1. Blood and immune system pag. 2

I

INDEX

Introduction pag. 1

1. Blood and immune system pag. 2

1.1. Blood pag. 2

1.2. Corpuscular fraction pag. 2

1.3. Plasma: general aspects pag. 5

1.4. Plasma proteins pag. 6

1.4.1. Albumin pag. 7

1.4.2 a-globulins pag. 7

1.4.3. ß-globulins pag. 9

1.4.4. ?-globulins pag. 10

1.4.6. Structure of in the heavy chains pag. 11

1.4.7. Association between heavy chain and light chain pag. 13

1.4.8. Distribution of antibodies pag. 13

2. The immunological system pag. 13

2.1. The innate immunological system pag. 13

2.2. The acquired immunological system pag. 14

2.2.1 Cell-mediated immunity pag. 14

2.2.2. Humoral immunity pag. 15

2.3. Effector functions of the antibodies pag. 15

2.4. Immunodeficient syndromes pag. 17

3. Production of gammaglobulins G for therapeutic use pag. 28

3.1. Plasma collection pag. 22

3.1.1. General aspects pag. 22

3.1.2. Donations pag. 23

3.1.2.1. Collection centres pag. 23

3.1.2.2. Collection procedures pag. 23

3.1.2.3. Equipment for plasma collection pag. 24

3.1.2.4. Traceability of donations and procedures after the collection pag. 25

4. IgG manufacturing methods pag. 26

4.1. General aspects pag. 27

4.2.. Precipitation pag. 28

4.2.1 Cohn’s and Oncley method pag. 28

II

4.3. Cohn’s method disadvantages pag. 32

4.4. Alternative IgG manufacturing techniques pag.33

4.4.1. Chromatography pag.33

4.4.1.1. Experimental approach for chromatographic purification pag.34

4.4.1.2. General remark8(IEX) pag.36

4.4.1.3. Ion exchange chromatography pag.38

4.4.1.4. Hydrophobic interaction chromatography (HIC) pag.41 4.4.1.5. Reversed phase chromatography (RPC) pag. 41 4.4.1.6. Size exclusion chromatography (RPC) pag. 42

4.4.1.7. Affinity chromatography (AC) pag. 42

4.4.1.9. Mixed mode chromatography pag. 45

4.4.1.9. Mixed mode chromatography pag. 45

4.4.2. New techniques pag. 46

4.4.2.2. Membrane chromatography pag. 48

4.5. Use of chromatography in the IVIG manufacturing pag. 49

5. Industrial manufacturing pag. 50

5.1. Guidelines pag. 50

5.2 Facilities pag. 51

5.2.1. Equipment for water production pag. 51

5.2.2. Solid-liquid separation systems pag. 52

5.2.3. Containment equipments pag. 55

5.2.4. Cleaning systems pag. 55

5.2.5. Gases pag. 55

5.2.6. Safety of biological products pag. 55

5.2.7. Viral inactivation methods pag. 56

5.2.8. Viral removal methods pag. 58

6. Pharmacological applications pag. 60

7. Immunoglobulin market context pag. 61

III

Materials and methods pag. 63

1. Materials and methods - Analytical section pag. 64

1.1. Equipment pag. 64

1.2. Materials pag. 65

1.3. Preparation of solutions pag. 68

1.4. Reconstitution of kits and reagents pag. 71

1.5. Method and description of analytical methods pag. 73 1.5.1. Colorimetric methods for total protein content determination pag. 73

1.5.1.1. Biuret method pag. 73

1.5.1.2. Bradford method pag. 74

1.5.2. Immunonephelometric method for

plasmatic protein content determination pag. 75

1.5.3. Electrophoresis method for molecular characterization pag. 75

1.5.3.1. Miniprotean II procedure pag. 76

1.5.3.2. SAS–1 procedure pag. 76

1.5.4. HPLC analysis for molecular characterization pag. 77 1.5.5. Gas chromatography method for TnBp analysis pag. 78 1.5.6. HPLC analysis for determination of Triton X-100 analysis pag. 79 1.5.7. Haemolytic assay for the determination of

