Therapeutic
`Peptides and
`Proteins
`
`Formulation, Processing, and Delivery Systems
`
`Aiay K. Banga
`
`Taylor &Francis
`Taylor &Francis Group
`
`_
`_
`MAIA EXhlblt 1044
`MAIA V. BRACCO
`
`IPR PETITION
`
`
`
`
`MAIA Exhibit 1044
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`Therapeutic
`Peptides and
`Proteins
`Formulation, Processing, and Delivery Systems
`Second Edition
`
`
`
`
`

`

`Therapeutic
`Peptides and
`Proteins
`Formulation, Processing, and Delivery Systems
`Second Edition
`
`Ajay K. Banga, Ph.D.
`Mercer University
`Atlanta, Georgia
`
`Boca Raton London New York
`
`A CRC title, part of the Taylor & Francis imprint, a member of the
`Taylor & Francis Group, the academic division of T&F Informa plc.
`
`
`
`
`

`

`Published in 2006 by
`CRC Press
`Taylor & Francis Group
`6000 Broken Sound Parkway NW, Suite 300
`Boca Raton, FL 33487-2742
`
`© 2006 by Taylor & Francis Group, LLC
`CRC Press is an imprint of Taylor & Francis Group
`
`No claim to original U.S. Government works
`Printed in the United States of America on acid-free paper
`10 9 8 7 6 5 4 3 2 1
`
`International Standard Book Number-10: 0-8493-1630-8 (Hardcover)
`International Standard Book Number-13: 978-0-8493-1630-2 (Hardcover)
`Library of Congress Card Number 2005043743
`
`This book contains information obtained from authentic and highly regarded sources. Reprinted material is
`quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts
`have been made to publish reliable data and information, but the author and the publisher cannot assume
`responsibility for the validity of all materials or for the consequences of their use.
`
`No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
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`Library of Congress Cataloging-in-Publication Data
`
`Banga, Ajay K.
`Therapeutic peptides and proteins : formulation, processing, and delivery systems / Ajay K. Banga.-
`-2nd ed.
`
`p. cm.
`Includes bibliographical references and index.
`ISBN 0-8493-1630-8
`1. Protein drugs. I. Title.
`
`RS431.P75B36 2005
`615'.3--dc22
`
`2005043743
`
`Visit the Taylor & Francis Web site at
`http://www.taylorandfrancis.com
`
`Taylor & Francis Group
`is the Academic Division of T&F Informa plc.
`
`and the CRC Press Web site at
`http://www.crcpress.com
`
`
`
`
`

