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Vaccines, Blood & Biologics
  
    
  
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Mid-Cycle Review of GTCs BLA - ATryn




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Memorandum

To: Pratibha Rana, HFM-380 & File of STN 125284 

From: Roman Drews, HFM-392 

Through: Timothy Lee, HFM-392
 Acting Chief, Laboratory of Hemostasis/Division of Hematology 

Subject: Mid-cycle review of GTC’s Biological License Application for 
recombinant Antithrombin III (rATIII) manufactured in the milk of transgenic 
goats; GTC Biotherapeutics, Inc. 

Background

Recombinant Antithrombin III (rATIII) produced in the milk of transgenic goats 
is a single-chain glycoprotein composed of 432 amino acid. The molecule has six 
cysteine residues which form three disulfide bridges between Cys 8-128, 21-95, 
and 247-430, and four N-linked glycosylation sites at asparagines 96, 135, 155, 
and 192. The amino-acid sequence, disulfide linkages, and glycosylation sites of 
rATIII are identical to those of human plasma-derived ATIII. The carbohydrate 
composition and profile of rATIII differ from those of human plasma-derived 
ATIII that result in an approximately 4-fold increase in heparin affinity as 
demonstrated in in vitro experiments. The structural scheme of the protein is 
provided below.



rATIII is a serine protease inhibitor that is principal inhibitor of the blood 
coagulation system serine proteases – thrombin, and Factor Xa, and to a lesser 
extent, factors IXa, XIa, XIIa, trypsin, plasmin, and kallikrein. ATIII inhibits 
the activity of these serine proteases by forming a 1:1 stoichiometric complex 
between enzyme and inhibitor. This formation occurs at a relatively slow rate in 
the absence of heparin. When heparin is present, it binds to lysyl residues on 
ATIII and dramatically accelerates the rate of complex formation (around 1000 
times).

GTC’s rATIII is expressed in the milk of transgenic goats. The herd is 
maintained in a closed bio-secured system. The bio-security system addresses 
both external and internal control of diseases that may affect the herd. The 
health and welfare of the herd is under supervision of GTC’s full-time 
veterinary staff.

Milk (source material) is collected from qualified female animals and frozen 
until further processing. The frozen milk is stored by 
-------b(4)-------------------. The manufacture of rATIII bulk drug substance 
(BDS) is performed ------b(4)----------------------. rATIII is recovered from 
pools of milk via filtration system linked with affinity chromatography (heparin 
resin) and two additional chromatography columns. The most of analytical testing 
required for the manufacture and release of final material is performed by GTC 
facility located in Framingham, MA. In terms of product safety, the animals and 
source material are tested for the presence of adventitious viruses. The 
manufacturing process has been validated for the removal/inactivation of panel 
of four model viruses including acceptable variation regarding physico-chemical 
and structural properties. In addition, the firm validated the ability of the 
manufacturing process to clear prion protein.

The rATIII final drug product (FDP) is manufactured in ------b(4)--------------- 
facility located in the Netherlands. rATIII FDP is a lyophilized product 
formulated with glycine, sodium chloride and sodium citrate with 
-----b(4)-------- in each vial. The product is intended for intravenous infusion 
to patients with congenital ATIII deficiency prior to, during and following 
surgical or obstetrical procedures.

Bulk Drug Substance

Manufacturing Process – Development and Validation

Transgenic herd – general description

Female transgenic goats are the expression platform for the manufacturing of 
rATIII. The genetic material (transgene) that comprises of goat beta casein gene 
(regulatory elements directing expression in the mammary gland) and human cDNA 
coding sequence for ATIII was microinjected into goat embryos. The microinjected 
embryos were transferred to surrogate mothers. Goat progeny were born 5 months 
later and were analyzed for the presence of the ATIII transgene by 
-------b(4)----------. A male founder (155-92) was selected and mated with 
resulting transgene female offspring being bred to lactate following 
parturition. The positive transgenic animals pass the transgene to subsequent 
generations in a Mendelian fashion. Genetic and milk tests are performed to 
assess the presence of rATIII in the production animals. Transgenic offspring 
were further expanded using standard goat breeding techniques to create a herd 
of production animals. For details, please see the graph attached below.



