IIR International Generics Strategy:Pre-Conference Symposium: Comparable Biotech Products

Multisource Biologics: Demonstrating Therapeutic Equivalence of Biotech-derived

Products

Robert ZeidPrincipal ConsultantTLI Development113 SE 5th St.Oak Island, North Carolina 28465USA

[phone]: (910) 278-9176[FAX]: (910) 278-9022[e-mail]: robert_zeid@msn.com

Demonstrating Therapeutic Equivalence of Biotech-derived Products

Therapeutic Equivalence Components: Analytical, PK/PD, and Surrogate EndpointsStructure-Activity Relationships (SAR)Analytical Equivalence:

Methods OverviewCharacterization TestingBioassay Equivalence: Points to ConsiderComparability Protocols – Characterization Testing vs. Release Testing

Bioequivalence:Overview of Classic Drug Model ApplicationsPharmacokinetic/ Pharmacodynamic (PK/PD) RelationshipsSAR Links to PK/PD

Surrogate Endpoint EquivalenceManufacturing-Related IssuesIntegration of Analytical Testing Profile to Manufacturing and Stability ArenasClasses of ProductsSummary of Key Points

Therapeutic Equivalence Components: Analytical, PK/PD, and Surrogate Endpoints

Analytical ComparabilityPhysico-chemical Comparability

Confirmation of Primary, Secondary, and Tertiary StructuresStructure-Activity Relationships (SAR) Database Analysis of Differences: Purity vs. Impurities for Product Classes

SAR Links to Manufacturing and StabilitySAR Links to Bioassay Equivalence and Surrogate EndpointsCompendial reference methods: Cross-over studiesBioassay Equivalence

Bioequivalence: PK/PDPharmacokinetic/ Pharmacodynamic (PK/PD) RelationshipsSAR Links to PK/PD and Dose-response Curve

Surrogate EndpointsUse of Validated Surrogate EndpointsEstablishing New Surrogate EndpointsSAR Links to Endpoints

Structure-Activity Relationships (SAR)

SAR databases established for unique product classes (e.g., interferon, interleukin, tissue plasminogen activator, colony stimulating factors, etc.)

Gather public information (e.g., SBA, EPARs, published articles, etc.)Compare your product against innovator/ reference standard

Differences in physico-chemical profiles correlated to impact on purity, potency, and safety

What is sensitivity of your assay method/ analytical method?Establish sensitivity criteria via forced degradation studies and correlate specifications to product failure

SAR database grows with additional links to purity/ impurity profiles assessed for various manufacturing schemes, stability, surrogateendpoints, etc.Quantitative SAR (QSAR) databases established from sound modeling correlated with SAR data

Analytical Equivalence: Methods Overview

Products often assessed for purity, potency, and safety – and testing reflects this holy trinity, such as:Purity (physico-chemical characterization) of drug substance assessed by:

Amino acid analysis (e.g., AA sequencing (entire sequence or amino- and carboxy-terminal sequences)Peptide mapping via restriction enzymatic digestion/ fragment analysisDetermination of sulfide linkageCircular DichroismChromatography: HPLC (chiral, reverse phase, size exclusion, ion-exchange), GC (GC-MS, GC-FID), TLC, etc.Spectroscopy: MS, MALDI-TOF, EI, CI, FAB, ESI, NMR, FT-NIR,Electrophoresis (reduced and non-reduced): SDS PAGEImmunochemical analysis (e.g., ELISA, RIA, EIA, RID (radial immunodiffusion), flow cytometry, Western Blot, Southern Blot, Northern Blot, etc.)Microheterogeneity analysis (e.g., glycosylation profiling, IgG sub-types)

Potency assessed via bioassays (compendial or proprietary) and PK/PD linksSafety assessed via sterility, bioburden, LAL, residual host DNA, impurities, immunogenicity, product-specific toxicities, etc.

