U.S. patent application number 17/116587 was filed with the patent office on 2021-04-08 for aqueous pharmaceutical formulation comprising anti-pd-l1 antibody avelumab.
The applicant listed for this patent is MERCK PATENT GMBH, PFIZER INC.. Invention is credited to Alessandra DEL RIO, Silvia FRATAR-CANGELI, Gianluca RINALDI, Senta VOSS, Markus WEIGANDT.
Application Number | 20210100903 17/116587 |
Document ID | / |
Family ID | 1000005290074 |
Filed Date | 2021-04-08 |
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United States Patent
Application |
20210100903 |
Kind Code |
A1 |
RINALDI; Gianluca ; et
al. |
April 8, 2021 |
AQUEOUS PHARMACEUTICAL FORMULATION COMPRISING ANTI-PD-L1 ANTIBODY
AVELUMAB
Abstract
The present invention relates to a novel anti-PD-L1 antibody
formulation. In particular, the invention relates to an aqueous
pharmaceutical formulation of the anti-PD-L1 antibody Avelumab.
Inventors: |
RINALDI; Gianluca;
(Monterotondo, IT) ; DEL RIO; Alessandra; (Roma,
IT) ; FRATAR-CANGELI; Silvia; (Ceprano, IT) ;
VOSS; Senta; (Mainz, DE) ; WEIGANDT; Markus;
(Mannheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MERCK PATENT GMBH
PFIZER INC. |
Darmstadt
New York |
NY |
DE
US |
|
|
Family ID: |
1000005290074 |
Appl. No.: |
17/116587 |
Filed: |
December 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16060319 |
Jun 7, 2018 |
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PCT/EP2016/002040 |
Dec 5, 2016 |
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17116587 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/54 20130101;
C07K 2317/21 20130101; C07K 2317/76 20130101; A61K 39/39591
20130101; C07K 16/2827 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 7, 2015 |
EP |
15198233.7 |
Claims
1-42. (canceled)
43. A method of treating cancer comprising administering an aqueous
pharmaceutical antibody formulation to a patient, wherein the
formulation comprises: (i) Avelumab in a concentration of 1
milligram/milliliter (mg/mL) to 30 mg/ml, as the antibody; (ii)
acetate or histidine in a concentration of 5 millimolar (mM) to 15
mM as the buffering agent; (iii) D-mannitol or trehalose in a
concentration of 240 mM to 320 mM, or a combination of arginine HCl
in a concentration of 50 to 150 mM and glutamic acid in a
concentration of 25 mM to 75 mM as a stabiliser; and (iv) Poloxamer
188 or Polysorbate 20 in a concentration of 0.25 mg/mL to 0.75
mg/mL, as surfactant, or no surfactant; wherein the formulation
does not comprise methionine, and further wherein the formulation
has a pH of 5.0 to 6.0.
44. The method of claim 43, wherein the formulation has a pH of 5.0
to 5.6.
45. The method of claim 43, wherein the formulation comprises
Avelumab at a concentration of about 10 mg/mL to about 20
mg/mL.
46. The method of claim 43, wherein the formulation comprises
acetate or histidine at a concentration of about 10 mM.
47. The method of claim 43, wherein the formulation comprises
D-mannitol or trehalose at a concentration of about 280 mM, or for
the combination of arginine HCl and glutamic acid, the formulation
comprises arginine HCl at a concentration of about 150 mM and
glutamic acid at a concentration of about 50 mM.
48. The method of claim 43, wherein the formulation comprises
Poloxamer 188 or Polysorbate 20 at a concentration of about 0.5
mg/mL.
49. The method of claim 43, wherein the formulation has a pH of 5.2
(.+-.0.1) to 5.5 (.+-.0.1).
50. The method of claim 43, wherein the formulation comprises
acetate in a concentration of about 10 mM, and not comprising any
other buffering agent.
51. The method of claim 43, wherein the formulation comprises
D-mannitol or trehalose in a concentration of about 280 mM, and not
comprising any other stabiliser.
52. The method of claim 43, wherein the formulation comprises
Polysorbate 20 or Poloxamer 188 in a concentration of about 0.5
mg/mL and does not comprise any other surfactant.
53. The method of claim 51, wherein the formulation comprises: (i)
Avelumab in a concentration of 10 mg/mL; (ii) acetate in a
concentration of 10 mM; (iii) D-mannitol or trehalose in a
concentration of 280 mM; and (iv) Polysorbate 20 or Poloxamer 188
in a concentration of 0.5 mg/mL; wherein the formulation has a pH
of 5.5 (.+-.0.1).
