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(BQ) Part 2 book “Update on production of recombinant therapeutic protein - Transient gene expression” has content: Optimisation of transient gene expression for therapeutic protein production, clinical applications of the transient gene expression.
3 Optimisation of Transient Gene Expression for Therapeutic Protein Production Jianwei Zhu Transient gene expression (TGE) is a well-established technology for rapid generation of recombinant proteins with human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cell lines Many TGE protocols have been published over the last ten years A number of representative protocols are described in the last section of Chapter for producing therapeutic proteins at different scales (from 10 mL -100 L), facilitated by calcium phosphate or polyethylenimine (PEI), using viral or non-viral vectors The protocol using 25 kDa linear PEI (Table 2.3) as a transfection component to form a plasmid/ PEI complex for transfecting CHO and HEK cells has been widely accepted by many laboratories and the biopharmaceutical industry Scalability and expression levels of TGE have improved significantly in the past few years to the extent that it is now feasible to produce 100 g of protein for preclinical studies or even early phase clinical development of therapeutics Successful reports from the past few years are listed and summarised in Table 3.1 Table 3.1 presents the representative productivity level with the TGE technology platform Since 2008, when the production titre first reached the g/L milestone [1], there have been repeatedly successful results either through TGE [2] or stable transfection pools [3] at, or over, g/L expression levels, making it feasible to use the TGE system to produce therapeutic proteins in sufficient quantity for preclinical studies or even early stage clinical trials In the successful examples (Table 3.1) both CHO and HEK293 cells were predominantly used as host cell lines Great effort has been invested into optimisation of many aspects of the TGE system in coexpression of antiapoptosis genes [1], the use of new additives in the media [4, 5], and transfection 81 82 Expression 81 mg/L 1000 mg/L 50 mg/L 80 mg/L Cell HEK293E HEK293E HEK293E CHOK1SV Mab r-protein Mab Mab Product 4) Process scaled up to 20 L in Wave bioreactor 3) Product showed similar glycosylation patterns from batch to batch 2) Fed-batch process maintained for 14 days with post-transfections at 2-4 day intervals 1) DMSO and lithium acetate increased expression Ten-fold increase in human SEAP expression was obtained in HEK293E cells compared with pcDNA3.1 vector 3) Process scaled-up to 2L 10% hp21, 5% FGF resulting in g/L expression level 2) Cotransfection with 37.5% HC, 10% LC, 10% hp18 1) Human CMV + Intron + WPRE (40 mg/L) Enhancement by cotransfection of WPRE by five-fold Expression and process description [5] [75] [1] [53] Reference Table 3.1 Recombinant therapeutics produced by transient gene expression technology platform Update on Production of Recombinant Therapeutic Protein 90 mg/L 3.1 mg/L 4-9.1 mg/L 50-60 mg/L 40-50 mg/L 30-60 mg/L 69-165 mg/L CHO-DG44 CHO-S HEK293 CHO-S HEK293F CHO-DG44 HEK293 Human IgG Antibody Mab Mab Fc-Fusion protein Mab 2) Transfection at a high density 250 mg/L (at 500 mL scale) Large scale in Wave bioreactor Disposable shake bioreactor at 30 L working volume Flask at L scale using FreeStyle TM MAX Human erythropoietin and human blood coagulation factor IX were expressed in both HEK293 and CHO cells Comparison of two hosts: CHO versus HEK293 VPA increased mRNA and protein levels 3) Eliminating the culture dilution step after