Analytical Equivalence: Characterization Testing

Testing may be compartmentalized into three regimens: (1) intact structure, (2) post-enzymatic digestion, and (3) individual peptide sequences.Entire/ Intact Structure:

Chromatography: HPLC (chiral, reverse phase, size exclusion, ion-exchange), GC (GC-MS, GC-FID), TLC, etc.Circular DichroismDetermination of sulfide linkageSpectroscopy: MS, MALDI-TOF, EI, CI, FAB, ESI, NMR, FT-NIR,Immunochemical analysis (e.g., ELISA, RIA, radial immunodiffusion, flow cytometry, etc.)Electrophoresis (reduced and non-reduced): SDS PAGEMicroheterogeneity analysis (e.g., glycosylation profiling, IgG sub-types)

Post-enzymatic Digestion:Chromatography: HPLC (chiral, reverse phase, size exclusion, ion-exchange), GC (GC-MS, GC-FID), TLC, etc.Electron scatter (ESI-LS/MS)Circular DichroismSpectroscopy: MS, MALDI-TOF, EI, CI, FAB, ESI, NMR, FT-NIR,Collisionally Induced Dissociation (CID)

Peptide Sequences:Amino acid analysis (e.g., AA sequencing (entire sequence or amino- and carboxy-terminal sequences)Peptide mapping via restriction enzymatic digestion/ fragment analysis

DNA Sequencing: Mutations and Revertants

Recommended by FDA for characterization of cell lines, expression vectors, and plasmidsStability of plasmid boosted through some redundancyManual (radioactive) vs. automated (fluorescent), cycle vs. isothermal methods, end label vs. internal labelsSophisticated software available for analysis and trending; some with structural projectionsMutation detection and localization kits also commercially availableTemplates/ kits also available for single-stranded (ss) DNA

Bioassay Equivalence: Points to Consider

Binding is linked to signal transduction: Need to use bioassays that assess an effect via signal transduction – not just binding alone

Compendial assays vs. non-compendial or proprietary assays: Need to assess if compendial methods (if applicable) are sensitive enough for distinguishing slight variations in critical product parameters; if compendial methods exist, do crossover studies for comparison

Establishing critical product parameters: Need to assess/ correlate with functional activity/ stability in bioassays (e.g., antigen content vs. immunogenicity)

Relative Standard Deviation (RSD): What is noise-to-signal ratio for bioassay? > 25% RSD? Need to use statistical modeling to ensure adequate sample size for assessing wide variability (see Juran’s Handbook of Quality, Chapter 25 – Acceptance Sampling).

Use “gold standards” in assays: Compare product to compendial reference standards or lots of innovator product. Establish what physico-chemical differences relate to bioassay activity.

Process Development: Using neural nets to (1) help establish critical process parameters with impact on product quality and (2) develop an “edge of failure” for any given process and establishing ranges within those critical areas. Don’t depend on early stage material for market needs.

Statistical Process Control (SPC): Sampling: continued analysis of product quality against long-term manufacturing history

Combining Tests into a Panel: Complex moieties may require several tests combined into a conglomerate panel to assess potency (e.g., RP-HPLC, SDS PAGE, and bioassay)

Express Potency as Quantifiable Units: Need to express potency in some quantifiable measure -units [of activity]/ mg of compound (anhydrous weight)

Analytical Equivalence: Comparability Protocols –Characterization Testing vs. Release Testing

Characterization Testing: Establishing a “Gold” reference standard of internal product – comparison to external standards (if available)

Establishing critical product parameters: assessing correlation with functional activity/ stability

Release Testing: refinement of characterization testing required to assess product identity, safety, and purity.

Scale of Manufacturing: Pilot vs. Commercial scale changes – what to assess?

Process Development: Using neural nets to help establish critical process parameters with impact on product quality

Process Development: Using neural net analysis to develop an “edge of failure” for any given process and establishing ranges within those critical areas.