54. The method of claim 52, wherein the formulation consists of:
(i) Avelumab in a concentration of 10 mg/mL; (ii) sodium acetate
trihydrate in a concentration of 10 mM; (iii) D-mannitol or
trehalose in a concentration of 280 mM; (iv) Polysorbate 20 or
Poloxamer 188 in a concentration of 0.5 mg/mL; (v) HCl to adjust
the pH; and (vi) water (for injection) as the solvent; wherein the
formulation has a pH of 5.5 (.+-.0.1).
55. The method of claim 54, wherein the formulation consists of:
(i) Avelumab in a concentration of 10 mg/mL; (ii) sodium acetate
trihydrate in a concentration of 10 mM; (iii) trehalose dihydrate
in a concentration of 280 mM; (iv) Polysorbate 20 in a
concentration of 0.5 mg/mL; (v) HCl to adjust the pH; and (vi)
water (for injection) as the diluent; wherein the formulation has a
pH of 5.5 (.+-.0.1).
56. The method of claim 43, wherein the formulation comprises: (i)
Avelumab in a concentration of about 20 mg/mL as the antibody; (ii)
acetate in a concentration of about 10 mM as the buffering agent;
(iii) D-mannitol or trehalose in a concentration of about 280 mM as
a stabiliser; and (iv) Polysorbate 20 or Poloxamer 188 in a
concentration of about 0.5 mg/mL as surfactant; wherein the
formulation has a pH of 5.2 (.+-.0.1).
57. The method of claim 56, wherein the formulation comprises: (i)
Avelumab in a concentration of 20 mg/mL; (ii) acetate in a
concentration of 10 mM; (iii) D-mannitol or trehalose in a
concentration of 280 mM; and (iv) Polysorbate 20 or Poloxamer 188
in a concentration of 0.5 mg/mL; wherein the formulation has a pH
of 5.2 (.+-.0.1).
58. The method of claim 43, wherein the formulation does not
comprise an antioxidant.
59. The method of claim 56, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 10 mM; (iii) D-mannitol or trehalose dihydrate in
a concentration of 280 mM; (iv) Polysorbate 20 or Poloxamer 188 in
a concentration of 0.5 mg/mL; (v) sodium acetate to adjust the pH;
and (vi) water (for injection) as the diluent; wherein the
formulation has a pH of 5.2 (.+-.0.1).
60. The method of claim 59, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 10 mM; (iii) D-mannitol in a concentration of 280
mM; (iv) Polysorbate 20 in a concentration of 0.5 mg/mL; (v) sodium
acetate to adjust the pH; and (vi) water (for injection) as the
diluent; wherein the formulation has a pH of 5.2 (.+-.0.1).
61. The method of claim 59, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 10 mM; (iii) trehalose dihydrate in a
concentration of 280 mM; (iv) Polysorbate 20 in a concentration of
0.5 mg/mL; (v) sodium acetate to adjust the pH; and (vi) water (for
injection) as the diluent; wherein the formulation has a pH of 5.2
(.+-.0.1).
62. The method of claim 59, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 10 mM; (iii) D-mannitol in a concentration of 280
mM; (iv) Poloxamer 188 in a concentration of 0.5 mg/mL; (v) sodium
acetate to adjust the pH; and (vi) water (for injection) as the
diluent; wherein the formulation has a pH of 5.2 (.+-.0.1).
63. The method of claim 59, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 10 mM; (iii) trehalose dihydrate in a
concentration of 280 mM; (iv) Poloxamer 188 in a concentration of
0.5 mg/mL; (v) sodium acetate to adjust the pH; and (vi) water (for
injection) as the diluent; wherein the formulation has a pH of 5.2
(.+-.0.1).
64. The method of claim 56, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 10 mM; (iii) D-mannitol in a concentration of 280
mM; (iv) Polysorbate 20 in a concentration of 0.5 mg/mL; (v) sodium
hydroxide in a concentration of 7.5 mM; and (vi) water (for
injection) as the diluent; wherein the formulation has a pH of 5.2
(.+-.0.1).
65. The method of claim 64, wherein the formulation is made by
combining: (i) 20 mg/mL of Avelumab; (ii) 0.6 mg/mL of glacial
acetic acid; (iii) 51 mg/mL of D-mannitol; (iv) 0.5 mg/mL of
Polysorbate 20; (v) 0.3 mg/mL of sodium hydroxide; and (vi) water
(for injection) as the diluent.