transfection 1) Reduced pDNA by 50% 300 mg/L Mab (at mL scale) Process scaled up to 110 L bioreactor with 80 L working volume CHO Mab 22 mg/L CHO-DG44 [13] [127] [123] [124] [4] [6] [20] Optimisation of Transient Gene Expression 83 84 r-protein 60 pg/cell/day 100-400 mg/L 40 mg/L 235 mg/L 160 mg/L 206 mg/L PER.C6 CHOK1SV HKB-11 CHO 1.0 g/L 1.4 g/L Chimeric antibody 300-500 mg/L PER.C6 CHO-GS Fc-Fusion protein 63 mg/L CHO Mab Various Mab Mab Mab r-protein Human IgG 1000 mg/L HEK293F Mab 140 mg/L Epi-CHO Stable transfection pool at × 25 L scale Stable transfection pool at L scale CHO and lentiviral vector expression system Produced in 10 L wave bioreactor Seven different antibodies were expressed in the system Cells were transfected by eletroporation and stable pools were selected Productions were at 100 L scale Roller bottle scale Roller bottle scale Adenovirus-based expression at small scale High density cell culture using Expi293 Medium EBVNA1 and oriP in expression plasmid [3] [130] [131, 132] [25] [129] [129] [128] [2] [78] Update on Production of Recombinant Therapeutic Protein 121-405 mg/L Mab Stable transfection pools at 20-200 L WAVE or 300 L stirred-tank CMV: Cytomegalovirus DMSO: Dimethylsulfoxide DNA: Deoxyribonucleic acid EBVNA1: Epstein-Barr virus nuclear antigen Epi-CHO: Transient expression system in Chinese hamster ovary Fc: Fragment crystallisable FGF: Fibroblast growth factor HC: Heavy chain HKB-11: Hybrid of human embryonic kidney 293 and a human B cell line hp: Human cell cycle regulatory protein Ig: Immunoglobulin IgG: Immunoglobulin G LC: Light chain Mab: Monoclonal antibodies mRNA: Messenger ribonucleic acid oriP: Plasmid origin of viral replication pDNA: Plasmid deoxyribonucleic acid RNA: Ribonucleic acid r-protein: Recombinant protein SEAP: Secreted alkaline phosphatase VPA: Valproic acid WPRE: Woodchuck hepatitis virus post-transcriptional regulation element CHO-GS [25] Optimisation of Transient Gene Expression 85 Update on Production of Recombinant Therapeutic Protein at high cell density [6, 2] However, compared with an optimised stable transfection expression, TGE showed that the volumetric titres using TGE are lower and this is still a limiting factor The overall expression yield is at least five- to ten-fold lower than that obtained with stable expression cell lines for the production of therapeutic recombinant proteins and Mab The majority of published titres in a TGE system are in the range of 10-1000 mg/L with a specific productivity range of 1-10 pg/cell/day, whilst an optimised stable gene expression (SGE) process could reach 100 mg-10 g/L with a specific productivity of 10-80 pg/cell/day (Table 1.1, Table 2.3 and Table 3.1) As time- and resource-consuming cell line development is eliminated, every batch production run has to start with plasmid preparation and transfection Low productivity causes the manipulation of increasing quantities of culture media and vector DNA Optimisation of the productivity through higher transfection rate, better culture conditions for cell growth leading to better expression, simplification of the procedure, and extension of production duration have been highly desirable Ultimately, the technology platform is aimed at large scale production to supply clinical grade material for early phase human clinical trials In this chapter we will present recent developments in the optimisation of TGE methodology in order to maximise the production capacity for therapeutic applications 3.1 Optimisation of the Transient Gene Expression Conditions Several strategies have worked well to improve overall yields using CHO cells for TGE including transfection media optimisation, high