`

`Table of Contents
`
`Chapter 1 Pharmaceutical Biotechnology: The Arrival of
`Recombinant Proteins................................................................... 1
`1.1 What is biotechnology?................................................................................ 1
`1.1.1 Recombinant DNA technology ...................................................... 2
`1.1.2 Monoclonal antibodies .................................................................... 4
`1.1.3 Expanding scope of biotechnology ............................................... 7
`1.1.3.1 Antisense agents and RNAi............................................. 8
`1.1.3.2 Transgenic therapeutics .................................................... 9
`1.1.3.3 Gene therapy ...................................................................... 9
`1.1.3.4 Human Genome Project ................................................. 10
`1.2 The biotechnology industry ...................................................................... 11
`1.2.1 Biotechnology products on the market ...................................... 11
`1.2.1.1 Human insulins and analogues .................................... 12
`1.2.1.2 Growth hormone ............................................................. 14
`1.2.1.3 Interferons......................................................................... 14
`1.2.1.4 Dornase alfa...................................................................... 15
`1.2.1.5 Antimicrobial peptides ................................................... 16
`1.2.1.6 Tissue plasminogen activator ........................................ 16
`1.2.1.7 Fusion proteins................................................................. 16
`1.2.1.8 Miscellaneous ................................................................... 17
`1.3 Regulatory aspects of biotechnology-derived drugs ............................ 17
`1.3.1 Preclinical and clinical studies..................................................... 19
`1.3.2 Pharmacoeconomics and the regulatory process...................... 20
`1.3.3 Characterization and purity of biotechnology-derived
`proteins ............................................................................................ 21
`1.3.4 Patents.............................................................................................. 22
`1.4 Immunogenicity of proteins...................................................................... 22
`1.5 Structure-based drug design..................................................................... 24
`1.6 Conclusions.................................................................................................. 25
`
`Chapter 2 Structure and Analysis of Therapeutic Peptides
`and Proteins .................................................................................. 33
`2.1 Amino acids: building blocks of proteins............................................... 33
`2.2 Structure of peptides and proteins .......................................................... 34
`2.2.1 Primary structure ........................................................................... 35
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`Secondary structure ........................................................................35
`2.2.2
`2.2.3 Tertiary structure and protein folding........................................ 36
`2.2.4 Quaternary structure ..................................................................... 38
`2.3 Protein conformation and other structural features ............................. 38
`2.4 Analytical methods..................................................................................... 41
`2.4.1 Electrophoresis................................................................................ 41
`2.4.2
`Spectroscopy ................................................................................... 43
`2.4.2.1 Ultraviolet spectroscopy................................................. 43
`2.4.2.2 Fluorescence spectroscopy ............................................. 43
`2.4.2.3 Circular dichroism spectroscopy .................................. 44
`2.4.2.4 Infrared spectroscopy ..................................................... 45
`2.4.2.5 Light scattering ................................................................ 46
`2.4.2.6 Colorimetric assay ........................................................... 47
`2.4.3 Chromatography ............................................................................ 48
`2.4.3.1 Reverse-phase chromatography.................................... 48
`2.4.3.2 Size exclusion chromatography .................................... 49
`2.4.3.3 Other chromatographic techniques .............................. 50
`2.4.4 Thermal analysis ............................................................................ 50
`2.4.5
`Immunoassays and bioassays ...................................................... 51
`2.4.6 Other quality control procedures ................................................ 53
`2.4.6.1 Peptide mapping ............................................................. 54
`2.4.6.2 Analytical ultracentrifugation ....................................... 55
`2.4.6.3 Analysis of glycoproteins............................................... 55
`2.5 Conclusions.................................................................................................. 56
`
`Chapter 3 Stability of Therapeutic Peptides and Proteins.................... 67
`3.1 Introduction ................................................................................................. 67
`3.2 Physical instability...................................................................................... 68
`3.2.1 Denaturation ................................................................................... 68
`3.2.2 Aggregation..................................................................................... 69
`3.2.2.1 Mechanism of aggregation............................................. 70
`3.2.2.2 Aggregation behavior in liquid and solid states........ 72
`3.2.3 Adsorption....................................................................................... 73
`3.3 Chemical instability.................................................................................... 75
`3.3.1 Oxidation ......................................................................................... 75
`3.3.2 Hydrolysis ....................................................................................... 77
`3.3.3 Deamidation.....................................................................................78
`b-Elimination and disulfide exchange........................................ 79
`3.3.4
`3.3.5 Racemization................................................................................... 80
`3.3.6 Thermal stability ............................................................................ 80
`3.3.7 Multiple pathways of chemical instability ................................ 80
`3.4 Well-characterized products and comparability protocols ..................82
`3.5 Conclusions.................................................................................................. 83
`
`Chapter 4 Preformulation and Formulation of Therapeutic
`Peptides and Proteins ................................................................. 91
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`4.1 Introduction ................................................................................................. 91
`4.2 Preformulation studies............................................................................... 98
`4.3 Formulation development......................................................................... 99
`4.3.1 Buffer system ................................................................................ 100
`4.3.2 pH of the vehicle.......................................................................... 101
`4.3.3 Protein solubility .......................................................................... 101
`4.3.4
`Selection of solvent system......................................................... 103
`4.3.5 Preservation of a formulation .................................................... 103
`4.3.6 Choice of container ...................................................................... 104
`4.4 Pharmaceutical excipients in formulations .......................................... 104
`4.4.1 Albumin......................................................................................... 106
`4.4.2 Amino acids .................................................................................. 107
`4.4.3 Carbohydrates............................................................................... 108
`4.4.4 Chelating and reducing agents.................................................. 108
`4.4.5 Cyclodextrins ................................................................................ 109
`4.4.6 Polyhydric alcohols...................................................................... 110
`4.4.7 Polyethylene glycol...................................................................... 111
`4.4.8
`Salts................................................................................................. 111
`4.4.9
`Surfactants ..................................................................................... 112