Process Validation: confirmation of product vs. process with release testing + stability data to meet SUPAC/ BIOSUPAC

Statistical Process Control (SPC): Sampling: continued analysis of product quality against long-term manufacturing history

Bioequivalence: Overview of Classic Drug Model Applications

21 CFR 320.21 specifies BA/BE requirements for NDA/ ANDA and/or waivers based on chemical equivalence (or pharmaceutical equivalence)BA/BE study requirements typically waived (21 CFR 320.22) for parenteral, ophthalmic, or otic drug products based on assumed 100% BA of chemically equivalent materialException to BA/BE waiver is for pro-drugs which may have different ADME profiles prior to activationBA measured as: AUC and Cmax, ; Tmax collected for informationBA/BE may also be established via in vitro testing when correlated to in vivoparameters Recent proposed BCS classifications may allow expanded BA/BE waivers for solid oral dosage forms on the basis of demonstrated in vitro characterization (e.g., dissolution, solubility, permeability, etc.)Are BA/BE studies sensitive enough to measure impact of subtle physico-chemical changes for biologics? If not, then are comparable results from such a BA/BE study (comparator vs. innovator) definitive?

Bioequivalence: Pharmacokinetic/ Pharmacodynamic (PK/PD) Relationships

Scenarios for PK/PD relationships: PK < PDPK = PDPK > PD

Current drug model PD/PD study design may be insufficient for biotherapeutics with long-lasting effects due to downstream cascade or impact on secondary systems.Depending on product class and PK/PD relationship, biostudies may need to add safety evaluations (e.g., immunogenicity) and surrogate endpoints.Some biotherapeutics may show similar/ different AUC profiles but have same impact on in vitro tests that are closely correlated to in vivo parameters of efficacy.

Bioequivalence: SAR Links to PK/PD

SAR databases must include links to both preclinical and clinical PK/PD data:

Can relate PK/PD to SAR purity and impurity levels for safety qualificationCan relate PK/PD to potency evaluation in vivo when observed in vitro changes happenCan relate SAR activity to observed activity ex vivo or in vitro testing at various receptor sites/ tissues – or impact on surrogate endpoints in PK/PD study

SAR databases should include links to manufacturing processes:Can relate PK/PD to manufacturing changes and the sensitivity of PK/PD studies to discern significant product profile changes

When finished, the SAR database may be used in conjunction with some computer modeling, PCA analysis, or extrapolation software to evaluate structural changes and the necessity of certain types of testing.Given expense of PK/PD studies, some forced degradation of product may be used to evaluate SAR in preclinical studies first – then evaluate correlative value of observed effects in human studies.

Immunogenicity: Evaluation of Impact on Safety or Clinical Efficacy

Immunogenicity studies will be virtually standard for pioneer and comparator companies wanting to do major changes. It’s prudent to have protocols and assays for FDA/EMEA review prior to doing work.

Antibody formation? (YES or NO). If yes, what type of antibodies are formed? Binding only or neutralizing or both? What rates of formation occur? Does the antibody profile change over time or duration of exposure? What is clinical impact on safety or efficacy?

Note that detection of binding and neutralizing antibodies do not automatically mean some clinical impact – needs clinical correlation.Issue over neutralizing Abs and how they could complicate efficacy is still debatable. Could they continue to work even after the treatment period is over? Other safety issue could be the deposition of immune complexes in lung (e.g., CIC). Sample antibody profiles reported for major product categories are cited below.Fortunately, anaphylaxis to proteins is a rare reaction in humans.