Transgenic Construct

------------------------------------------b(4)

 .

-------------------------------------b(4) 

15 Pages determined to be not releasable:

b(4)

Information Request (September 14, 2008)
  Regarding --b(4)---- stability, please provide: 
    Information about non-specific (not mammary gland) expression of the rATIII 
    transgene in the transgenic goats.
    Information confirming that DNA sequencing of the rATIII transgene coding 
    region was performed for every new generation of goats qualified for the 
    production sub-group. If the sequencing has not been performed, please 
    provide DNA sequence representative of the last generation of goats that are 
    currently used in the manufacture of rATIII.

   Regarding qualification of production goats and milk pools, 
    Please establish in-process limits specifying the maximum duration of 
    lactation and number of lactations per animal that is allowed in the 
    manufacture of rATIII. Alternatively, please submit data to demonstrate that 
    your source material, milk, is suitable for the manufacture of rATIII 
    regardless of the duration of lactation and number of lactations for each 
    production animal.
    Please propose an upper limit of rATIII content in the milk of each 
    individual goat qualified for the production sub-group. The limit should be 
    based on your developmental studies and manufacturing history.
    Please establish means to control the level of rATIII expression throughout 
    the duration of lactation.

   Regarding specification limits for composite milk pools: 
    Please provide justifications for the specification limit of --b(4)-- for 
    ---b(4)---- while the highest observed level of ----b(4)---- in the 
    composite milk pools was 4.2%.
    Please revise the specification for b(4) casein -b(4)- based on your 
    manufacturing experience. In addition, please provide the following:
    Data demonstrating that rATIII batches used in the clinical studies were 
    manufactured using composite milk pools containing b(4) casein
    Data demonstrating clearance of b(4) casein by the purification process of 
    rATIII
    Data showing that the assay for colloidal goat milk proteins in rATIII drug 
    substance is validated for the detection of b(4) casein

  4 Pages determined to be not releasable:

  b(4)

  [ b(4) ]

  In addition, the purification steps are controlled for the level of bioburden 
  and bacterial endotoxin. The maximum number of runs for chromatography resin 
  and usage of filter membranes have been established based on the data 
  collected from the small and commercial scales runs. So far, GTC has 
  manufactured b(4) lots at the proposed commercial manufacturing site using the 
  manufacturing process described in this BLA. Since the initiation of 
  development of rATIII,b(4) lots of product have been manufactured ( b(4) at 
  laboratory scale and b(4) at different scales). Lots of the final drug 
  products that were manufactured with the significant process changes have been 
  used in non-clinical and clinical studies.

  Development and Validation

  Development of rATIII processing first occurred at ---b(4)---------------- and 
  included scaled down and commercial scale validation studies. The purification 
  process was later transferred to --b(4)--------- where rATIII clinical batches 
  were purified and used in hereditary deficiency studies. Transfer of the 
  process to -b(4)- included modifications involving closure of the -b(4)-. The 
  process has been re-validated in -b(4)- facility. The process equipment, 
  chromatography resins and analytical methods have not been changed. 
  Furthermore, GTC introduced nano-filtration step (after heparin affinity 
  column) to the transferred process. The introduction of nano-filtration 
  required comparability studies that included biochemical tests, pre-clinical, 
  and human pharmacokinetic study in normal volunteers (The results of these 
  studies have been submitted to CBER review during the IND process and/or into 
  this BLA).