66. The method of claim 44, wherein the formulation consists of:
(i) Avelumab in a concentration of 20 mg/mL; (ii) acetic acid in a
concentration of 0.6 mg/mL; (iii) D-mannitol in a concentration of
51 mg/mL; (iv) Polysorbate 20 in a concentration of 0.5 mg/mL; (v)
sodium hydroxide in a concentration of 0.3 mg/mL; and (vi) water
(for injection) as the diluent; wherein the formulation has a pH of
5.0 to 5.6
67. The method of claim 43, wherein the Avelumab has the heavy
chain sequence of either (SEQ ID NO:1) or (SEQ ID NO:2), the light
chain sequence of (SEQ ID NO:3), and carries a glycosylation on
Asn300 comprising FA2 and FA2G1 as the main glycan species, having
a joint share of more than 70% of all glycan species.
68. The method of claim 67, wherein in the Avelumab glycosylation
the FA2 has a share of 44% to 54% and the FA2G1 has a share of 25%
to 41% of all glycan species.
69. The method of claim 68, wherein in the Avelumab glycosylation
the FA2 has a share of 47% to 52% and the FA2G1 has a share of 29%
to 37% of all glycan species.
70. The method of claim 67, wherein in the Avelumab glycosylation
the FA2 has a share of about 49% and the FA2G1 has a share of about
30% to about 35% of all glycan species.
71. The method of claim 67, wherein the Avelumab glycosylation
further comprises as minor glycan species A2 with a share of less
than 5%, A2G1 with a share of less than 5%, A2G2 with a share of
less than 5% and FA2G2 with a share of less than 7% of all glycan
species.
72. The method of claim 71, wherein in the Avelumab glycosylation
the A2 has a share of 3% to 5%, the A2G1 has a share of less than
4%, the A2G2 has a share of less than 3% and the FA2G2 has a share
of 5% to 6% of all glycan species.
73. The method of claim 72, wherein in the Avelumab glycosylation
the A2 has a share of about 3.5% to about 4.5%, the A2G1 has a
share of about 0.5% to about 3.5%, the A2G2 has a share of less
than 2.5% and the FA2G2 has a share of about 5.5% of all glycan
species.
74. The method of claim 67, wherein the Avelumab has the heavy
chain sequence of (SEQ ID NO:2).
75. The method of claim 43, wherein the formulation is administered
by intravenous (IV) administration.
76. The method of claim 43, wherein the cancer is selected from
non-small cell lung cancer, urothelial carcinoma, bladder cancer,
mesothelioma, Merkel cell carcinoma, gastric or gastroesophageal
junction cancer, ovarian cancer, breast cancer, thymoma,
adenocarcinoma of the stomach, adrenocortical carcinoma, head and
neck squamous cell carcinoma, renal cell carcinoma, melanoma,
and/or classical Hodgkin's lymphoma.
77. A method of treating cancer comprising administering an aqueous
pharmaceutical antibody formulation to a patient, wherein the
formulation comprises: (i) Avelumab in a concentration of about 10
milligram/milliliter (mg/mL) as the antibody; (ii) acetate in a
concentration of about 10 millimolar (mM) as the buffering agent;
(iii) D-mannitol or trehalose in a concentration of about 280 mM as
a stabiliser; and (iv) Polysorbate 20 or Poloxamer 188 in a
concentration of about 0.5 mg/mL as surfactant; wherein the
formulation does not comprise methionine, and further wherein the
formulation has a pH of 5.5 (.+-.0.1).
78. The method of claim 77, wherein the formulation consists of:
(i) Avelumab in a concentration of 10 mg/mL; (ii) sodium acetate
trihydrate in a concentration of 10 mM; (iii) D-mannitol in a
concentration of 280 mM; (iv) Polysorbate 20 in a concentration of
0.5 mg/mL; (v) HCl to adjust the pH; and (vi) water (for injection)
as the diluent; wherein the formulation has a pH of 5.5
(.+-.0.1).
79. The method of claim 77, wherein the formulation is administered
by intravenous (IV) administration.
