchemometrics. Biotechnol. Bioeng. 106:432-442.

187.

Sellick CA, Croxford AS, Maqsood AR, Stephens G, Westerhoff HV, Dickson(2011) Metabolite profiling of recombinant CHO cells: Designing tailored feedingregimes that enhance recombinant antibody production. Biotechnol. Bioeng.108:3025-3031.

212.

Sengupta N, Rose ST, Morgan JA (2011) Metabolic flux analysis of CHO cellmetabolism in the late non-growth phase. Biotechnol. Bioeng. 108:82-92.

202.

Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M (2000) Influence of Bcl-2on cell death during the cultivation of a Chinese hamster ovary cell line expressinga chimeric antibody. Biotech. Bioeng. 68:31-43.

Transfection with the control expression vector (Neo marker) used in this studyand exposure to the selective drug G418 led to upregulation of endogenousBcl-2. This led to an increase in cell viability in cell cultures and prolongedsurvival. There was no influence on antibody titre.

99.

Veraitch FS, Al-Rubeai M (2005) Enhanced growth in NS0 cells expressingaminoglycoside phosphotransferase is associated with changes in metabolism,productivity, and apoptosis. Biotechnol. Bioeng. 92:589-599.

123.

Zhou W, Bibila T, Glazomitsky K, Montalvo J, Chan C, Di Stefano D, Munshi SRobinson D, Buckland B, Aunins J (1996) Large-scale production of recombinantmouse and rat growth hormone by fed-batch GS-NS0 cell cultures. Cytotechnol.22:239-250.

Transfected NS0 cells with GS vector containing growth hormone genes andsequence for targeting integration by homologous recombination (for optimalexpression). Operated fed-batch cultures at 250 litre scale and information onoperating conditions is given. Yields of mouse and rat growth hormone were580 and 240 mg/L respectively. Data presented on cell metabolism. Glucoseconsumption rate decreased during transition to stationary phase and lactatewas consumed during stationary phase.

77.

SECTION 9: Virology of NS0 Cell Lines

Froud SJ, Birch JR, McLean C, Shepherd AJ, Smith KT (1997) Viral contaminantsfound in mouse cell lines used in the production of biological products. In: AnimalCell Technology: From Vaccines to Genetic Medicine (Eds: Carrondo MJT, GriffithsJB, Moreira JL), Kluwer Academic Publishers, Dordrecht, pp681-686.

Authors present data showing that, whilst it may not be possible to detectinfectious retrovirus in final bulk harvest of GS-NS0 fermentations, it may havebeen present at high titre up to this point..

139.

Taylor FR, Ferrant JL, Foley SF, Zeng C, Sernatinger J, Juffras R, Pepinsky B(2000) Biochemical analysis of retroviral structural proteins to identify and quantify

103.


retrovirus expressed by an NS0 murine myeloma cell line. J. Biotech. 84:33-43.

Large numbers of retroviral particles were observed in an NS0 subclone usingEM. Titres by infectivity assay were low and had the host range expected for amurine amphotropic retrovirus. Analysis of viral Gag Proteins indicated thepresence of at least two closely related viruses N-terminal sequencing indicatesthat the viruses belong to the murine leukemia retrovirus family. A western blotassay using an antibody for the capsid protein was developed and may beuseful for monitoring viral titre and clearance.

SECTION 10: ‘Omic Studies of GS Cell Lines and SystemsBiology

Ahmad N, Zhang J, Brown PJ, James DC, Birch JR, Racher AJ, Smales CM(2006) On the statistical analysis of the GS-NS0 cell proteome: imputation,clustering and variability testing. Biochem. Biophys. Acta 1764:1179-1187.

132.

Alete DE, Racher AJ, Birch JR, James DC, Smales CM (2005) The functionalcompetence of animal cells: analysis of the secretory pathway. In: Animal CellTechnology meets Genomics (Eds: Gòdia F, Fussenegger M) Springer, Dordrecht,pp71-74.

129.

Alete DE, Racher AJ, Birch JR, Stansfield SH, James DC, Smales CM (2005)Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myelomacells with varying secreted recombinant monoclonal antibody productivities.Proteomics 5:4689-4704.

122.

Charaniya S, Karypis G, Hu W-S (2009) Mining transcriptome data for function-traitrelationship of hyper productivity of recombinant antibody. Biotechnol, Bioeng.102:1654-1669.

172.

Dadehbeigi N; Dickson AJ. (2013) Application of a nonradioactive method ofmeasuring protein synthesis in industrially relevant chinese hamster ovary cells.Biotechnol. Prog. 29:1043-1049.

235.

Dinnis DM, Stansfield SH, Schlatter S, Smales CM, Alete D, Birch JR, Racher AJ,Marshall CT, Nielsen LK, James DC (2006) Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibodyproduction rate. Biotechnol Bioeng 94:830-841.