  Validation Studies

  The validation studies for rATIII purification process have been conducted at 
  commercial and small scales. The scope and outcome of the validation studies 
  are adequate. GTC submitted data deriving from the following studies:
    Commercial scale validation process performed at -b(4)--- facility
    Small scale developmental studies demonstrating robustness of the 
    manufacturing steps and removal of goat milk proteins
    A side-by-side summary of the biochemical analyses for three batches 
    manufactured at -b(4)- and -b(4)- facilities

  Three consecutive lots of rATIII BDS (lots 
  ----------------b(4)-----------------------) have been manufactured at -b(4)- 
  facility and met current release requirements for BDS. The manufacturing runs 
  for these three lots were subjected to the additional testing for purity, 
  yield, and impurities content. The testing was performed at the level of each 
  unit operation. In addition, GTC performed extended characterization of milk 
  pool and monitored rATIII glycosylation throughout the process. The data 
  characterizing manufacturing milk pools and composite samples are acceptable 
  and have been already reviewed in the paragraph of this review concerning 
  qualification of source material and production goats.

  Process yield and purity of rATIII BDS

  The yield of rATIII for three conformance lots ranged from -b(4)- meeting 
  specification for process yield of -b(4)-.

  [ b(4) ]

  -------------------------b(4)--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.

  10 Pages determined to be not releasable:

  b(4)





  9 Pages determined to be not releasable:

  b(4)

  -------------------------b(4)-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.

  [ b(4) ]

  ---------------------------------------b(4)--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.

  [ b(4) ]

  Control of rATIII Bulk Drug Substance – Proposed Specifications

  In general, the firm proposed adequate array of analytical assays to assess 
  the identity, purity, strength, potency, and quality attributes of rATIII BDS. 
  The analytical methods and proposed acceptance limits are based on the data 
  obtained from the developmental studies and manufacture experience of the 
  firm. The acceptance limits were based on mean values and + 3 Standard 
  Deviation statistical calculation deriving from b(4) lots manufactured. The 
  acceptance limits for process related impurities are mainly based on a limit 
  of detection of the assay. The proposed specifications were confirmed by the 
  non-clinical and clinical studies and I found them acceptable. However, based 
  on the heightened biochemical characterization studies and tests that were 
  performed on release of conformance batches of rATIII BDS, I am recommending 
  to include the following additional analytical tests:
    -------------------------b(4)-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.
    ----------------------------b(4) .
    -------------------------b(4)-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.
    ------------------------------b(4)-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.
    ------------------------------b(4)------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.

  [ b(4) ]

  3 Pages determined to be not releasable:

  b(4)

  ----------------------------b(4)--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.

  The validation of the manufacturing process for ATryn® FDP and container 
  closure issues are reviewed by Dr. Chiang Syin, Division of Manufacture and 
  Product Quality. A flow diagram for the ATryn® FDP manufacturing process is 
  attached below.

  [ b(4) ]

  Proposed Final Release Specifications

  The action limits for specification analyzes were established based on the 
  data collected from b(4) lots of ATryn® FDP, of which 17 met the pre-set 
  release specifications. These include three conformance lots 
  --------b(4)------------------------------ manufactured consecutively. Date of 
  manufacture has been on: --------------b(4)------------------ , and 
  -----b(4)-------------------, respectively. Conformance lot --b(4)----- did 
  not meet release specification failed as a result of out-of-specification 
  results for ----b(4)---------- (due to the operator error) and to 
  corresponding result for percent of --b(4)--- rATIII. However, it met the 
  in-process control requirement and remaining final release limit. The tests 
  performed during the release of the conformance batches are presented in the 
  tables attached below.

  1 Page determined to be not releasable:

  b(4)

  Over the course of product development, the additional analytical tests were 
  performed for the release of rATIII FDP lots. Lots deriving from the all 
  stages of commercial product development were used in the non-clinical and 
  clinical studies performed by the firm.

  Table attached below illustrates the historical development of analytical 
  tests used for release of ATryn® lots manufactured with different 
  manufacturing changes.