Surrogate Endpoint Equivalence: Use of Validated Endpoints

Vaccines: Geometric Mean Titer (GMT) and seroconversion (vs. demonstrated prophylaxis from long-term studies)Fibrinolytics: in vitro clot lysis, post-MI (myocardial infarction) patency, ventricular function testing (ECG), etc. (vs. survival rates)Somatotropins: hyphosectomized rat model and comparison to external reference standards (vs. long-term growth confirmation)Cystic Fibrosis Rx: pulmonary function testing (PFT), days in ICU, days on intravenous antibiotics (vs. reduced number of exacerbations and long-term survival)HIV/ AIDS: reduction in CD4 count, viral burden, reduced rate of opportunistic infections (vs. survival rates)Arthritis: radiological imaging of joint damage, reduction of inflammatory mediators, improved joint mobility, etc.Chemotherapeutics: immunological mediators, carcinogenic antigen levels, lymph node involvement (vs. long-term survival)

Surrogate Endpoint Equivalence:Establishing New Endpoints

Vaccines: Instead of GMT or seroconversion, prophylaxis may be correlated with other blood proteins or cell typesFibrinolytics: May be able to apply latest non-invasive real-time assessments of patency and post-MI ventricular functionSomatotropins: Replace expensive and variable hyphosectomized rat model with short-term biochemical stimulation assay in comparison to external reference standardsCystic Fibrosis Rx: Ex vivo testing of sputum samples may allow precise measurements of pulmonary milieu more than pulmonary function testing (PFT), days in ICU, days on IV antibiotics, etc.HIV/ AIDS: Assess most current markers used in efficacy assessment (that have been correlated with survival, quality of life, reduced opportunistic infection rate, etc.)Arthritis: Expand upon the existing radiological, CAT scan, PET, or other non-invasive imaging of joint damage – correlate to reduction of inflammatory mediators, improved joint mobility, etc.Chemotherapeutics: immunological mediators, carcinogenic antigen levels, lymph node involvement, ex vivo assessments, reduced infections, reduced recurrence rates, etc.

Surrogate Endpoint Equivalence:SAR Links to Endpoints

SAR links of your compound to clinical endpoints

SAR links of your compound to the innovator/ compendial reference material also used in clinical surrogate endpoint analysis

SAR links of your compound to manufacturing changes and correlation to clinical endpoints

SAR links of your compound’s stability profile to innovator/ compendial reference material stability profile and correlation to impact on clinical endpoints:

Manufacturing-Related Issues

Process Design - Choice of System ComponentsCharacterization TestingScale of Manufacturing: Pilot vs. CommercialProcess Development: Neural Nets and Edge of FailureProcess Development: Overview of Mammalian CellsBioprocessing Overview: Bioreactor ConsiderationsBioreactor DesignDownstream PurificationComparability Studies

Manufacturing-Related Issues

Process Design - Choice of System Components:Eukaryotic vs. Prokaryotic Organisms

Microbial (e.g., E. coli): inexpensive and versatile in bioprocessing scenarios, but limited utility for products with post-translational changes such as glycosylation or certain types of protein foldingMammalian cells (e.g., Chinese Hamster Ovary): while tissue cultures are more expensive to maintain, grow, and harvest – it has advantages in efficiency for post-translational changes and secretion of desired product: limitations in that mammalian cells grow on surfaces or must be adapted for suspended cultures, making large scale production trickyInsect cells (e.g., Drosophila species): can be used in continuous circulation tanks since they can take more abuse in shear pressureYeast (e.g., Saccharomyces): limited utility of plasmid vectors in yeast due to difficulty inintegration; Yeast Artificial Chromosomes (YAC) used to mimic chromosome – mostly used in R&D but not too much for commercial Plant cell cultures can also be used (e.g., large scale fermentation of Taxol). Increased taxol levels from of taxol are < 0.1% (dry weight basis from Pacific Yew tree bark) and now by cell culture, can be up to 120 mg/ gram of dry weight basis.

Metabolic Engineering vs. Expressions SystemsMetabolic engineering is continuous (cost-effective); expression systems are more on a batch basis (typically what FDA has seen)Focus is to minimize burden of plasmid on organism by using inducible enzyme system

Comparison of Protein Expression Systems

Manufacturing-Related Issues

Characterization Testing:Host cell systems

Source, relevant genotype and phenotype, as well as stability, purity, etc.

