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B-domain deleted recombinant factor VIII formulation and stability
B-Domain Deleted Recombinant Factor VIII Formulation and Stability T. &ten4erg, A. Fatouros, E. Neidhdrdt, N. Wayne, and M. Mikaelsson B-domain deleted recombinant factor VIII (BDDrFVIII) is a deletion form of human coagulation factor VIII. A lyophilized formulation of highly purified BDDrFVlll has been developed that does not require the use of blood-derived products such as human serum albumin (HSA). By avoiding the use of blood-derived products, the BDDrFVlll formulation minimizes the risk of transmitting blood-borne pathogens that may be present in plasma-derived factor VIII or in other recombinant factor VIII products that contain HSA in their formulation. Upon reconstitution with saline (4 mL), the composition of the reconstituted product (62.5 to 250 IU/mL BDDrFVIII) is 18 mg/mL sodium chloride, 3.0 mg/mL sucrose, 1.5 mg/ml. r-histidine, 0.25 mg/mL calcium chloride dihydrate, and 0.1 mg/mL polysorbate 80. The optimal combination of these excipients in the lyophilized BDDrFVlll formulation provides long-term stability, as measured by a variety of analytical methods. The formulation preserves factor VIII activity of lyophilized BDDrFVlll during storage for at least 24 months at WC, and for up to 6 months at room temperature (25°C). The reconstituted product retains its factor VIII potency for at least 100 hours at 25”C, which would allow it to be continuously administered via an infusion pump, assuming the product is handled under aseptic conditions. Semin Hematol38(suppl4):40-43. Copyright 0 2001 by W-B. Saunders Company.
ONCERN OVER THE potential transmission of viral disease by plasma-derived human factor VIII products,l as well as periodic shortages of both plasma-derived and recombinant human factor VIII products, has underscored the need for the development of new recombinant products for treating patients with hemophilia A. Recently, a B-domain deleted form of human coagulation factor VIII (BDDrFVIII; ReFacto, Wyeth/Genetics Institute, Cambridge, MA) has been developed. The plasma-derived factor VIII molecule exists in several active forms ranging in size from 170 kd to 280 kd.2 The BDDrFVIII gene has been genetically engineered to correspond to the smallest (170 kd) of the multiple active forms of factor VIII found in plasma-derived factor VIII concentrates. The rationale for the design of BDDrFVIII was the observatipn that a large region within the full-length factor VIII molecule, the heavily glycosylated B-domain consisting of 980 amino acids, is not required for
C
From Pharmucia AB, Stockholm, Sweden; and the Genetics Institute, Cambridge, MA. Address reprint requests to N. Wame, PhD, Genetics Institute, One Burtt Rd, Andover, MA 01810. Copyright 0 2001 by W.B. Saunders Company 0037-1963/01/3802-4006$35.00/O doi:l0.1053lshenz.2001.25892
40
Seminars
in Hematology,
expression of biologic activity.* Like the activated full-length form, the BDDrFVIII molecule is composed of two polypeptide chains, domains Al and A2 (90 kd) and domains A3, Cl, and C2 (80 kd), held together by a metal ion bridge.5 The goal for the BDDrFVIII formulation was to develop an easily reconstituted, freeze-dried formulation that maintains the integrity and potency of factor VIII without requiring the addition of human serum albumin (HSA) or other macromolecular stabilizers.
Formulation
Strategy
Development of a formulation for BDDrFVIII that preserves factor VIII activity during prolonged storage of the lyophilized product and for appropriate time periods after reconstitution required extensive testing of a large number of candidate formulations. A broad range of discriminating analytical methods for assessingthe stability of BDDrFVIII was utilized. These analytical methods include the following6: 1. The chromogenic substrate assay for factor VIII activity using the Coatest Factor VIII kit (Chromogenix AB, MBlndal, Sweden). 2. Size-exclusion high-performance liquid chromatography to detect soluble aggregates and degradation products. 3. Sodium dodecyl sulfate-polyacrylamide gel
Vol 38, No 2, Suppl 4 (April),
2001:
pp 40-43
BDDrFVlll
electrophoresis for visualization tion products. 4. The coulometric Karl Fisher sure moisture content. 5. Visual inspection for clarity coloration.
