therapeutic.peptides.and.proteins.formulation.processing.and.delivery.systems.third.edition (1)

Ajay K. Banga

Therapeutic Peptides and Proteins

Formulation, Processing, and Delivery Systems

Third Edition

ISBN: 978-1-4665-6606-4

9 781466 566064

90000

K16132

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PHARMACEUTICAL SCIENCE & REGULTATION

There are more than 500 biopharmaceuticals on the market, including more than 200 therapeutic proteins, making biologics the fastest growing sector in the biopharmaceutical market. These products include more than 40 monoclonal antibodies, for indications ranging from treatment or mitigation of various types of cancer to rheumatoid arthritis.

The clinical application of these therapeutic peptides and proteins is limited by several problems, such as lack of physical and chemical stability or the lack of desirable attributes for adequate absorption or distribution. Thus, as these therapeutic peptides and proteins are made available, it will be essential to formulate these drugs into safe, stable, and efficacious delivery systems. The pharmaceutical scientist involved in this effort needs to call upon the knowledge of several disciplines, such as pharmaceutics, medicinal chemistry, biochemistry, microbiology, and chemical engineering and needs to keep abreast with the latest research in the published literature.

Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Third Edition provides a comprehensive overview of the field for scientists in industry and academia and for students, while also providing practical information on the challenges facing the formulation and delivery aspects of these unique macromolecules. In particular, the book:

Explains how recombinant DNA techniques now allow us to produce therapeutic proteins in a commercially viable form

Discusses the physical and chemical pathways of peptide and protein degradation

Includes a detailed discussion of protein formulation and lyophilization

Overviews the pharmacokinetic aspects of therapeutic peptides and proteins and discusses controlled delivery systems for parenteral administration, including microsphere formulations

Discusses research progress on oral, transdermal, mucosal, and topical delivery systems

Third Edition

K16132_Cover_PubGr.indd All Pages 3/13/15 10:07 AM



Third Edition

Ajay K. Banga



Third Edition

CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742

2015 by Taylor & Francis Group, LLCCRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government worksVersion Date: 20150309

International Standard Book Number-13: 978-1-4665-6607-1 (eBook - PDF)

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This book is dedicated to my wife, Saveta, and my children, Anshul and Manisha, for their support and understanding during the preparation of this book.

vii

ContentsPreface......................................................................................................................xvAuthor ....................................................................................................................xvii

Section i Formulation and Processing

Chapter 1 Pharmaceutical Biotechnology: The Arrival of Recombinant Proteins ..........................................................................3

1.1 What Is Biotechnology? ............................................................31.1.1 Recombinant DNA Technology ...................................41.1.2 Monoclonal Antibodies ................................................61.1.3 Expanding Scope of Biotechnology ........................... 10

1.1.3.1 AntibodyDrug Conjugates ........................ 111.1.3.2 Antisense Agents and RNAi ....................... 111.1.3.3 Transgenic Therapeutics ............................. 121.1.3.4 Gene Therapy ............................................. 131.1.3.5 Human Genome Project.............................. 13

1.2 Biotechnology Industry ........................................................... 141.2.1 Biotechnology Products on the Market ...................... 15

1.2.1.1 Human Insulins and Analogs ..................... 151.2.1.2 Growth Hormone ........................................ 171.2.1.3 Interferons ................................................... 171.2.1.4 Dornase Alfa (rhDNase) ............................. 181.2.1.5 Antimicrobial Peptides ............................... 191.2.1.6 Tissue Plasminogen Activator .................... 191.2.1.7 Fusion Proteins ...........................................201.2.1.8 Miscellaneous .............................................20

1.3 Regulatory Aspects of Biotechnology-Derived Drugs ............ 211.3.1 Preclinical and Clinical Studies .................................221.3.2 Pharmacoeconomics and the Regulatory Process .....231.3.3 Characterization and Purity

of Biotechnology-Derived Proteins ............................241.3.4 Patents ........................................................................25

1.4 Immunogenicity of Proteins ....................................................251.5 Structure-Based Drug Design .................................................271.6 Conclusions ..............................................................................28References ..........................................................................................29

viii Contents

Chapter 2 Structure and Analysis of Therapeutic Peptides and Proteins ........... 37

2.1 Amino Acids: Building Blocks of Proteins ............................. 372.2 Structure of Peptides and Proteins .......................................... 38

2.2.1 Primary Structure ....................................................... 382.2.2 Secondary Structure ................................................... 392.2.3 Tertiary Structure and Protein Folding ......................402.2.4 Quaternary Structure.................................................. 41

2.3 Protein Conformation and Other Structural Features ............. 422.4 Analytical Methods ................................................................. 45

2.4.1 Electrophoresis ........................................................... 452.4.2 Spectroscopy .............................................................. 47

2.4.2.1 UV Spectroscopy ........................................ 472.4.2.2 Fluorescence Spectroscopy .........................482.4.2.3 Circular Dichroism Spectroscopy ...............482.4.2.4 Infrared Spectroscopy ................................502.4.2.5 Light Scattering .......................................... 512.4.2.6 Colorimetric Assay ..................................... 52

2.4.3 Chromatography ......................................................... 522.4.3.1 Reversed-Phase Chromatography ............... 522.4.3.2 Size-Exclusion Chromatography ................ 532.4.3.3 Liquid ChromatographyMass

Spectrometry...............................................562.4.3.4 Other Chromatographic Techniques ...........56

2.4.4 New Techniques to Characterize Subvisible Particles .................................................... 56

2.4.5 Thermal Analysis ....................................................... 572.4.6 Immunoassays and Bioassays ..................................... 582.4.7 Other Quality Control Procedures .............................60

2.4.7.1 Peptide Mapping .........................................602.4.7.2 Analytical Ultracentrifugation ................... 612.4.7.3 Analysis of Glycoproteins ........................... 62

2.5 Conclusions .............................................................................. 62References .......................................................................................... 63

Chapter 3 Stability of Therapeutic Peptides and Proteins .................................. 73

3.1 Introduction ............................................................................. 733.2 Physical Instability .................................................................. 74

3.2.1 Denaturation ............................................................... 743.2.2 Aggregation ................................................................ 75

3.2.2.1 Mechanism of Aggregation ........................ 763.2.2.2 Protein Fibrillation...................................... 783.2.2.3 Aggregation Behavior in Liquid and

Solid States ................................................. 78

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3.2.3 Adsorption ..................................................................803.3 Chemical Instability ................................................................82

3.3.1 Oxidation .................................................................... 833.3.2 Hydrolysis ...................................................................843.3.3 Deamidation ............................................................... 853.3.4 -Elimination and Disulfide Exchange ......................863.3.5 Racemization ..............................................................873.3.6 Thermal Stability .......................................................873.3.7 Multiple Pathways of Chemical Instability ................87

3.4 Biosimilars and Comparability Protocols ...............................883.5 Conclusions ..............................................................................90References ..........................................................................................90

Chapter 4 Preformulation and Formulation of Therapeutic Peptides and Proteins ........................................................................................97

4.1 Introduction .............................................................................974.2 Preformulation Studies ............................................................974.3 Formulation Development ..................................................... 105

4.3.1 Buffer System ........................................................... 1064.3.2 pH of the Vehicle ...................................................... 1074.3.3 Protein Solubility ...................................................... 1084.3.4 Selection of Solvent System ..................................... 1094.3.5 Preservation of a Formulation .................................. 1094.3.6 Choice of Container ................................................. 110

4.4 Pharmaceutical Excipients in Formulations .......................... 1114.4.1 Albumin ................................................................... 1134.4.2 Amino Acids ............................................................ 1144.4.3 Carbohydrates........................................................... 1144.4.4 Chelating and Reducing Agents ............................... 1154.4.5 Cyclodextrins............................................................ 1164.4.6 Polyhydric Alcohols ................................................. 1174.4.7 Polyethylene Glycol .................................................. 1174.4.8 Salts .......................................................................... 1174.4.9 Surfactants ................................................................ 1184.4.10 Miscellaneous ........................................................... 1204.4.11 Selection of Excipient ............................................... 120

4.5 Aggregation in Protein Formulations .................................... 1224.5.1 Aggregation Behavior of Insulin .............................. 1224.5.2 Aggregation Behavior of Human

Growth Hormone ..................................................... 1254.5.3 Aggregation Behavior of Alpha1-Antitrypsin ........... 1254.5.4 Aggregation Behavior of Human Interferon ............ 1264.5.5 Aggregation of Monoclonal Antibodies ................... 1264.5.6 Aggregation Behavior of Some Other Proteins ........ 127

x Contents

4.6 Novel Formulation Approaches ............................................. 1274.6.1 Liposomes ................................................................ 1284.6.2 Reverse Micelles ...................................................... 1304.6.3 Emulsions ................................................................. 1304.6.4 Genetic Engineering or Chemical Modification ...... 131