density transfections, mild hypothermia, and vector design improvement [1, 7-12, 123] A widely acceptable protocol for transfection using linear 25 kDa PEI with CHO or HEK cells to generate milligram to gram quantities of therapeutic proteins and monoclonal antibodies (Mab) are available However, to maximise the capacity of the technology platform, studies are still needed to optimise overall cell growth to increase the transfection efficiency, to simplify the protocols, and ultimately to improve the expression 86 Optimisation of Transient Gene Expression and productivity In the following sections the optimisation of TGE cell culture conditions including medium components, additives for the medium, optimisation of construction and TGE procedure will be updated and discussed 3.1.1 Medium Optimisation Over the last several years, extensive research into media formulations for the manufacture of recombinant therapeutics has led to serumfree cultures becoming the standard procedure for mammalian cell growth in suspension Hence a number of chemically defined media which support high cell densities and facilitate the expression and purification of recombinant proteins have been developed [13, 14] The following are commonly used: FreeStyle 293, OptiMEM, OptiPRO SFM, and chemically-defined CHO (CD-CHO) from Invitrogen; D/H from Biochrom; UltraCHO and ProCHO5 from Lanza, as shown in Table 3.2, which summarises a list of media which have been used successfully for TGE since 2007 In most cases, these media have been developed specifically for the cultivation of HEK293 or CHO cells, the major hosts for TGE The choice of culture medium has a significant impact on transfection efficiency and productivity Many serum-free media formulations may not support good transfection efficiency [18, 21] For example, transfection with calcium phosphate requires the presence of serum, therefore transfections with this compound was performed in media such as Dulbecco's modified Eagle’s medium (DMEM)/F-12 (Ham’s nutrient mixture formulation) plus 1% fetal bovine serum (FBS) [16, 17] Whilst newly developed media greatly improved cell growth and DNA transfection, it is also possible to perform PEI-mediated serum-free transfection in a minimal medium such as Roswell Park Memorial Institute 1640 (RPMI 1640) [15] As stated previously, linear PEI with a molecular weight of 25,000 has been a standard key reagent to facilitate plasmid delivery [18-20] Transfection competent polycation-DNA complexes have net positive charges and are thought to bind cells through 87 Update on Production of Recombinant Therapeutic Protein ionic interaction with negatively-charged membrane-associated proteoglycans [22] As heparin or dextran sulfate is usually present in serum-free formulations to reduce cell aggregation, the low transfection efficiency observed when using such formulations is most likely a result of polyplex neutralisation by these polyanionic molecules Indeed, it has been reported that heparin strongly inhibits polycation-mediated TGE [22] To overcome this inhibition, a complete medium exchange for a transfection-competent medium is often performed prior to transfection [23, 24] Alternatively, neutralisation of the polyanions in the medium could probably be achieved by using higher PEI:DNA ratios, or by adding free PEI to the culture prior to transfection Table 3.2 shows a summary of culture media used in TGE and their suppliers As mentioned previously, a cell culture medium is often not able to support high transfection efficiency, so changing the medium is a common procedure in order to reach optimum transfection efficiency In Table 3.2, two sets of media are listed