Anti-complementary activity (ACA) pag. 80

1.6. Pretreatment of plasmatic intermediates pag. 81

2. Materials and methods-Process section pag. 83

2.1. Materials pag.83

2.1.1. Chromatographic media pag. 83

2.1.2. Chemicals pag. 83

2.1.3. Water pag. 84

2.1.4. Plasmatic intermediates (starting materials) pag. 84

2.2. Equipment pag. 84

2.2.1. Chromatography equipment pag. 84

2.2.2. Expanded bed chromatography equipment pag. 84

2.2.3. Dead end filtration equipment pag. 85

2.2.4. Tangential filtration equipment pag. 85

2.2.5. Accessory equipment pag. 85

IV

2.2.6. Systems descriptions pag.86

2.2.6.1. Chromatographic systems pag.86

2.2.6.2. Batch mode chromatographic system pag.86

2.2.6.3. Packed bed chromatographic system pag.87

2.2.6.4. Automated chromatographic system

(Akta Explorer, GE Healthcare) pag.87

2.2.6.5. Manual chromatographic system pag.88

2.2.6.6. Membrane chromatography system pag.89

2.2.6.7. Packing of the columns and verification of the packing pag.90

2.2.6.8. Radial column pag.91

2.2.6.9. Theoretical plates calculations pag.92

2.2.6.10. Expanded bed Adsorption (EBA)

chromatographic system and packing pag. 93

2.2.7. Dead end filtration systems pag. 94

2.2.8. Tangential filtration systems pag. 94

2.2.8.1. TFF Labscale Millipore pag. 94

2.2.8.2. Minimate system with LV Centramate holder pag. 95

2.2.8.3. Tangential filtration operations pag. 95

2.2.9. Planova nanofiltration devices pag. 96

2.3. Data elaboration pag. 98

2.3.1. Formulas pag. 98

2.3.2. Yields and other parameters pag. 99

2.4. Preparation of buffers for process procedures pag. 100

2.4.1. Batch trials for capture step pag. 100

2.4.1.1. Conditioning buffers pag. 100

2.4.1.2. Washing buffers pag. 100

2.4.1.3. Elution buffers pag. 100

2.4.2. Step trials on axial column for capture pag. 100

2.4.2.1. Conditioning buffers pag. 100

2.4.2.2 Washing buffers pag. 101

2.4.2.3 Elution buffers pag. 101

2.4.3. Step trials on radial column for capture pag. 102

2.4.3.1. Conditioning buffers pag. 102

2.4.3.2. Washing buffers pag. 102

V

2.4.3.3. Elution buffers pag. 102

2.4.4. Expanded bed trials pag. 102

2.4.4.1 Conditioning buffers pag. 102

2.4.4.2. Washing buffers pag. 103

2.4.4.3. Elution buffers pag. 103

2.4.5. Step trials for polishing pag. 103

2.4.5.1. Conditioning buffer pag. 103

2.4.5.2. Washing buffers pag. 103

2.4.5.3. Elution buffers pag. 103

2.4.6. Cleaning and sanitisation buffers pag. 104

2.4.7. Solutions used for the pH correction pag. 104

2.5. Cromatographic procedures pag. 105

2.5.1. Operating conditions of batch experiments pag. 105 2.5.2. Operating conditions of gradient experiments pag. 106 2.5.3. Operating conditions of step experiments pag. 107

2.5.3.1. Capture step protocols pag. 107

2.5.3.2. Step trials on axial column for polishing pag. 112

2.5.3.2.1. MEP Hypercell pag. 112

2.5.3.2.2. Ceramic HyperD Q pag. 113

2.5.3.2.3. Step trials on membrane chromatography for polishing pag.114 2.5.3.3.4. Expanded Bed Adsorption (EBA) chromatography pag. 117