4.7 Accelerated Stability Testing ................................................. 1324.8 Quality by Design for Formulation Development ................. 1334.9 Conclusions ............................................................................ 134References ........................................................................................ 135

Chapter 5 Lyophilization, Pharmaceutical Processing, and Handling of Therapeutic Peptides and Proteins ................................................... 147

5.1 Introduction ........................................................................... 1475.2 Protein Destabilization Induced by

Pharmaceutical Processing .................................................... 1475.2.1 Adsorption Induced by

Pharmaceutical Processing ..................................... 1475.2.2 Aggregation Induced by

Pharmaceutical Processing ...................................... 1495.2.2.1 Shaking ..................................................... 1495.2.2.2 Hydrophobic Surfaces ............................... 1495.2.2.3 Heating ...................................................... 1505.2.2.4 Shear-Induced Aggregation ...................... 1505.2.2.5 Miscellaneous Factors .............................. 151

5.3 Lyophilization as a Pharmaceutical Process ......................... 1515.3.1 Process Details ......................................................... 1515.3.2 Maximum Allowable Product Temperature ............. 1535.3.3 Measurement of Lyophilization-Induced

Thermal Changes ..................................................... 1545.3.4 Protein Denaturation during Lyophilization ............ 1545.3.5 Freeze-Thaw Stability .............................................. 1555.3.6 Formulation Components for Lyophilization ........... 156

5.3.6.1 Bulking Agents ......................................... 1575.3.6.2 Tonicity Modifiers .................................... 1575.3.6.3 Cryoprotectants and Lyoprotectants ......... 157

5.3.7 Lyophilization Cycle ................................................ 1595.4 Alternative Pharmaceutical Processes .................................. 161

5.4.1 Spray Drying ............................................................ 1625.4.2 Use of Nonaqueous Solvents .................................... 1635.4.3 Compaction and Tablets ........................................... 1645.4.4 Aerosolization .......................................................... 165

5.5 Handling of Therapeutic Peptides and Proteins .................... 1665.5.1 Sterilization .............................................................. 1665.5.2 Sanitizing Agents and Protein Stability ................... 166

xiContents

5.5.3 Preparation of a Typical Batch ................................. 1675.5.4 Storage in Solid State ............................................... 1695.5.5 Handling of Recombinant Proteins in a

Hospital Setting ........................................................ 1705.5.5.1 Reconstitution ........................................... 1705.5.5.2 Incompatibilities ....................................... 1725.5.5.3 Adsorption ................................................ 1735.5.5.4 Other Considerations ................................ 174

5.6 Conclusions ............................................................................ 174References ........................................................................................ 175

Section ii Drug Delivery Systems

Chapter 6 Parenteral Controlled Delivery and Pharmacokinetics of Therapeutic Peptides and Proteins ................................................... 185

6.1 Introduction ........................................................................... 1856.2 Pharmacokinetics of Peptide and Protein Drugs ................... 186

6.2.1 Pharmacokinetic Parameters .................................... 1886.2.1.1 Volume of Distribution ............................. 1886.2.1.2 Clearance .................................................. 1896.2.1.3 Half-Life ................................................... 190

6.2.2 Other Pharmacokinetic/Pharmacodynamic Considerations .......................................................... 1906.2.2.1 Pharmacodynamics ................................... 1906.2.2.2 Antibody Induction ................................... 1906.2.2.3 Interspecies Scaling .................................. 1916.2.2.4 Chemical Modification to Control

Protein Disposition ................................... 1926.2.2.5 Transport of Peptides across the

BloodBrain Barrier ................................. 1936.3 Polymers Used for Controlled Delivery of Peptides

and Proteins ........................................................................... 1956.3.1 Nondegradable Polymers .......................................... 1956.3.2 Biodegradable Polymers ........................................... 196

6.4 Parenteral Controlled-Release Systems ................................. 1996.4.1 Microspheres ............................................................ 1996.4.2 Release of Drugs from Microspheres .......................2006.4.3 Preparation of Microspheres .................................... 2016.4.4 Microsphere Formulations of LHRH

and Analogs .............................................................2036.4.5 Implants ....................................................................2046.4.6 Liposomes ................................................................206

xii Contents

6.4.7 Nanoparticles ............................................................2066.4.8 Vaccines ...................................................................2076.4.9 Pulsatile Drug Delivery Systems .............................208

6.4.9.1 Externally Regulated or Open-Loop Systems .................................208

6.4.9.2 Pumps .......................................................2096.4.9.3 Self-Regulated or

Closed-Loop Systems ............................... 2106.5 Innovations in Parenteral Administration of Proteins ........... 211

6.5.1 PEGylated Proteins .................................................. 2126.5.2 Protein Crystals ........................................................ 2136.5.3 Prefilled Syringes and Needle-Free Injections ......... 213

6.6 Examples of Protein Pharmacokinetics................................. 2146.6.1 Interferon .................................................................. 2146.6.2 Interleukins ............................................................... 2156.6.3 Insulin ....................................................................... 2156.6.4 Epoetin ..................................................................... 2176.6.5 Miscellaneous ........................................................... 218

6.7 Conclusions ............................................................................ 219References ........................................................................................ 219

Chapter 7 Oral Delivery of Peptide and Protein Drugs .................................... 233

7.1 Introduction ........................................................................... 2337.2 Barriers to Protein Absorption and Pathways

of Penetration ........................................................................2347.2.1 Extracellular Barriers ............................................... 2357.2.2 Cellular Barriers ....................................................... 236

7.3 Approaches to Improve Oral Delivery .................................. 2387.3.1 Site-Specific Drug Delivery ..................................... 2387.3.2 Chemical Modification .............................................2407.3.3 Bioadhesive Drug Delivery Systems ........................2447.3.4 Penetration Enhancers ..............................................2447.3.5 Protease Inhibitors....................................................2467.3.6 Carrier Systems ........................................................ 2477.3.7 Other Formulation Approaches ................................ 250

7.4 Methods to Study Oral Absorption .......................................2507.4.1 Intestinal Segments ..................................................2507.4.2 In Vivo Studies ......................................................... 2517.4.3 Diffusion Cells ......................................................... 2517.4.4 Cell Cultures............................................................. 2517.4.5 Brush-Border Membrane Vesicles ............................ 253

7.5 Oral Immunization ................................................................2547.6 Conclusions ............................................................................ 255References ........................................................................................ 255

xiiiContents

Chapter 8 Transdermal and Topical Delivery of Therapeutic Peptides and Proteins .......................................................................263

8.1 Introduction ...........................................................................2638.1.1 Skin Structure...........................................................2648.1.2 Pathways of Transdermal Delivery .......................... 2678.1.3 Enzymatic Barrier of Skin .......................................268

8.2 Approaches to Enhance Transdermal Peptide Delivery ........2688.2.1 Skin Microporation ..................................................269

8.2.1.1 Microneedles ............................................2698.2.2 Phonophoresis........................................................... 2728.2.3 Prodrug Approach .................................................... 2738.2.4 Permeation Enhancers .............................................. 2738.2.5 Protease Inhibitors.................................................... 2768.2.6 Other Technologies ................................................... 277

8.3 Iontophoresis .......................................................................... 2788.3.1 Factors Affecting Iontophoretic Delivery ................280

8.3.1.1 Molecular Weight .....................................2808.3.1.2 Drug Charge .............................................2808.3.1.3 Current Density ........................................ 2818.3.1.4 Electrode Material .................................... 281

8.3.2 Examples of Iontophoretic Delivery of Therapeutic Peptides ................................................2828.3.2.1 Amino Acids and Small Peptides ............. 2828.3.2.2 Oligopeptides ............................................ 2838.3.2.3 Polypeptides ..............................................2848.3.2.4 Insulin .......................................................284

8.3.3 Commercialization of Iontophoretic Delivery .........2858.4 Topical Delivery of Therapeutic Peptides and Proteins ........286

8.4.1 Growth Factors .........................................................2878.4.2 Liposomes ................................................................2888.4.3 Iontophoresis and Phonophoresis .............................289

8.5 Conclusions ............................................................................ 289References ........................................................................................290

Chapter 9 Pulmonary and Other Mucosal Delivery of Therapeutic Peptides and Proteins ....................................................................... 301

9.1 Introduction ........................................................................... 3019.2 Pulmonary Delivery ..............................................................302

