Stabilizing agent for pharmaceutical proteins

Abstract

A method for stabilising a human blood protein or human blood plasma protein with a molecular weight of >10 KDa by adding melezitose to a solution comprising the human blood protein or human blood plasma protein with a molecular weight of >10 KDa.

Claims

1. A method for the preparation of a human factor VIII-containing product, wherein the method comprises the following steps: (a) providing a human factor VIII-containing solution; (b) adding between 10 mM and 200 mM of melezitose to the human factor VIII-containing solution of step (a), wherein the amount of melezitose per amount of human factor VIII is in the range of 735:1 to 2500:1 when calculated on a weight by weight basis, (c) lyophilizing the solution of step (b) to prepare a lyophilized human factor VIII-containing product, and (d) resuspending the lyophilized human factor VIII-containing product of step (c) to prepare a resuspended human factor VIII-containing solution; wherein the human factor VIII is a human B-domain deleted factor VIII protein recombinantly produced in cell culture, and wherein the human factor VIII is stabilized by the addition of the melezitose in the human factor VIII-containing solution of step (b), in the lyophilized human factor VIII-containing product of step (c), and/or in the resuspended human factor VIII-containing solution of step (d).

2. The method of claim 1 wherein at least one surface active agent selected from the group consisting of recombinant albumin, Polysorbate 80, Polysorbate 20 and a Poloxamer is present.

3. The method of claim 1 wherein at least one buffering agent selected from the group consisting of histidine, sodium citrate, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ehtanesuflonic acid (HEPES), Tris, 3-morpholinorpoane-1-sulfonic acid (MOPS) and 1,4Piperazinediethanesulfonic acid (PIPES) is present.

4. The method of claim 1 wherein at least one further stabilizer selected from the group consisting of sugars, amino acids, polyols, and combinations thereof is present; and/or wherein at least one tonicity modifier selected from the group consisting of sodium chloride, arginine, glycine, potassium chloride, sugars and sugar alcohols is present; and/or wherein at least one bulking agent selected from the group consisting of glycine, mannitol, sodium chloride, arginine and sucrose is present.

5. The method of claim 1 wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 6 months.

6. The method of claim 1, wherein the melezitose is present in an amount from about 10 mM to about 100 mM.

7. The method of claim 2, wherein the Poloxamer is Poloxamer 188.

8. The method of claim 5, wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 12 months.

9. The method of claim 5, wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 24 months.

10. The method of claim 5, wherein the lyophilized human factor VIII-containing product of step (c) has improved long-term stability and a shelf-life of at least 36 months.

11. The method of claim 1 wherein the human factor VIII protein is not a chimeric human factor VIII protein, a human factor VIII fusion protein or a human factor VIII derivative coupled with one or more chemical compounds.

Description

EXAMPLES

(1) Recombinant Factor VIII

(2) The factor VIII used in the experiments is a recombinant human B-domain deleted factor VIII protein, produced in the human cell line HEK293F according to the process described in EP-A-1 739 179 (Schrder et al). The purification process consisted of five chromatography steps and generated a highly pure factor VIII protein preparation (Winge et al, WO-A-2009/156430) with a human glycosylation like pattern (Sandberg et al, PCT/EP2009/060829).

(3) Plasma Derived Factor IX

(4) The material used in these experiments origins from the commercially available product Nanotiv, which is a high purity SD treated and nanofiltered factor IX concentrate. Before use in these experiments the material has been further purified over a gel filtration column where the factor IX monomer peak was used for further experiments.

(5) Recombinant HESylated G-CSF

(6) The cell line used is a derivative of human embryonic kidney cell 293 (HEK 293), which was adapted to serum-free growth. This host, HEK 293F, was stably transfected with an expression cassette carrying the cDNA coding sequence for G-CSF. The strong promoter was used for the cassette. The general process is also described in EP 1739179 (Schrder et al).

(7) The purification process consisted of four chromatography steps and generated a highly pure G-CSF protein preparation. The G-CSF protein was coupled to a hydroxylethyl starch (HES) derivative of a molecular weight of approximately 100 KDa. Finally, the HES-G-CSF was purified from the non reacted HES derivative and G-CSF by one chromatography step, resulting in a molecule with a total molecular weight of approximately 120 KDa.

Example 1

Stabilisation of rFVIII by Melezitose in Solution

(8) Preparation

(9) The recombinant factor VIII (rFVIII) was prepared according to the description in the experimental section above. This experiment compared the stabilizing effect of melezitose on rFVIII in solution, with that of the commonly used stabilizer trehalose. The concentration of rFVIII was 100 IU/ml. The compositions of the formulations investigated in this experiment are displayed in Table 1.

