IBC Final 43004

1
Recombinant Anti-Hemophilic Factor - A Case Study
in Post Licensure Evolution of Specifications

• Mehrshid Alai, Ph.D
• Baxter Healthcare Corporation
• October 2004

2
Factor VIII Structure
Synthesized as a polypetide chain of 2,332 amino
acids which is cleaved into a dimer as it is
produced in cells.
3
Factor VIII Structure and Function
Adapted from Lenting PJ et al. Blood 1998
923983-96
4
Factor VIII Biological Activity
5
Hemophilia A

• Hemophilia A is an X-linked bleeding disorder
  associated with a deficiency of functional factor
  VIII.
• Over 200 different mutations known.
• Clinical Severity
• Mild 5 factor level Bleeding only
  withsignificant trauma or surgery only
  occasionalhemarthroses, with trauma
• Moderate 15 factor level Bleeding with mild
  trauma hemarthroses with trauma occasionally
  spontaneous hemarthroses
• Severe hemarthroses and soft tissue bleeding

6
Hemophilia Therapy

• Replacement therapy with factor VIII corrects the
  clotting deficiency.
• Typical dosage is 30-50 IU/Kg
• 1 µg factor VIII 5 IU
• Therapeutic dose 6-10 µg/Kg
• Prior to the advent of the technology for the
  commercial production of recombinant proteins
  factor VIII concentrates were plasma (human or
  porcine) derived.

7
Factor VIII Immunogenicity

• The incidence of inhibitor formation is related
  to the severity of the disease and the nature of
  the mutation.
• 25 for severe hemophiliacs
• 5 to 15 for mild to moderate hemophiliacs
• Higher in patients whose mutation interferes with
  factor VIII biosynthesis
• Inhibitor development is the most significant
  adverse event associated with factor VIII
  therapy. Alterations in factor VIII
  manufacturing process have been associated with
  increases in immunogenicity.

8
Factor VIII Inhibitors

• A2 and C2 domains are antigenic hotspots
• A significant portion of factor VIII inhibitors
  are
  proteolytic antibodies
• Inhibitory anti-C2 antibodies also block vWF and
  membrane binding
• Lacroix-Desmazes
  S et al. NEJM 2002 346 662-667

9
RECOMBINATE rAHF Manufacturing Process
Fed Batch Bioreactor
Harvest / Clarification
Process Notes CHO Host Cell Continuous Culture -
24/7 Immunoaffinity Mass Capture
Affinity and Ion Exchange Purification
Bulk
Formulation and Lyophilization
10
RECOMBINATE rAHF Cell Culture Production Process
11
RECOMBINATE rAHF Purification and Finishing
Processes
12
RECOMBINATE Specifications
13
RECOMBINATE rAHF Licensure

• Approved in 1992 as the first recombinant factor
  VIII product.
• 1993 Licensure in Europe and other geographies.
• Specifications for bulk drug substance and final
  product aggregate of all geographic requirements.

14
History of Licensure

• It is one of the first complex recombinant
  products being licensed.
• It is manufactured in two facilities, by two
  entities.
• Extensive release testing is part of ensuring
  product quality.
• Due to higher product concentration and purity,
  the product is extensively tested and
  characterized at the BDS stage.

15
Characterization vs. Release Testing

• Tests such as amino acid (a.a.) sequence analysis
  are typically considered as characterization
  tests and unless there is a reason to suspect
  changes in the a.a. sequence, it is not necessary
  to test the a.a. for release of every lot.
• A photospectrum analysis should be done as a
  characterization tool to define appropriate
  wavelength to measure protein concentration and
  for routine release of product.

16
Process Related Impurities

• Many upstream process related impurities are
  removed in the downstream purification process.
  During process validation data can be generated
  to assure removal of such impurities.
• Even though, Baxter had generated data on removal
  of process related impurities, due to lack of
  history with recombinant products release testing
  of impurities was suggested/required.