9.2.1 Drug Deposition in the Respiratory Tract ................3049.2.2 Drug Absorption from the Respiratory Tract ...........3059.2.3 Drug Delivery Devices .............................................3069.2.4 Formulation of Peptides/Proteins for

Pulmonary Delivery .................................................3079.2.5 Commercialization and Regulatory Considerations.....310

xiv Contents

9.3 Nasal Delivery ....................................................................... 3129.3.1 Approaches to Overcome Barriers

to Nasal Absorption .................................................. 3149.3.1.1 Penetration Enhancers .............................. 314

9.4 Rectal Delivery ...................................................................... 3199.5 Buccal Delivery ..................................................................... 3209.6 Ocular Delivery ..................................................................... 322

9.6.1 Mechanism of Ocular Penetration ............................ 3239.6.2 Ocular Route for Systemic Delivery ........................ 323

9.6.2.1 Insulin ....................................................... 3239.6.2.2 Enkephalins ..............................................324

9.6.3 Ocular Route for Topical Delivery ...........................3249.7 Comparative Evaluation of Mucosal Routes ......................... 3259.8 Conclusions ............................................................................ 326References ........................................................................................ 326

Chapter 10 Recombinant Protein Subunit Vaccines and Delivery Methods ...... 339

10.1 Introduction ........................................................................... 33910.1.1 Vaccine Adjuvants ....................................................34410.1.2 Subunit Vaccines ...................................................... 34510.1.3 DNA Vaccines .......................................................... 34510.1.4 Heat Shock Proteins .................................................346

10.2 Immunology Relevant to Vaccines ........................................34610.2.1 Immune Responses to Vaccines ...............................348

10.3 Routes of Administration ...................................................... 34910.3.1 Intranasal Immunization .......................................... 35010.3.2 Transcutaneous Immunization ................................. 35210.3.3 Oral Immunization ................................................... 35410.3.4 Other Mucosal Routes .............................................. 354

10.4 Delivery Approaches for Administration of Vaccines .......... 35510.4.1 Nanoparticles ............................................................ 35610.4.2 Particle-Mediated Immunization ............................. 35710.4.3 Microspheres ............................................................ 357

10.5 Case Example: Hepatitis B .................................................... 35810.6 Future Outlook and Regulatory Status .................................. 35910.7 Conclusions ............................................................................ 359References ........................................................................................360

xv

PrefaceDuring the publication of the first edition of this book, there were only 19 biotechnol-ogy medicines on the market, and that number grew to 100 when the second edition came out. Now, there are more than 500 biologics on the market, including more than 200 therapeutic proteins, making the predictions in the preface to the first two editions come true. These products include more than 40 monoclonal antibodies, for indications ranging from treatment or mitigation of various types of cancer to rheu-matoid arthritis. More than 900 more biologics are in development, targeting more than 100 diseases, with monoclonal antibodies and vaccines being the predominant category. More than 8000 companies are working in the biotechnology field in the United States alone. The global market for these products is more than $160 billion.

However, the clinical application of these therapeutic peptides and proteins is limited by several problems, such as lack of physical and chemical stability or the lack of desirable attributes for adequate absorption or distribution. Thus, as these therapeutic peptides and proteins are made available, it will be essential to formulate these drugs into safe, stable, and efficacious delivery systems. The pharmaceutical scientist involved in this effort needs to call upon the knowledge of several dis-ciplines, such as pharmaceutics, medicinal chemistry, biochemistry, microbiology, and chemical engineering and needs to keep abreast with the latest research in the published literature. This book provides a comprehensive overview of the field for scientists in industry and academia and for students, while also providing practi-cal information on the challenges facing the formulation and delivery aspects of these unique macromolecules. The book should also be useful in a hospital setting to understand potential physicochemical stability problems that may result during the reconstitution or administration of the new recombinant proteins.

This third edition has thoroughly updated the information under different topics with references to recent literature, and several new figures have been added. In addi-tion, new topics have been added or expanded, such as subvisible particles, advances in our understanding of protein aggregation and fibrillation, recent regulatory approval of inhaled insulin, the principles of quality by design approach, antibodydrug conjugates, biosimilars, and microneedle delivery systems. More than 20 of the top-selling biopharmaceuticals will come off patent by 2020, creating significant opportunity for biosimilars, with an estimated market size for biosimilars in excess of $50 billion. This is an exciting time for realizing the promise of recombinant pro-teins and other biotechnology medicines, with the biotechnology industry enjoying a return of investor enthusiasm.

The book has been divided into two main sections. Section I (Chapters 1 through 5) introduces the reader to therapeutic peptides and proteins and discusses several practical aspects of relevance to the pharmaceutical industry, which may be broadly categorized as formulation and pharmaceutical processing. Chapter 1 provides insights into the field of pharmaceutical biotechnology and explains how recombinant DNA techniques now allow us to produce therapeutic proteins in a

xvi Preface

commercially viable form. Chapter 2 introduces the issues unique to these drugs with respect to the structure and analysis aspects. The physical and chemical pathways of peptide and protein degradation are discussed, along with methods of stabilization (Chapters 3). Chapters 4 and 5 are devoted to the formulation develop-ment and pharmaceutical processing aspects for therapeutic peptides and proteins. Chapter 5 includes a detailed discussion of lyophilization. Section II (Chapters 6 through 10) of this book discusses the various drug delivery routes for these drugs. Chapter 6 provides an overview of the pharmacokinetic aspects of therapeutic pep-tides and proteins and discusses controlled delivery systems for parenteral adminis-tration, including microsphere formulations. Chapter 7 discusses research progress on oral delivery systems while Chapter 8 discusses transdermal and topical delivery. Chapter 9 focuses on mucosal delivery, including products marketed for pulmonary or nasal delivery. Finally, Chapter 10 discusses recombinant protein subunit vac-cines and delivery methods.

Since this book is the work of a single author, I was able to minimize overlap between chapters and provide extensive cross-referencing to direct the reader to rel-evant parts of the book for related topics. I thank Pooja Bakshi and Saveta Banga for their extensive help with the preparation of this book. Finally, I would like to acknowledge the staff of CRC Press (a member of the Taylor & Francis Group) for their efforts in bringing this book to the readers.

xvii

AuthorDr. Ajay K. Banga, PhD, is professor, department chair, and endowed chair in the Department of Pharmaceutical Sciences at the College of Pharmacy, Mercer University, Atlanta, Georgia. He holds a PhD in pharmaceutical sciences from RutgersThe State University of New Jersey. He has more than 250 peer-reviewed publications and research abstracts. Dr. Banga currently serves on the editorial board of 10 journals, serves as associate editor for 1 journal, and has served as the editor-in-chief for a drug delivery journal. He serves as codirector for the Center for Drug Delivery Research at the College of Pharmacy and is a fellow of the American Association of Pharmaceutical Scientists.

Section I

Formulation and Processing

31 Pharmaceutical BiotechnologyThe Arrival of Recombinant Proteins

Advances in biotechnology have resulted in a significant increase in the number of therapeutic peptides and proteins that are reaching the market. This trend is expected to continue and escalate in the future. Applications of biotechnology are not lim-ited to pharmaceuticals; they extend to agriculture, food processing, and chemicals, among others. However, the most significant activity to date has been in human health care, both therapeutics and diagnostics (Assenberg, Wan, Geisse, & Mayr, 2013; Montague, 1992; Sterling, 1990; Wordell, 1992). Although a detailed discus-sion of biotechnology is not within the scope of this book, this chapter provides a very brief overview of some of the principles of biotechnology that can be critical in the formulation and delivery of peptides and proteins. For example, the presence of host-cell protein (HCP) as impurity in the final product may be responsible for sta-bility or immunogenicity problems of a formulation. Two things need to be pointed out here. First, biotechnology principles go beyond the production of proteins by recombinant DNA (rDNA) technology. For example, antisense agents are oligonucle-otides, not proteins. Similarly, gene therapy is of direct benefit to the patient but does not involve the development of a drug as conventionally understood. Some of the biotechnology principles and products are briefly reviewed in this chapter, but the focus of this book in the remaining chapters is on the formulation and delivery of therapeutic peptides and proteins. Second, it must be realized that the basic concepts of formulation and delivery of these drugs are applicable to any peptide or protein, whether of biotechnology origin or not.

1.1 WHAT IS BIOTECHNOLOGY?

Biotechnology is the use of biological systems or living organisms to produce or modify materials or products. Biotechnology-derived drugs are produced by rDNA technology, known as genetic engineering or gene cloning, whereby human genes responsible for the production of a protein are transferred to an organism such as an Escherichia coli bacterium. The E. coli is then cultured by fermentation to achieve a large-scale production of therapeutic proteins in a commercially viable scale (Emtage, 1986; Harford, 1985).