for cell culture and transfection Most of these media are used in published experiments and are commercially available; however exact components may not be disclosed except for the popularly used media DMEM and F12 From the production scale-up point of view, if a medium change could be avoided by using a single formulation to support growth and transfection, the target of realising 1g/L expression level at production scale would be quite promising 3.1.1.1 Peptones Protein hydrolysates (peptones) are used in serum-free cell culture media for biosafety reasons and to facilitate downstream processing Peptones provide substantial nutrients including trace elements for a serum-free cell culture medium Supplementation of standard protein-free media with peptones yielded a significant increase in TGE productivity in HEK293E cells [9, 10] The effects of many protein hydrolysates were evaluated on cell proliferation, transfection 88 Culture medium Ex-cell 293 FreeStyle + SFX4HEK Ex-cell 293 FreeStyle 293 HyQSFM4TransFx293 M11V3 FreeStyle Ex-cell V-Pro DMEM/F12 or EpiSerf Ex-cell 293 Protein Expression Medium + supplement DMEM/F12 (D/H) or EpiSerf Cell HEK293E HEK293E HEK293E HEK293F HEK293 SF-3F6 HEK293T HEK293E HEK293E HEK293E HEK293 CAP-T VERO Biochrom or Invitrogen Invitrogen Sigma-Aldrich Biochrom or Invitrogen SAFC Biosciences Invitrogen Novartis Proprietary Hyclone Invitrogen Invitrogen Invitrogen/HyClone SAFC Biosciences Supplier Invitrogen Invitrogen Invitrogen Invitrogen Supplier DMEM/F12 (D/H) or EpiSerf OptiMEM RPMI 1640 + supplement DMEM/F12 or EpiSerf DHI/SAFC medium 293 SFM II M11V3 Biochrom or Invitrogen Invitrogen Lonza Biochrom or Invitrogen SAFC Biosciences Invitrogen Novartis proprietary HyQSFM4TransFx293 Hyclone OptiPRO SFM DMEM + FBS FreeStyle 293 FreeStyle 293 Transfection medium Table 3.2 Culture media used for cell growth and DNA transfection [26] [133] [15] [26] [19] [134] [13] [135] [123] [34] [24] [1] Reference Optimisation of Transient Gene Expression 89 90 CD-CHO/DMEM/F12 CD-CHO + supplement CHO-S-SFMII + ACA* Invitrogen CD-CHO CD-CHO DMEM CHO-S CHO-S CHO-T CHOK1SV CHOK1SV CHO DUKX-B11 Lonza CHO-DG44 ProCHO5 Invitrogen ProCHO5 CHO-S-SFMII DMEM DMEM/FBS/GS supplements UltraCHO CHO-S-SFMII CD-CHO + supplement DMEM Lonza Invitrogen Biochrom JRH Biosciences Lonza Invitrogen Invitrogen Invitrogen DMEM + supplements Introgen OptiPRO SFM [4, 6, 11, 12, 20, 30] [27] [136] [5, 38] [5] [78] [32] [16] [34] [123] ACA: Anticlumping agent CHOM: A CHO cell growth medium developed by Chiang and co-worker (Chiang GG, Sisk WP 2005 Bcl-xL which mediates increased production of humanised Mab in Chinese hamster ovary cells [85] GS: Glutamine synthetase VERO: Cell line derived from kidney of an African green monkey Chiang and Sisk CHO-DG44 CHOM Biochrom Invitrogen Invitrogen Invitrogen Invitrogen Lonza ProCHO5 CHO-S Invitrogen FreeStyle CHO + supplements CHO-S Update on Production of Recombinant Therapeutic Protein Abbreviations GC Guanine-cytosine GFP Green fluorescent protein GHT Glycine + hypoxanthine + thymidine GlcNAc N-Acetylglucosamine GMCSF Granulocyte macrophage colony-stimulating factor GMP Good manufacturing practice GOI Gene of interest Grp78 Glucose-regulated protein GS Glutamine synthetase HBPL Hyperbranched polylysine HCl Hydrochloric acid hCMV Human Cytomeglovirus HEK Human embryonic kidney hES Human embryonic stem HIC Hydrophobic interaction chromatography HIV Human immunodeficiency virus HKB Hybrid of human kidney cell and B cells hp Human cell cycle regulatory protein HP High performance HPLC High performance liquid chromatography HT Sodium hypoxanthine + thymidine Huh-7 Human hepatocyte-derived carcinoma