(10) TABLE-US-00002 TABLE 1 Compositions of the formulation. 1A 1B Melezitose, mM 48 Trehalose dihydrate, mM 63 NaCl, mg/ml 30 30 Calcium chloride dihydrate, 0.5 0.5 mg/ml Poloxamer 188, mg/ml 2 2 Histidine, mg/ml 3 3

(11) The formulations were stored for up to 7 days at +25 C. to evaluate the protein activity over time. Samples were taken at regular intervals and analyzed with the chromogenic assay, as described in the experimental section above. The results are summarized in Table 2, as percentage of the initial value.

(12) TABLE-US-00003 TABLE 2 Results. Factor VIII activity over time (days), (% of initial value) 0 1 7 1A +25 C. 100 86 85 1B +25 C. 100 82 86

Conclusions of Example 1

(13) This experiment shows that, surprisingly, melezitose, despite of its lower molar concentration, has a stabilizing effect on rFVIII in solution equal to that of trehalose.

Example 2

Stabilisation of rFVIII by Melezitose in Lyophilized Form

(14) Preparation

(15) The recombinant factor VIII (rFVIII) was prepared according to the description in the experimental section above. This experiment compared the stabilizing effect of melezitose with that of the commonly used stabilizer trehalose, over the freeze-drying process and in lyophilized formulations. The concentration of rFVIII was 100 IU/ml. The compositions of the formulations investigated in this experiment are displayed in Table 3.

(16) TABLE-US-00004 TABLE 3 Compositions of the formulations. 2A 2B Trehalose, mM 63 Melezitose, mM 48 NaCl, mg/ml 30 30 Calcium chloride dihydrate, 0.5 0.5 mg/ml Poloxamer 188, mg/ml 2 2 Histidine, mg/ml 3 3

(17) 1.5 ml aliquots of the solutions were lyophilized in a laboratory scale freeze-drier. The protein recovery over the lyophilisation step was 93% for formulation 2B and 86% for formulation 2A. The lyophilized samples were stored for up to 4 weeks at +25 C. and +40 C. to evaluate the protein activity over time. The samples were reconstituted in 1.5 ml water for injections and analyzed with the chromogenic assay, described in the experimental section above. Results are summarized in Table 4.

(18) TABLE-US-00005 TABLE 4 Results Factor VIII activity over time (weeks), (% of initial value) 0 1 2 4 2A +25 C. 100 97 89 * +40 C. 100 98 90 93 2B +25 C. 100 117 95 * +40 C. 100 104 97 99 * no significant change

Conclusions of Example 2

(19) Surprisingly, this experiment shows that melezitose is able to protect rFVIII in lower molar concentration than trehalose over the lyophilisation step, and that it stabilizes rFVIII better than trehalose during storage.

Example 3

Stabilisation of rFVIII by Melezitose in Lyophilized Form

(20) Preparation

(21) The recombinant factor VIII (rFVIII) was prepared according to the description in the experimental section above. This experiment compared the stabilizing effect of melezitose at different concentrations over the freeze-drying process and in lyophilized formulations, and also compared the stabilizing effect with the tetrasaccharide Stachyose. The concentration of rFVIII was 170 IU/ml. The compositions of the formulations investigated in this experiment are displayed in Table 5.

(22) TABLE-US-00006 TABLE 5 Compositions of the formulations. 3A 3B 3C 3D Melezitose, mM 48 36 24 Stachyose, mM 30 NaCl, mg/ml 30 30 30 30 Calcium chloride dihydrate, 0.5 0.5 0.5 0.5 mg/ml Poloxamer 188, mg/ml 2 2 2 2 Sodium citrate, mg/ml 2 2 2 2

(23) 1.5 ml aliquots of the solutions were lyophilized in a laboratory scale freeze-drier. The protein recovery over the lyophilisation step was 91 to 100% for formulations containing melezitose, while the recovery was 84% for formulation 3D containing stachyose as stabilizer. The lyophilized samples were stored for up to 12 months at +25 C. and +40 C. to evaluate the protein activity over time.

(24) The samples were reconstituted in 1.5 ml water for injections and analyzed with the chromogenic assay, described in the experimental section above. Results are summarized in Table 6.

(25) TABLE-US-00007 TABLE 6 Results. Factor VIII activity over time (months), (% of initial value) 0 1 2 3 6 12 3A 25 C. 100 * n.a. * * 91 40 C. 100 96 93 93 n.a. n.a. 3B 25 C. 100 92 n.a. 96 95 78 40 C. 100 90 79 73 n.a. n.a. 3C 25 C. 100 91 n.a. 86 86 67 40 C. 100 70 58 48 n.a. n.a. 3D 25 C. 100 95 n.a. 79 65 n.a. 40 C. 100 74 n.a. 51 n.a. n.a. n.a. = not analysed; * no significant change

Conclusions of Example 3

(26) This experiment shows that melezitose functions exceptionally well as a stabilizer for rFVIII over the lyphilization step and in lyophilized form. Also, it shows that stachyose is not a preferable stabilizer for lyophilized formulations, as it shows very unsatisfactory results during storage at both 25 C. and at 40 C., compared to the melezitose containing formulations.

