Over the last decade, biologic agents have revolutionized the treatment of rheumatoid arthritis (RA) and seronegative spondyloarthropathies. Although many promising interventions failed, these new therapies have taught us much, not only about the underlying immunopathophysiology of inflammatory arthritis but also about their pragmatic use in clinical practice. Future interventions hold promise for addressing the remaining unmet need in inflammatory arthritis, and also systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren’s syndrome (SS), vasculitis, and inflammatory myopathies.
The introduction of a biologic agent into the clinic poses challenges that are different from those posed by a traditional “small molecule” therapeutic. As proteins or peptides, they require more complex manufacturing and characterization processes. Biologics are “designer” drugs whose mode of action in an underlying disease pathophysiology is frequently well understood. Their target specificity means that data derived from animal models can support a more rational clinical development program, facilitating better predictions of dosing, efficacy, and safety profiles compared with small molecule therapeutics. Nonetheless, preclinical data and relevant toxicology studies may not be possible in two species, as required by International Conference on Harmonisation (ICH) guidelines, owing to lack of sufficient homology of the target between species. Also, toxicology studies may be limited in duration owing to immunogenicity observed in the species used (usually primate), resulting in increased clearance of the biologic agent and reduced exposure in the animals. Throughout the development program of a biologic agent, guidances are available from the US Food and Drug Administration (FDA), which offer useful information regarding most aspects ( Tables 33-1 and 33-2 ).
|Traditional Products||Biologic Agents|
|Low molecular weight (<1 kDa)||High molecular weight|
|Typically well-defined physicochemical properties||Complex physicochemical properties; tertiary structure; glycosylation|
|Chemically synthesized||Produced recombinantly in engineered cells|
|Stable; not heat sensitive||Heat and shear-sensitive, may aggregate|
|Well characterized; typically homogeneous; High chemical purity with standards well established||Characterization may be challenging; typically heterogeneous composition; Need to define potency, purity, identity, stability|
|Mechanism of action often not known||Hypothesized mechanism of action usually well characterized|
|Generic and related products are common||Typically unique based on binding epitope, cell line and FcγR structure|
|Rapidly enter systemic circulation through blood capillaries||Larger molecules, primarily reach circulation through lymphatic system; May undergo proteolysis during interstitial and lymphatic transit|
|Distribution to organs/tissues||Distribution usually limited to plasma and/or extracellular fluids|
|Oral administration typical||Parenteral administration: intravenously, subcutaneously, or intraarticularly|
|Linear dose response||Nonlinear dose response which may be relatively flat or inverted|
|Metabolized to nonactive and active metabolites||Catabolized to endogenous amino acids|
|Cyp 450 involvement||Typically Cyp450 independent|
|Toxicities may be “off target”, e.g., not associated with primary pharmacologic effect||Receptor-mediated or mechanistically related toxicity; toxicities may be species specific “pharmacologic” and “industrial strength” dosing|
|Typically a single analytical method required for pharmacokinetic studies||Multiple assays to assess pharmacokinetics (bioassay, enzyme-linked immunosorbent assay, and ligand-binding assay)|
|Draft Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products – 10/9/2008|
|International Conference on Harmonisation (ICH); Guidance for Industry: S1C(R2) Dose Selection for Carcinogenicity Studies (PDF – 185 KB) – 9/17/2008|
|Concept Paper: Animal Models — Essential Elements to Address Efficacy Under the Animal Rule (PDF) – 9/9/2008|
|Draft Guidance for Industry: Integrated Summary of Effectiveness – 8/27/2008|
|Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs) – 4/9/2008|
|Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) – 4/9/2008|
|International Conference on Harmonisation (ICH); Guidance for Industry: E15 Definitions for Genomic Biomarkers, Pharmacogenomics, Pharmacogenetics, Genomic Data and Sample Coding Categories – 4/7/2008|