`4.4.10 Miscellaneous................................................................................ 114
`4.4.11 Selection of excipient ................................................................... 114
`4.5 Aggregation in protein formulations..................................................... 116
`4.5.1 Aggregation behavior of insulin................................................ 116
`4.5.2 Aggregation behavior of human growth hormone................ 119
`4.5.3 Aggregation behavior of a1-antitrypsin ................................... 119
`4.5.4 Aggregation behavior of human interferon ............................ 120
`4.5.5 Aggregation of monoclonal antibodies .................................... 120
`4.5.6 Aggregation behavior of some other proteins ........................ 121
`4.6 Novel formulation approaches............................................................... 121
`4.6.1 Liposomes...................................................................................... 122
`4.6.2 Reverse micelles ........................................................................... 123
`4.6.3 Emulsions ......................................................................................123
`4.6.4 Genetic engineering or chemical modification ....................... 124
`4.7 Accelerated stability testing .................................................................... 125
`4.8 Statistical design for formulation development .................................. 127
`4.9 Conclusions................................................................................................ 128
`
`Chapter 5 Lyophilization, Pharmaceutical Processing, and
`Handling of Therapeutic Peptides and Proteins ................ 139
`5.1 Introduction ............................................................................................... 139
`5.2 Protein destabilization induced by pharmaceutical processing ....... 140
`5.2.1 Adsorption induced by pharmaceutical processing .............. 140
`5.2.2 Aggregation induced by pharmaceutical processing............. 141
`5.2.2.1 Shaking............................................................................ 141
`5.2.2.2 Hydrophobic surfaces................................................... 142
`5.2.2.3 Heating ............................................................................ 142
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`5.2.2.4 Shear-induced aggregation .......................................... 142
`5.2.2.5 Miscellaneous factors.................................................... 143
`5.3 Lyophilization as a pharmaceutical process......................................... 143
`5.3.1 Process details ...............................................................................144
`5.3.2 Maximum allowable product temperature.............................. 145
`5.3.3 Measurement of lyophilization-induced thermal changes ... 146
`5.3.4 Protein denaturation during lyophilization ............................ 147
`5.3.5
`Freeze–thaw stability................................................................... 147
`5.3.6
`Formulation components for lyophilization............................ 149
`5.3.6.1 Bulking agents................................................................ 149
`5.3.6.2 Tonicity modifiers ..........................................................149
`5.3.6.3 Cryoprotectants and lyoprotectants ........................... 150
`5.3.7 The lyophilization cycle .............................................................. 151
`5.4 Alternative pharmaceutical processes................................................... 154
`5.4.1
`Spray-drying ................................................................................. 155
`5.4.2 Use of nonaqueous solvents....................................................... 156
`5.4.3. Compaction and tablets .............................................................. 156
`5.4.4 Aerosolization ............................................................................... 157
`5.5 Handling of therapeutic peptides and proteins .................................. 159
`5.5.1
`Sterilization.................................................................................... 159
`5.5.2
`Sanitizing agents and protein stability..................................... 159
`5.5.3 Preparation of a typical batch.................................................... 160
`5.5.4
`Storage in solid state ................................................................... 161
`5.5.5 Handling of recombinant proteins in a hospital setting ....... 163
`5.5.5.1 Reconstitution................................................................. 163
`5.5.5.2 Incompatibilities ............................................................ 165
`5.5.5.3 Adsorption...................................................................... 166
`5.5.5.4 Other considerations ..................................................... 167
`5.6 Conclusions................................................................................................ 167
`
`Chapter 6 Parenteral Controlled Delivery and Pharmacokinetics of
`Therapeutic Peptides and Proteins........................................ 177
`6.1 Introduction ............................................................................................... 177
`6.2 Pharmacokinetics of peptide and protein drugs ................................. 179
`6.2.1 Pharmacokinetic parameters ...................................................... 180
`6.2.1.1 Volume of distribution.................................................. 180
`6.2.1.2 Clearance......................................................................... 181
`6.2.1.3 Half-life.............................................................................182
`6.2.2 Other pharmacokinetic/pharmacodynamics
`considerations ................................................................................182
`6.2.2.1 Pharmacodynamics ....................................................... 183
`6.2.2.2 Antibody induction....................................................... 183
`6.2.2.3 Interspecies scaling........................................................ 183
`6.2.2.4 Chemical modification to control protein
`disposition....................................................................... 184
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`6.2.2.5 Transport of peptides across the blood–brain
`barrier .............................................................................. 186
`6.3 Polymers used for controlled delivery of peptides and proteins..... 187
`6.3.1 Nondegradable polymers ........................................................... 188
`6.3.2 Biodegradable polymers ............................................................. 189
`6.4 Parenteral controlled release systems.................................................... 191
`6.4.1 Microspheres ................................................................................. 191
`6.4.2 Release of drugs from microspheres......................................... 192
`6.4.3 Preparation of microspheres ...................................................... 193
`6.4.4 Microsphere formulations of luteinizing
`hormone-releasing hormone and analogues ........................... 195
`Implants ......................................................................................... 197
`6.4.5
`6.4.6 Liposomes...................................................................................... 199
`6.4.7 Nanoparticles ................................................................................200
`6.4.8 Vaccines.......................................................................................... 201
`6.4.9 Pulsatile drug delivery systems................................................. 201
`6.4.9.1 Externally regulated or open-loop systems .............. 202
`6.4.9.2 Pumps ............................................................................. 202
`6.4.9.3 Self-regulated or closed-loop systems........................ 203
`6.5 Innovations in parenteral administration of proteins ........................ 205
`6.5.1 Pegylated proteins........................................................................ 205
`6.5.2 Protein crystals ............................................................................. 206
`6.5.3 Prefilled syringes and needle-free injections........................... 207
`6.6 Examples of protein pharmacokinetics................................................. 208
`6.6.1
`Interferon ....................................................................................... 208
`6.6.2
`Interleukins.................................................................................... 208
`6.6.3
`Insulin ............................................................................................ 209
`6.6.4 Epoetin ........................................................................................... 211
`6.6.5 Miscellaneous................................................................................ 211
`6.7 Conclusions................................................................................................ 212
`
`Chapter 7 Oral Delivery of Peptide and Protein Drugs ...................... 229
`7.1 Introduction ............................................................................................... 229
