Assessing Higher Order Structure and Comparability of Protein Therapeutics

Transcription

1 Assessing Higher Order Structure and Comparability of Protein Therapeutics a Regulator s Perspective Evi Struble, PhD Pharmacologist CBER/OBRR/DH 1

2 Disclaimer This presentation reflects the views of the author and should not be construed to represent FDA s views or policies. Examples are provided for illustration purpose only. 2

3 Outline Products we regulate Product characteristics Complex o pe pocess process Example Complex structure Example FDA Guidance 3

4 OBRR Divisions OFFICE OF BLOOD RESEARCH & REVIEW DIVISION OF BLOOD APPLICATIONS DBA DIVISION OF HEMATOLOGY DH DIVISION OF EMERGING AND TRANSFUSION TRANSMITTED DISEASES DETTD Blood and plasma center licenses Blood establishment t software Blood grouping and HLA reagents Next Slide Blood donor screening tests for infectious agents Retroviral diagnostics 4

5 Biologics in Division iii of Hematology Laboratory of Lb Laboratory of Laboratory of Biochemistry and Plasma Derivatives Hemostasis Vascular Biology (LPD) (LH) (LBVB) Laboratory of Cellular Hematology (LCH) Immunoglobulins Clotting factors Vascular proteins Cellular products Antitoxins Antivenoms Alpha-1 p PI Plasma derived Recombinant Wound sealants Device/biologics Volume expanders Oxygen carriers Enzyme inhibitors Plasma Storage devices Anticoagulants Collection devices 5

6 Complex Manufacturing Process Products are derived from highly complex starting material Blood/Plasma (human or animal), mammalian and yeast cell culture, milk Process is aseptic/sterile and includes robust viral removal and deactivation High temperature, solvent/detergent treatment Production includes many steps with very tightly controlled operating parameters to ensure purity Very slight changes in ph, temperature can change the impurity profile/product attributes 6

7 Complex Manufacturing Example Cohn Fractionation of Plasma Conditions Fraction I Fraction II Fraction III Fraction IV Fraction V Ethanol %: ph: Temperature ( C) Protein fraction (%): Supernatant II+III Supernatant IV Supernatant V Precipitate V Supernatant I Precipitate IV Plasma Precipitate II+III Precipitate I Fibrinogen Factor VIII IgG A1 Antitrypsin Antithrombin III Albumin Diafiltration; Chromatography Purification; Viral inactivation; Limited proteolysis; Lyophilization Cohn et al, J Am Chem Soc Mar;68: K.Hiltbrunner, Genetic Engineering & Biotechnology News, Feb 1, 2010 Wikipedia

8 Complex Products Example: Factor VIII (~300 kda) Heavy Chain Ca 2+ Light Chain Stabilization by vwf N A1 A2 B A3 C1 C2 C Courtesy of Dr. Andrey Sarafanov LH, DH,OBRR Cleavage by Thrombin (activation) A2 FVIII / vwf Complex Thrombin A3 A1 C2 C1 8

9 Complex Biologically Active Products Example: Coagulation Pathway Highly complex macromolecules, involved in tightly regulated pathways Coagulation Cascade Slight modifications in structure or changes in product attributes such as impurity profile can have grave consequences Thrombosis? Bleeding Sources of activation: Artificial surfaces Protein aggregates Denatured proteins Plasma storage Certain plasma fractionation steps cont tact pathwa ay nsic pathw way extri PK FXII TF VIIa Ka HK XIIa HK FXI FVII TFPI FIX common pathway Courtesy of Dr. Mikhail V. Ovanesov LH, DH,OBRR HK XIa FX C1INH A2AT IXaVIIIa FII Xa Va fibrinogen PC PS FVIIITM IIa FV fibrin ATIII a2mg HCII 9

10 Complex Products Example: vwf in KO Mouse Enzyme deficiency recapitulates TTP-like pathohistologic hi t i changes following administration of a recombinant von Willenbrand Factor preparation in knock-out mice 2012 by American Society of Hematology 10 Schiviz A et al. Blood 2012;119:

11 ICH Q5B Structural Characterization, Purity, Stability and Consistency of New Products Identify nucleic acid coding sequence or the transcription products, as appropriate. ICH M4Q Important to ensure no drifting/mutation Submit details on primary, secondary and higher order structure for the desired product and product related substances. In addition to information on biological activity of the product, the guidance recommends the submission of information on the molecular mass, details of posttranslational forms (e.g.,,gy glycoforms), and a schematic amino acid sequence showing glycosylation sites or other post translational modifications. ICH Q6B Physicochemical characterization molecular weight, isoform pattern, extinction coefficient, electrophoretic pattern, liquid chromatography patterns and spectroscopic profiles. Primary structural characterization amino acid composition, terminal sequencing, peptide mapping, sequence, disulfide bonding patterns, carbohydrate content and structure. 11

12 ICH Q6B, continued Demonstrate product purity Use a combination i of analytical ltechniques. Small conformational changes in the protein structure may be difficult to identify Results may depend on the method used Examples of impurities iti that t may be found din a product Process related impurities (filter aid added during in depth filtration, detergent added during solvent detergent treatment ) Product related ltdimpurities iti Degradation products, protein aggregates inherent degree of structural heterogeneity is expected in proteins the pattern of heterogeneity shouldbe defined and lot to lot consistency and comparability between lots should be demonstrated Impurities can have known structures, may be partially characterized, or remain unidentified When adequate quantities are present/can be generated they should be characterized to the greatest possible extent their biological activity evaluated Product characterization sets a basis for establishing specifications 12

13 Defined in ICH Q6B Specifications A set of test methods and acceptance criteria that are used in product testing to ensure that product is acceptable for its intended use Linked to the preclinical and clinical studies Develop specifications that provide assurance that changes in purity and potency are detected. Methods used need to be validated 13

14 Stability Throughout Shelf Life ICH Q1A and ICH Q5C Evaluate attributes at risk ikfor change during storage and likely to influence quality, safety, and efficacy Use validated d methods Q2(R1) 14

15 Manufacturing Changes and Comparability ICH Q5E Comparing gquality data on the pre and post change product Comparing data to predefined criteria should allow an objective assessment of whether or not the pre and postchange product are comparable Qualified methods : scientifically soundand provide results that are reliable Evaluate all data collected routine batch analyses, in process control, process validation/evaluation, characterization and stability More than one analytical technique may be needed for the same quality attribute. Orthogonal methods 15

16 Manufacturing Changes and Comparability Determine higher order structure (secondary, tertiary, and quaternary structure) is maintained If higher order structural information cannot be obtained, a relevant biological i l activity ii assay with high hprecision and accuracy could indicate that changes in conformational structure have not occurred. However biological assays may not be sensitive to detect the small relative amounts of aggregates Initiation of real time/real temperature as well as accelerated stability studies on the product to detect if quality is potentially affected by the change. Stability studies performed under accelerated conditions, such as increased temperature, light intensity, agitation, freeze thaw cycles... 16

17 ICH Q6B Comparability, Continued Under certain circumstances physicochemical tests may replace a biological assay to measure the biological activity wheresufficient physicochemical information exists when the higher order structure can be thoroughly established by qualified methods a relevant physicochemical h i correlation lti with biologic i activity it is demonstrated, t d and a well established manufacturing history exists if a consistent pattern of product heterogeneity is demonstrated, an evaluation of the activity, efficacy and safety (including immunogenicity) of individual forms may not be necessary if not, the significance of such alterations should be evaluated 17

18 References FDA Guidance ceregulatoryinformation/guidances/default.h tm Thank you 18