4 Therapeutic Peptides and Proteins

Calcitonin can be considered as an example of the cost-effectiveness of the biotechnology process. Chemical synthesis of calcitonin would need linking one amino acid at a time. The 32-amino-acid sequence requires hundreds of individual steps to complete the synthesis. In contrast, recombinant technology can reduce the cost of the large-scale calcitonin production by about 20-fold (Levy, 1993). Recombinant technology has been very critical to meet the high demand for pro-teins, which is partly due to the low bioavailability of the new drug delivery sys-tems, which may slowly replace parenteral delivery (Devogelaer et al., 1994; Levy, 1993). However, short peptides are generally prepared by chemical synthesis, and automated peptide synthesizers are also available. Impurity profile of peptides made by solid-phase synthesis would be different from that of proteins made by recombinant technologies (DHondt et al., 2014).

1.1.1 Recombinant Dna technology

rDNA technology, or genetic engineering, involves combining genetic material from two sources in vitro and then introducing the recombined material into a host cell.

One aspect of rDNA technology (Figure 1.1) involves the transfer of a gene from a plant or animal cell into a bacterial plasmid. Enzymes called restriction endonucleases are used to cut DNA from the cell. For example, a DNA fragment equal to one gene that codes for a particular protein can be cut from a human cell for transfer to bacteria. The same enzyme is used to open a site in the DNA molecule for the bacterial cell, thus allowing easy integration of human genes into bacteria. A new hybrid, called rDNA molecule, results and is then incorporated into a new bacterium. The new strain is selectively grown and purified (cloned), and if the incorporated gene is expressed, then the bacteria would produce human protein. Human protein may then be manufactured through large-scale fermenta-tion and purification. The native structure of the protein should be maintained during downstream processing. The advent of rDNA technology continues to drive the trend toward development of plasma-free protein therapeutics. This trend is also driven by the concern on the part of regulatory agencies about the inher-ent potential for transmission of infectious agents through plasma-derived pro-teins or additives, as well as their heterogeneity and lack of reliability of serum supply (Grillberger, Kreil, Nasr, & Reiter, 2009). Challenges experienced during the development of high-purity, plasma-derived factor VIII concentrate have been described in the literature (Schulte, 2013).

There are several disadvantages associated with the use of E. coli, and more sophisticated organisms such as yeasts and mammalian cells have also been uti-lized (Emtage, 1986; Walsh, 2010). These disadvantages primarily relate to glyco-sylation, the addition of sugar residue to a peptide backbone (Sola & Griebenow, 2010). Proteins produced in E. coli are not glycosylated. However, in eukaryotic cells, glycosylation is a common posttranslational modification made to pro-teins. Also, when E. coli is used, lysis of the bacteria is necessary to recover the protein, and since lysis releases many other proteins and cellular components, significant purification is required. Plant and insect cells have also been used as bio-reactors for protein production (Cox, 2012; Crosby, 2003; Hellwig, Sack, Spiegel,

5Pharmaceutical Biotechnology

Drossard, & Fischer, 2003; Kwon, Verma, Singh, Herzog, & Daniell, 2013; Smith, Richter, Arntzen, Shuler, & Mason, 2003).

When yeast or cell cultures are used, protein may be released directly into the medium, and purification will be simpler, starting with removal of the cells themselves. However, mammalian cells do have drawbacks also related to bio-processing and scale-up, which result in long processing times and elevated costs. Mammalian cell expression in Chinese hamster ovary (CHO) cells is used for the majority of products on the market today as well as for those in clinical develop-ment (Zhu, 2012). A strategy of in vitro evolution has been suggested by isolating protein variants with improved expression, solubility, and stability, including resis-tance to aggregation (Buchanan et al., 2012). The glycosylation status of a protein must be clearly established (Jenkins & Curling, 1994). Although most physiologi-cal proteins are glycoproteins, glycosylation is not necessarily required for bioac-tivity. Recombinant human interleukin 2 produced in E. coli is not glycosylated but retains full biological potency. However, immunogenicity issues should also be considered.

Human cell

NucleusBacterial cell

PlasmidHuman DNA

Restrictionendonucleases

Plasmid containing human gene

Fermentation

Cleaved plasmid

Bacteria withhuman gene

DNA segments(genes)

FIGURE 1.1 rDNA technology for the production of a human protein by a bacterial cell.


1.1.2 monoclonal antiboDies

The normal antibody response to antigenic challenge is polyclonal since a large num-ber of different immunoglobulin (Ig) molecules are produced by different lympho-cytes. Polyclonals derived from the plasma obtained from nonimmunized donors are known as standard intravenous immunoglobulins (IVIGs). For persons specifi-cally immunized with an antigen, high titers of antibodies specific for that anti-gen are produced and obtained as specific IVIGs or specific polyclonal antibodies. Immunoglobulin products are also available for intramuscular administration. While IVIGs are well established and continue to carry a prominent role in medicine, it is possible to produce immunoglobulin molecules that all possess the same structure (monoclonal). Monoclonal antibodies are produced by the fusion of an individual B lymphocyte with a cancer cell (usually from a myeloma). The fusion produces a hybrid cell that has both the antibody-producing character of a B lymphocyte and the immortality or continuous reproduction of a myeloma cell, allowing the commercial production of a unique antibody molecule.

Monoclonal antibodies are finding widespread use in diagnosis and therapeutics. The monoclonal antibody Orthoclone OKT3 was approved for use in kidney, heart, and liver transplant rejection in 1986. There was then a lag period of several years when not much was approved, but in recent years, several monoclonal antibodies have been approved for varying indications. Monoclonal antibodies constitute the fastest-growing segment of biopharmaceuticals, with many already on the market and many more in clinical trials. Table 1.1 lists the monoclonal antibodies currently on the market. Unlike most other therapeutic proteins, monoclonal antibodies have relatively high doses and long half-lives (Daugherty & Mrsny, 2006). Therefore, they are generally not suited to be delivered by some of the alternate delivery systems discussed in this book and are rather given by parenteral administration.

Monoclonal antibodies have many possible diagnostic and therapeutic applica-tions in the management of human carcinomas, including colorectal, gastric, ovar-ian, endometrial, breast, lung, and pancreatic cancers. Cytotoxic drugs can be linked to monoclonal antibodies for targeting specific tumor-associated antigen (Schlom, 1988). Similarly, radioisotopes can also be directed to specific sites by the use of monoclonal antibodies. A monoclonal antibody has also been developed for imag-ing myocardial necrosis associated with acute myocardial infarction. The product (Myoscint, Janssen Biotech, Inc.) was on the market and had a high degree of sensitivity for detecting infarction and specificity for excluding a recent ischemic event in patients admitted with chest pain syndrome but is now withdrawn from the market (Spada & Walsh, 2005). Monoclonal antibodies have also been linked to enzymes for the detection of other antibodies or antigens by enzyme-linked immunosorbent assay, which will be discussed in Chapter 2. Typically, monoclo-nal antibodies are prepared using mouse lymphocytes. and this had limited their effectiveness since there is often a human immunogenic response to the mouse anti-body. Using genetic engineering, the mouse antibody can be humanized to produce a chimeric antibody, which is part human and part mouse in structure, and sev-eral of these have now been approved for marketing with several more in develop-ment. A stable human myeloma that may be useful to generate human monoclonal


TAB

LE 1

.1Ex

ampl

es o

f FD

A-A

ppro

ved

Mon

oclo

nal A

ntib

odie

s

Prod

uct

Nam

e C

ompa

ny/I

mm

unog

lobu

lin

Type

D

rug

Con

c.; I

nact

ives

In

dica

tion

A

ppro

val

Dat

e

Ort

hocl

one

OK

T3

(Mur

omon

ab-C

D3)

Jans

sen-

Cila

g/m

ouse

IgG

2a1

mg/

mL

sol

n co

ntg.