`7.2 Barriers to protein absorption and pathways of penetration............ 231
`7.2.1 Extracellular barriers ................................................................... 231
`7.2.2 Cellular barriers............................................................................ 232
`7.3 Approaches to improve oral delivery ................................................... 235
`7.3.1
`Site-specific drug delivery .......................................................... 235
`7.3.2 Chemical modification ................................................................ 237
`7.3.3 Bioadhesive drug delivery systems .......................................... 239
`7.3.4 Penetration enhancers ..................................................................240
`7.3.5 Protease inhibitors........................................................................ 242
`7.3.6 Carrier systems............................................................................. 243
`7.3.7 Other formulation approaches ..................................................245
`7.4 Methods to study oral absorption ......................................................... 245
`7.4.1
`Intestinal segments ...................................................................... 245
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`In vivo studies ............................................................................... 246
`7.4.2
`7.4.3 Diffusion cells ............................................................................... 246
`7.4.4 Cell cultures .................................................................................. 246
`7.4.5 Brush border membrane vesicles .............................................. 248
`7.5 Oral immunization ................................................................................... 249
`7.6 Conclusions................................................................................................ 250
`
`Chapter 8 Transdermal and Topical Delivery of Therapeutic Peptides
`and Proteins ................................................................................ 259
`8.1 Introduction ............................................................................................... 259
`8.2 Skin Structure ............................................................................................ 260
`8.2.1 Pathways of transdermal delivery ............................................ 262
`8.2.2 Enzymatic barrier of skin ............................................................263
`8.3 Approaches to enhance transdermal peptide delivery ...................... 263
`8.3.1
`Skin microporation....................................................................... 263
`8.3.2 Phonophoresis............................................................................... 265
`8.3.3 Prodrug approach ........................................................................ 266
`8.3.4 Permeation enhancers ................................................................. 266
`8.3.5 Protease inhibitors........................................................................ 268
`8.3.6 Other technologies ....................................................................... 269
`8.4 Iontophoresis ............................................................................................. 269
`8.4.1
`Factors affecting iontophoretic delivery................................... 271
`8.4.1.1 Molecular weight........................................................... 271
`8.4.1.2 Drug charge.................................................................... 272
`8.4.1.3 Current density .............................................................. 272
`8.4.1.4 Electrode material.......................................................... 273
`8.4.2 Examples of iontophoretic delivery of therapeutic
`peptides.......................................................................................... 274
`8.4.2.1 Amino acids and small peptides ................................ 274
`8.4.2.2 Oligopeptides ................................................................. 275
`8.4.2.3 Polypeptides ................................................................... 275
`8.4.2.4 Insulin.............................................................................. 276
`8.4.3 Commercialization of iontophoretic delivery ......................... 277
`8.5 Topical delivery of therapeutic peptides and proteins....................... 278
`8.5.1 Growth factors .............................................................................. 279
`8.5.2 Liposomes .....................................................................................279
`8.5.3
`Iontophoresis and phonophoresis ............................................. 281
`8.6 Conclusions................................................................................................ 281
`
`Chapter 9 Pulmonary and Other Mucosal Delivery of Therapeutic
`Peptides and Proteins ............................................................... 291
`9.1 Introduction ............................................................................................... 291
`9.2 Pulmonary delivery.................................................................................. 293
`9.2.1 Drug deposition in the respiratory tract .................................. 294
`9.2.2 Drug absorption from the respiratory tract............................. 295
`9.2.3 Drug delivery devices ................................................................. 296
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`9.2.4
`
`Formulation of peptides/proteins for pulmonary
`delivery .......................................................................................... 297
`9.2.5 Commercialization and regulatory considerations ................ 300
`9.3 Nasal delivery............................................................................................ 301
`9.3.1 Approaches to overcome barriers to nasal absorption.......... 302
`9.3.1.1 Penetration enhancers................................................... 302
`9.4 Rectal delivery........................................................................................... 307
`9.5 Buccal delivery .......................................................................................... 308
`9.6 Ocular delivery.......................................................................................... 310
`9.6.1 Mechanism of ocular penetration.............................................. 310
`9.6.2 Ocular route for systemic delivery ........................................... 311
`9.6.2.1 Insulin.............................................................................. 311
`9.6.2.2 Enkephalins .................................................................... 311
`9.6.3 Ocular route for topical delivery............................................... 312
`9.7 Comparative evaluation of mucosal routes ......................................... 312
`9.8 Conclusions................................................................................................ 313
`
`Chapter 10 Recombinant Protein Subunit Vaccines and Delivery
`Methods...................................................................................... 327
`10.1 Introduction ................................................................................................327
`10.1.1 Vaccine adjuvants......................................................................... 330
`10.1.2 Subunit vaccines........................................................................... 331
`10.1.3 DNA vaccines ............................................................................... 331
`10.1.4 Heat shock proteins ..................................................................... 332
`10.2 Immunology relevant to vaccines.......................................................... 332
`10.2.1 Immune responses to vaccines .................................................. 334
`10.3 Routes of administration ......................................................................... 335
`10.3.1 Intranasal immunization............................................................. 337
`10.3.2 Transcutaneous immunization................................................... 338
`10.3.3 Oral immunization....................................................................... 339
`10.3.4 Other mucosal routes .................................................................. 340
`10.4 Delivery approaches for administration of vaccines .......................... 341
`10.4.1 Liposomes.......................................................................................342
`10.4.2 Particle-mediated immunization ............................................... 342
`10.4.3 Microspheres ..................................................................................343
`10.5 Case example: Hepatitis B....................................................................... 343
`10.6 Future outlook and regulatory status.................................................... 344
`10.7 Conclusions................................................................................................ 344
`
`Index .....................................................................................................................351
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`

