A Report Prepared by BioProcess Technology Consultants, Inc.Editors:Howard L. Levine, PhDBrendan R. Cooney

The Development ofTherapeutic MonoclonalAntibody ProductsA Comprehensive Guide to CMC Activities from Clone to Clinic

ii

Table of Contents

List of Tables iv

List of Figures vi

Foreword viii

CHAPTER 1: The Therapeutic Monoclonal Antibody Market 1

CHAPTER 2: Overview of Chemistry, Manufacturing, and Control Activities for Monoclonal Antibody Product Development

33

CHAPTER 3: Quality by Design 65

CHAPTER 4: Analytical Development 95

CHAPTER 5: Cell Line Development and Engineering 155

CHAPTER 6: Cell Culture Development and Scale-up 195

CHAPTER 7: Purification Development 241

CHAPTER 8: Formulation Development and Stability 313

CHAPTER 9: Drug Product Manufacturing 370

CHAPTER 10: Comparability 393

CHAPTER 11: Process Validation 423

CHAPTER 12: Manufacturing Strategies 473

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List of Tables Table 1.1 Applications of Therapeutic Monoclonal Antibody Products 7

Table 1.2 2015 Sales of the Top Ten Selling Biopharmaceutical Products 9

Table 1.3 Commercially Marketed Therapeutic Monoclonal Antibody Products 11

Table 1.4 Patent Expiration Dates for Key Monoclonal Antibody Products 23

Table 2.1 Estimated CMC-Related Costs for Monoclonal Antibody Development 56

Table 3.1 ICH Guidelines Related to Quality by Design 66

Table 3.2 Control Strategy Elements 86

Table 4.1 ICH Guidance Documents Covering the Testing and Characterization of Monoclonal Antibody Products

99

Table 4.2 Minimum AMV Characteristics from ICH Q2(R1) 107

Table 4.3 Some Methods Used for Identity Testing of Monoclonal Antibody Products 109

Table 4.4 Some Methods Used for Determination of Purity and Product-Related Impurities of Monoclonal Antibody Products

113

Table 4.5 Some Methods Used for Measurement of Some Process Related Impurities 119

Table 4.6 Some Methods Used for Safety Testing of Monoclonal Antibody Products 121

Table 4.7 Methods Used for Potency Testing of Monoclonal Antibody Products 122

Table 4.8 Methods Used for Testing General Attributes of Monoclonal Antibody Drug Substance and Drug Product

124

Table 4.9 Analytical Methods Used to Characterize Monoclonal Antibody Drugs 126

Table 4.10 Common Release Tests for Monoclonal Drug Substance and Drug Product 143

Table 4.11 An Example of QC Release Methods and Specifications for a Monoclonal Antibody Product in Early Clinical Development

145

Table 5.1 CHO Species Used in Monoclonal Antibody Production 160

Table 5.2 Commercially Available Expression Systems 164

Table 5.3 Expression Vector Construction 168

Table 5.4 Transfection and Selection 170

Table 5.5 Single Cell Cloning 172

Table 5.6 Testing of Mammalian Cell Banks 184

Table 7.1 Parameters to be Considered in Chromatography Step Development 278

Table 7.2 Comparison of High Throughput Methods for the Development of Chromatographic Separations

281

Table 7.3 Guidelines for Linear Scale-up of Chromatography 294

Table 8.1 Formulation Details for Currently Marketed Therapeutic Monoclonal Antibody Products

314

iv

Table 8.2 Potential Degradation Pathways of Monoclonal Antibody Products and Analytical Methods to Detect Them

322

Table 8.3 Example of a Forced Degradation Matrix for a Monoclonal Antibody Product 332

Table 8.4 Typical Analytical Methods Used in Monoclonal Antibody Stability Studies 333

Table 8.5 Commonly Used Buffers in Monoclonal Antibody Formulations 336

Table 8.6 Example of Design of Experiments Study Investigating Four or Five Components of a Potential Monoclonal Antibody Product Formulation