80. The method of claim 77, wherein the cancer is selected from
non-small cell lung cancer, urothelial carcinoma, bladder cancer,
mesothelioma, Merkel cell carcinoma, gastric or gastroesophageal
junction cancer, ovarian cancer, breast cancer, thymoma,
adenocarcinoma of the stomach, adrenocortical carcinoma, head and
neck squamous cell carcinoma, renal cell carcinoma, melanoma,
and/or classical Hodgkin's lymphoma.
81. A method of treating cancer comprising administering an aqueous
pharmaceutical antibody formulation to a patient, wherein the
formulation consists of Avelumab in a concentration of 20
milligram/milliliter (mg/mL) as the active ingredient; and glacial
acetic acid, D-mannitol, Polysorbate 20, sodium hydroxide and water
for injection as the excipients; wherein the formulation has a pH
of 5.0 to 5.6.
82. The method of claim 81, wherein the formulation has a pH of 5.2
(.+-.0.1).
83. The method of claim 81, wherein the formulation is administered
by intravenous (IV) administration.
84. The method of claim 81, wherein the cancer is selected from
non-small cell lung cancer, urothelial carcinoma, bladder cancer,
mesothelioma, Merkel cell carcinoma, gastric or gastroesophageal
junction cancer, ovarian cancer, breast cancer, thymoma,
adenocarcinoma of the stomach, adrenocortical carcinoma, head and
neck squamous cell carcinoma, renal cell carcinoma, melanoma,
and/or classical Hodgkin's lymphoma.
Description
[0001] The present invention relates to a novel anti-PD-L1 antibody
formulation. In particular, the invention relates to an aqueous
pharmaceutical formulation of the anti-PD-L1 antibody Avelumab.
BACKGROUND OF THE INVENTION
[0002] The programmed death 1 (PD-1) receptor and PD-1 ligands 1
and 2 (PD-L1, PD-L2) play integral roles in immune regulation.
Expressed on activated T cells, PD-1 is activated by PD-L1 and
PD-L2 expressed by stromal cells, tumor cells, or both, initiating
T-cell death and localized immune suppression (Dong H, Zhu G,
Tamada K, Chen L. B7-H1, a third member of the B7 family,
co-stimulates T-cell proliferation and interleukin-10 secretion.
Nat Med 1999;5:1365-69; Freeman G J, Long A J, Iwai Y, et al.
Engagement of the PD-1 immunoinhibitory receptor by a novel B7
family member leads to negative regulation of lymphocyte
activation. J Exp Med 2000;192:1027-34; Dong H, Strome S E, Salomao
D R, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a
potential mechanism of immune evasion. Nat Med 2002; 8:793-800.
[Erratum, Nat Med 2002;8:1039; Topalian S L, Drake C G, Pardoll D
M. Targeting the PD-1/B7-H1(PD-L1) pathway to activate anti-tumor
immunity. Curr Opin Immunol 2012;24:207-12), potentially providing
an immune-tolerant environment for tumor development and growth.
Conversely, inhibition of this interaction can enhance local T-cell
responses and mediate antitumor activity in nonclinical animal
models (Dong H, Strome S E, Salomao D R, et al. Nat Med 2002;
8:793-800. [Erratum, Nat Med 2002;8:1039; Iwai Y, Ishida M, Tanaka
Y, et al. Involvement of PD-L1 on tumor cells in the escape from
host immune system and tumor immunotherapy by PD-L1 blockade. Proc
Natl Acad Sci USA 2002;99:12293-97). In the clinical setting,
treatment with antibodies that block the PD-1-PD-L1 interaction
have been reported to produce objective response rates of 7% to 38%
in patients with advanced or metastatic solid tumors, with
tolerable safety profiles (Hamid O, Robert C, Daud A, et al. Safety
and tumor responses with lambrolizumab (Anti-PD-1) in melanoma. N
Engl J Med 2013;369:134-44; Brahmer J R, Tykodi S S, Chow L Q, et
al. Safety and activity of anti-PD-L1 antibody in patients with
advanced cancer. N Engl J Med 2012;366(26):2455-65; Topalian S L,
Hodi F S, Brahmer J R, et al. Safety, activity, and immune
correlates of anti-PD-1 antibody in cancer. N Engl J Med
2012;366(26):2443-54; Herbst R S, Soria J-C, Kowanetz M, et al.
Predictive correlates of response to the anti-PD-L1 antibody
MPDL3280A in cancer patients. Nature 2014;515:563-67). Notably,
responses appeared prolonged, with durations of 1 year or more for
the majority of patients.