2.5.3.4.5. Polishing on TMAE Fractogel pag. 119

2.5.3.5. Packing and preparation of the columns

for the chromatographic trials pag. 121

2.5.3.5.1. CM Sepharose pag. 121

2.5.3.5.2. SP Sepharose pag. 121

2.5.3.5.3 MEP Hypercell pag. 122

2.5.3.5.4. MBI Hypercell pag. 122

2.5.3.5.5. IgSelect pag. 123

2.5.3.5.6. Capto S pag. 123

2.5.3.5.7. Ceramic Hyper D Q pag. 124

2.5.3.5.8. Expanded bed pag. 124

2.5.3.5.9. TMAE packing pag. 124

VI

Results pag. 126

1. Experimental approach pag. 127

2. Identification of the starting material pag. 127 3. Identification of a suitable system for

the intermediate purification of IgG pag. 128

3.1. Batch experiments pag. 128

3.2. Gradient experiments pag. 132

3.3. Step chromatography experiments pag. 135

3.3.1. Chromatographic trials on CM Sepharose resin pag. 135 3.3.2. Chromatographic trials on SP Sepharose resin pag. 136 3.3.3. Chromatographic trials on MEP Hypercel resin pag. 138 3.3.4. Chromatographic trials on MBI Hypercel resin pag. 140

3.3.5. Industrial scal up hypothesis pag. 150

3.3.6. Chromatographic trials on IgSelect resin pag. 152 3.3.7. Chromatographic trials on Capto S resin pag. 154

3.3.8. Industrial scale up hypothesis pag. 158

4. Identification of a suitable system for the final purification (polishing) of IgG solutions

obtained from the capture step pag. 160

4.1. Polishing on MEP Hypercel pag. 161

4.2. Polishing on Ceramic Hyper D Q pag. 165

4.3. Polishing on Mustang chromatographic membranes pag. 169 4.3.1. Polishing on Mustang Q (MBI eluate) pag. 170 4.3.2. Experiments with Mustang Q + Mustang S (MBI eluate pag. 172 4.3.3. Experiments with Mustang Q (Capto S eluate) pag. 181

4.3.4. Mustang scale up pag. 183

5. IgG purification by Expanded Bed Adsorption

(EBA) chromatography pag. 187

5.1. Capture on Fast Line IVIG pag. 187

5.1.1. Trials on (II+III)S/D sample pag. 188

5.2. Polishing on fast Line PEI pag. 191

5.3. Industrial scale up hypothesis pag. 194

6. Polishing on TMAE Fractogel chromatographic resin pag. 196

VII

6.1. Conclusions pag. 203

7. Process verification trials pag. 204

7.1. Trial 1PVT pag. 204

7.2. Trial 2PVT pag. 209

7.3. Trial 3PVT pag. 214

7.4. Trial 4PVT pag. 219

7.5. Trial 5PVT pag. 223

7.6. Trial 6PVT pag. 228

7.6. Comments and conclusions pag. 233

8. Scale up trials pag. 234

8.1. Trial 1ST pag. 235

8.2. Trial 2ST pag. 242

8.3. Trial 3ST pag. 249

8.4. Comments and conclusions pag. 256

9. Stability experiment pag. 257

9.1. Stability program and results pag. 259

10. Analytical characterization by electrophoresis pag. 260

11. Analytical characterization by HPLC pag. 265

Discussion pag.267

1. Chromatography versus Cohn’s fractionation: better and faster? pag. 269

2. Time to choose pag. 270

2.1. Starting material pag. 270

2.2. Capture resin pag. 272

2.3. Polishing resin pag. 274

3. Connecting the capture and the polishing, ending the process pag. 276

4. The transferrin issue pag. 278

5. The albumin line pag. 279

6. Planning the pilot scale pag. 280

7. Conclusions and future perspectives pag. 287

VIII

References pag. 288

Appendix 1 Chromatograms

Appendix 2 Tables of results

IX

Glossary

aa: Aminoacid

AC: Affinity chromatography

ACCS: Axial chromatography capture step

ACCS: Axial chromatography Capture step ACPS: Axial Chromatography Polishing Step