343

Table 8.7 Typical Stability Study Design for a Monoclonal Antibody Drug Substance to Support Early Stage Clinical Development

354

Table 8.8 Typical Stability Study Design for a Monoclonal Antibody Drug Product to Support Early Stage Clinical Development

359

Table 9.1 Improvements in Rubber Stopper Formulations 378

Table 9.2 Typical Monoclonal Antibody Drug Product Specifications 386

Table 10.1 Regulatory Submissions Worldwide Supporting Process Changes 399

Table 10.2 Risk Assessment and Comparability Requirements in Early Development 403

Table 10.3 Typical Monoclonal Antibody Product Release Tests Used in Comparability Protocols 408

Table 10.4 Characterization Tests used in Monoclonal Antibody Product Comparability Protocols 411

Table 11.1 Typical Stage 1 Process Design Activities 431

Table 11.2 Typical Stage 2 Process Qualification Activities 440

Table 11.3 Potential Cell Culture Critical Process Parameters 448

Table 11.4 Sample VMP Table of Contents 464

Table 12.1 Typical Contents of a Request for Proposal for CMO Services 485

Table 12.2 Operating Costs for Stainless Steel and Single-Use Facilities 492

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List of Figures Figure 1.1 Antibody Structure 2

Figure 1.2 IgG Oligosaccharide Structure 4

Figure 1.3 Annual Approvals of Monoclonal Antibody Products 10

Figure 1.4 Sales of Biopharmaceutical Products by Product Type and Class 17

Figure 1.5 Sales Growth for Commercial Monoclonal Antibody Products 19

Figure 2.1 Typical CMC Timeline for Monoclonal Antibody Development 54

Figure 3.1 The Quality by Design Approach 68

Figure 3.2 CQA Risk Assessment 72

Figure 3.3 Prior Knowledge Elements 73

Figure 3.4 Example of a Design Space 76

Figure 3.5 Specifications Settings 78

Figure 3.6 Relationship of Process Characterization Studies to Design Space 79

Figure 3.7 Development of a Process Control Strategy 84

Figure 4.1 Analytical Methods Lifecycle 101

Figure 4.2 Method Validation Readiness Flow Path 104

Figure 5.1 Representative Cell Line Development Workflow 169

Figure 7.1 Typical Unit Operations Used in Monoclonal Antibody Purification 252

Figure 7.2 Basic Elements of a Platform Purification Processes 274

Figure 7.3 Effect of Processing Time on Membrane Area for a UF/DF Process 292

Figure 7.4 Principle of Linear Scale-up of a Chromatography Column 293

Figure 8.1 Structure of a Monoclonal Antibody 320

Figure 8.2 Mechanism of Methionine Oxidation 323

Figure 8.3 Mechanism of Deamidation of Asparagine Residues 324

Figure 8.4 Disulfide Rearrangement 325

Figure 8.5 Mechanism of β-Elimination and Rearrangement or Hydrolysis 326

Figure 8.6 Hydrolysis of Asp-Gly Peptide Bonds 327

Figure 8.7 Aggregation Pathways for Monoclonal Antibody Products 329

Figure 8.8 Liquid and Lyophilized Formulations for Currently Marketed Therapeutic Monoclonal Antibody Products

344

Figure 9.1 Steps in the Manufacture of a Monoclonal Antibody Drug Product 371

Figure 10.1 Typical Stability Study Design for a Monoclonal Antibody Drug Product to Support Early Stage Clinical Development

396

Figure 10.2 Comparability Decision Tree 404

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Figure 11.1 Overall Sequence of Process Validation Activities 426

Figure 11.2 Overview of Quality Risk Management 427

Figure 11.3 An example of an Ishikawa or Fishbone Diagram 430

Figure 11.4 Unit Operation-based Approach to Risk Assessment 435

Figure 11.5 Relationship between the Phases of Product Development and the Process Validation Lifecycle