  [ b(4) ]

  Table attached below lists of specifications is proposed for the product 
  licensure. The acceptance limits, where applicable, are based on the following 
  three criteria: mean value and plus/minus three standard deviations (data 
  from-b(4)-lots), established values of the compendial tests, and limit of 
  detection of the assay.

  [ b(4) ]

  Based on ATryn® FDP characterization and developmental data, I found that 
  proposed specification analysis is not fully adequate to assess consistency of 
  production and quality attributes of the product. Therefore, I recommend the 
  following changes to the proposed specifications:
    To maintain specification acceptance limit for the --b(4)---- form of rATIII 
    expressed as percent of total purity established by the --b(4)--- assay
    To maintain ----b(4)--------- assay to reduce potential heterogeneity of 
    rATIII active ingredient due to the glycosylation composition and/or effect 
    of the terminal heat treatment
    To maintain specification limits for ----b(4)------------ calculated based 
    on a ratio of results for Potency and Strength
    To include specification criteria for Appearance of the reconstituted ATryn® 
    assessing its clarity and color

  In addition, GTC should submit the additional data clarifying outcome of the 
  --b(4)---- chromatogram, i.e. to identify content of the ---b(4)----- on the 
  ascending arm of ---b(4)---- resolved by the ----b(4)----- assay

  Description and Composition of the Drug Product

  ATryn® is a sterile lyophilized powder intended for intravenous infusion 
  following reconstitution with Sterile Water for Injection (WFI). The product 
  is filed into a -b(4)------b(4)------------- glass container and closed with a 
  ------------b(4)---------- closure. Following lyophilization, the --b(4)- 
  closure is fully seated and the container is sealed wit a ---b(4)--------- 
  seal with a ----b(4)------------- cap. WFI recommended for reconstitution is 
  not provided or packaged with the product.

  ---------------------------------------------b(4) .

  The details regarding product composition are provided in the table attached 
  below. With the exception of rATIII; there are no novel or human derived or 
  animal derived materials added directly to the formulated drug product. The 
  reconstituted product is stable when stored at ambient room temperature for up 
  to 12 hours, as demonstrated in Stability (3.2.P.8) part of the BLA. 


Information Request (October 14, 2008)
  Regarding the Proposed Specification for the Bulk Drug Substance (BDS): 
    Please retain your established acceptance limits for the following 
    parameters ---b(4)------
      --------------b(4)---------------

    In addition to total --b(4)----- content, please also establish acceptance 
    limits for the -b(4)- detected species of ----------b(4)------------------.
    Please retain your specification for the content of the ---b(4)---- form of 
    rATIII expressed as percentage of total purity measured by the --b(4)---- 
    assay.
    Please establish an acceptance limit for ---b(4)-----, based on the ratio of 
    the Potency and Strength of rATIII.
    Please establish a criterion for visual inspection of the BDS solution that 
    includes assessment of color and clarity.

  Regarding the Proposed Specification for the Final Drug Product (FDP): 
    Please retain your specification for the content of the --b(4)--- form of 
    rATIII expressed as percentage of total Purity measured by the --b(4)----- 
    assay.
    Please retain your established acceptance criteria assessing the 
    heterogeneity of rATIII by ----------b(4)--------.
    Please establish an acceptance limit for ----b(4)--------, based on the 
    ratio of Potency and Strength of rATIII.
    Please establish a criterion for the visual inspection of the FDP solution 
    after reconstitution that includes assessment of its color and clarity.
    Please establish the -----b(4)---------- specification with respect to 
    ATryn® potency.

  Regarding the chromatogram from the --b(4)--- assay, please identify the 
  proteins eluted in the ---b(4)----- on the 
  ---------------b(4)----------------------------- peak.
  Please follow the CBER convention for non-proprietary names of blood products. 
  For example, the non-proprietary name for plasma-derived Antithrombin III 
  product is Antithrombin IIII (Human). Therefore, please use Antithrombin III 
  (Recombinant) as the non-proprietary name for your product.
 

 


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