17
Two Specific Examples

• Residual Host Cell DNA
• Residual Host Cell Protein (HCP)

18
Residual Host Cell DNA

• At the time of development and registration of
  RECOMBINATE, residual Host cell DNA was
  considered to be a potential safety risk to the
  patients.
• In 1987, a World Health Organization consultative
  group recommended that the safety risk was
  negligible in products containing less than 100
  pg/dose of cellular DNA.

19
Residual Host Cell DNA

• In an FDA/CBER-sponsored workshop on Well
  Characterized Biotechnology Products held in
  December 1995, it was concluded that process
  validation could provide a more appropriate and
  effective means of demonstrating product
  consistency and purity.
• At this meeting it was proposed and is generally
  accepted that appropriate validation of DNA
  removal could obviate the need for routine DNA
  testing.

20
Residual Host Cell DNA

• As part of routine release testing for further
  manufacturing use, each batch of rAHF concentrate
  was assayed for residual host cell DNA.
• The specification for this test was set at a
  maximum of 10 pg/1000 unit dose.

21
Experience with Host Cell DNA

• After years of experience with recombinant
  products, residual host cell DNA is not
  considered a major safety concern.

22
Rationale for Elimination of Residual DNA Testing

• Baxter submitted a pre-approval supplement
  requesting elimination of routine lot-to-lot
  testing for residual host cell DNA based on
• concurrent evaluation of DNA in samples of
  in-process pools from full scale manufacturing
  batches
• prospective-scale challenge studies using
  radioactive labeled DNA spiked into the process
  intermediates
• experience with 1000 batches of rAHF concentrate
  consistently meeting specifications which was
  significantly below the safety threshold set for
  host cell DNA.

23
Rationale for Elimination of Residual DNA Testing

• The results of the studies showed that
• the DNA challenge to the purification process is
  predictable and consistent in terms of size and
  the amount of DNA
• the purification process is robust and has
  capacity to remove DNA to levels below the
  specification
• the purification process can reproducibly remove
  DNA to acceptably low levels.

24
The Outcome

• The supplement was approved by the agency with
  the commitment of using the residual DNA testing
  for monitoring process changes.

25
Residual Host Cell Protein

• As part of routine testing, for further
  manufacturing, each batch of rAHF Concentrate is
  tested for residual host cell protein levels,
  using the Chinese Hamster Ovary (CHO), by
  immunoligand assay (ILA).
• The licensed specification was less than or equal
  to 1.0 ug/1000 IU rAHF.
• Baxter prepared a pre-approval supplement to
  increase the residual level to 1.5 ug/1000 IU
  rAHF.

26
Rationale for Changing the Specifications

• Original specification was based on limited data,
  consisting of 13 batches.
• After extensive experience with the process,
  1000 batches, specification was found to be to
  close to the capability of the process.
• Retrospective evaluation of in-process parameters
  including chromatographic separation of CHO
  proteins and rAHF was made.
• 99.9 of total CHO proteins are removed in the
  first step of the purification process.

27
Rationale for Changing the Specifications (contd)

• The analysis of CHO proteins showed that the
  differences between the batches below the
  specification and above the specifications were
  only quantitative and not qualitative.
• All other physcio-chemical parameters showed
  process consistency.
• Calculation of the specification involves two
  biological assays which bring variability to the
  results.

28
Clinical Data in Support of Change of HCP
Specification

• Based on two prospective clinical trials and
  post-licensure pharmacovigliance activities, it
  was concluded that RECOMBINATE has a high level
  of tolerability and safety when administered at a
  very high dose (as for immune tolerance) over
  long time intervals.

29
Clinical Data in Support of Change of HCP
Specification

• There is no relationship between residual CHO
  protein and the clinical adverse experience.
• Baxter made a commitment to conduct a prospective
  study with moderately increased levels of CHO
  protein levels (i.e. 1.2 and 1.5 ug/1000 IU).

30
Change in the HCP Specifications

• The proposed change of CHO specifications was
  approved by FDA on October 22, 1998 and by EU
  countries on August 30, 1999.
• Baxter has successfully conducted the prospective
  Pharmaco-surveillance Study of Anti-Hemophilic
  Factor, RecombinateTM, Manufactured with Modestly
  Increased CHO cell Protein Levels.