5 m

ga; s

od. p

hosp

hate

, NaC

l, po

lyso

rbat

e 80

(pH

7.0

)A

cute

allo

graf

t rej

ectio

n in

tr

ansp

lant

pat

ient

s19

86

Rev

ersa

l of

hear

t and

live

r tr

ansp

lant

rej

ectio

n19

93

Reo

Pro

(ab

cixi

mab

)E

li L

illy/

chim

eric

hum

an

mur

ine

F ab

2 m

g/m

L s

oln

cont

g. 1

0 m

g; s

od. p

hosp

hate

, NaC

l, po

lyso

rbat

e 80

(pH

7.2

)C

ompl

icat

ion

of c

oron

ary

angi

opla

sty

1994

Rit

uxan

(ri

tuxi

mab

)B

ioge

n Id

ec/G

enen

tech

ch

imer

ic m

urin

e/hu

man

IgG

1

10 m

g/m

L c

once

ntra

te c

ontg

. 100

or

500

mg;

NaC

l, so

d.

citr

ate,

pol

ysor

bate

80

(pH

6.5

)N

on-H

odgk

ins

lym

phom

a19

97

Zen

apax

(da

cliz

umab

)R

oche

/Gen

ente

ch/h

uman

ized

Ig

G1

5 m

g/m

L s

oln

cont

g. 2

5 m

g; s

od. p

hosp

hate

, NaC

l, po

lyso

rbat

e 80

(pH

6.9

adj

uste

d w

ith H

Cl/N

aOH

)Pr

ophy

laxi

s of

acu

te k

idne

y tr

ansp

lant

rej

ectio

n19

97

Sim

ulec

t (ba

silix

imab

)N

ovar

tis/c

him

eric

IgG

1Ly

ophi

lized

(10

mg/

2.5

mL

or

20 m

g/5

mL

); m

onob

asic

po

t. ph

osph

ate,

dis

od. h

ydro

gen

phos

phat

e (a

nhyd

rous

), N

aCl,

sucr

ose,

man

nito

l, gl

ycin

e

Ren

al tr

ansp

lant

rej

ectio

n19

98

Rem

icad

e (i

nflix

imab

)Ja

nsse

n B

iote

ch/M

erck

/ch

imer

ic I

gG1

Lyop

hiliz

ed p

owde

r (1

00 m

g re

cons

t. to

10

mg/

mL

);

sucr

ose,

pol

ysor

bate

80,

sod

. pho

spha

te (

pH 7

.2)

Cro

hns

dis

ease

Rhe

umat

oid

arth

ritis

1998

1999

200

2

Her

cept

in (

tras

tuzu

mab

)G

enen

tech

/hum

aniz

ed I

gG1

Lyop

hiliz

ed p

owde

r (4

40 m

g re

cons

t. to

21

mg/

mL

with

B

acte

rios

tatic

Wat

er f

or I

njec

tion

(BW

FI))

; l-h

istid

ine

HC

l, l-

hist

idin

e,

, -t

reha

lose

deh

ydra

te a

nd

poly

sorb

ate

20, b

enzy

l alc

ohol

in B

WFI

(pH

app

rox.

6)

Met

asta

tic b

reas

t can

cer

1998

Syna

gis

(pal

iviz

umab

)M

edIm

mun

e/hu

man

ized

IgG

1Ly

ophi

lized

pow

der

(100

mg/

mL

cle

ar o

r sl

ight

ly

opal

esce

nt s

oln.

); h

istid

ine,

gly

cine

, and

man

nito

lR

espi

rato

ry s

yncy

tial v

irus

1998

Myl

otar

g (g

emtu

zum

ab

ozog

amic

in)b

Wye

th/h

uman

ized

IgG

4

Lyop

hiliz

ed p

owde

r (5

mg/

20 m

L);

dex

tran

40,

suc

rose

, N

aCl,

sod.

pho

spha

teA

cute

mye

loid

leuk

emia

2000

(Con

tinu

ed)


TAB

LE 1

.1 (

con

tinu

ed )

Exam

ples

of F

DA

-App

rove

d M

onoc

lona

l Ant

ibod

ies

Prod

uct

Nam

e C

ompa

ny/I

mm

unog

lobu

lin

Type

D

rug

Con

c.; I

nact

ives

In

dica

tion

A

ppro

val

Dat

e

Cam

path

(al

emtu

zum

ab)

Gen

zym

e/hu

man

ized

IgG

130

mg/

3 m

L s

olut

ion;

NaC

l, so

d. p

hosp

hate

, KC

l, po

t ph

osph

ate,

pol

ysor

bate

80,

dis

odiu

m e

deta

te (p

H 6

.87

.4)

Lym

phoc

ytic

leuk

emia

2001

Zev

alin

(ib

ritu

mom

ab

tiuxe

tan)

Spec

trum

Pha

rmac

eutic

als/

mur

ine

IgG

1

3.2

mg/

14 m

L s

olut

ion;

NaC

l, N

a ac

etat

e, H

SA, s

od.

phos

phat

e, p

ot. p

hosp

hate

, KC

l, pe

ntet

ic a

cid

(pH

7.1

ad

just

ed w

ith H

Cl o

r N

aOH

)

Non

-Hod

gkin

s ly

mph

oma

2002

Hum

ira

(ada

limum

ab)

Abb

Vie

/rhI

gG1

mA

B s

peci

fic

for

hum

an T

NF

40 m

g/0.

8 m

L s

oln;

NaC

l, so

d. p

hosp

hate

Na

citr

ate,

ci

tric

aci

d m

onoh

ydra

te, m

anni

tol,

poly

sorb

ate

80 a

nd

Wat

er f

or I

njec

tion

(WFI

), U

SP. N

aOH

add

ed to

adj

ust

pH to

5.2

Rhe

umat

oid

arth

ritis

2002

Xol

air

(om

aliz

umab

)G

enen

tech

/Nov

artis

IgG

1Ly

ophi

lized

pow

der

(202

.5 m

g re

cons

t. w

ith S

WFI

to

150

mg/

1.2

mL

); s

ucro

se, l

-his

tidin

e, p

olys

orba

te 2

0A

llerg

y-re

late

d as

thm

a20

03

Rap

tiva

(ef

aliz

umab

)G

enen

tech

/Mer

ck S

eron

o/re

com

bina

nt h

uman

ized

IgG

1

Lyop

hiliz

ed p

owde

r; r

econ

st. t

o 10

0 m

g/m

l; su

cros

e,

l-hi

stid

ine,

pol

ysor

bate

20

(pH

6.2

)Pl

aque

pso

rias

is c

andi

date

s fo

r sy

stem

ic th

erap

y20

03

Bex

xar

(tos

itum

omab

and

I1

31 to

situ

mom

ab)

Gla

xoSm

ithK

line/

mou

se I

gG2a

14 m

g/m

L s

ingl

e-us

e vi

als

with

10%

mal

tose

, 145

mM

N

aCl,

10m

M p

hosp

hate

, and

WFI

Non

-Hod

gkin

s ly

mph

oma

2003

Ava

stin

(be

vaci

zum

ab)

Gen

ente

ch/R

oche

/hum

aniz

ed

IgG

1C

olor

ecta

l can

cer

2004

Erb

itux

(cet

uxim

ab)

Bri

stol

-Mye

rs S

quib

b/E

li L

illy/

Mer

ck K

GaA

/chi

mer

ic I

gG1

2 m

g/m

L p

rese

rvat

ive-

free

inje

ctab

le li

quid

Col

orec

tal c

ance

r20

04

Vec

tibix

(pa

nitu

mum

ab)

Am

gen/

hum

an I

gG20

mg/

mL

pre

serv

ativ

e-fr

ee s

olut

ion

Col

orec

tal c

ance

r20

06L

ucen

tis (

rani

bizu

mab

)G

enen

tech

/Nov

artis

/hum

aniz

ed

IgG

1

Mac

ular

deg

ener

atio

n20

06

Tysa

bri (

nata

lizum

ab)

Bio

gen

Idec

/Ela

n/hu

man

ized

Ig

G4

Mul

tiple

scl

eros

is a

nd C

rohn

s

dise

ase

2006

(Con

tinu

ed)


TAB

LE 1

.1 (

con

tinu

ed )

Exam

ples

of F

DA

-App

rove

d M

onoc

lona

l Ant

ibod

ies

Prod

uct

Nam

e C

ompa

ny/I

mm

unog

lobu

lin

Type

D

rug

Con

c.; I

nact

ives

In

dica

tion

A

ppro

val

Dat

e

Solir

is (

ecul

izum

ab)

Ale

xion

Pha

rmac

eutic

als/

hum

aniz

ed I

gG2

Paro

xysm

al n

octu

rnal

he

mog

lobi

nuri

a20

07

Cim

zia

(cer

toliz

umab

pe

got)

UC

B/h

uman

ized

Fab

fra

gmen

tLy

ophi

lized

pow

der

with

suc

rose

, lac

tic a

cid,

and

po

lyso

rbat

e or

pre

fille

d sy

ring

e w

ith s

od. a

ceta

te, s

od.

chlo

ride

, and

WFI

Cro

hns

dis

ease

2008

Ilar

is (

cana

kinu

mab

)N

ovar

tis/h

uman

IgG

1In

ject

ion

with

suc

rose

, l-h

istid

ine,

and

pol

ysor

bate

80

Cry

opyr

in-a

ssoc

iate

d pe

riod

ic

synd

rom

e20

09

Sim

poni

(go

limum

ab)

J&J/

Mer

ck/h

uman

IgG

1Si

ngle

-dos

e pr

efille

d sy

ring

e or

aut

oinj

ecto

r pr

ovid

ing

50 m

g pe

r 0.