`

`chapter 2
`
`Structure and Analysis
`of Therapeutic Peptides
`and Proteins
`
`This chapter discusses the unique structural and analytical aspects of pep-
`tides and proteins. A detailed discussion of these topics is beyond the scope
`of this book. Instead, the discussion emphasizes the basic concepts that will
`have application to the rest of this book as formulation, pharmaceutical
`processing, and delivery of therapeutic peptides and proteins are discussed.
`
`2.1 Amino acids: building blocks of proteins
`The 20 different naturally occurring amino acids are the building blocks of
`peptides and proteins. Amino acids are commonly represented by three-let-
`ter abbreviations, but one-letter abbreviations are also sometimes used.
`Although the human body needs all 20 amino acids, it cannot synthesize
`some amino acids, known as essential amino acids. Essential amino acids must
`be obtained from the diet. As the name implies, an amino acid contains an
`amino group and an acid group in the same molecule. Amino acids have a
`central carbon atom (a-carbon) to which are attached a carboxyl group, an
`amino group, a hydrogen atom, and a side chain. Different amino acids differ
`with respect to the side chain (R) only:
`
`33
`
`H
`|
`(cid:31)—(cid:31) (cid:31)—(cid:31)
`C
`COO
`|
`NH
`
`+3
`
`R
`
`
`
`
`

`

`34
`
`Therapeutic Peptides and Proteins
`
`Gly is the simplest amino acid, with no side chain. The side chain of Pro
`is unique in that it is bonded covalently to the nitrogen atom of the peptide
`group. The amide forms of Asp and Glu (Asn and Gln, respectively) occur
`naturally and are incorporated into proteins. Ionizable side chains of amino
`acids vary from acidic to basic. Aspartic and glutamic acid have a negative
`charge; lysine and arginine have a positive charge at the physiological pH
`of 7.4. The imidazole group in histidine carries a partial positive charge at
`pH 7.4. Serine and threonine have side chains that carry no charge at any
`pH but are polar in nature. In contrast, trypto