|International Conference on Harmonisation (ICH); Draft Guidance: S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use – 3/25/2008|
|Draft Guidance for Industry: Validation of Growth-Based Rapid Microbiological Methods for Sterility Testing of Cellular and Gene Therapy Products – 2/11/2008|
|International Conference on Harmonisation (ICH); Draft Guidance: Q8(R1) Pharmaceutical Development Revision 1 – 1/10/2008|
|International Conference on Harmonisation (ICH); Guidance for Industry – Q8 Pharmaceutical Development – 5/19/2006|
|Guidance for Industry: Providing Regulatory Submissions to the Center for Biologics Evaluation and Research (CBER) in Electronic Format – Lot Release Protocols – 11/27/2007|
|Draft Guidance for Industry: Drug-Induced Liver Injury: Premarketing Clinical Evaluation – 10/24/2007|
|Guidance for Industry: Manufacturing Biological Intermediates and Biological Drug Substances Using Spore-Forming Microorganisms – 9/6/2007|
|Draft Guidance for Industry: Pharmacogenomic Data Submissions – Companion Guidance – 8/28/2007|
|Guidance for Industry: Pharmacogenomic Data Submissions – 3/22/2005|
|Attachment to Guidance on Pharmacogenomic Data Submissions – 3/22/2005|
|Guidance for Industry: Regulation of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) – Small Entity Compliance Guide – 8/24/2007|
|Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) – 8/8/2007|
|Guidance for Industry: Exports Under the FDA Export Reform and Enhancement Act of 1996 – 7/24/2007|
|Draft Guidance for Industry: Cooperative Manufacturing Arrangements for Licensed Biologics – 7/20/2007|
|International Conference on Harmonisation (ICH); Draft Guidance: Q10 Pharmaceutical Quality System – 7/12/2007|
|Draft Guidance for Industry: Preparation of IDEs and INDs for Products Intended to Repair or Replace Knee Cartilage – 7/6/2007|
|Draft Guidance for Industry: Integrated Summaries of Effectiveness and Safety: Location Within the Common Technical Document – 7/2/2007|
|Draft Guidance for Industry: Providing Regulatory Submissions in Electronic Format – Receipt Date – 6/5/2007|
|Guidance for Industry: Computerized Systems Used in Clinical Investigations – 5/10/2007|
|Draft Guidance for Industry: Protecting the Rights, Safety, and Welfare of Study Subjects – Supervisory Responsibilities of Investigators – 5/10/2007|
|Draft Guidance for Clinical Investigators, Sponsors, and IRBs: Adverse Event Reporting – Improving Human Subject Protection – 4/17/2007|
|Draft Guidance for Industry: Dosage and Administration Section of Labeling for Human Prescription Drug and Biological Products – Content and Format – 4/9/2007|
|Guidance for Industry: Gene Therapy Clinical Trials – Observing Subjects for Delayed Adverse Events – 11/28/2006|
|Guidance for Industry: Biological Product Deviation Reporting for Licensed Manufacturers of Biological Products Other than Blood and Blood Components – 10/18/2006|
|Draft Guidance for Industry: Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling – 9/11/2006|
|International Conference on Harmonisation (ICH); Guidance for Industry: S8 Immunotoxicity Studies for Human Pharmaceuticals – 4/12/2006|
|Guidance for Clinical Trial Sponsors: Establishment and Operation of Clinical Trial Data Monitoring Committees – 3/27/2006|
|Guidance for Industry: Using a Centralized IRB Review Process in Multicenter Clinical Trials – 3/15/2006|
|Draft Guidance for Industry: Patient-Reported Outcome Measures: Use in Medical Product Development to Support Labeling Claims – 2/2/2006|
|Guidance for Industry: Adverse Reactions Section of Labeling for Human Prescription Drug and Biological Products – Content and Format – 1/18/2006|
|Guidance for Industry: Clinical Studies Section of Labeling for Human Prescription Drug and Biological Products – Content and Format – 1/18/2006|
|Draft Guidance for Industry: Warnings and Precautions, Contraindications, and Boxed Warning Sections of Labeling for Human Prescription Drug and Biological Products – Content and Format – 1/18/2006|
|Draft Guidance for Industry: Labeling for Human Prescription Drug and Biological Products – Implementing the New Content and Format Requirements – 1/18/2006|
|Guidance for Industry: Fast Track Drug Development Programs – Designation, Development, and Application Review – 1/11/2006|
|Guidance for Industry: Collection of Race and Ethnicity Data in Clinical Trials – 9/19/2005|