Fomzdation and Stability -
of degradaassay to meaand
degree
of
The two main problems that had to be addressed in formulating BDDrFVIII were its relative instability in solution and its potential for nonspecific adsorption to surfaces. Loss of active protein due to these factors can be considerable. Lyophilization is an appropriate means to stabilize such molecules, provided the components of the formulation are carefully selected.
Formulation
Components
The final formulation,6 after reconstitution of the freeze-dried product with 4 mL sodium chloride (9 mg/mL), contains 62.5 to 250 IU/mL BDDrFVIII, 18 mg/mL sodium chloride, 3.0 mg/mL sucrose, 1.5 mg/mL z-histidine, 0.25 mg/mL calcium chloride dihydrate, and 0.1 mg/mL polysorbate 80; and is adjusted to pH 7.0 (Table 1). Sodium chloride acts as a bulking agent during lyophilization and enhances the solubility of BDDrFVIII. Sucrose acts as a “water substitute” stabilizer in the amorphous phase of the lyophilized product.’ L-Histidine serves to buffer the formulation around pH 7.0, thereby minimizing aggregate formation and maintaining the factor VIII potency of BDDrFVIII during lyophilization and storage. Unlike other buffering agents that may crystallize during freezing and cause pH shifts, L-histidine does not crystallize around pH 6.0, which coincides with the pK of the weakly
Table
1.
Composition
of the
Final
BDDrFVlll
Concentration After Reconstitution*
Amount/Vial BDDrFVlll
250,
500,
1,000 Sodium
chloride
Sucrose
Polysorbate * With
62.5, IU
250
chloride
dihydrate
36
mg
18
mg/mL
mg
3.0
mg/mL
1 mg 0.4 mg
80 4 mL sodium
chloride
diluent
125, IU/mL
12
6 mg
L-Histidine Calcium
Formulation
1.5 0.25
(9 mg/mL).
0.1
mg/mL mg/mL mg/mL
41
basic imidazolium function.* Calcium ions preserve the metal ion bridge linking the two peptides required for the biologic activity of the heterodimer.” The concentration is kept as low as possible (0.25 mg/mL), since calcium salts decrease the glass transition temperature (TL) of the amorphous phase surrounding the ice crystals. The glass transition temperature should be as high as possible in order to shorten the freeze-drying process. 6 Polysorbate 80 is as effective as HSA for stabilizing the factor VIII activity of BDDrVIII (Fig l), and also protects the BDDrFVIII molecule from surface denaturation and adsorption.6 Formulations buffered without polysorbate 80 lose a substantial amount of factor VIII activity (>50%), whereas the addition of appropriate amounts of polysorbate 80 reduces the loss of factor VIII activity during freeze-drying (Fig l).”
Lyophilization
Temperature
Thermal analysis of the final formulation of BDDrFVIII showed a TL of -42” to -38°C. Freeze-drying microscopy revealed maintenance of the structural integrity of the freeze-dried product during freeze-drying at temperatures of -35°C and structural collapse of the product at temperatures higher than - 30°C. These data indicated that formulations of BDDrFVIII had to be lyophilized at a product temperature below - 32°C.”
PH Candidate formulations ranging in pH from 4.0 to 8.0 were freeze-dried and stored at temperatures of 7°C 25°C and 37”C, for up to 12 months. A pH range of 6.5 to 7.5 was found to be optimal for preserving factor VIII activity. Gel filtration detected no aggregates in BDDrFVIII formulations for this pH range. There was a consistent loss of approximately 10% of factor VIII activity of BDDrFVIII during the freeze-drying process, regardless of the pH of the formulation (Fig 2).6
Stability The final formulation preserves the potency integrity of BDDrFVIII over a prolonged
and pe-
42
isstwbwg et al
300
0 0 q n
250
B
Before filtration After filtration Thawed 5 times Freeze-dried
200
2 0 5
150
100 50
!u Buffer only
PEG 4000
HSA
riod of time. The factor VIII potency of the freeze-dried product remains stable for at least 24 months when stored at 8°C (Fig 3), and for at least 6 months when stored at room temperature, 25°C (Fig 4). Aggregates or fragments were below 5% during the storage period. In addition, storage stability of BDDrFVIII was unaffected over the pH range of 6.0 to 8.0.6 The reconstituted product remains stable for at least 100 hours when stored at 25’C, which would allow the product to be continuously
lemaccel@
Figure 1. BDDrFVlll activity before and after sterile filtration, freeze-thawing, and freezedrying, when buffered with different stabilizers. VIII:C, factor VIII activity. Reprinted with permis-
Haesesterile
sion.6
administered via infusion pump.9 However, in keeping with the International Conference on Harmonisation guidelines on the use of products not containing preservatives, it is recommended that BDDrFVIII be used within 3 hours after reconstitution.
