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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Mid-Cycle Review of GTC’s BLA - ATryn


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.


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.

Table shows the structural scheme of protein.

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 offspring were further expanded using standard goat breeding techniques to create a herd of production animals.

Transgenic Construct




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Information Request (September 14, 2008)

 1. Regarding --b(4)---- stability, please provide:

  1. Information about non-specific (not mammary gland) expression of the rATIII transgene in the transgenic goats.
  2. 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.

2. Regarding qualification of production goats and milk pools,

  1. 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.
  2. 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.
  3. Please establish means to control the level of rATIII expression throughout the duration of lactation.


3. Regarding specification limits for composite milk pools:

  1. 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%.
  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

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[ 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) ]


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Comparison of hpAT to Anthithrombin Alfa

Comparison of hpAT to Anthithrombin Alfa


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[ 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) ]

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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.

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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.


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)

  1. Regarding the Proposed Specification for the Bulk Drug Substance (BDS):
    1. Please retain your established acceptance limits for the following parameters
      1. ---b(4)------
      2. --------------b(4)---------------
    2. In addition to total --b(4)----- content, please also establish acceptance limits for the -b(4)- detected species of ----------b(4)------------------.
    3. 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.
    4. Please establish an acceptance limit for ---b(4)-----, based on the ratio of the Potency and Strength of rATIII.
    5. Please establish a criterion for visual inspection of the BDS solution that includes assessment of color and clarity.
  2. Regarding the Proposed Specification for the Final Drug Product (FDP):
    1. 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.
    2. Please retain your established acceptance criteria assessing the heterogeneity of rATIII by ----------b(4)--------.
    3. Please establish an acceptance limit for ----b(4)--------, based on the ratio of Potency and Strength of rATIII.
    4. Please establish a criterion for the visual inspection of the FDP solution after reconstitution that includes assessment of its color and clarity.
    5. Please establish the -----b(4)---------- specification with respect to ATryn® potency.
  3. Regarding the chromatogram from the --b(4)--- assay, please identify the proteins eluted in the ---b(4)----- on the ---------------b(4)----------------------------- peak.
  4. 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.