133.

Dorai H; Santiago A; Campbell M; Tang QM; Lewis MJ; Wang Y; Lu QZ; Wu SL;Hancock W. (2011) Characterization of the proteases involved in the N-terminalclipping of glucagon-like-peptide-1-antibody fusion proteins. Biotechnol. Prog.27:220-231.

228.

Edros RZ; McDonnell S; Al-Rubeai M. (2013) Using Molecular Markers toCharacterize Productivity in Chinese Hamster Ovary Cell Lines. PLoS One8:e75935.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075935

247.

García Münzer DG; Kostoglou M; Georgiadis MC; Pistikopoulos EN; Mantalaris A.(2013) Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0.Biochem. Eng. J. 81:97-107.

http://www.sciencedirect.com/science/article/pii/S1369703X13002787

240


Khoo SHG, Falciani F, Al-Rubeai M (2007) A genome-wide transcriptional analysisof producer and non-producer NS0 myeloma cell lines. Biotechnol. Appl. Biochem.47:85-95.

152.

Khoo SHG, Al-Rubeai M (2009) Metabolic characterization of a hyper-productivestate in an antibody producing NS0 myeloma cell line. Metabol. Eng. 11:199-211.

178.

Krampe B, Swiderek H, Al-Rubeai M (2008) Transcriptome and proteome analysisof antibody-producing mouse myeloma NS0 cells cultivated at different celldensities in perfusion culture. Biotechnol. Appl. Biochem. 50:133-141.

165.

Krampe B, Fagan A, Gaora PÓ, Al-Rubeai M (2011) Chemostat-basedtranscriptional analysis of growth rate change and BCL-2 over-expression in NS0cells. Biotechnol. Bioeng. 108:1603-1615.

The GS-NS0 cell line described by Al-Rubeai and co-workers as ‘6A1’ is in factthe cell line 6A1(100)3 (Bebbington et al 1992, reference #3).

199.

Kyriakopoulos S,; Polizzi KM; Kontoravdi C. (2013) Comparative analysis ofamino acid metabolism and transport in CHO variants with different levels ofproductivity. J. Biotechnol. 168:543-551.


237.

Mead EJ, Chiverton LM, Spurgeon SK, Martin EB, Montague GA, Smales CM, vonder Haar T. (2012) Experimental and in silico modelling analyses of the geneexpression pathway for recombinant antibody and by-product production in NS0cell lines. PLOS One 7(10):e47422.


221.

McLeod J, O'Callaghan PM, Pybus LP, Wilkinson SJ, Root T, Racher AJ, JamesDC (2011) An empirical modeling platform to evaluate the relative control discreteCHO cell synthetic processes exert over recombinant monoclonal antibodyproduction process titer. Biotechnol. Bioeng. 108:2193-2204.

197.

O'Callaghan PM, Berthelot ME, Young RJ, Graham JWA, Racher AJ, Aldana D(2015) Diversity in host clone performance within a Chinese hamster ovary cell line.Biotechnol. Prog. 31:1187-1200.


257.

Sellick CA, Hansen R, Jarvis RM, Maqsood AR, Stephens GM, Dickson AJ,Goodacre R (2010) Rapid monitoring of recombinant antibody production bymammalian cell cultures using fourier transform infrared spectroscopy andchemometrics. Biotechnol. Bioeng. 106:432-442.

187.

Sellick CA, Croxford AS, Maqsood AR, Stephens GM, Westerhoff HV, GoodacreR, Dickson AJ (2015) Metabolite profiling of CHO cells: Molecular reflections ofbioprocessing effectiveness. Biotechnol. J. 10:1434-1445.

http://dx.doi.org/10.1002/biot.201400664

255.

Shirsat N; Avesh M; English NJ; Glennon B; Al-Rubeai M. (2013) Application ofstatistical techniques for elucidating flow cytometric data of batch and fed-batch

242.


cultures. Biotechnol. Appl. Biochem. 60:536-545.

http://dx.doi.org/10.1002/bab.1138

Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ,Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murinemyeloma cell lines with varying recombinant monoclonal antibody production rate.Biotechnol. Bioeng. 88:474 – 488.

Proteomic analysis of four different NS0 cell lines, with different levels ofantibody production, identified a number of proteins with altered abundances.Amongst these proteins a number of molecular chaperones involved in foldingand assembly of immunoglobulins were identified. The authors also report anabundance of light chain protein over heavy chain protein a likely prerequisitefor efficient MAb production.

118.

Smales CM, Birch JR, Racher AJ, Marshall CT, James DC (2003) Evaluation ofindividual protein errors in silver-stained two-dimensional gels. Biochem. Biophys.Res. Comm. 306:1050-1055.