1250
1000 = 9 + 2 3
0 After freeze-drying 0 Before freezedrying
500
gl
80 70
400
0 I
2
60
0
50
: e f g
40
:
5
750
0 z z 500
250
3oo 0
200
III 0
30
3
I 9
6
I 12
I 15
I 18
I 21
I 24
Time (months)
100
0 PH
Figure 2. Recovery of factor VIII activity of BDDrFVlll in the final formulation before and after freeze-dt-ying at different pHs (500 W/vial). (-¤-) Percent recovery of VIII:C after freeze-drying.
25OlU
8~
5OOIU
8 *Batch
1000
IU
Batch
--+--Batch
1
. *es
1
-*-Batch2
1
-O-
Batch
2
Batch
2
Figure 3. Stability of 3 batches of lyophilized BDDrFVlll for 250, 500, and 1,000 IU stored at 8°C. Values for 500 and 1,000 IU batches at 0 and 3 months have been multiplied by 1.07 to correct for change of international standard.
BDDrFVllI
‘ii sY
0=
60 i 40
5: 20
0-I0
-o..a-. -=+.= e
1
8°C (ambi-RH) 25°C (60% RH) 30°C (60% RH) 40°C (75% RH) * Not tested.
2
3
4
5
43
Fwnudation and Stability
6
Time (months) Figure 4. Stability of lyophilized BDDrWlll (250 IU) at different temperatures. Data represent mean i SD (N = 3). There was no significant difference in factor VIII activity between BDDrFVlll stored at 8” and 25°C at month 6. ambi-RH, ambient relative humidity.
Solubility The ease of reconstitution of the BDDrFVIII formulation is comparable to that of other commercially available recombinant factor VIII products.
Conclusions BDDrFVIII is a highly purified recombinant derivative of human plasma-derived factor VIII. Extensive testing of a large number of candidate formulations using highly discriminatory analytical methods has shown that BDDrFVIII can be formulated for freeze-drying without the addition of HSA or other macromolecular stabilizers. Extensive experiments led to a formulation using a combination of sodium chloride, sucrose, L-histidine, calcium chloride dihydrate, and polysorbate 80 that effectively stabilizes BDDrFVIII during freeze-drying and storage. The factor VIII potency of the lyophilized BDDrFVIII formulation remains stable for at least 24 months when stored at S”C, and for at least 6 months when stored at room temperature. Although the data indicate that the recon-
stituted product remains stable for at least 100 hours when stored at room temperature,” it is recommended that BDDrFVIII be infused within 3 hours after reconstitution, unless the solution is handled under aseptic conditions. The small volume (4 mL saline) needed to reconstitute the product, combined with the ease of reconstitution, is an obvious advantage for BDDrFVIII as compared to the first generation of recombinant human factor VIII replacement products used to treat patients with hemophilia A. Because the formulation of BDDrFVIII avoids the use of human-derived (or animal-derived) components, the associated potential risk of introducing pathogens has been reduced.