437

Figure 11.6 Risk Assessment for Classifying Process Parameter Criticality 444

Figure 11.7 Defining Operating Parameter Ranges 444

Figure 12.1 Manufacturing Strategy Considerations 475

Figure 12.2 Pilot Plant for Production of Monoclonal Antibody Bulk Drug Substance 489

Figure 12.4 Cost Breakdown for a Simple Monoclonal Antibody Pilot Plant 491

Figure 12.4 Monoclonal Antibody Pilot Plant Construction Timeline 493

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CHAPTER 1: The Therapeutic Monoclonal Antibody Market

n 1984 Kohler and Milstein received the Nobel Prize in Medicine for their pioneering work

on the production of monoclonal antibodies.1 One of the most significant advantages of this

new technology over traditional techniques for producing antibodies was the creation of an

immortalized cell line creating a continuous source of the same antibody with a single antigen

specificity. This enabled the development of highly specific monoclonal antibodies directed

toward a single epitope on the target antigen. Initially monoclonal antibodies were used as

laboratory reagents but their use was quickly adopted as clinical diagnostic reagents. In the early

1980s, commercial development of monoclonal antibodies as therapeutic agents commenced so

that by 1986 the first therapeutic monoclonal antibody, Orthoclone OKT3, was approved for

prevention of kidney transplant rejection.

1. Antibody Structure

Antibodies are a component of the immune system whose ability to bind targets, activate other

immune system functions, and reside in the circulation for weeks has been harnessed to create

effective therapeutic products. The power of antibodies as effective therapeutics resides in their

specificity, their bivalency, and their modular structure, which has enabled this category of

therapeutic products to emerge as a leading component of the biopharmaceutical market. All

antibodies have the same basic structure, which consists of two identical heavy chains and two

identical light chains as shown in Figure 1.1. Early structural studies revealed that antibodies

could be enzymatically digested into two regions, the Fab region which contains the antigen

I

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binding site, and the Fc region, which contains sequences that interact with other components of

the immune system to activate additional functions.2 This modularity is the basis for many of

today’s discovery methods, which focus on discovery of suitable antigen binding sequences and

then use molecular techniques to add an Fc region that is appropriate for the intended indication.

Antibodies in human sera can be divided into five different classes based on the sequence of the

heavy chain. These classes are known as IgA, IgD, IgE, IgG, and IgM, and each class has a

different function within the overall immune system. Some classes can be further subdivided,

such as the IgG class, which contains the four subclasses IgG1, IgG2, IgG3, and IgG4. Most

therapeutic monoclonal antibodies are IgG, with most approved antibodies falling in the IgG1 or

IgG4 subclass.3 Some products in development are IgM, which consist of pentamers or

hexamers of the four chain basic antibody structure, but most discovery and development efforts

continue to focus on IgG antibodies and this class is the primary focus of this report. Two types

of light chain are also found in human antibodies, kappa (κ) and lambda (λ) with the κ chain

being far more common in therapeutic monoclonal antibodies. A typical IgG antibody with either

type of light chain contains approximately 1,080 amino acids and has a total molecular weight of

approximately 146 kDa prior to post-translational modification.

Figure 1.1 Antibody Structure.

VH=heavy chain variable region; VL=Light chain variable region; CH1, CH2, CH3=Heavy chain constant regions, CL= Light chain constant region

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As shown in Figure 1.1, IgG antibodies usually have four inter-chain disulfide bonds, two

connecting each light chain with a heavy chain and two connecting the heavy chains to enable

dimerization. This feature of the Fc region of the heavy chain can be utilized to form dimers of

other therapeutic proteins by creating a fusion between the protein of interest and the IgG heavy

chain Fc sequence. Among the potential therapeutic benefits of these fusion proteins is a longer

serum half-life of the fusion protein compared to the monomer used without linkage to the Fc

region and bivalent functionality.