Gene ConstructGene construct and restriction enzyme digestion map, complete nucleotide sequencing with regulatory elements identified

VectorSource and function of vector components, origins of replication, antibiotic resistance, restriction enzyme digestion map, critical genetic markers identified

Expression system: Final Gene ConstructDescription of assembly, restriction enzyme digestion map, identity of critical sites, etc.

Cloning & Establishment of Cell Lines (MCB/ MWCB)Mechanism of transfer of final gene construct, copy number, physical state of final product (e.g., integrated vs. extra-chromosomal), selection criteria, etc.

Resulting Product & End-of-Production (EPC) CellsAAA, AA sequencing, peptide mapping, determination of disulfide linkage, SDS PAGE (reduced and non-reduced), isoelectric focusing, HPLC, SEC HPLC, RP HPLC, mass spectroscopy, assays to detect proteins including deamidated, oxidized, cleaved, and aggregated forms, AA substitutions, adducts/ derivatives, assays to detect DNA, residual host cell proteins, and reagents, immunochemical analyses, bioburden, endotoxin, sterility; Post-translational modification testing such as glycosylation, sulfation, phosphorylation, or formylation; Additional testing for derivatization from/ with toxins, conjugates, radionuclides, etc.

Manufacturing-Related Issues

Scale of Manufacturing: Pilot vs. CommercialPilot scale:

use for process development (e.g., neural nets, edge of failure vs. process optimization)Commercial scale:

may be done at same site or with similar equipment but on larger scale; should be material used in regulatory filing; should support filing with at least 3 months stability from commercial runcompare against 6 months (or more) from pilot scale lots;compare both against innovator stability profile

Manufacturing-Related Issues

Process Development: Neural Nets & Edge of FailureProcess focus is on biobatch parameters such as pH, time, temperature, fed-batch characteristics for translation of proteins, etc.Watch copy number, growth rates, and expressed product vs. plasmid stabilityProcess Control: PID (Proportional Integral Detector) is integrated into commercial fermentation technology as programmable logic controller (PLC) languagePrinciple Component Analysis (PCA): is part of the modeling used in neural net analysisPCA/ PLS (projection to latent structure) used to analyze several variables at once and assess covariance of each – relevance to “good vs. bad” batches

Principle Component Analysis (PCA)

Manufacturing-Related Issues: Overview of Mammalian Cells

CHO is the most commonly used tissue culture line, but mouse myeloma cell lines are also becoming more popularAttachment-dependent cell lines are not the most optimal for production, since the attachment requirement is hard to implement on large scaleThe demand for non-essential amino acids becomes greater when you have TC under rapid growth, so most media come with a combined essential and non-essential amino acids.When shifting to serum-free media, you need to assess the minimum concentration of added components (e.g., insulin, transferrin) that will get you to the near maximal levels of cell growth.Osmolality: when adding nutrients in a fed batch scenario, it’s important to use osmotically balanced solutions so that the continuous addition of solutes don’t impact cell viability.Oxygen saturation is not toxic to the TC cells when done for a short time; useful to determine the duration of oxygen tension.Cell culture bioreactors: batch, fed batch, continuous, or perfusion it’s important to simultaneously feed glucose and glutamine! Glutamine prolongs the stationary phase of growth – less cell die off.Productivity (e.g., Mab production) can be higher when cells are grown at specific growth rates less than the maximal. The theory is that Mab production is primarily in G1 growth phase of the cell and the slower growth rate allows a prolonged G1 cycle.