cell line Ig Immunoglobulin ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IL Interleukin IND Investigational new drug ins Human insulin 163 Update on Production of Recombinant Therapeutic Protein LAL Limulus amoebocyte lysate assay LR3-IGF Recombinant insulin-like growth factor LT Large-T antigen LV Lentiviral vectors Mab Monoclonal antibodies MAR Matrix attachment regions MCB Master cell bank MOI Multiplicity of infection MPSV Myeloproliferative sarcoma virus mRNA Messenger ribonucleic acid MW STD Molecular weight standard NaCl Sodium chloride NANA N-Acetylneuraminic acid Neo Neomycin-resistant gene Neu5Ac N-Acetylneuraminic acid Neu5Gc N-Glycosylneuraminic acid NIH United States National Institutes of Health NS0 Mouse myeloma cell line oriP Plasmid origin of viral replication PB PiggyBac PBS Phosphate buffer saline pbv Packed bed column volume pDNA Plasmid DNA pEGFP-N1 Plasmid expressing green fluorescent protein PEI Polyethylenimine PETG Polyethylene terephthalate PGK Phosphoglycerate kinase-1 pO2 Dissolved oxygen tension pPyEBV Expression plasmid encoding the Polyomavirus origin of replication 164 Abbreviations PTRE Post-transcriptional regulatory elements Py Polyomavirus PyOri Polyomavirus origin of replication Qp Specific productivity RNA Ribonucleic acid RP Reverse phase rpm Revolutions per minute RPMI Roswell Park Memorial Institute r-protein Recombinant protein RP-HPLC Reverse phase high performance liquid chromatography SB Sleeping beauty SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEAP Secreted alkaline phosphatase SEC Size exclusion chromatography SFV Semliki forest virus SGE Stable gene expression STP Stable transfection pool SV40 Simian virus 40 or Simian vacuolating virus 40 TFF Tangential flow filtration TGE Transient gene expression tPA Tissue plasminogen activator TNFR/Fc Tumor necrosis factor receptor as an Fc fusion Tris Tris(hydroxymethyl)aminomethane UbC Ubiquitin C UCOE Ubiquitous chromatin opening elements VERO Cell line derived from kidney of African green monkey VLP Virus-like particle 165 Update on Production of Recombinant Therapeutic Protein VP Viral particles VPA Valproic acid WCB Working cell bank WFI Water for injection WPRE Woodchuck hepatitis virus post-transcriptional regulation element 166 I ndex A Absorbance, 63, 69 at the wavelength of 280 nm, 63, 67-69 Acidic fibroblast growth factor, 103-104 Additives, 81, 87, 91-92, 94-95 Adenosine triphosphate, 120-121 Adenoviral, 11, 20, 37, 40-41, 44 African green monkey kidney cell line, 37 Aggregation, 88, 156 Alkaline phosphatase, 20, 44, 85 American Type Culture Collection, 36-41, 50, 80 Ampicillin-resistant gene, 24 Anionic, 32-33 Anti-inflammatory, 137 Antibiotic, 105-107 Antibody-dependent cellular cytotoxicity, 120, 137 Antigen, 25, 36-40, 44, 85, 113-114, 136 Apoptosis, 114-117, 119 Arthritis, 70, 159 B Baby hamster kidney, 22-23, 38, 43, 115, 121 Backbone, 18, 22, 53, 96, 142 Bacteria, 29 Baculovirus expression vector system, 9-10 Bioactivity, 107, 136, 142, 144-145 Biological activity, 10, 63, 69 Bovine serum albumin, 91 167 Update on Production of Recombinant Therapeutic Protein C Calcium phosphate, 25, 27, 30-31, 35, 47-49, 58, 81, 87 Capillary electrophoresis, 145, 158 Carcinoma cell, 36, 45, 121 Cationic, 28-29, 32-33, 50, 120 Cell line derived from kidney of African green monkey, 37, 46, 89-90 Cell membrane, 29, 33, 92, 99 Cell proliferation, 88, 94 Cell viability, 30, 92, 94, 115, 117, 142, 157 Ceramic hydroxyapatite, 144, 154 Chemically defined chinese hamster ovary medium, 52, 87, 90 Chinese hamster ovary, 2, 7, 13, 17, 20-21, 23, 30, 33-35, 38-45, 48-49, 51-53, 75-77, 81, 83-87, 90-95, 98, 102-104, 106108, 110-112, 