# Presentation: 881708

## Case Study for Comparability and Efficacy – rec FIX versus Plasma derived

**Bob Adamson Ph.D.**
**Vice President, Biopharmaceutical Process Development,**
**Wyeth BioPharma**

## Coagulation Cascade

***Activated FIX is required for activation of FVIII.***
***FIX deficiency leads to hemophilia B***

_**Intrinsic**_
_**pathway**_

_**Extrinsic**_
_**pathway**_

**FXII**

**FXI**

**FIX**

**FXIIa**

**FXIa**

**Ca****++**

**FVIII**

**T**

**FVIIIa**

**FIXa**

**or**

**FVIIa  TF**

**TF   **

**B**

**FVII**

**Cell**
**Injury**

**FVa**

**FXa**

**FX**

**Ca****++**

**FV**

**T**

**PT**

**T**

**Ca****++**

**FG**

**Fibrin Clot**

## BeneFIX 

- LC

- AP

- HC

- S-S

- LC

- AP

- HC

- S-S

**rFIX**

**rFIXa**

**Activation**

## Human Factor IX (415 amino acids, multiple post-translational modifications)

## Analytical Parameters Selected for Drug Substance Release Specifications

**Potency**
- Potency (IU/mL, CS assay)
**Identity**
- Specific Activity
- SDS-PAGE, reducing
- Peptide map
- Carbohydrate fingerprint
- Gla domain map
**Safety**
- Bioburden
- Endotoxin
**Quality**
- Appearance
- pH

## Structural Characterization of rFIX

**	****TOP-DOWN Approach**
- Structural analysis of  intact rFIX
- Peptide mapping: 100% sequence coverage
- Carbohydrate fingerprinting: Fine structure established
- Structural analysis of rFIX domains
**Gla domain**
- Activation peptide
- Techniques include: Preparative ion exchange, peptide mapping, carbohydrate mapping and LC-MS

## Comparative Posttranslational Modifications

## Purity by Size Exclusion HPLC(circa mid-90s)

- 0

- 0.4

- 0

- 0.4

- 0

- 0.4

- 0.8

**rFIX**

**Mononine**

**Alphanine SD**

- ABS (214nm)

- 0

- 0.4

- 0.8

- 0

- 0.5

- 1.0

- 4

- 6

- 8

- 10

- MINUTES

**Replenine**

**Immunine**

**Agg.  (<3%)**

**FIX Monomer**

**10%**

**30%**

**26%**

**50%**

- Abs (214 nm)

## In Vivo Recovery of rFIX and pdFIX

## Relative Recovery

**1.0**

**0.9**

**0.8**

**0.7**

**0.6**

**0.5**

**0.4**

**0.3**

**0.2**

**0.1**

**0**

**pdFIX**

**rFIX**

**rFIX (10GLA)**

**rFIX (11GLA)**

**rFIX (12GLA)**

**rFIX + HMW**

**rFIX (FT1)**

**rFIX (FT2)**

**rFIX + SO****3**

**pdFIX + PO****4**

**pdFIX + HMW**

**pdFIX**

## Posttranslational Modifications in the Activation Peptide

**Posttranslational Modifications in the Activation Peptide**

## Sulfation in rFIX Is Consistent (11-14%)

- 0

- 10

- 20

- 5A21M043

- 6D21M003

- 6D21M014

- 6D21M017

- 6D21M018

- 6D21M019

- 6D21M020

- 6D21M022

- 6D21M030

- 7D21M004

- 7D21M009

- 7D21M012

- 7D21M017

- 7D21M021

- 7D21M024

- 7D21M047

- 7D21M048

- 7D21M049

- 7A21M003

- 7A21M011

- 7A21M017

- 7A21M019

- 8A21M001

- 8A21M002

- 8A21M003

- Bulk Drug Substance Batch

**% Sulfation**

**95-4**

**96-1**

**96-2**

**96-3**

**97-1**

**97-2**

**97-3**

**97-4**

- % Sulfation

## Summary & Conclusions from BeneFIX Case Study

**Comparability established between rFIX and high purity plasma derived product.**
**Reduced bioavailability was observed for rFIX  in  in-vivo models**
**In depth characterization was required assign cause for reduced bioavailability **
**Awarded  Orphan Drug Status on the basis of improved viral safety over incumbent therapeutics**
**Reduced bioavailability resulted in a specified dosing regimen outlined in package insert **
**Clinical studies were as stringent and comprehensive as high purity plasma derived products**

## What Constitutes Innovator Company Know-how?  

**Production Cell Line/Clone**
**Critical raw materials (media, resins, formulation excipients)**
**Internal Standards**
**In-process controls**
**Product Specifications **
**Analytical Characterization database**
**Product structure-function database**

**Clinical database, Preclinical database and toxicology studies**

**Critical issue is the connectivity between clinical studies and product characteristics**

## What FOBs Manufacturers Need (and cannot obtain) to produce a comparable product

**Production Cell Line/Clone**
**Critical raw materials (media, resins, formulation excipients)**
**Internal Standards**
**In-process controls**
**Product Specifications **
**Analytical Characterization database**
**Product structure-function database**

**Clinical database, Preclinical database and toxicology studies**

**Critical issue is the connectivity between clinical studies and product characteristics**

## Follow on Biologics  

**FOBs manufacturers will be able to develop and manufacture FOBs.  ****However....**
**Given the complexity of Biologics processes and products, a detailed database will be required to ensure safety and efficacy, including:**
-  In-depth characterization, including identification of critical structural elements of the product (structure/function)
- Product specifications and in-process controls (relative to internal standards)
- Clinical studies appropriate to establish safety and efficacy
- Since innovator know-how will be unavailable to FOBs manufacturers it is expected that clinical studies would be as rigorous as that demanded from innovators