5 m

L o

f so

lutio

nR

heum

atoi

d ar

thri

tis, p

sori

atic

ar

thri

tis, a

nd a

nkyl

osin

g sp

ondy

litis

2009

Arz

erra

(of

atum

umab

)G

SK/h

uman

IgG

120

mg/

mL

in 5

or

50 m

L s

ingl

e-us

e vi

als

Chr

onic

lym

phoc

ytic

leuk

emia

2009

Prol

ia (

deno

sum

ab)

Am

gen/

hum

an I

gG2

60 m

g/1

mL

in s

ingl

e-us

e pr

efille

d sy

ring

e or

via

lPo

stm

enop

ausa

l ost

eopo

rosi

s,

solid

tum

ors

bon

y m

etas

tasi

s20

10

Act

emra

and

RoA

ctem

ra

(toc

ilizu

mab

or a

tlizu

mab

)H

uman

ized

IgG

1R

heum

atoi

d ar

thri

tis20

10

Yer

voy

(ipi

limum

ab o

r M

DX

-101

)B

rist

ol-M

yers

Squ

ibb/

hum

an

IgG

1

5 m

g/m

L in

sin

gle-

use

50 m

g or

200

mg

vial

Mel

anom

a20

11

Adc

etri

s (b

rent

uxim

ab

vedo

tin)

Chi

mer

ic I

gG1

Ana

plas

tic la

rge

cell

and

Hod

gkin

s ly

mph

oma

2011

Ent

yvio

(ve

doliz

umab

)Ta

keda

/hum

aniz

ed I

gG1

Cro

hns

dis

ease

, ulc

erat

ive

colit

is20

14

a M

ay c

onta

in a

few

fine

tran

sluc

ent p

rote

in p

artic

les.

b D

rug

sens

itive

to li

ght.

(c

ompi

led

from

info

rmat

ion

in r

efer

ence

, PD

R S

taff

, [20

14],

Ho

and

Gib

aldi

[20

03],

and

Spa

da a

nd W

alsh

[20

05])

.


antibodies has also been described (Ho & Gibaldi, 2003). As just discussed, the failure of many antibodies in clinic in the past was due to human antimouse antibody response to mouse monoclonals. Chimeric antibodies solved this problem to a large extent. Also, companies like Abgenix (now Amgen) have used their Xenomouse transgenic mice to produce fully human antibody, panitumumab (Table 1.1). Human antibody genes have replaced the mice genes in these transgenic mice so that their genome can now produce fully human antibodies, and it is no longer required to humanize each individual antibody produced in mice. More recently, there has been increasing attention on fragments of monoclonal antibodies. These are not glycosyl-ated but have the antigen-binding properties and be produced economically in easier to handle microbial organisms rather than in mammalian cells (Spadiut, Capone, Krainer, Glieder, & Herwig, 2014; Weisser & Hall, 2009). Similarly, chemically programmed antibodies are being explored, and they involve covalent conjugation of small molecules to monoclonal antibodies to enable targeting multiple target mol-ecules, due to the chemical diversity of small molecules (Rader, 2014). Cocktails of monoclonals, each with unique binding and specificity, are also being investigated to improve clinical efficacy, a so-called recombinant polyclonal approach (Wang, Coljee, & Maynard, 2013).

1.1.3 expanDing scope of biotechnology

Although earlier developments were restricted to rDNA and monoclonal antibodies, the field of biotechnology is currently expanding into other areas. These new areas include proteomics, antibodydrug conjugates (ADCs), antisense technology, gene therapy, transgenic technology, and drug delivery technologies. The scope of bio-technology now runs the spectrum from designer organisms to tissue regeneration. The end products of some of the newer applications of biotechnology are oligonucle-otides, and not therapeutic peptides and proteins. In this sense, they are outside the scope of this book, but a brief overview of these new applications will be provided.

Protein engineering techniques can also improve the clinical utility of therapeu-tic proteins (Carter, 2011). For example, site-directed mutagenesis can be used to achieve a precise alteration of the protein sequence by a point mutation of the DNA that encodes the amino acid sequence (McPherson & Livingston, 1989). Gene diag-nostics is another promising area, and genetic counseling will become a routine part of the health-care system. The ability to amplify specific segments of DNA with a polymerase chain reaction has enhanced the analytical limits so that genetic defects can be determined with just a few cells (Sadee, 1988). An ideal situation for the deliv-ery of a recombinant protein would be where a cell from the recipient can be isolated and grown. Recombinant genes can be introduced into this cell, and it can then be reimplanted into the host organism (Barr & Leiden, 1991; Dhawan et al., 1991).

Although many of the new therapeutic peptides and proteins are products of bio-technology, synthetic peptides or proteins continue to be important (Craik, Fairlie, Liras, & Price, 2013). For example, Salmon et al. (1993) have described a system for production and screening of large libraries of peptides to speed up drug discov-ery. All the peptides in this library were produced by solid-phase chemical synthe-sis. Techniques are now available to routinely generate synthetic peptide libraries


with millions of sequence combinations, with minimal investment in equipment. Biologically active peptides or proteins can then be identified in this combinato-rial library by using a monoclonal antibody, soluble receptors, or directed evolution approaches (Medynski, 1994; Urvoas, Valerio-Lepiniec, & Minard, 2012). Small peptides (2 to 50 residues) and especially peptides containing unnatural amino acids will continue to be made by synthetic methods rather than by rDNA technol-ogy. The use of some of the more common new areas of biotechnology will now be discussed.

1.1.3.1 AntibodyDrug ConjugatesADCs consist of a mAb chemically linked to a cytotoxic drug with a linker. ADCs take advantage of the specificity of mAbs to deliver potent cytotoxic drugs selec-tively to antigen-expressing tumor cells (Alley, Okeley, & Senter, 2010; Casi & Neri, 2012; Perez et al., 2014; Trail, 2013). They are designed to stay stable in circulation and reach target site where they undergo antigen-specific binding, internalization, and intracellular drug release. This reduces the systemic toxicity of the cytotoxic drugs. Though simple in concept, design of ADCs poses many challenge, including those related to mAb specificity, stability in circulation, complex pharmacokinetics, stoichiometry, and linker chemistry/technology. The reactive linker molecule needs to be carefully analyzed to ensure the purity of the conjugate (Li, Medley, Zhang, Wigman, & Chetwyn, 2014). The high cost of goods makes ADCs more relevant for severe diseases, and most successful applications are currently in the oncology area (Casi & Neri, 2012; Trail, 2013). Several advances made in the past few decades led to the first FDA approval in 2000 (Mylotarg, gemtuzumab ozogamicin) for acute myeloid leukemia though the product was withdrawn a decade later for lack of improvement in overall survival. In recent years, two ADCs have been approved: Adcetris (brentuximab vedotin) for the treatment of Hodgkins and anaplastic large cell lymphomas and Kadcyla (ado-trastuzumab emtansine) for the treatment of patients with HER2-positive breast cancer. This has generated considerable interest and investment, and currently, 30 ADCs are in clinical development (Perez et al., 2014). Other approaches to target monoclonal antibodies include immunotoxins and immunoliposomes (Sapra & Shor, 2013). In addition to monoclonals, other macro-molecules such as polysaccharides have also been conjugated to cytotoxic drugs for cancer therapy (Goodarzi, Varshochian, Kamalinia, Atyabi, & Dinarvand, 2013).

1.1.3.2 Antisense Agents and RNAiThe principles involved in antisense technology exploit the genetic sequence of events in the cell. As is well known, the double-helix DNA molecule does not directly serve as an immediate template for protein synthesis. Instead, it directs the synthesis of a complementary messenger RNA in a process called transcription. The mRNA then directs protein synthesis by ribosomes, in a process called transla-tion. The sequence of events by which a DNA genetic sequence produces a protein is called gene expression. If an RNA complementary to mRNA is introduced into a cell, it will pair up with the mRNA and prevent it from translating a gene into a protein. This agent, called an antisense RNA, will thus form an antisense duplex that will block protein synthesis and can have therapeutic applications in human


diseases (Alper, 1993; Chrisey & Hawkins, 1991; Lares, Rossi, & Ouellet, 2010; Misra et al., 1993; Wu-Pong, 1994).