114, 116, 118-121, 124, 133, 138, 140-143, 149, 151, 156 with glutamine synthetase system, 48, 84-85 Chromatography, 27, 49, 55, 61-69, 99, 141-145, 154-157 Coefficient, 67-68 Combination of the cytomegalovirus early enhancer element and chicken β-actin promotor, 24, 96-97, 143 Conductivity, 62, 65-66, 68 Contamination, 8-9, 18, 31, 46, 157-158 Cyclooxygenase, 22-23 Cytomegalovirus, 18, 24-26, 45, 82, 85, 96-98, 110, 143 Cytotoxicity, 23, 33, 93, 120-121, 137 D Deoxyribonucleic acid, 3-4, 8-10, 12, 18-19, 21-23, 26-34, 37-38, 40-41, 45, 50-55, 57, 59, 61, 64-65, 67, 73, 85-89, 91-94, 98-99, 102-103, 106, 109, 112-114, 121, 126, 136, 143-144, 148, 150, 152, 154-155, 157 Depletion, 120 Design of experiments, 93 Differentiation, 6, 11, 20, 120 Dihydrofolate reductase, 19, 39, 106 168 Index deficient marker, 39 Dimethylsulfoxide, 82, 85, 92 Dissolved oxygen tension, 32, 53 Dulbecco’s modified Eagle’s medium, 31-32, 52-53, 87-90 E Enhanced green fluorescent protein, 55, 112 Enzyme linked immunosorbent assay, 49, 53 Epstein-Barr virus, 36-37, 39-41, 85, 112-114 nuclear antigen, 1, 36, 39-41, 84-85, 91, 112-114 Equilibration buffer, 61-62, 65-66, 68, 71 Erythropoietin, 20, 45, 83, 120 Escherichia coli, 18, 24, 26-28, 31, 97, 154 Ethylenediaminetetraacetic acid, 31, 61, 65-66, 68 European Medicines Agency, 4, 147 Expression augmenting sequence elements, 8, 23, 29, 40, 51, 105, 110-111, 121 Expression plasmid encoding the polyomavirus origin of replication, 114 Extracellular domain, 44-45 F Fast flow, 64-67, 144 Ferritin heavy chain, 96-97 Fibroblast growth factor, 43, 82, 85, 103-104 Final vialled product, 63, 69, 155, 157 Flow cytometry, 58 Foetal bovine serum, 87, 89-90 Food and Drug Administration (US), 1, 4, 8-9, 14-15, 147, 149, 151-153, 157, 160 Formulation, 28, 31, 50, 87-88, 155 Fragment crystallisable, 6-7, 10, 20, 83-85, 91, 104, 112, 118, 120, 137 Fragmentation, 120 Fucosyltransferase, 120 169 Update on Production of Recombinant Therapeutic Protein G Galactose, 21, 138 Gelatin, 91 Gene for dihydrofolate reductase, 19, 38-39, 42, 106 Geneticin, 50, 58 Glucose-regulated protein, 96-97 Glutamine synthetase, 48, 84-85, 90, 106-107, 137, 141 Glycine + hypoxanthaine + thymidine, 42 Good manufacturing practice, 8, 146, 151-154, 158 Granulocyte macrophage colony-stimulating factor, 99 Green fluorescent protein, 10, 29, 32, 55, 57-58, 98, 100, 112 Guanine-cytosine, 26, 99 H Ham’s nutrient mixture formulation, 31-32, 52, 87-90 Heterogeneous, 105-106, 148 High performance liquid chromatography, 49, 55, 62-63, 67-69, 99, 101, 145-146, 154-155 High throughput, 15, 92 Homogeneous, 142 Human cell cycle regulatory protein, 67, 85 Human cytomeglovirus, 18, 21, 25-26 Human embryonic kidney, 2, 17, 40-41, 43-45, 50, 53, 81, 85-86, 102, 114, 138 Human embryonic stem, 11-12 Human growth hormone, 114 Human hepatocyte-derived carcinoma cell line, 36, 40, 43, 45-46 Human immunodeficiency virus, 20 Human insulin, 45 Hybrid of human kidney cell and B cells, 37, 43-45, 84-85 Hydrochloric acid, 50, 52, 65-66, 68 Hydrophobic interaction chromatography, 154-155 Hydroxyapatite, 144 Hyperbranched polylysine, 35 170 Index I Identification, 144, 149 Immobilisation, 46 Immunoglobulin G, 26, 31, 52, 55, 61-63, 83-85, 91, 106-107, 112, 119-120, 137, 140, 142-143 Impurities, 8, 61, 64-66, 68, 101, 144-145, 154, 156 Infection, 10, 19-20, 41, 153 Insect, 6, 9-11, 21-22 Instability, 109, 111 Insulin-like growth factor, 95 Integration, 8, 26, 46, 103, 105, 109, 111, 147-148, 150 Interleukin, 18, 26, 43, 99, 104, 144 International Conference of Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 147, 152 Investigational new drug, 28, 55, 135, 157 Iscove’s modified