References 1. Fricke WA, Lamb MA: Viral safety of clotting factor concentrates. Semin Thromb Hemost 19:54-61, 1993 2. Berntorp E: Second generation, B-domain deleted recombinant factor VIII. Thromb Haemost 77:256260, 1997 3. Lind P,. Larsson K, Spira J, et al: Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization. Eur J Biochem 232:19-27, 1996 4. Toole JJ, Pittman DD, Orr EC, et al: A large region (-95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Nat1 Acad Sci USA 83:5939-5942, 1986 5. Sandberg H, Almstedt A, Brandt J, et al: Structural and functional characteristics of a B-domain deleted recombinant factor VIII molecule, r-VIII SQ. Thromb Haemost 85:93-100, 2001 6. ijsterberg T, Fatouros A, Mikaelsson M: Development of a freeze-dried albumin-free formulation of recombinant factor VIII SQ. Pharm Res 14:892-898, 1997 Arakawa T, Kita Y, Carpenter J: Protein-solvent interactions in pharmaceutical formulations. Pharm Res 8:285-291, 1991 ijsterberg T, Wadsten ‘I’: Physical state of L-histidine after freeze-drying and long term storage. Eur J Pharm Sci 8:301-308, 1998 Juhlin F, Moberg U, Oswaldsson U, et al: Stability and compatibility of reconstituted recombinant factor VIII SQ (r-VIII SQ) in a system for continuous infusion. XXII International Congress of the World Federation of Haemophilia, Dublin, Ireland, June 23-28, 1996 (abstr)
ONCERN OVER THE potential transmission of viral disease by plasma-derived human factor VIII products,l as well as periodic shortages of both plasma-derived and recombinant human factor VIII products, has underscored the need for the development of new recombinant products for treating patients with hemophilia A. Recently, a B-domain deleted form of human coagulation factor VIII (BDDrFVIII; ReFacto, Wyeth/Genetics Institute, Cambridge, MA) has been developed. The plasma-derived factor VIII molecule exists in several active forms ranging in size from 170 kd to 280 kd.2 The BDDrFVIII gene has been genetically engineered to correspond to the smallest (170 kd) of the multiple active forms of factor VIII found in plasma-derived factor VIII concentrates. The rationale for the design of BDDrFVIII was the observatipn that a large region within the full-length factor VIII molecule, the heavily glycosylated B-domain consisting of 980 amino acids, is not required for
C
From Pharmucia AB, Stockholm, Sweden; and the Genetics Institute, Cambridge, MA. Address reprint requests to N. Wame, PhD, Genetics Institute, One Burtt Rd, Andover, MA 01810. Copyright 0 2001 by W.B. Saunders Company 0037-1963/01/3802-4006$35.00/O doi:l0.1053lshenz.2001.25892
40
Seminars
in Hematology,
expression of biologic activity.* Like the activated full-length form, the BDDrFVIII molecule is composed of two polypeptide chains, domains Al and A2 (90 kd) and domains A3, Cl, and C2 (80 kd), held together by a metal ion bridge.5 The goal for the BDDrFVIII formulation was to develop an easily reconstituted, freeze-dried formulation that maintains the integrity and potency of factor VIII without requiring the addition of human serum albumin (HSA) or other macromolecular stabilizers.
Formulation
Strategy
Development of a formulation for BDDrFVIII that preserves factor VIII activity during prolonged storage of the lyophilized product and for appropriate time periods after reconstitution required extensive testing of a large number of candidate formulations. A broad range of discriminating analytical methods for assessingthe stability of BDDrFVIII was utilized. These analytical methods include the following6: 1. The chromogenic substrate assay for factor VIII activity using the Coatest Factor VIII kit (Chromogenix AB, MBlndal, Sweden). 2. Size-exclusion high-performance liquid chromatography to detect soluble aggregates and degradation products. 3. Sodium dodecyl sulfate-polyacrylamide gel
Vol 38, No 2, Suppl 4 (April),
2001:
pp 40-43
BDDrFVlll
electrophoresis for visualization tion products. 4. The coulometric Karl Fisher sure moisture content. 5. Visual inspection for clarity coloration.
Fomzdation and Stability -
of degradaassay to meaand
degree
of
The two main problems that had to be addressed in formulating BDDrFVIII were its relative instability in solution and its potential for nonspecific adsorption to surfaces. Loss of active protein due to these factors can be considerable. Lyophilization is an appropriate means to stabilize such molecules, provided the components of the formulation are carefully selected.