|International Conference on Harmonisation (ICH); Guidance for Industry: Q5E Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process – 6/29/2005|
|Reviewer Guidance: Evaluating the Risks of Drug Exposure in Human Pregnancies – 4/27/2005|
|International Conference on Harmonisation (ICH); Guidance for Industry: E2E Pharmacovigilance Planning – 3/31/2005|
|Guidance for Industry: Good Pharmacovigilance Practices and Pharmacoepidemiologic Assessment – 3/25/2005|
|Guidance for Industry: Continuous Marketing Applications: Pilot 2 – Scientific Feedback and Interactions During Development of Fast Track Products Under PDUFA – 10/6/2003|
|Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice – 9/29/2004|
|Draft Guidance for Industry and FDA: Current Good Manufacturing Practice for Combination Products – 9/29/2004 – (PDF – 134 KB)|
|Guidance for Industry: Available Therapy – 7/21/2004|
|Draft Guidance for Industry: Information Program on Clinical Trials for Serious or Life-Threatening Diseases and Conditions (Revision 1) – 1/26/2004|
|Guidance for Industry: Information Program on Clinical Trials for Serious or Life-Threatening Diseases and Conditions – 3/18/2002|
|Draft Guidance for Industry: Comparability Protocols – Protein Drug Products and Biological Products – Chemistry, Manufacturing, and Controls Information – 9/3/2003|
|Guidance for Industry: Exposure-Response Relationships – Study Design, Data Analysis, and Regulatory Applications – 5/5/2003|
|Guidance for Industry: Source Animal, Product, Preclinical, and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans – 4/3/2003|
|Draft Guidance for Industry; Comparability Protocols – Chemistry, Manufacturing, and Controls Information – 2/20/2003|
|Draft Guidance for Industry and Reviewers on Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers – 1/15/2003|
|Draft Guidance for Industry: Drugs, Biologics, and Medical Devices Derived from Bioengineered Plants for Use in Humans and Animals – 9/6/2002|
|Guidance for Industry: Container Closure Systems for Packaging Human Drugs and Biologics; Questions and Answers – 5/13/2002|
|Guidance for Industry: Container Closure Systems for Packaging Human Drugs and Biologics; Chemistry, Manufacturing, and Controls Documentation – 7/7/1999|
|Guidance for Industry: IND Meetings for Human Drugs and Biologics; Chemistry, Manufacturing and Controls Information – 5/25/2001|
|ICH Guidance for Industry: E 10 Choice of Control Group and Related Issues in Clinical Trials – 5/11/2001|
|Guidance for Industry: Q & A Content and Format of INDs for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-Derived Products – 10/3/2000|
|Guidance for Industry: Content and Format of Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-derived Products – 11/1995|
|ICH Guidance on Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products – 8/18/1999 – (PDF – 58 KB)|
|ICH Guidance on the Duration of Chronic Toxicity Testing in Animals (Rodent and Nonrodent Toxicity Testing); Availability – 6/25/1999 – (PDF – 22 KB)|
|Guidance for Industry: For the Submission of Chemistry, Manufacturing and Controls and Establishment Description Information for Human Plasma-Derived Biological Products, Animal Plasma or Serum-Derived Products – 2/17/1999|
|Guidance for Industry: Population Pharmacokinetics – 2/10/1999|
|Draft Guidance for Industry: General Considerations for Pediatric Pharmacokinetic Studies for Drugs and Biological Products – 11/30/1998|
|ICH Guidance on Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin – 9/24/1998 – (PDF – 89 KB)|
|ICH Guidance on Quality of Biotechnological/Biological Products: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products – 9/21/1998 – (PDF – 47 KB)|
|Guidance for Industry: Providing Clinical Evidence of Effectiveness for Human Drugs and Biological Products – 5/15/1998|
|Guidance for Industry – Changes to an Approved Application: Biological Products – 7/24/1997|
|Guidance for Industry – Changes to an Approved Application for Specified Biotechnology and Specified Synthetic Biological Products – 7/24/1997|
|International Conference on Harmonisation (ICH); Guidance for Industry: S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals (PDF) – 7/1997|
|Guidance for Industry for the Submission of Chemistry, Manufacturing, and Controls Information for a Therapeutic Recombinant DNA-Derived Product or a Monoclonal Antibody Product for In Vivo Use – 8/1996 – (PDF – 44 KB)|