Dulbecco’s medium, 58-60, 89 L Large-T antigen, 38 Lentiviral vectors, 11, 20-21 Ligand, 18 Light scattering, 158 Limulus amoebocyte lysate assay, 63, 69, 145, 154-1544, 157 Lipid, 10, 19, 30 Liquid chromatography, 49, 63, 65, 69, 99, 143, 145 M Macrophage, 99 Manufacturing, 1, 8-9, 22-23, 33, 144, 146, 150-154, 156, 158, 160 Mass spectrometry, 143 Master cell bank, 27, 152-154, 156 Matrix attachment regions, 101-103 Membrane, 21-22, 29, 33, 54, 88, 92, 99, 120-121, 155 171 Update on Production of Recombinant Therapeutic Protein Messenger ribonucleic acid, 83, 85, 91-92, 94, 102 Metabolism, 132 Methodology, 6, 30, 86, 93, 108 Micelle, 32 Mix, 32, 50, 54 Mixed, 31-32, 50, 54, 57, 108, 110, 156 Mixing, 30, 54 Mixture, 31, 50-51, 54, 57, 87 Model, 91, 96 Modification, 7, 10, 42, 44, 109, 136 Modified, 22, 31-32, 41-42, 45, 58, 87, 121 Module, 54 Molecular structure, 34 Molecular weight, 34, 55, 68, 87, 101, 143, 146, 151 standard, 101, 146 Monoclonal antibodies, 1, 3-4, 6-8, 17-18, 28, 30, 34-35, 47, 49, 53, 55, 61, 63-64, 82-86, 90-91, 94-95, 105-107, 117, 136, 138-141, 154-158 Mouse myeloma cell line, 38 Multiplicity of infection, 19-20 Myeloproliferative sarcoma virus, 26, 98 N N-Acetylglucosamine, 138 N-Acetylneuraminic acid, 119, 138 Neomycin-resistant gene, 24 Neutralisation, 88 P Packed bed column volume, 61-62, 64-68 Particle(s), 9, 10, 17, 19, 32-33, 150 Pharmacology, 75-76, 157 Phase separation, 140 Phosphate buffer saline, 58, 61 Phosphoglycerate kinase-1, 96-97 Phosphorylation, 10 172 Index PiggyBac, 111-112 Plasmid deoxyribonucleic acid, 3-4, 10, 23, 26-28, 30-33, 38, 52-54, 57, 83, 85, 91-92, 94, 105-106, 113, 121, 144, 148, 154-155, 157 Plasmid expressing green fluorescent protein, 32 Plasmid origin of viral replication, 24, 36, 39, 84-85, 113-114, 149 Polyacrylamide gel, 63, 69, 96, 142 Polyethylene, 62 terephthalate, 62, 67-68 Polyethylenimine, 10-11, 25, 27-29, 33-35, 45, 47-55, 57, 59, 81, 86-88, 92-94, 146 Polyomavirus, 38, 114 origin of replication, 38, 114 Polystyrene, 140 Post-transcriptional regulatory elements, 101-102 Proliferation, 11, 88, 94 Promoter, 18, 21-22, 24-26, 45, 96-100, 102, 143 Properties, 6, 103, 108, 137 Protein synthesis, 21, 115, 142 Proton, 33 Protonation, 33 Purification, 3, 5, 12-15, 19, 23, 26-28, 31, 47, 55, 61-67, 69, 71-72, 79, 87, 125-126, 132, 143-145, 154-158 Q Quality control, 6, 63, 69, 156-158 R Recombinant insulin-like growth factor, 95 Reductase, 19, 39, 106 Reproducibility, 135-136, 142-143, 146-147, 149, 158 Resin, 61-62, 64-66, 154-155 Retention, 17, 112, 114 Reverse phase, 63, 69, 140-141, 145, 154-155 Reverse phase high performance liquid chromatography, 69, 145, 154-155 173 Update on Production of Recombinant Therapeutic Protein Rheumatism, 159 Ribonucleic acid, 22, 29, 64-65, 67, 85, 98, 104 Roswell Park Memorial Institute, 57, 87, 89 S Safety, 9, 20, 22-23, 43, 63, 69, 121, 135-136, 145, 152, 156-158 Saturation, 53-55 Secreted alkaline phosphatase, 44-45, 82, 85, 91, 114, 121 Semliki forest virus, 22-23 Simian virus 40 or Simian vacuolating virus, 36-37, 39-40, 44, 96, 98 Size exclusion chromatography, 55, 62-63, 67-69, 99, 101, 142, 146, 154-155 Sleeping beauty, 11, 111 Sodium chloride, 61-62, 65-66, 68, 95 Sodium dodecyl sulfate polyacrylamide gel electrophoresis, 63-65, 67-69, 96-97, 99-101, 142-143, 145-146, 154-155 Sodium hypoxanthine + thymidine, 52 Specific productivity, 86, 92, 104, 108, 116-119, 142 Spectrometry, 141, 143 Spectroscopy, 158 Stability, 92, 98, 105, 109-112, 147, 153, 157 Stable gene expression, 2-3, 5, 86, 105, 108, 117, 136, 143, 147-148, 152-153 Stable transfection pool, 24, 48, 84, 105, 107, 109, 112, 147 Strain, 22, 26, 31, 42, 112 