Formulation
Components
The final formulation,6 after reconstitution of the freeze-dried product with 4 mL sodium chloride (9 mg/mL), contains 62.5 to 250 IU/mL BDDrFVIII, 18 mg/mL sodium chloride, 3.0 mg/mL sucrose, 1.5 mg/mL z-histidine, 0.25 mg/mL calcium chloride dihydrate, and 0.1 mg/mL polysorbate 80; and is adjusted to pH 7.0 (Table 1). Sodium chloride acts as a bulking agent during lyophilization and enhances the solubility of BDDrFVIII. Sucrose acts as a “water substitute” stabilizer in the amorphous phase of the lyophilized product.’ L-Histidine serves to buffer the formulation around pH 7.0, thereby minimizing aggregate formation and maintaining the factor VIII potency of BDDrFVIII during lyophilization and storage. Unlike other buffering agents that may crystallize during freezing and cause pH shifts, L-histidine does not crystallize around pH 6.0, which coincides with the pK of the weakly
Table
1.
Composition
of the
Final
BDDrFVlll
Concentration After Reconstitution*
Amount/Vial BDDrFVlll
250,
500,
1,000 Sodium
chloride
Sucrose
Polysorbate * With
62.5, IU
250
chloride
dihydrate
36
mg
18
mg/mL
mg
3.0
mg/mL
1 mg 0.4 mg
80 4 mL sodium
chloride
diluent
125, IU/mL
12
6 mg
L-Histidine Calcium
Formulation
1.5 0.25
(9 mg/mL).
0.1
mg/mL mg/mL mg/mL
41
basic imidazolium function.* Calcium ions preserve the metal ion bridge linking the two peptides required for the biologic activity of the heterodimer.” The concentration is kept as low as possible (0.25 mg/mL), since calcium salts decrease the glass transition temperature (TL) of the amorphous phase surrounding the ice crystals. The glass transition temperature should be as high as possible in order to shorten the freeze-drying process. 6 Polysorbate 80 is as effective as HSA for stabilizing the factor VIII activity of BDDrVIII (Fig l), and also protects the BDDrFVIII molecule from surface denaturation and adsorption.6 Formulations buffered without polysorbate 80 lose a substantial amount of factor VIII activity (>50%), whereas the addition of appropriate amounts of polysorbate 80 reduces the loss of factor VIII activity during freeze-drying (Fig l).”
Lyophilization
Temperature
Thermal analysis of the final formulation of BDDrFVIII showed a TL of -42” to -38°C. Freeze-drying microscopy revealed maintenance of the structural integrity of the freeze-dried product during freeze-drying at temperatures of -35°C and structural collapse of the product at temperatures higher than - 30°C. These data indicated that formulations of BDDrFVIII had to be lyophilized at a product temperature below - 32°C.”
PH Candidate formulations ranging in pH from 4.0 to 8.0 were freeze-dried and stored at temperatures of 7°C 25°C and 37”C, for up to 12 months. A pH range of 6.5 to 7.5 was found to be optimal for preserving factor VIII activity. Gel filtration detected no aggregates in BDDrFVIII formulations for this pH range. There was a consistent loss of approximately 10% of factor VIII activity of BDDrFVIII during the freeze-drying process, regardless of the pH of the formulation (Fig 2).6
Stability The final formulation preserves the potency integrity of BDDrFVIII over a prolonged
and pe-
42
isstwbwg et al
300
0 0 q n
250
B
Before filtration After filtration Thawed 5 times Freeze-dried
200
2 0 5
150
100 50
!u Buffer only
PEG 4000
HSA
riod of time. The factor VIII potency of the freeze-dried product remains stable for at least 24 months when stored at 8°C (Fig 3), and for at least 6 months when stored at room temperature, 25°C (Fig 4). Aggregates or fragments were below 5% during the storage period. In addition, storage stability of BDDrFVIII was unaffected over the pH range of 6.0 to 8.0.6 The reconstituted product remains stable for at least 100 hours when stored at 25’C, which would allow the product to be continuously
lemaccel@
Figure 1. BDDrFVlll activity before and after sterile filtration, freeze-thawing, and freezedrying, when buffered with different stabilizers. VIII:C, factor VIII activity. Reprinted with permis-
Haesesterile
sion.6
administered via infusion pump.9 However, in keeping with the International Conference on Harmonisation guidelines on the use of products not containing preservatives, it is recommended that BDDrFVIII be used within 3 hours after reconstitution.