|International Conference on Harmonisation: Final Guidance on Stability Testing of Biotechnological/Biological Products – 7/10/1996 – (PDF – 26 KB)|
|FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, Including Therapeutic Biotechnology-Derived Products – 4/1996 – (PDF – 25 KB)|
|Points to Consider in the Manufacture and Testing of Therapeutic Products for Human Use Derived from Transgenic Animals – 1995|
|International Conference on Harmonisation (ICH); Guideline for Industry: E1A The Extent of Population Exposure to Assess Clinical Safety: For Drugs Intended for Long-term Treatment of Non-Life-Threatening Conditions (PDF) – 3/1995|
|International Conference on Harmonisation (ICH); Guideline for Industry: S3A Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies (PDF) – 3/1995|
|International Conference on Harmonisation (ICH); Guideline for Industry: S3B Pharmacokinetics: Guidance for Repeated Dose Tissue Distribution Studies (PDF) – 3/1995|
CHARACTERIZATION OF THE PRODUCT
Because of their targeted mechanisms of action, means of manufacture, and species specificity, biologic agents must be well characterized by potency, identity/quality, purity, and stability. Typically, ex vivo assays are used to assess potency, and it can be challenging to adapt one or multiple assays to be practical or feasible for routine use in a commercial manufacturing process. Requirements for identity and purity must be well defined, and these as well as stability may change as the manufacturing process is refined. Because of the potential variability introduced by changes in the working cell bank, it is ideal to define the process and initiate scale-up of manufacturing as soon as proof of concept of clinical activity has been obtained. Finally, formulation and route of administration should be decided early in the development process. FDA may allow manufacturers of biologic products to make manufacturing changes without conducting additional clinical efficacy studies but only if “comparability” data demonstrate that the product after the manufacturing change is still identical by assays for purity, potency, and biologic activity. Changes in either formulation or manufacturing process may force “bridging” studies to demonstrate that the newer product is not only identical by potency, purity, and identity as well as pharmacokinetic (PK) profile, but that it also performs similarly in the clinic. Such bridging studies may introduce significant delays in clinical development programs.
Although manufacturing and testing practices typically evolve during product development, scale up is risky if it changes the product characteristics. Often a source of regulatory concern, it should be performed sooner rather than later, for example, before phase 3, and must be dealt with proactively ( Fig. 33-1 ).
Glycosylation occurs in the endoplasmic reticulum and Golgi apparatus as a post-translational modification of protein production. It is absent in Escherichia coli but present in yeast and highly conserved among mammalian cells (mouse myeloma, baby hamster, Chinese hamster ovary [CHO] and human), according to the cell line selected for manufacture and working cell bank. Of the tumor necrosis factor-α (TNF-α) inhibitors, adalimumab and etanercept are manufactured in CHO cells, whereas the aglycosylated F(ab)’-PEG product, certolizumab pegol, is manufactured in E. coli .
LESSON 1: DIFFERENCES IN GLYCOSYLATION PATTERNS DUE TO PRODUCTION IN MICROBIAL VERSUS MAMMALIAN CELL LINES: GRANULOCYTE MACROPHAGE/GRANULOCYTE COLONY STIMULATING FACTOR
Five different granulocyte macrophage (GM)/granulocyte colony stimulating factor (G-CSF) molecules have been produced in various expression systems. Bacterial expression ( E. coli ) yields aglycosylated molecules (molgramostim and filgrastim), yeast expression ( Saccharomyces cerevisiae ) yields O-glycosylated molecules (sargramostim) and mammalian expression (CHO cell line) yields fully glycosylated molecules (regramostim and lenograstim).
In prospective randomized placebo controlled trials, only glycosylated CSFs have been associated with improved patient survival (sargramostim and lenograstim).
In vitro and in vivo data generated from comparative studies have documented differences in biologic activity among CSFs.
A literature review of clinical trials evaluating sargramostim (glycosylated) and molgramostim (aglycosylated) concluded that molgramostim was associated with a higher incidence of adverse effects than sargramostim, as shown in the following table.