Downstream Purification

Process Sequence and Engineering PrinciplesProduct purity constraintsCost of production/ yieldScalabilityReproducibility/ ease of implementation

Downstream Purification

Use processes with high purity factor, such as:Affinity chromatographyInorganic adsorptionGel filtrationHydrophobic chromatographyIon exchangeDetergent extractionAqueous separationPrecipitation

Comparability Studies

Links: Impact of Manufacturing Controls to -Analysis of Product Characteristics: Comparability testingSurrogate Equivalence: Impact on activityStability: Emergence of undetected impuritiesAnalytical Methods: Consistency of methodology/ dataSetting Rational Specifications

Integration of Analytical Testing Profile to Manufacturing and Stability

FDA Guidance on Drug & Biologics Stability (6/98)Length of stability studies with pilot scale materialLength of stability studies with commercial scale material (or representative of commercial scale)Analytical links to manufacturingAnalytical links to innovator stability performanceAdequate sampling

Classes of Products

fibrinolytics (e.g., Activase, Abbokinase)human insulin (e.g., Humulin)monoclonal antibodies (e.g., ReoPro, Herceptin, Oncoscint)polyclonal antibodies (e.g., CytoGam)interferon (e.g., Intron A, Roferon A, Alferon N)interleukins (e.g., Proleukin)vaccines; monovalent vs. polyvalent; unconjugated vs. conjugated; adjuvant vs. none (e.g., Engerix B, DPT, polio, measles, chicken pox, mumps, etc.)somatotropins (e.g., Protropin, Nutropin)

Fibrinolytics: Activase (alteplase)

Physico-chemical Description: a serine protease (glycoprotein of 527 AA) which enhances the fibrin conversion of plasminogen to plasmin; see USP reference for compendial testing profileClinical Indications: acute myocardial infarction (AMI), acute ischemic stroke, pulmonary embolismApproved Products: Activase (alteplase) (Genentech); Method(s) of Manufacture: complementary DNA (cDNA) from a human melanoma cell line introduced into a Chinese Hamster Ovary(CHO) cell line + gentamycin (selection pressure)Analytical Testing Considerations: biological potency assessed by in vitro clot lysis assay vs. WHO standard; alteplase activity = 580,000 IU/mg; bulk sterile material assayed for viral, mycoplasma, DNA contamination, and molecular identity (e.g., tryptic mapping, specific activity, & protein content); finished product tested for appearance, sterility, safety, pyrogenicity, identity, purity, potency, pH, inorganic phosphate content, arginine content, polysorbate content, moisture, and fill volume.

Fibrinolytics: Abbokinase (urokinase)

Physico-chemical Description: a serine protease (glycoprotein of 527 AA) which enhances the fibrin conversion of plasminogen to plasmin;Clinical Indications: acute myocardial infarction (AMI), pulmonary embolism, and IV catheter clearanceApproved Products: Abbokinase (urokinase) (Abbott Labs); Method(s) of Manufacture: cultured from human kidney cells (removed from newborns who died)Analytical Testing Considerations: biological potency assessment –also by in vitro clot lysis assay vs. WHO standard ? Bulk sterile material probably assayed for viral and mycoplasma contamination, molecular identity (e.g., tryptic mapping, specific activity, & protein content). Finished product tested probably tested for appearance, sterility, safety, pyrogenicity, identity, purity, potency, pH, moisture, and fill volume.

Human Insulin

Physico-chemical Description: a protein (MW = 5807.69) with two chains; Chain A (21 AA) is linked to Chain B (30 AA) via cysteine linkages at positions # 7 and 20 (on Chain A) to positions # 7 and 19 (on Chain B). Insulin available in 6 formulations: R (regular), N (NPH), L (Lente), U (Ultralente), 50/50 NPH:R, and 70/30 NPH:R Clinical Indications: treatment of diabetesApproved Products: Humulin R (Lilly) and Humalog (lispro injection) (Lilly)Method(s) of Manufacture: rDNA expression in E. coli or by chemical modification of pork insulin; re-folding efficiencies only about 30% but still cheaper to do via E. coli than mammalian cell lines; arginine used a lot in formulations to protect proteinAnalytical Testing Considerations: see USP standards for Insulin, Insulin Injection, Insulin Human, Insulin Human Injection, Isophane Insulin Suspension, Insulin Zinc Suspension, Extended Insulin Zinc Suspension, and Prompt Insulin Zinc Suspension