Intra-chain disulfide bonds are also found in the variable and constant regions. The intra-chain

bonds in the variable regions help create the three dimensional structure that enables proper

antigen binding. Low levels of free sulfhydryl groups from disulfide bonds that did not form

properly can be found in recombinant antibodies and can create product stability problems.4

Antigen Binding

The antigen binding function of an antibody is located within the 110 amino acid variable region

at the N-terminus of each chain. Within the variable regions, three surface-exposed hypervariable

amino acid loops, known as complementarity determining regions (CDR), are embedded in a

relatively conserved framework structure.5 The six combined CDRs from the heavy and light

chains form the antigen binding site, and slight changes to CDR sequences can significantly alter

affinity and specificity for the target antigen.6 Because the antigen binding function of an

antibody is localized in such a specific region of the protein, molecular engineering tools can be

used to introduce novel variability in the CDRs of one or both chains followed by in vitro

selection for improvements in target binding.7 Binding at the antigen binding sites on each arm

of the antibody can occur independently so that the antibody can also be engineered to contain

two different antigen binding domains. Such bi-specific antibodies are currently under

development by several companies.8 Also, if the variable region of an antibody is cloned

independently and expressed as a soluble monomer it will retain the ability to bind to the target

antigen.9 These monovalent products are also under development by several companies.10

Effector Functions

In addition to antigen binding function, antibodies contain oligosaccharides on the constant

region that can interact with other components of the immune system to activate effector

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functions such as antibody dependent cellular cytotoxicity (ADCC) or complement dependent

cytotoxicity (CDC). If immune system activation is important for the therapeutic activity of an

antibody, the oligosaccharide structure is often critical to the clinical behavior of the molecule.11

For IgG antibodies, an N-linked biantennary oligosaccharide is attached to a highly-conserved

asparagine. The core structure contains three mannose residues and two N-acetyl-glucosamine

residues (GlcNAc) as shown in Figure 1.2.12 In some monoclonal antibodies, the carbohydrate

structure may also contain fucose. If present, the fucose residue is linked to the proximal

GlcNAc residue, and additional terminal sugar residues including galactose and sialic acid are

also present. Occasionally GlcNAc is added to the central mannose to form a structure known as

a bisecting GlcNAc, which has a significant impact on antibody function.13

Figure 1.2. IgG Oligosaccharide Structure.

The oligosaccharide structure of N-linked glycans in the CH2 domains is shown. Individual sugar moieties may or may not be present in all molecules (indicated by ± in the figure), however sialic acid can only be present if galactose is also present in the oligosaccharide structure. (Figure adapted and reprinted with permission from References 14 and 15)

Variation in the terminal sugar residues is the basis of most of the glycan heterogeneity seen in

purified, recombinant monoclonal antibodies. This can influence which, if any, effector functions

are activated. For example, the oligosaccharide can contain either no (G0), one (G1), or two (G2)

terminal galactose residues (see Figure 1.2); increased galactose content can increases CDC

activity while ADCC activation is not known to be affected by the galactose content of the

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oligosaccharide.16 Likewise, if fucose is not present on the core GlcNAc, the antibody exhibits

enhanced ADCC activity compared to the fully fucosylated form but no impact on CDC has been

observed.17 In addition, variation in the oligosaccharide structure in the binding protein of an

Fc-fusion may greatly impact overall half-life in a way not generally seen with whole antibodies.

For example, sialic acid content in the binding protein may greatly affect half-life or efficacy of

the product.

Glycan variability is primarily influenced by clone selection and cell culture conditions, but

should also be considered during discovery and lead candidate identification, especially when

choosing a heavy chain constant region for a particular target product profile. If effector

functions are not required for the intended therapeutic mode of action, it may be most effective

to develop an IgG4 antibody that has less effector function. For example, for monoclonal

antibodies whose therapeutic activity is entirely based on blocking another protein from binding

to the target, effector function and oligosaccharide structure are not critical to therapeutic

function.

