# Presentation: 516218

## 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

## Hemostasis

- <date/time>

**Fibrinogen**

**Fibrin**

**XL-Fibrin**

**XIIIa**

**Thrombin**

**Prothrombin**

**X**

**IXa**

**VIIIa**

**Ca, PL**

**Xa**

**Va**

**Ca, PL**

**V**

**Ca, PL**

**XIa + Ca**

**XIIa + HMWK**

**XII**

**Contact**

**Kallikrein**

**PK**

**VIIa**

**TF**

**Ca**

**VII + Tissue Factor**

**Thrombin**

**IXa or Xa**

**Vascular Injury**

**Ca**

***Extrinsic Pathway***

***Intrinsic Pathway***

## FVIII

- <date/time>

**A1**

**A2**

**B**

**A3**

**C1**

**C2**

- N

- C

- 90-210 kDa

- 50 kDa

- 43 kDa

- 740/741

- 372/373

- 72 kDa

- 1689/1690

**Thrombin**

- 210 kDa

- 1648/1649

- IC Protease

**Heavy Chain**

**Light Chain**

- vWF

**A1**

**A2**

**A3**

**C1**

**C2**

- N

- C

**Heavy Chain**

**Light Chain**

- 372/373

- 1689/1690

- vWF

**FVIII**

**Activated FVIII**

**B-Domain Deleted FVIII**
**Toole *****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 ProductsSchilow 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

- 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

- 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.