Pooled Median Frequency (%)
Other manufacturing platforms include transgenic plants and animals as well as live animal platforms such as mammary glands of living goats. Glycosylation largely determines tertiary, even quaternary structure; binding to the selected epitope as well as immunogenicity. Several examples exist in which changes in the working cell bank due to scale up issues resulted in products with differing glycosylation profiles, resulting in loss of efficacy despite identity by high performance liquid chromatography (HPLC) and other in vitro assays.
LESSON 2: RISKS OF CHANGING MANUFACTURING PROCESSES DURING CLINICAL DEVELOPMENT: PRIMATIZED ANTI CD4 MONOCLONAL ANTIBODY AND LENERCEPT
Monoclonal antibody (mAb) effector function and clearance are affected by glycosylation patterns, which depend on the production cell line [working cell bank] and culture media.
This was illustrated with a nondepleting primatized anti-CD4 mAb following a change in the working cell bank due to scale up requirements, as well as removal of products of bovine and human origin in the manufacturing process. Although the inserted coding construct remained identical, a new CHO cell line adapted to growth in cell-free media was used, resulting in sustained rather than transient T-cell depletion, infusion reactions, and less efficacy.
Similarly, attempts to improve manufacturing yield of the p55 soluble TNF-α receptor I (sTNF RI-Fc) construct lenercept resulted in inter-batch differences in glycosylation patterns with loss of efficacy, despite identity in assays of purity, potency, and specificity.
Differences in glycosylation patterns accounted for rapid initial phases of clearance, altering the half life and analytical ultracentrifugation (AUC) of the molecule.
Production processes can also affect immunogenicity, when aggregates form after lyophilization (see Lesson 3 ).
Changes in manufacturing processes, and even the final formulation, may also introduce variability, which is exemplified by the alterations in final manufacture of erythropoietin, which resulted in cases of pure red cell aplasia ascribed to increased immunogenicity of the altered product.
LESSON 3: SUBTLE CHANGES IN MANUFACTURING PROCESSES CAN SIGNIFICANTLY AND DETRIMENTALLY ALTER IMMUNOGENICITY, DEMONSTRATED BY AGGREGATE FORMATION WITH RECOMBINANT ERYTHROPOIETIN
Since 1998, approximately 200 suspected cases of pure red cell aplasia (PRCA) due to neutralizing antibodies have been reported in patients with chronic renal failure receiving erythropoietin treatment. The majority of cases were associated with the use of epoetin alfa distributed outside of the United States, manufactured at a different site than that distributed within the United States. Immunogenicity of this preparation appeared to occur when polysorbate 80 and glycine were substituted for human serum albumin in the formulation to comply with new regulations from the European regulatory authorities.
It is believed that the new formulation was less stable, so that aggregates might form under adverse storage circumstances, such as prolonged exposure to increased temperatures. The presence of aggregates increase the likelihood of antibody formation following subcutaneous administration, and antierythropoietin antibodies observed in patients with PRCA appear to be directed against the protein moiety of the molecule. It is not known whether subtle differences between the carbohydrate moieties of epoetin alfa and beta have had any impact on this observed immunogenicity.
With a well-characterized mechanism of action, animal models are considered to have high relevance to clinical use of biologic agents. Nonetheless they must be viewed with caution in terms of their interpretability and predictive value to human disease. Owing to the lack of species homology for many products, murine-specific analogs may be used in disease models in rodents. With sufficient homology regarding target binding or mechanism of action, pharmacodynamic (PD) effects in primates may be informative. Clearly, differences in the underlying immune systems may result in unexpected or difficult to interpret findings. Most products designed for treatment of RA have been studied in adjuvant-induced (AIA) or collagen-induced (CIA) arthritis in murine systems: rat or mouse. CIA in rhesus monkey offers the potential opportunity to use the human product, but the arthritis in this model is often unpredictable and very severe—and forces use of a valuable animal resource. Gene therapy products have been extensively studied in antigen-induced arthritis in rabbits; B-cell targeted therapies by demonstrating B-cell depletion in non-human primates ( Table 33-3 ).