136.

Stansfield SH, Allen EE, Dinnis DM, Racher AJ, Birch JR, James DC (2007)Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibodyduring fed-batch culture. Biotechnol. Bioeng. 97:410-424.

151.

Swiderek H, Al-Rubeai M (2007) Functional genome-wide analysis of antibodyproducing NS0 cell line cultivated at different temperatures. Biotechnol. Bioeng.98:616-630.

Erratum: Biotechnol. Bioeng. (2008) 100:838

153.

Swiderek H, Logan A, Al-Rubeai M (2008) Cellular and transcriptomic analysis ofNS0 cell response during exposure to hypoxia. J. Biotechnol. 234:103-111.

166.

Tait AS; Tarrant RDR; Velez-Suberbie ML; Spencer DIR; Bracewell DG. (2013)Differential response in downstream processing of CHO cells grown under mildhypothermic conditions. Biotechnol. Prog. 688-696.

231.

Wu MH, Dimopoulos G, Mantalaris A, Varley J (2004) The effect of hyperosmoticpressure on antibody production and gene expression in the GS-NS0 cell line.Biotechnol. Appl. Biochem. 40:41-46.

Describes growth and productivity kinetics as well as gene expression, usingmicroarray technology, at various osmolalities.

128.

SECTION 11: Cell Line Engineering

Astley K, Naciri M, Racher AJ, Al-Rubeai M. (2007) The role of p21cip1 inadaptation of CHO cells to suspension and protein-free culture. J Biotechnol130:282-290

158.

Astley K, Al-Rubeai M (2008) The role of Bcl-2 and its combined effect with p21CIP1

in adaptation of CHO cells to suspension and protein-free culture. Appl. Microbiol.Biotechnol. 78:391-399.

169.

Bell SL, Bebbington CR, Bushell ME, Sanders PG, Scott MF, Spier RE, Wardell JN(1991) Genetic engineering of cellular physiology. In: Production of Biologicals

4.


from Animal Cells in Culture. Eds: Spier RE, Griffiths JB, Meignier B. Butterworth-Heinemann, pp304-306.


Bell SL, Bushell ME, Scott MF, Wardell JN, Spier RE, Sanders PG (1992) Geneticmodification of hybridoma glutamine metabolism: physiological consequences. In:Animal Cell Technology: Developments, Processes and Products. Eds: Spier RE,Griffiths JB, MacDonald C. Butterworth-Heinemann, pp 180-182.


5.

Bell SL, Bebbington C, Scott MF, Wardell JN, Spier RE, Bushell ME, Sanders PG(1995) Genetic engineering of hybridoma glutamine metabolism. Enzyme andMicrobial Technol. 17:98-106.


65.

Bi J-X, Shuttleworth J, Al-Rubeai M (2004) Uncoupling of cell growth andproliferation results in enhancement of productivity in p21CIP1-arrested CHO cells.Biotechnol. Bioeng. 85:741-749.

150.

Browne SM, Al-Rubeai M (2011) Analysis of an artificially selected GS-NS0 variantwith increased resistance to apoptosis. Biotechnol. Bioeng. 108:880-892.

201.

Cairo JJ, Paredes C, Godia F, Prats E, Azorin F, Cornudella L (1998) Modificationof hybridoma cells metabolism. In: New Developments and New Applications inAnimal Cell Technology. Eds: Merten O-W, Perrin P, Griffiths B. Kluwer AcademicPublishers, pp167-174.

Transfection of hybridoma with GS gene eliminated glutamine requirement andsuppressed ammonia production. Also resulted in halving of glucose uptakerate whilst maintaining similar growth pattern. Discusses metabolic fluxes incells with and without GS.

86.

Contie M, Leger O, Fouque N, Poitevin Y, Kosco-Vilbois M, Mermod N, Elson G.(2013) IL-17F co-expression improves cell growth characteristics and enhancesrecombinant protein production during CHO cell line engineering. Biotechnol.Bioeng. 110:1153-1163.

223.

Dorai H. Kyung YS, Ellis D, Kinney C, Lin C, Jan D, Moore G, Betenbaugh MJ(2009) Expression of anti-apoptosis genes alters lactate metabolism of Chinesehamster ovary cells in culture. Biotechnol. Bioeng. 103:592-608.

177.

Dorai H, Ellis D, Keung YS, Campbell M, Zhuang M, Lin C, Betenbaugh MJ (2010)Combining high-throughput screening of caspase activity with anti-apoptosis genesfor development of robust CHO production cell lines. Biotechnol. Prog. 26:1367-1381.

192.

Fan L, Kadura I, Krebs LE, Hatfield CC, Shaw MM, Frye C (2012) Improving theefficiency of CHO cell line generation using glutamine synthetase gene knockoutcells. Biotechnol. Bioeng. 109:1007-1015.