Monoclonal Antibodies

Physico-chemical Description: Several classes with varying properties for each: IgA, IgG, IgM, IgE, etc., plus sub-types.Clinical Indications: colorectal cancer imaging (Oncoscint), treatment of certain patients with metastatic breast cancer (Herceptin), prevention of ischemic complications post-PTCA (ReoPro), non-Hodgkin’s lymphoma (Rituxan)Approved Products: Herceptin (Genentech); Rituxan (Genentech); ReoPro (Centocor); Oncoscint (Cytogen)Method(s) of Manufacture: murine hybridoma cell lines; surface epitope masking (SEM) may be used to identify and develop certain Ab sub-typesAnalytical Testing Considerations: functional assays such as antigen-binding, neutralization assays, PK profiles; biochemical tests such as cyanogen bromide (CNBr) cleavage following N-terminal sequencing, peptide mapping, comparison of impurity & degradation profiles (via SDS PAGE), carbohydrate analysis, SEC,DSC, capillary electrophoresis, etc.

Polyclonal Antibodies

Physico-chemical Description: gamma globulinsClinical Indications: adjunct to antibiotics in treatment or prevention of infectious diseases (e.g., respiratory syncitial virus [RSV], P. aeruginosa infection in cystic fibrosis patients, S. aureus infections in renal dialysis patients, treatment of ITP in AIDS patients, etc.)Approved Products: RespiGam & Cytogam (Medimmune), WinRho SD (Cangene), Gamimmune (Bayer), Sandoglobulin (Sandoz)Method(s) of Manufacture: gamma globulins isolated by fractionation from pooled source plasma donors (either vaccinated or naturally high-titered individuals). See GMP requirements for blood and blood component collection facilities (21 CFR 606 & 640(j)).Analytical Testing Considerations: From the early 30’s to the 60’s, source plasma had high Fc fragments that precluded IV use (thus limited to IM administration). In the ’70’s, improved manufacturing allowed low enough Fc fragment levels for IV Rx – now known as IGIV. Antibody characterization testing for activity/ specificity.

Interferon (cytokines)

Physico-chemical Description: Three major classes – alpha, beta, and gamma (most potent phagocytic activity); proteins with antiviral and anti-proliferative activity; binds to cell membranes. Actions include enhancement of phagocytosis to augmented cytotoxicity. See notes below for specifics of each interferon typeClinical Indications: multiple sclerosis, chronic granulomatous disease, hairy cell leukemia and AIDS-related Kaposi’s sarcoma, genital warts, hepatitis, etc.Approved Products: Alferon N (Purdue Frederick); Intron A (Schering Plough); Roferon A (Roche); Actimmune A (Genentech); and Betaseron A (Chiron/ Berlex)Method(s) of Manufacture: rDNA in E. coli or CHO cells or induction from human leukocytes following incubation with viral challenges; purification with immunoaffinity chromatography, acidification, and gel filtration chromatography.Analytical Testing Considerations: See details below.

Interleukins

Physico-chemical Description: lymphokines with multiple effects on cell-mediated and humoral immune responses; IL-2 molecular weight about 15,300 Da.Clinical Indications: adjuncts to chemotherapy (e.g., metastatic renal carcinoma), AIDS, off-label uses, etc. Approved Products: Proleukin (aldesleukin) (Chiron Therapeutics); IL-4 and IL-10 under clinical development by several firmsMethod(s) of Manufacture: (for Proleukin) rDNA production in E. coli resulting in a non-glycosylated version of IL-2; rDNA in CHO or suspended tissue culturesAnalytical Testing Considerations: Assess biological potency against external reference standard (e.g., native human or WHO reference).

