Pilot-scale process development for the purification of the recombinant antibody 2G12 from transgenic tobacco [Elektronische Ressource] / vorgelegt von Martin Lobedann

Pilot-scale process development for the
purification of the recombinant antibody
2G12 from transgenic tobacco



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Ingenieurwissenschaften genehmigte Dissertation


vorgelegt von

Diplom-Ingenieur
Martin Lobedann

aus Görlitz




Berichter: Universitätsprofessor Dr. Rainer Fischer
Universitätsprofessor Dr.-Ing. Winfried Hartmeier



Tag der mündlichen Prüfung: 10. Juli 2009
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
TABLE OF CONTENTS
Table of contents
I Introduction..............................................................................................................1
I.1 Downstream processing of recombinant proteins............................................3
I.2 Regulatory considerations for production of PMPs..........................................4
I.3 The Pharma-Planta project ..................................................................................5
I.4 The recombinant antibody 2G12.........................................................................5
I.5 Objectives6
II Materials and Methods............................................................................................8
II.1 nd Equipment .....................................................................................8
II.1.1 Chemicals and Consumables..............................................................................8
II.1.2 Buffers and solutions ...........................................................................................8
II.1.3 Plant material dispersion units...........................................................................10
II.1.4 Further equipment .............................................................................................11
II.2 Protein production methods13
II.2.1 Production host Nicotiana tabacum cv. SR-1 ....................................................13
II.2.1.1 Transformation and expression of 2G12 ........................................................13
II.2.1.2 Cultivation and harvest of Nicotiana tabacum cv. SR-1..................................13
II.2.2 Small and medium scale protein extraction techniques.....................................14
II.2.3 Pilot scale dispersion and extraction .................................................................14
II.2.4 Clarification strategies .......................................................................................15
II.2.4.1 Dead end filtration...........................................................................................15
II.2.4.2 Hollow-fiber cross-flow filtration ......................................................................16
II.2.5 Chromatographic processing.............................................................................17
II.2.5.1 Multimodal cation exchange chromatography ................................................20
II.2.5.2 Multimodal anion exchange chromatography .................................................20
II.2.5.3 Affinity chromatography ..................................................................................20
II.2.5.4 Ceramic hydroxyapatite21
II.2.6 Virus reduction...................................................................................................22
II.2.7 Concentration and diafiltration...........................................................................22
II.2.8 Methods for DsRed purification .........................................................................23
II.3 Stability studies ..................................................................................................23
II.4 Analytical assays................................................................................................23
II.4.1 Surface plasmon resonance measurement .......................................................23
II.4.2 Enzyme linked immunosorbent assay (ELISA)..................................................24
II.4.3 SDS-PAGE and staining....................................................................................24
II.4.4 Western blot.......................................................................................................25
II.4.5 Analytical size exclusion chromatography .........................................................25
I TABLE OF CONTENTS
II.4.6 Nicotine analytics...............................................................................................25
II.4.7 DNA analytics ....................................................................................................25
II.4.8 Protein quantification .........................................................................................26
II.4.9 Turbidity determination ......................................................................................26
II.4.10 Mass spectroscopy............................................................................................26
II.4.11 N-glycosylation analytics ...................................................................................26
II.4.12 Syncytium inhibition assay.................................................................................26
III Results and Discussion ........................................................................................27
III.1 Screening of extraction buffers.........................................................................27
III.2 Vacuum filtration and plate pressing................................................................27
III.3 2G12 expression levels......................................................................................28
III.4 Approaches for the dispersion of plant biomass............................................30
III.4.1 Optimization of RT50 Megatron and Polytron....................................................32
III.5 Approaches for fiber removal............................................................................36
III.5.1 Fiber removal by custom-made filter capsules ..................................................37
III.5.2 Fiber removal by bag filter .................................................................................38
III.6 Approaches for extract clarification .................................................................39
III.6.1 Extract clarification by hollow-fiber filtration.......................................................39
III.6.2 ication by depth filtration ................................................................41
III.7 Approaches for capturing 2G12 from clarified tobacco extract.....................42
III.7.1 Capture by packed-bed chromatography ..........................................................42
III.7.1.1 Capture on a Protein A column.......................................................................42
III.7.1.2 Capture on a non-affinity column....................................................................44
III.7.2 Capture on affinity membranes..........................................................................45
III.7.2.1 Capture by the tangential flow affinity membrane technique ..........................45
III.7.2.2 Capture on a normal flow affinity membrane adsorber...................................47
III.7.2.3 Pilot-scale antibody capture............................................................................49
III.8 Intermediate purification....................................................................................52
III.8.1 Intermediate purification by Capto adhere.........................................................52
III.8.2 n by ceramic hydroxyapatite..........................................53
III.9 Polishing for virus reduction.............................................................................55
III.10 Ultrafiltration and diafiltration ...........................................................................55
III.11 Stability results...................................................................................................56
III.12 Production of clinical-grade material from 216 kg of tobacco leaves ...........58
III.13 Characteristics of the final bulk product..........................................................61
III.13.1 Analytical gel filtration........................................................................................62
III.13.2 Protein quantification .........................................................................................62
II TABLE OF CONTENTS
III.13.3 Residual DNA quantification..............................................................................62
III.13.4 SDS-PAGE and LC/MS analysis .......................................................................63
III.13.5 Nicotine content.................................................................................................64
III.13.6 N-glycan profile..................................................................................................64
III.13.7 In vitro binding of gp120 ....................................................................................65
III.13.8 N