This technology can be used to turn off disease-related genes. Since the antisense agent targets the primary cause of the disease, it could potentially provide a cure. Antisense agents may suppress disease-causing damaged or mutated human genes or foreign genetic material such as viral or bacterial genes. Promising application would be to suppress (or downregulate) oncogenes, which are implicated in devel-opment of cancer (Chrisey & Hawkins, 1991) or treatment of tumors by blocking the overexpression of growth factor genes (Lenz & Mansson, 1991). However, anti-sense oligonucleotides may often be unsuitable for therapeutic application as they are susceptible to enzymatic degradation by various nucleases, and thus stabilization techniques such as by chemical modification, encapsulation into liposomes, or other approaches may be required (Chrisey & Hawkins, 1991; Sueishi et al., 1994).

Another problem relates to the low efficiency of penetration of oligonucleotides into intact cells. An oligonucleotide with 17 bases will have a molecular weight of about 5000, which is smaller than most therapeutic proteins but larger than most con-ventional drugs. The size places a limitation on oral absorption of oligonucleotides as well (Sherman, 1993). Interest later shifted to double-stranded short- interference RNA molecules (siRNAs or RNAi) that exist naturally in the cells of many species. Like antisense agents, RNAis interfere with mRNA, but they provide a more natural way to silence genes unlike the totally synthetic antisense agents. However, deliv-ery of RNAi is also very challenging, and thus the promise may be slow to realize. Conjugation of a SPACE peptide (see Section 8.2.4) to siRNA has been reported to cause enhanced absorption via skin and lead to the knockdown of the correspond-ing protein targets (Hsu & Mitragotri, 2011). Similarly, microneedles and carbon nanotubes have been shown to enhance delivery of siRNA into skin and lead to gene silencing in tumor tissue or silencing of reporter gene expression in skin (Gonzalez-Gonzalez et al., 2010; Siu et al., 2014).

1.1.3.3 Transgenic TherapeuticsThe term transgenic applies to the transfer of genes between species. For example, human genes can be introduced into an animal model at an embryonic level, by microinjection of purified DNA into the pronucleus of a fertilized egg. The new genetic information gets integrated into the chromosomal DNA of the host organism, and the gene will be passed on to the offspring by the normal laws of heredity. By identifying the gene of interest in the offsprings and continued matings, a transgenic line of animals can be produced. The use of transgenic animals has the potential to limit the total number of animals used in research. Transgenic technology can be used for studying human genetic diseases and for the production of a human protein by the animal. A human gene expressed in the animal will produce a protein that will appear in all its body fluids. The protein can then be isolated from milk or blood, and the live animal becomes a living factory for the synthesis of a human protein (Chew, 1993; Garthoff, 1994; Houdebine, 2009). Human antithrombin III, isolated from milk of farm transgenic mammals, has received approval from the European Agency for the Evaluation of Medicinal Products (Houdebine, 2009). Since the clon-ing of a sheep (Dolly) in 1997, transgenic technology has received more attention and


proteins have been produced in transgenic animals using mice, rabbits, pigs, goats, sheep, or other animals. Site-specific gene transfer may also potentially generate immune-tolerant humanized tissues.

1.1.3.4 Gene TherapyGene therapy involves the transfer of genetic material to the cells of an individual for therapeutic benefit, such as the cure for a human genetic disease. The introduced gene must then become a part of the system and respond to physiological changes. For example, an engineered insulin gene must respond to changes in blood glucose levels for gene therapy to be successful for diabetes (Morgan, 1993). Genetic mate-rial, usually cloned DNA, can be transferred to cells by physical, chemical, or biolog-ical means. Biological transfer by means of viruses is the most widely used method. Advances in vaccine technology have laid the groundwork for the development of viral vector gene therapy delivery systems (Summers & Cooney, 1994). However, currently there are certain barriers that need to be overcome. Nonviral vectors are being developed to use gene therapy to treat incurable diseases such as cancer and genetic disorders (Canine & Hatefi, 2010; Ibraheem, Elaissari, & Fessi, 2014).

In principle, an antisense oligonucleotide can be designed to target any single gene within the entire human genome. Thus, any disease whose causative gene is known may benefit from gene therapy. The barriers relate to the ability of the oligo-nucleotide to cross the cellular membrane in order to reach the cytoplasm or nucleus. Once inside the cell, the oligonucleotide must not degrade and must bind specifically and with high affinity to the RNA target. Several attempts that are being investi-gated to overcome these barriers are discussed in the literature (Apaolaza, Delgado, Pozo-Rodriguez, Gascon, & Solinis, 2014; Milligan, Matteucci, & Martin, 1993; Suk et al., 2014). Plasmids containing gene of interest or viral vectors are now being investigated. The field of gene therapy suffered a setback because of an unfortunate death in 1999, which changed the political and social climate with intense public attention. However, we have seen success stories in recent years (Wirth, Parker, & Yla-Herttuala, 2013) as well as a marketed product (alipogene tiparvovec, Glybera).

The treatment of adenosine deaminase (ADA) deficiency is an ideal candidate for gene therapy. ADA deficiency is a rare genetic disease in which afflicted children lack the ADA enzyme necessary for the normal function of the immune system, resulting in severe combined immunodeficiency syndrome. The process of gene therapy for this disease involves the isolation of lymphocytes from a severely immu-nodeficient patient. These lymphocytes are then grown in the laboratory, when new genes are introduced into them. Finally, they are safely returned to the patient, and results have shown that patients have responded favorably to the therapy (Morgan, 1993; Moseley & Caskey, 1993). Gene therapy protocols have been investigated for diseases such as cystic fibrosis, HIV infection, type 1 diabetes, various hemoglobin disorders, and several cancers and cardiovascular diseases (Nwanguma, 2003).

1.1.3.5 Human Genome ProjectThe human genome project was launched in 1988 by the National Institute of Health (NIH, Bethesda, MD) and the Department of Energy (Washington, DC). The goal of the project was to sequence the entire six-billion-base-pair human genome by


the year 2005. These base pairs were expected to code for approximately 100,000 genes in the human body. The project had brought together molecular biologists, geneticists, and medical researchers, who, with the aid of robotics and other tools, worked on this project (Pestka, 1994; Summers & Cooney, 1994; Uber, 1994). Based on this work, NIH had filed patents on many gene fragments but later dropped the claim. Such patenting of the human genome may allow some companies, universi-ties, or governments to control the use of as yet undiscovered uses related to patented DNA sequences (Wuethrich, 1993). The project was completed ahead of schedule in 2003 with the unraveling of the entire human DNA sequence and the surprise observation that there are only about 30,000 genes in the human genome. Also, it was noted that interindividual variations are attributed to only 0.1% of the genome. Genetic variability among individuals is now being realized as a major determinant of variable drug effect, and the field of pharmacogenomics, which has some applica-tions already, has been predicted to have far-reaching effects on medical practice in the coming years (Mesko, Zahuczky, & Nagy, 2012; Nwanguma, 2003). Though complete genome sequencing was done quickly, the functional characterization of the sequenced genome has proceeded more slowly (Galperin & Koonin, 2010). Antibodies for any protein encoded by these genes are being generated by in vitro methods to complete the human proteome (Dubel, Stoevesandt, Taussig, & Hust, 2010). Proteomics is expected to bridge the gap between genomics and drug discov-ery in the coming years.

1.2 BIOTECHNOLOGY INDUSTRY

The first biotechnology company was founded in 1971. Since then, the industry has made steady progress, with over a thousand biotechnology companies in the United States alone. In addition, the United Kingdom has several hundred companies, and other countries active in this area include Japan and the European Economic Community (Burrill, 1992b; Dibner, 1986; Dibner & White, 1989; Itoh, 1987; Small, 1992; Szkrybalo, 1987). Other countries are also active in the biotechnology area, and many are now getting active in the biosimilar area (see Section 3.4). Although it is often believed that biotechnology-derived products have been slow to arrive, their total development time, at least for the early products, was about 7years. This is less than the average development time of 12years for conventional drugs (Bienz-Tadmor & Brown, 1994).

Although biotechnology companies provide efficient discovery and develop-ment, they often need to collaborate with the pharmaceutical industry to bring a product to the market. Pharmaceutical companies provide superior distribution channels and clinical and regulatory assistance. These complementary strengths create a good atmosphere for strategic alliances (Burrill, 1992b; Lyons, Lang, & Guzzo, 1993). Similar collaborative arrangements between universities and the biotechnology industry also exist. Traditionally, such collaborative efforts existed between the universities and the pharmaceutical industry (Swann, 1986). The bio-technology industry has generally been able to maintain its independence and raise capital from the public. Some of the biggest players in the United States include Amgen, Gilead Sciences, Celgene, and Biogen Idec. Consolidation, mergers,


and acquisitions have been commonly part of an industry-wide trend (Burrill, 1990; Das, 2003; Dibner, 1992; Spalding, 1993).