207.

Hayes NVL, Smales CM, Klappa P (2010) Protein disulfide isomerase does notcontrol recombinant IgG4 productivity in mammalian cell lines. Biotechnol. Bioeng.105:770-779.

186.


Ibarra N, Watanabe S, Bi JX, Shuttleworth J, Al-Rubeai M (2003)Modulation of cell cycle for enhancement of antibody production in perfusion cultureof NS0 cells. Biotechnol. Prog. 19:224-228.


111.

Jossé L, Smales CM, Tuite MF (2010) Transient expression of human TorsinAenhances secretion of two functionally distinct proteins in cultured Chinese hamsterovary (CHO) cells. Biotechnol. Bioeng. 105:556-566.

184.

Kiparissides A; Pistikopoulos EN; Mantalaris A. (2015) On the model-basedoptimization of secreting mammalian cell (GS-NS0) cultures. Biotechnol, Bioeng.112:536-548.


224.

Krampe B, Fagan A, Gaora PÓ, Al-Rubeai M (2011) Chemostat-basedtranscriptional analysis of growth rate change and BCL-2 over-expression in NS0cells. Biotechnol. Bioeng. 108:1603-1615.

The GS-NS0 cell line described by Al-Rubeai and co-workers as ‘6A1’ is in factthe cell line 6A1(100)3 (Bebbington et al 1992, reference #3).

199.

O'Connor KC, Muhitch JW, Lacks DJ, Al-Rubeai M (2006) Modelling suppressionof cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells. Biotechnol. Lett.28:1919-1924.

The GS-NS0 cell line described by these authors is actually the cell line6A1(100)3 described by Bebbington et al in 1992.

154.

Paredes C, Prats E, Cairo JJ, Azoris F, Cornudella Ll, Godia F (1999) Modificationof glucose and glutamine metabolism in hybridoma cells through metabolicengineering. Cytotechnol. 30:85-93.

Transfected a hybridoma cell line with GS. Transfected cells had a reducedgrowth rate and a lower glucose utilisation rate. Ammonia and alanineproduction was eliminated. Alanine was required for optimal growth.

101.

Tey BT, Singh RP, Al-Rubeai M (1999) Influence of bcl-2 over-expression on NS0and CHO culture viability and chimeric antibody productivity. In: Animal CellTechnology: Products from Cells, Cells as Products. Eds: Bernard A et al. pp 59-61Kluwer.

Over-expression of bcl-2 significantly reduced the rate of cell death in a GS-CHO and in GS-NS0 cell line and resulted in a 19% and 25% increase,respectively in antibody production.

98.

Tey BT, Singh RP, Piredda L., Piacentini M, Al-Rubeai M (2000) Influence of bcl-2on cell death during the cultivation of a Chinese hamster ovary cell line expressinga chimeric antibody. Biotech. Bioeng. 68:31-43.

Transfection with the control expression vector (Neo marker) used in this study

99.


and exposure to the selective drug G418 led to upregulation of endogenousbcl-2. This led to an increase in cell viability in cell cultures and prolongedsurvival. There was no influence on antibody titre.

Tey BT, Singh RP, Piredda L, Piacentini MP, Al-Rubeai M (2000) Bcl-2 mediatedsuppression of apoptosis in myeloma NS0 cultures. J. Biotechnol. 79:147-159.

In batch culture, no difference was seen in product concentration between cellline over-expressing Bcl-2 and control cell line. However, in fed-batch cultureusing a concentrated amino acid feed, antibody concentration was increased60% in the Bcl-2 over-expressing cell line..

147.

Tey BT, Al-Rubeai M. (2004) Suppression of apoptosis in perfusion culture ofMyeloma NS0 cells enhances cell growth but reduces antibody productivity.Apoptosis 9:843-852.

160

Tey BT, Al-Rubeai M (2005) Effect of Bcl-2 overexpression on cell cycle andantibody productivity in chemostat cultures of myeloma NS0 cells. J Biosci Bioeng100:303-310.

159

Tey BT, Al-Rubeai M. (2005) Bcl-2 over-expression reduced the serumdependency and improved the nutrient metabolism in a NS0 cells culture.Biotechnol. Bioprocess Eng. 10:254-261.

225.

Underhill MF; Coley C; Birch JR; Findlay A; Proud CG; James DC.(2003) Engineering mRNA translation initiation to enhance transient gene

expression in Chinese hamster ovary cells. Biotechnol. Prog. 19:121-129.

227.

Watanabe S, Shuttleworth J, Al-Rubeai M (2002) Regulation of cell cycle andproductivity in NS0 cells by the over-expression of p21CIP1. Biotechnol. Bioeng.77:1-7.

148.

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