2. Therapeutic Applications of Monoclonal Antibodies

Following the approval of Orthoclone OKT3, there was a long gap before any new antibody

products were approved. During this time, new approaches to discovering and developing

antibody products emerged and enthusiasm for therapeutic monoclonal antibodies returned.

Several additional monoclonal antibody products were approved in the US and Europe in the mid

to late 1990’s, while the 2000’s ushered in the next wave of antibody products generally being

developed as anti-cancer and anti-inflammatory agents. Today, monoclonal antibody products,

including fragments, conjugates, and full length entities are a mainstay in the pharmaceutical

industry. Utilizing today’s novel technologies and enhanced targeting, they continue to be

discovered, developed, and approved to treat many different diseases18.

As of October 31, 2016, there were 71 monoclonal antibody-related products on the market in

the US and/or Europe for the treatment of a variety of diseases including autoimmune disorders,

cardiovascular indications, infectious diseases, and oncology (see Table 1). These approved

monoclonal antibody products, which include full length monoclonal antibodies as well as

antibody fragments (Fab fragments), Fc-fusion proteins, antibody-drug conjugates, and other

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conjugated antibody products, have been approved for diseases with patient populations ranging

from a few thousand or fewer for such orphan indications as paroxysmal nocturnal

hemoglobinuria, or the cryopyrin-associated periodic syndromes, to hundreds of thousands of

patients for some cancers and multiple sclerosis, to millions of patients for diseases such as

asthma and rheumatoid arthritis. In some cases, the market penetration of monoclonal antibody

products in the US and Europe is quite large with most of the potentially treatable patients

receiving the appropriate antibody therapy. However, for some diseases such as asthma, there are

a large number of potentially treatable patients who are not receiving monoclonal antibody

therapy due to the variety of treatment options currently available and the fact that the approved

monoclonal antibody product in this indication is not the typical first line of treatment for the

disease. On the other hand, access to monoclonal antibody therapies in emerging or developing

countries is often much lower than in the US or Europe due to lack of availability of the

monoclonal antibody products, higher prices, or other regulatory hurdles.

Table 1.1 Applications of Therapeutic Monoclonal Antibody Products

Disease Category

Example Product Specific Indication(s) Additional

Productsa

Allergy Xolair Asthma, moderate to severe, chronic idiopathic urticaria

2

Bone disease Prolia Osteoporosis 1

Cardiovascular Praluent Primary hyperlipidemia 1

Hematologic Reopro Anti-platelet prevention of blood clots in high-risk percutaneous transluminal angioplasty, and in refractory angina when percutaneous coronary intervention is planned

3

Immune and autoimmune diseases

Humira Rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis ankylosing spondylitis, Crohn’s disease, ulcerative colitis, plaque psoriasis, hidradenitis suppurativa and uveitis

27

Infectious diseases

Synagis Prevention of respiratory syncytial virus (RSV) infections in children

3

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Table 1.1 Applications of Therapeutic Monoclonal Antibody Products

Disease Category

Example Product Specific Indication(s) Additional

Productsa

Macular degeneration

Lucentis Neovascular (wet) age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema and diabetic retinopathy in patients with diabetic macular edema

1

Oncology Rituxanb Non-Hodgkin’s lymphoma, chronic lymphocytic leukemia (also for rheumatoid arthritis and Wegener’s granulomatosis)

25

a Number of other approved products in each therapeutic indication area b Rituxan is approved for both oncology and immune and autoimmune

indications

Among the monoclonal antibody-related products, one very important class is the Fc-fusion

proteins, which incorporate or contain the Fc region of an antibody within their structure.