1250
1000 = 9 + 2 3
0 After freeze-drying 0 Before freezedrying
500
gl
80 70
400
0 I
2
60
0
50
: e f g
40
:
5
750
0 z z 500
250
3oo 0
200
III 0
30
3
I 9
6
I 12
I 15
I 18
I 21
I 24
Time (months)
100
0 PH
Figure 2. Recovery of factor VIII activity of BDDrFVlll in the final formulation before and after freeze-dt-ying at different pHs (500 W/vial). (-¤-) Percent recovery of VIII:C after freeze-drying.
25OlU
8~
5OOIU
8 *Batch
1000
IU
Batch
--+--Batch
1
. *es
1
-*-Batch2
1
-O-
Batch
2
Batch
2
Figure 3. Stability of 3 batches of lyophilized BDDrFVlll for 250, 500, and 1,000 IU stored at 8°C. Values for 500 and 1,000 IU batches at 0 and 3 months have been multiplied by 1.07 to correct for change of international standard.
BDDrFVllI
‘ii sY
0=
60 i 40
5: 20
0-I0
-o..a-. -=+.= e
1
8°C (ambi-RH) 25°C (60% RH) 30°C (60% RH) 40°C (75% RH) * Not tested.
2
3
4
5
43
Fwnudation and Stability
6
Time (months) Figure 4. Stability of lyophilized BDDrWlll (250 IU) at different temperatures. Data represent mean i SD (N = 3). There was no significant difference in factor VIII activity between BDDrFVlll stored at 8” and 25°C at month 6. ambi-RH, ambient relative humidity.
Solubility The ease of reconstitution of the BDDrFVIII formulation is comparable to that of other commercially available recombinant factor VIII products.
Conclusions BDDrFVIII is a highly purified recombinant derivative of human plasma-derived factor VIII. Extensive testing of a large number of candidate formulations using highly discriminatory analytical methods has shown that BDDrFVIII can be formulated for freeze-drying without the addition of HSA or other macromolecular stabilizers. Extensive experiments led to a formulation using a combination of sodium chloride, sucrose, L-histidine, calcium chloride dihydrate, and polysorbate 80 that effectively stabilizes BDDrFVIII during freeze-drying and storage. The factor VIII potency of the lyophilized BDDrFVIII formulation remains stable for at least 24 months when stored at S”C, and for at least 6 months when stored at room temperature. Although the data indicate that the recon-
stituted product remains stable for at least 100 hours when stored at room temperature,” it is recommended that BDDrFVIII be infused within 3 hours after reconstitution, unless the solution is handled under aseptic conditions. The small volume (4 mL saline) needed to reconstitute the product, combined with the ease of reconstitution, is an obvious advantage for BDDrFVIII as compared to the first generation of recombinant human factor VIII replacement products used to treat patients with hemophilia A. Because the formulation of BDDrFVIII avoids the use of human-derived (or animal-derived) components, the associated potential risk of introducing pathogens has been reduced.
References 1. Fricke WA, Lamb MA: Viral safety of clotting factor concentrates. Semin Thromb Hemost 19:54-61, 1993 2. Berntorp E: Second generation, B-domain deleted recombinant factor VIII. Thromb Haemost 77:256260, 1997 3. Lind P,. Larsson K, Spira J, et al: Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization. Eur J Biochem 232:19-27, 1996 4. Toole JJ, Pittman DD, Orr EC, et al: A large region (-95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Nat1 Acad Sci USA 83:5939-5942, 1986 5. Sandberg H, Almstedt A, Brandt J, et al: Structural and functional characteristics of a B-domain deleted recombinant factor VIII molecule, r-VIII SQ. Thromb Haemost 85:93-100, 2001 6. ijsterberg T, Fatouros A, Mikaelsson M: Development of a freeze-dried albumin-free formulation of recombinant factor VIII SQ. Pharm Res 14:892-898, 1997 Arakawa T, Kita Y, Carpenter J: Protein-solvent interactions in pharmaceutical formulations. Pharm Res 8:285-291, 1991 ijsterberg T, Wadsten ‘I’: Physical state of L-histidine after freeze-drying and long term storage. Eur J Pharm Sci 8:301-308, 1998 Juhlin F, Moberg U, Oswaldsson U, et al: Stability and compatibility of reconstituted recombinant factor VIII SQ (r-VIII SQ) in a system for continuous infusion. XXII International Congress of the World Federation of Haemophilia, Dublin, Ireland, June 23-28, 1996 (abstr)