Licensed Biological Products with Structural Heterogeneity

Andrew C. Chang, Ph.D.Associate Director for Policy and Regulation

Division of Hematology, CBER, FDA

BPAC, Gaithersburg, MD November 4, 2005

TOPICS TO BE COVERED Structural Heterogeneity of Biological

Products: Case Studies Plasma derived products

- Andrew Chang, Ph.D. OBRR/CBER

Recombinant products and monoclonal antibodies

- Kurt Brorson, Ph.D. OPS/CDER

Structural Heterogeneity of Biological Products

Factors contributing to structural heterogeneity Biosynthetic processes used by living organisms Manufacture and/or storage of the drug substance

and drug product Control of structural heterogeneity

To demonstrate consistency of the heterogeneity pattern of commercial lots with that of the lots used in preclinical and clinical studies

To assure lot-to-lot consistency, the kind and extent of this heterogeneity should be characterized and controlled

Structural Heterogeneity of Biological Products (Cont)

Acylation Amidation or deamidation Carbamylation Carboxylation Formylation Formation of gamma

carboxyglutamic acid Methylation Succinimide forms Aspartate isomerization Disulfide linkage

Oxidation Phosphorylation Sulphation Proteolysis (terminal or

domain deletion) Glycosylation (N-linked, O-

linked, site occupancy, terminal groups, fucosylation)

Aggregation High order structural change

(conformational change or denaturization)

Examples of potential structural modifications

Case Study (I) Charge analysis of N-linked

Oligosaccharides from FVIII Standards, Recombinant and Plasma Derived FVIII Products

Fibrinogen Fibrin XL-FibrinXIIIa

Thrombin

Prothrombin

X

IXaVIIIa

Ca, PL

IX

XaVa

Ca, PLV

Ca, PL

VIIICa, PL

XIa + Ca

XI

XIIa + HMWK

XII

ContactKallikrein

PK

VIIaTFCa

VII + Tissue Factor

ThrombinIXa or Xa

Vascular Injury

Ca

Extrinsic Pathway

Intrinsic Pathway Hemostasis

A1 A2 B A3 C1 C2N C

80 kDa90-210 kDa

50 kDa 43 kDa

90 kDa

740/741

372/373

72 kDa

1689/1690

Thrombin

210 kDa 80 kDa

1648/1649IC Protease

Heavy Chain Light Chain

vWF

A1 A2 A3 C1 C2N C

90 kDa 80 kDa

Heavy Chain Light Chain

372/373 1689/1690 vWF

FVIII

Activated FVIII

B-Domain Deleted FVIIIToole et al., PNAS 1986; 83: 5939-5942

FVIII: • Multi-domain glycoprotein (Mr 264,763)

• Potential 25 N-linked Oligosaccharides

Charge Analysis of N-linked Oligosaccharides from FVIII Standards, Recombinant and Plasma Derived FVIII ProductsSchilow et al., Thromb Haemost 2004; 92:427-428

Conclusion The charge analysis demonstrated

heterogeneity of the glycoforms of the tested FVIII products.

Case Study (II) Characterization of five von

Willebrand Factor (VWF) concentrates produced by five different manufacturing processes

von Willebrand Factor

Variation of vWF Multimers Among Five Different VWF Concentrates

IS P C-1 C-2 C-3 C-4 C-5 IS P C-1 C-2 C-3 C-4 C-5

1% Gel 2% Gel

Chang et al., Blood 2000; 96:567a, 2434

VWF Activities from 5 Different VWF Concentrates

Samples vWF:Ag vWF:RCof vWF:CBA(IU/vial) (IU/vial) (IU/vial) RCof/Ag (%) CBA/Ag (%)

C-1 10.43 10.03 10.23 96 98C-2 21.54 9.27 11.71 43 54C-3 10.92 8.19 8.45 75 77C-4 18.29 13.5 13.66 74 75C-5 13.4 8.63 9.74 64 73

Specific Activity

Chang et al., Blood 2000; 96:567a, 2434

Conclusion The characteristics (e.g., Multimeric

pattern and specific activity) of von Willebrand factor concentrates depend on the manufacturing processes.