ADCC: Antibody dependent cell-mediated cytotoxicity APC: Cell presenting the antigen

C: Constant regions of Immunoglobulin CBC: Complete blood count

CD: Cluster differentiation

CDR: Complementary determining regions

C

1 e C

2: First and second constant domain of heavy chain CIP: Cleaning in place

CIPP: Capture, Intermediate Purification, Polishing strategy CTL: T cytotoxic cells

CV: Column volume

CVID: Severe combined immunodeficiency D: Diversity regions

EBA: Expanded bed chromatography EBCS: Expanded Bed Capture Step

EBPS:

EMEA: European agency for evaluation og medicinal products Fab: Antigen binding fragment

Fas-Fas L: Fas- fas ligand Fc: Crystallizable fragment

FcR: Fc fragment membrane receptor GBS: Guillain-Barrè syndrome GMP. Good manufacturing practises HAV: Hepatitis A virus

HBV: Hepatitis B virus

X HCI: Hydrophobic charge induction chromatography

HCV: Hepatitis C virus

HIC: Hydrophobic induction chromatography HIV-1: Human Immunodeficiency virus type 1 IEX: Ion exchange cromatography

IFN. Interferon Ig: Immunoglobulins IgA Immunoglobulin A IgG: Immunoglobulin G

IgG1: Immunoglobulin G type 1 IgG2:Immunoglobulin G type 2 IgG3: Immunoglobulin G type 3 IgG4: Immunoglobulin G type 4 IgM: Immunoglobulin M IL: Interleukin

IVIG: immunoglobulin for inntravenous administration J: Junctional chain

k: Light chain of immunoglobulins K

: Distribution coefficient kDa: Kilodalton

kGy: Kgray

LAK: Lymphochine activated killer cells

MBISA: 2-mercapto-5-benzimidazole sulphonic acid MEP: Methyl ethyl pyridine

MG: Miastenia Gravis

MHC: Major complex of Histocompatibility MMN: Motoneuronal Multifocal Neuropathy mRNA : Messenger RNA

NK: Natural killer cells PEG: Poly-ethylen-glicole PM: Molecular weight

PW: Purified water

RPC: Reversed Phase Chromatography

XI

S/D: Solvent detergent SDS: Sodium-dodecil-sulfate SIP: Steam in place

SN/C: Cryo supernatant SN/I: Supernatant of Fraction I SP: Sulfo-propyle

ST: Scale up trial

SVID: Severe common variable immunodeficiency TCR:T receptor cells

TFF: Tangential flow filtration

TMAE: Tri-methyl-ammonium-ethyl group TnBP. Tri-n-butylphosphate

TnBp: Tri-normal-Butylphosphate Trf: Transferrin

UFC: UpFront Chromatography

V: Variable regions of Immunoglobulins

WFI: Water for injection

XII

Aim of the work

The aim of this research project is to develop a new chromatography-based manufacturing process of an immunoglobulin G (IgG) concentrate for intravenous administration, starting from human plasma.

The experimental work has been performed in the Research and Development laboratories of the italian company Kedrion Biopharmaceuticals, an international leader in the manufacture and supply of plasma derivative products at the highest standards of quality and safety, for the italian and foreign markets.

This commitment was determined by the growing worldwide and by the marketing request demand of more purified and safer IgG concentrates, in comparison to the products obtained by Cohn’s fractionation. Therefore the new manufacturing process was designed by taking into account the purity and pathogen safety requirements.

The overall IgG yield increase, the possible application of innovative technologies and

the feasibility at the industrial level were also important drivers during the research

work.