[0003] Avelumab (also known as MSB0010718C) is a fully human
monoclonal antibody of the immunoglobulin (Ig) G1 isotype. Avelumab
selectively binds to PD-L1 and competitively blocks its interaction
with PD-1.
[0004] Compared with anti-PD-1 antibodies that target T-cells,
Avelumab targets tumor cells, and therefore is expected to have
fewer side effects, including a lower risk of autoimmune-related
safety issues, as blockade of PD-L1 leaves the PD-L2-PD-1 pathway
intact to promote peripheral self-tolerance (Latchman Y, Wood C R,
Chernova T, et al. PD-L1 is a second ligand for PD-1 and inhibits T
cell activation. Nat Immunol 2001;2(3):261-68).
[0005] Avelumab is currently being tested in the clinic in a number
of cancer types including non-small cell lung cancer, urothelial
carcinoma, mesothelioma, Merkel cell carcinoma, gastric or
gastroesophageal junction cancer, ovarian cancer, and breast
cancer.
[0006] The amino acid sequences of Avelumab and sequence variants
and antigen binding fragments thereof, are disclosed in
WO2013079174, where the antibody having the amino acid sequence of
Avelumab is referred to as A09-246-2. Also disclosed are methods of
manufacturing and certain medical uses.
[0007] Further medical uses of Avelumab are described in
WO2016137985, PCT/IB2016/052748, PCT/US2016/037498,
PCT/US2016/053939, U.S. patent application Ser. No. 62/341,921.
[0008] WO2013079174 also describes in section 2.4 a human aqueous
formulation of an antibody having the amino acid sequence of
Avelumab. This formulation comprises the antibody in a
concentration of 10 mg/ml, methionine as an antioxidant and has a
pH of 5.5.
[0009] A formulation study for an aglycosylated anti-PD-L1 antibody
of the IgG1 type is described in WO2015048520, where a formulation
with a pH of 5.8 was selected for clinical studies.
DESCRIPTION OF THE INVENTION
[0010] As Avelumab is generally delivered to a patient via
intravenous infusion, and is thus provided in an aqueous form, the
present invention relates to further aqueous formulations that are
suitable to stabilize Avelumab with its post-translational
modifications, and at higher concentrations as disclosed in
WO2013079174.
[0011] FIG. 1a (SEQ ID NO:1) shows the full length heavy chain
sequence of Avelumab, as expressed by the CHO cells used as the
host organism.
[0012] It is frequently observed, however, that in the course of
antibody production the C-terminal lysine (K) of the heavy chain is
cleaved off. Located in the Fc part, this modification has no
influence on the antibody-antigen binding. Therefore, in some
embodiments the C-terminal lysine (K) of the heavy chain sequence
of Avelumab is absent. The heavy chain sequence of Avelumab without
the C-terminal lysine is shown in FIG. 1b (SEQ ID NO:2).
[0013] FIG. 2 (SEQ ID NO:3) shows the full length light chain
sequence of Avelumab.
[0014] A post-translational modification of high relevance is
glycosylation.
[0015] Most of the soluble and membrane-bound proteins that are
made in the endoplasmatic reticulum of eukaryotic cells undergo
glycosylation, where enzymes called glycosyltransferases attach one
or more sugar units to specific glycosylation sites of the
proteins. Most frequently, the points of attachment are NH.sub.2 or
OH groups, leading to N-linked or O-linked glycosylation.
[0016] This applies also to proteins, such as antibodies, which are
recombinantly produced in eukaryotic host cells. Recombinant IgG
antibodies contain a conserved N-linked glycosylation site at a
certain asparagine residue of the Fc region in the CH2 domain.
There are many known physical functions of N-linked glycosylation
in an antibody such as affecting its solubility and stability,
protease resistance, binding to Fc receptors, cellular transport
and circulatory half-life in vivo (Hamm M. et al., Pharmaceuticals
2013, 6, 393-406). IgG antibody N-glycan structures are
predominantly biantennary complex-type structures, comprising
b-D-N-acetylglucosamine (GlcNac), mannose (Man) and frequently
galactose (Gal) and fucose (Fuc) units.
[0017] In Avelumab the single glycosylation site is Asn300, located
in the CH2 domain of both heavy chains. Details of the
glycosylation are described in Example 1.
[0018] Since glycosylation affects the solubility and stability of
an antibody, it is prudent to take this parameter into account when
a stable, pharmaceutically suitable formulation of the antibody is
to be developed.
