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.
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