Example 4

Stabilisation of Plasma Factor IX by Melezitose in Lyophilized Form

(27) Preparation

(28) The plasma derived factor IX (pFIX) was prepared according to the description in the experimental section above. This experiment investigates the stabilizing effect of melezitose on pFIX. The concentration of pFIX was 100 IU/ml. The composition of the formulation investigated in this experiment is displayed in Table 7.

(29) TABLE-US-00008 TABLE 7 Composition of the formulation. 4A Melezitose, mM 42 NaCl, mg/ml 30 Polysorbate 80, mg/ml 0.1 Sodium citrate, mg/ml 2.35

(30) 1.5 ml aliquots of the solutions were lyophilized in a laboratory scale freeze-drier. The lyophilized samples were stored for up to 6 months at +5 C., +25 C. and +40 C. to evaluate the protein activity over time. The samples were reconstituted in 1.5 ml water for injections and analyzed with the chromogenic assay, described in the experimental section above.

(31) Results

(32) The protein recovery over the lyophilisation step was about 100%. The results of the stability study are shown in Table 8.

(33) TABLE-US-00009 TABLE 8 Results. Factor IX activity over time (months), (% of initial value) 0 1 3 6 4A 5 C. 100 88 87 85 25 C. 100 94 89 86 40 C. 100 93 92 n.a. n.a. = not analysed; * no significant change

Conclusions of Example 4

(34) This experiment shows that, surprisingly, melezitose functions well as a stabilizer for factor IX in lyophilized form.

Example 5

Stabilization of HESylated Recombinant G-CSF by Melezitose in Lyophilized Form

(35) Preparation

(36) The recombinant HESylated G-CSF (rHES-G-CSF) was prepared according to the description in the experimental section above. The experiment compared the stabilizing effect of melezitose on rHES-G-CSF in lyophilized form, with that of the commonly used stabilizer trehalose. The concentration of rHES-G-CSF was 0.3 mg/ml and the compositions of the formulations investigated are displayed in Table 9.

(37) TABLE-US-00010 TABLE 9 Compositions of the formulation. 5A 5B Melezitose, mM 70 Trehalose, mM 70 NaCl, mg/ml 30 30 Polysorbate 20, mg/ml 0.2 0.2 Histidine, mg/ml 3 3

(38) 1.5 ml aliquots of the solutions were lyophilized in a laboratory scale freeze-drier. The protein recovery was measured after 4 weeks storage at +40 C. by the Resource S method, described in the experimental section above. Results are summarized in Table 10.

(39) TABLE-US-00011 TABLE 10 Results. Gain of peak width (min) after 4 weeks 5A +40 C. 0.04 5B +40 C. 0.06

Conclusions of Example 5

(40) This experiment shows that melezitose has a stabilizing effect on rHES-G-CSF better than that of the commonly used stabilizer trehalose at equal molar concentration.

Example 6

Stabilisation of Plasma Factor IX by Melezitose Over the Freeze-Drying Step

(41) Preparation

(42) The plasma derived factor IX (pFIX) was prepared according to the description in the experimental section above. This experiment investigates the stabilizing effect of melezitose on pFIX over the freeze-drying step, compared to the tetrasaccharide stachyose. The concentration of pFIX was 100 IU/ml. The compositions of the formulations investigated in this experiment are displayed in Table 11.

(43) TABLE-US-00012 TABLE 11 Compositions of the formulations. 6A 6B Melezitose, mM 42 Stachyose, mM 30 NaCl, mg/ml 30 30 Polysorbate 80, mg/ml 0.1 0.1 Sodium citrate, mg/ml 2.35 2.35

(44) 1.5 ml aliquots of the solutions were lyophilized in a laboratory scale freeze-drier. The samples were analyzed with the chromogenic assay, described in the experimental section above, before and after the lyophilization step.

(45) Results

(46) The protein recovery over the lyophilisation step was about 100% for formulation 6A, while the corresponding recovery for formulation 6B was 84%.

Conclusions of Example 6

(47) This experiment shows that, melezitose functions well as a stabilizer for factor IX over the lyophilization step. However, stachyose is not a suitable candidate as stabilizer since a significant activity loss occurs over the freeze-drying step.