Vaccines

Physico-chemical Description: monovalent vs polyvalent; conjugated vs. not; formulated with adjuvants; etc.Clinical Indications: prevention of infectious disease (e.g., small pox, measles, polio, DPT, etc.); boosting titers as part of therapy (e.g., adjunct to cancer Rx); boosting antibody titers in sourceplasma donors for creation/ collection of specified high-titer antibodies (e.g., polyclonal Ab collection in passive immunotherapy) Approved Products: See PDR for complete listing Method(s) of Manufacture: microbial fermentation, tissue culture, egg culture, animal colonies. See May 1998 FDA guidance for details on vaccine CMC documentation.Analytical Testing Considerations: See May 1998 FDA guidance; testing varies according to vaccine type and manufacturing considerations (see below). See 21 CFR 610.53 for stability expiry dating periods. See 21 CFR 620 for additional testing for bacterial vaccines (e.g., pertussis, typhoid, anthrax, cholera, & BCG). See 21 CFR 630 for additional standards for vial vaccines.

Somatotropins

Physico-chemical Description: rDNA polypeptide of 191 amino acids (Humatrope); 192 AA (Protropin) – the extra AA being methionine; both with a molecular weight about 22,000 DaClinical Indications: long-term treatment of children with growth failure (Humatrope); AIDS-related complex; chronic renal insufficiencyApproved Products: Humatrope (Lilly); Nutropin or Protropin (Genentech); Bio-tropin (Bio-Technology General Corporation); Genotropin (Pharmacia); Nordiotropin (Novo Nordisk); Saizen (Serono Laboratories)Method(s) of Manufacture: rDNA in E. coli (Humatrope), Analytical Testing Considerations: bioactivity assessed in hypophysectomized rat model against WHO standard (3 IU)/ mg); neo-antigen testing; peptide maps, FAB MS, RP-HPLC, Raman and CD spectra, electrophoresis, tryptic mapping, and bioassay

Summary of Key Points

Demonstrating therapeutic equivalence is a mix of product complexity, clinical indications, clinical experience with product class, and regulatory pathways.Although regulatory strategy can be approximated, it is really defined after analytical and therapeutic equivalence is established (or not)Therapeutic equivalence criteria will probably be a mixture of innovator criteria + industry/ clinical experience with those classes of productsAnalytical equivalence must be established using the most current and holistic approachesAnalytical data must be extensive to support bioassay variability, manufacturing flexibility, microheterogeneity, stability changes, etc.Surrogate endpoints may be a repeat of innovator or a completelydifferent approach, depending upon FDA acceptance

Summary of Key Points

Manufacturing process controls must reflect product parameters well within ‘edge of failure’Stability data must be supported by extensive characterization testing to show subtle changes do not impact purity, potency, or safetyStability testing should include innovator/ reference product as well as comparator lotsManufacturing scale and process development changes must be supported by extensive comparability testing

List of References

FDA Guidelines, Guidances, & Points to Consider (PTC)CMC Information for a Therapeutic rDNA-derived Product or a Monoclonal Antibody Product for in vivo Use (August 1996)PTC in the Characterization of Cell Lines Used to Produce Biologicals (1993)PTC in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1994)PTC for the Evaluation of Combination Vaccines: Production, Testing, and Clinical Study (1995)PTC on Plasmid DNA Vaccines for Preventive Infectious Disease Indications (1996)CMC and Establishment Description Information for a Vaccine or Related Product (1998)CMC and Establishment Description Information for Human Blood and Blood Components Intended for Transfusion or for Further Manufacture (July 1998)CMC and Establishment Description Information for Human Plasma-derived Biological Products or Animal Plasma-derived Products (Dec 1997)Regulation of Placental/ Umbilical Cord Blood Stem Cell Products Intended for Transplantation or for Further Manufacture into Injectable Products (Dec 1995)Demonstration of Comparability of Human Biological Products, Including Therapeutic Biotechnology-derived ProductsStability Testing of Drug Substance and Drug Products (June 1998)

Code of Federal Regulations (CFR)21 CFR 600 - 680

Federal StatutesFood and Drug Administration Modernization Act of 1997 (FDAMA)Drug Price Competition and Patent Term Restoration Act of 1984 (Waxman-Hatch)Food Drug & Cosmetic Act