1.2.1 biotechnology pRoDucts on the maRket

Over 200 biotechnology-derived drugs are currently approved by the U.S. FDA. Follow-on products or biosimilars are discussed in Section 3.4. Many of these biotechnology-derived products are available in limited quantities, especially when first introduced. For example, when interferon--1b (Betaseron, Bayer HealthCare) was first introduced, supplies were limited and it was allocated to patients by lot-tery. However, eventually rDNA technology allows the production of sufficient quantities in a commercially viable manner. As discussed, different indications of the same drug may receive regulatory approval at different times. Also, it should also be pointed out that the same drug produced in a different cell line will need separate regulatory approval and may actually be structurally different. Considering the example of -interferon again, Betaseron discussed earlier is different from that developed by Biogen. Biogens molecule is glycosylated, has one amino acid differ-ent from Betaseron, and is produced in mammalian cell lines rather than E. coli. Many of the products introduced have been engineered in some way to improve their therapeutic or pharmacokinetic properties. Such second-generation biophar-maceuticals may have altered amino acid sequence, an altered glycocomponent, or a covalently attached chemical moiety such as polyethylene glycol (Walsh, 2004). Monoclonal antibodies was discussed in Section 1.1.2, and a few of the other recom-binant products will be briefly discussed below. Please also see Tables 4.1 and 4.2 in Chapter 4 for a listing of some of the products on the market.

1.2.1.1 Human Insulins and Analogs1.2.1.1.1 Human InsulinSeveral million patients currently require insulin, with a worldwide market in bil-lions. Human insulin (Humulin, Eli Lilly) was the first therapeutic recombinant polypeptide to be approved by the FDA in October 1982. Recombinant human insulin is synthesized in E. coli bacteria and is chemically and physically equiv-alent to pancreatic human insulin and biologically equivalent to human and pork insulin (Chance, Kroeff, Hoffmann, & Frank, 1981; Ladisch & Kohlmann, 1992). The advantage of human insulin lies in its lower antigenicity relative to animal-derived insulins (Brogden & Heel, 1987). Human insulin has now largely replaced the animal-derived insulins.

Before 1986, human insulin was prepared by the production of genetically engi-neered A and B chains in separate fermentations. These two chains were then iso-lated, purified, and joined together chemically. Recombinant insulin is currently produced by the production of proinsulin, followed by the enzymatic cleavage of the C-peptide to produce human insulin. Humulin produced by these two manufactur-ing processes has been shown to have no pharmacokinetic or clinical differences.

Recombinant human insulin can be used by diabetic patients in much the same way as insulin derived from animal sources. Clinical advantages that may be attrib-uted to the recombinant product are slight and may include less immunogenicity


and quicker absorption, but these depend on patient factors as well. Several for-mulations of Humulin are available today: regular (R), NPH (N), lente (L), ultra-lente (U), Humulin 50/50, and Humulin 70/30. Using one of these formulations, rapid or intermediate or long action can be achieved. In 1996, human recombinant insulin made utilizing Saccharomyces cerevisiae (bakers yeast) as the product organism was approved (Novolin, Novo Nordisk). Considerable effort has been directed to investigate noninvasive delivery systems for insulin and its analogs (Khafagy, Morishita, Onuki, & Takayama, 2007) as will be discussed in the sub-sequent chapters.

1.2.1.1.2 Human Insulin AnalogsIn addition to basal insulin needs that control glucose levels between meals, most diabetics need fast-acting insulin after meals. Insulin analogs with reduced tendency for self-association, which have a rapid onset of action and can thus better mimic meal-stimulated pharmacokinetics of insulin observed in nondiabetics, are highly desired (Defelippis, Chance, & Frank, 2001). Modifications in the insulin sequence in the B2630 region led to the approval of three rapid-acting recombinant insulins, insulin lispro (Humalog, Eli Lilly), insulin aspart (NovoLog, Novo Nordisk), and glulisine (Apidra, Sanofi-Aventis). Also, two long-acting analogs, insulin glargine (Lantus, Aventis) and detemir (Levemir, Novo Nordisk), have been marketed. Insulin glargine has a different isoelectric point, while insulin detemir has an added fatty acid residue (2005; Sheldon, Russell-Jones, & Wright, 2009). Insulin glargine had a U.S. market of $6.6 billion in 2012 (Bruno, Miller, & Lim, 2013).

The rapid-acting analogs mimic endogenous insulin profile closely and can be given at mealtime or even after a meal and thus provide an important advantage and are also less likely to cause hypoglycemia (Freeman, 2009). Both regular and glulisine insulins are equally effective during the treatment of diabetic ketoacido-sis (Umpierrez et al., 2009). Insulin lispro is chemically a Lys (B28), Pro (B29) analog that was created when the amino acids at positions 28 and 29 on the insu-lin B chain are reversed. The gene for insulin lispro is then inserted into E. coli bacteria. Production is carried out under conditions designed to minimize the degree of microbial contamination. As per European Pharmacopoeia, tests for host-cell-derived proteins and single-chain precursor should be carried out prior to release. The praline group at position B28 in regular insulin is important for -sheet con-formation and formation of dimer units. Reversal of the prolinelysine sequence in insulin lispro eliminates important sites for hydrophobic interactions, thereby decreasing the tendency for dimerization. Zinc is added to commercial insulin preparations for stability, and they exist as hexameric units in their containers as marketed. However, unlike regular insulin, insulin lispro dissociates more rapidly to monomers due to decreased tendency for dimerization (Kucera & Graham, 1998). It thus has more rapid absorption, faster onset, and shorter duration of action compared to regular insulin, allowing more flexibility in dosing and mealtime scheduling (Hermans, Nobels, & De, I, 1999; Koivisto, 1998; Kucera & Graham, 1998; Nobels, Hermans, & De, I, 1999). The inversion of the proline and lysine apparently does not increase the immunogenicity of insulin lispro (Hermans et al., 1999). Insulin aspart was made by a single substitution of the amino acid proline by aspartic acid


in position B28 and is produced utilizing baker yeast. Insulin aspart is also approved for use of subcutaneous insulin infusion via an external insulin pump.

Insulin glargine is made in E. coli and differs from human insulin in that the asparagine at position A21 is replaced by glycine and two arginines are added to the C-terminus of the B chain. Due to the addition of two amino acids, its molecular weight increases to 6.1 kDa. Insulin glargine is a long-acting analog and can pro-vide nearly constant basal levels by a once-daily subcutaneous injection at bedtime (Ho & Gibaldi, 2003). Basal insulin needs of type 1 diabetic patients can be met either by using NPH human insulin or by using one of the newer long-acting insulin analogs (Monami, Marchionni, & Mannucci, 2009). In addition to clinical studies, safety evaluations now include investigating molecular characteristics of the insulin analogs by detailed analysis of receptor binding, activation, and in vitro mutagenic-ity tests (Hansen et al., 2012). An ultralong-acting basal insulin (insulin degludec, Tresiba) is being developed by Novo Nordisk (Heise et al., 2011; Wang, Surh, & Kaur, 2012).

1.2.1.2 Growth HormoneRecombinant human growth hormone (GH) was first made available as somatrem, which includes an additional methionyl group at the N-terminus. This product is more immunogenic than the natural sequence that was produced later though the immunogenicity is believed to be due to E. coliderived impurities and not the extra methionine (Patten & Schellekens, 2003). Later, human GH with the natural sequence (somatotropin) was made available (Humatrope, Eli Lilly, and Nutropin AQ, Genentech). Both products are approved for the treatment of GH deficiency in children. New formulations have also been investigated and compared for bioequiva-lence in human volunteers (Dao, Jacobs, Kuebler, Bakker, & Lippe, 2010). Prior to the availability of the recombinant products, GH was extracted from pituitary glands obtained at autopsy. The supply was limited, and furthermore, contamination with an infectious agent was responsible for the development of a viral-like condition in some patients (JacobCreutzfeldt syndrome). This extremely serious side effect, which led to a slow mental deterioration and death (Powell-Jackson et al., 1985), was eliminated when GH is obtained using recombinant techniques. The recombinant hormones can now be produced in sufficient quantities, and studies are now possible to explore a wider therapeutic use of GH. GH treatment is proving promising for short children wh

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