Products in this category, which include Enbrel, AlprolIX and Nplate, generally combine the

antibody Fc region with a binding protein whose half-life in the body is usually too short to

allow the binding protein alone to be therapeutically beneficial. In these products, the primary

function of the Fc region is to prolong the half-life of the product thereby increasing its efficacy

and bioavailability rather than to activate the complement system. While these antibody-related

products combine two very different protein moieties in a single molecule, many of the

Chemistry, Manufacturing and Control (CMC) strategies, activities, costs, and timelines for the

development of Fc-fusion proteins are similar to those of full-length antibodies. For example, the

presence of the Fc region in the fusion protein allows Protein A affinity chromatography to be

used as a primary capture step in the downstream processing of these products (see Chapter 7).

As a result, the CMC strategies, activities, costs, and timelines for the development of these

products are similar to those for full-length monoclonal antibodies.

Similarly, the development, manufacturing, and quality control of the monoclonal antibody

portion of an antibody-drug conjugate follow much the same CMC strategies as for full-length

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antibodies. For these products, however, the preparation and conjugation of the toxic drug

moiety adds additional complexity and cost to the development of these products.

For monoclonal antibody fragments, many of which are produced in microbial hosts rather than

mammalian cell culture, the development strategies, costs, and timelines may vary compared to

full-length monoclonal antibodies. For example, since these products lack the Fc region of a full-

length antibody, they cannot be purified using Protein A affinity chromatography and much of

the discussion in this report regarding platform processes is not relevant to these products.

3. Growth of the Monoclonal Antibody Market

Following the approval of the first monoclonal antibody product in 1986, sales growth and

approval of additional products was slow until the late 1990s when the first chimeric monoclonal

antibodies were approved. With the approval of these products, followed by the approval of

humanized and then fully human monoclonal antibodies, the rate of product approvals and sales

of monoclonal antibody products has increased dramatically so that in 2015, global sales revenue

for all monoclonal antibody products was nearly $90 billion,19 representing nearly 60% of the

total sales of all biopharmaceutical products. Among biopharmaceutical products currently on

the market, seven of the top ten selling products in 2015 were monoclonal antibody products (see

Table 1.2).

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Table 1.2. 2015 Sales of the Top Ten Selling Biopharmaceutical Products a

US Product Name 2015 Sales ($ Billions) b

Humira 14.0

Enbrel 8.7

Remicade 8.4

Rituxan 7.0 b

Lantus 7.0 b

Avastin 6.7 b

Herceptin 6.5 b

Neulasta 4.7

Novolog 4.6 b

Eylea 4.0 b

a Non-monoclonal antibody products are listed in italics b Values converted to USD from reporting currency with a strong USD in 2015.

As shown in Figure 1., the number of monoclonal antibody products approved for commercial

sale in the US and Europe has grown steadily. Since 2011, between three and eleven products

have been approved, with an average of seven new products approved per year and since 2014 an

unprecedented average of ten products per year have been approved. While a total of

82 monoclonal antibody products have been approved in Europe and/or the US since 1986,

eleven of these products have been withdrawn for various reasons, leaving 71 approved

monoclonal antibody products currently on the market.19, 20, 21

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Figure 1.3. Annual Approvals of Monoclonal Antibody Products.20, 21

The number of monoclonal antibody products first approved for commercial sale in the US or Europe each year since 1982 is shown. The totals include all monoclonal antibody and antibody-related products. Products approved but subsequently removed from the market are denoted in blue; products currently marketed are denoted in green. For 2016, the figure includes the total number of products approved as of October 31.

Those products still on the market as of October 31, 2016, are listed in Table 1.3 along with the

year of first approval in the US or Europe. Of these products approved and marketed in the

United States and Europe, three are produced in E. coli while all of the other products are

produced in mammalian cells. Of the products produced in mammalian cell culture, 49 are full-

length naked monoclonal antibodies, including four biosimilars; two are bispecific antibodies,

two are antibody-drug conjugates, one is a radio-labeled antibody conjugate, two are

antigen-binding fragments (Fab), and 12 are Fc-fusion proteins containing the antibody constant

region fused to another non-antibody-related protein domain, including two biosimilars. Two of

the three products produced in E. coli are Fabs, one of which is a Fab conjugate while the third is

an Fc-fusion protein.