Synthesis, 21, 95, 104, 115, 142 T Tangential flow filtration, 144, 154-155 Tension, 32 Time, 1-4, 6-8, 11, 30, 49, 52-53, 56, 64, 68, 86, 93, 104-108, 110, 112, 115, 117, 119, 121, 140, 142, 145, 149 Tissue plasminogen activator, 26, 99, 107 Transformation, 36, 40 174 Index Transient gene expression, 1-3, 5-11, 13, 15, 17-21, 23-31, 33-37, 39-53, 55, 57-59, 61, 63-65, 67, 69, 71, 73, 75, 77, 79, 8183, 85-89, 91-97, 99-105, 107-109, 111-119, 121, 123-125, 127, 129, 131, 133, 135-139, 141-143, 145-159 Tris(hydroxymethyl)aminometane, 31 Tumor necrosis factor receptor as an fragment crystallisable fusion, 10, 112 Type adenovirus vector, 40, 46 U Ubiquitin C, 96-97 Ubiquitous chromatin opening elements, 105, 109-110 United States National Institutes of Health, 19, 21, 70-71 Untreated, 91 V Validation, 51, 136, 151 Valproic acid, 42-43, 56, 58, 83, 85, 91-92, 104 Viral particles, 19, 47 Virus-like particle(s), 9-10, 150 Volume, 2, 4, 17, 29-32, 45, 48-54, 56-57, 59-61, 66-68, 83, 94, 107-108, 122, 142-144, 154 W Water for injection, 65-66, 68 Western blotting, 96-97, 99-101, 145-146, 154-155, 157 Woodchuck hepatitis virus post-transcriptional regulation element, 82, 85, 94-95, 97, 101-102 Working cell bank, 27-28, 58, 152 175 Smithers Rapra Technology Ltd, 2013 Over the past decade, the transient gene expression (TGE) technology platform has been actively pursued to produce a wide range of therapeutic proteins, monoclonal antibodies, and vaccines for mainly preclinical assessment, due to its short development times and low overall cost This book updates the latest advances in the field, with focusing on systematic description of the technology from cell lines, cell culture conditions, vector construction, expression strategy, current protocols, optimisation of the procedure, and potential for clinical application As a conclusion, the author foresees that therapeutic biopharmaceutics will be manufactured for clinical development using TGE technology in the near future because of its fast development time, good protein expression, acceptable quality of product and due to the progress which has been made in analytical methodology and process quality control Update on Production of Recombinant Therapeutic Protein: Transient Gene Expression Published by The objectives of this book are to summarise current TGE protocols, to describe optimisation of the technology through the latest advances, and to explore clinical applications of the technology It gives the reader a good insight into the latest development and future application of the technology platform, including: • • • • Jianwei Zhu The current protocols from small to large scale for different cells Optimisation methods in construction designing, transfection procedures, and cell culture conditions Overall quality of the product from the transient gene expression Future clinical application of the technology platform Jianwei Zhu Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118 Web: www.polymer-books.com Update on Production of Recombinant Therapeutic Protein: Transient Gene Expression ... Optimisation of Transient Gene Expression 85 Update on Production of Recombinant Therapeutic Protein at high cell density [6, 2] However, compared with an optimised stable transfection expression,... number of DNA-PEI complexes, leading to a higher percentage of transfected cells [30] 92 Optimisation of Transient Gene Expression 3.1 .2 Optimisation of Transient Gene Expression Conditions and... expression system [1] 95 Update on Production of Recombinant Therapeutic Protein 3.1.3 Construction Optimisation Gene expression levels in mammalian TGE system largely depend, among other things, on