[0019] Surprisingly, it has been found by the inventors of the
present patent application that it is possible to stabilize
Avelumab, fully characterized by its amino acid sequence and its
post-translational modifications, in a number of aqueous
formulations without the presence of an antioxidant, at pH values
as low as 5.2.
FIGURES
[0020] FIG. 1a: Heavy chain sequence of Avelumab (SEQ ID NO:1)
[0021] FIG. 1b: Heavy chain sequence of Avelumab, lacking the
C-terminal K (SEQ ID NO:2)
[0022] FIG. 2: Light chain sequence of Avelumab (SEQ ID NO:3)
[0023] FIG. 3: Secondary structure of Avelumab
[0024] FIG. 4: 2AB HILIC-UPLC Chromatogram of Avelumab Glycans
[0025] FIG. 5: Numbering of the peaks of FIG. 4
[0026] FIG. 6: Total aggregates by SE-HPLC of DoE2 formulations
(40.degree. C.)
[0027] FIG. 7: Total aggregates by SE-HPLC of DoE2 formulations
(25.degree. C.)
[0028] FIG. 8: Fragments by Bioanalyzer of DoE2 formulations
(40.degree. C.)
[0029] FIG. 9: Fragments by Bioanalyzer of DoE2 formulations
(25.degree. C.)
[0030] FIG. 10: Acidic cluster and main peak abundance of DoE2
(25.degree. C.)
[0031] FIG. 11: Long Term Stability LMWs (%) at 2-8.degree. C.
[0032] FIG. 12: Long Term Stability Sub-visible particles
.gtoreq.10 .mu.m at 2-8.degree. C.
[0033] FIG. 13: Long Term Stability Sub-visible particles
.gtoreq.25 .mu.m at 2-8.degree. C.
[0034] FIG. 14: Long Term Stability Acidic cluster (%) at
2-8.degree. C.
[0035] FIG. 15: Long Term Stability Main peak (%) at 2-8.degree.
C.
[0036] FIG. 16: Long Term Stability Basic cluster (%) at
2-8.degree. C.
[0037] FIG. 17: Long Term Stability LMWs (%) at 25.degree. C.
[0038] FIG. 18: Long Term Stability Sub-visible particles
.gtoreq.10 .mu.m at 25.degree. C.
[0039] FIG. 19: Long Term Stability Sub-visible particles
.gtoreq.25 .mu.m at 25 .degree. C.
[0040] FIG. 20: Long Term Stability Acidic cluster (%) at
25.degree. C.
[0041] FIG. 21: Long Term Stability Main peak (%) at 25.degree.
C.
[0042] FIG. 22: Long Term Stability Basic cluster (%) at 25.degree.
C.
[0043] FIG. 23: Long Term Stability LMWs (%) at 40.degree. C.
[0044] FIG. 24: Long Term Stability Sub-visible particles
.gtoreq.10 .mu.m at 40 .degree. C.
[0045] FIG. 25: Long Term Stability Sub-visible particles
.gtoreq.25 .mu.m at 40.degree. C.
[0046] FIG. 26: Long Term Stability Acidic cluster (%) at
40.degree. C.
[0047] FIG. 27: Long Term Stability Main peak (%) at 40.degree.
C.
[0048] FIG. 28: Long Term Stability Basic cluster (%) at 40.degree.
C.
DEFINITIONS
[0049] Unless otherwise stated, the following terms used in the
specification and claims have the following meanings set out
below.
[0050] References herein to "Avelumab" include the anti-PD-L1
antibody of the IgG1 type as defined in WO2013079174 by its amino
acid sequence, and as defined in the present patent application by
its amino acid sequence and by its post-translational
modifications. References herein to "Avelumab" may include
biosimilars which, for instance, may share at least 75%, suitably
at least 80%, suitably at least 85%, suitably at least 90%,
suitably at least 95%, suitably at least 96%, suitably at least
97%, suitably at least 98% or most suitably at least 99% amino acid
sequence identity with the amino acid sequences disclosed in
WO2013079174. Alternatively or additionally, references herein to
"Avelumab" may include biosimilars which differ in the
post-translational modifications, especially in the glycosylation
pattern, herein disclosed.
[0051] The term "biosimilar" (also known as follow-on biologics) is
well known in the art, and the skilled person would readily
appreciate when a drug substance would be considered a biosimilar
of Avelumab. The term "biosimilar" is generally used to. describe
subsequent versions (generally from a different source) of
"innovator biopharmaceutical products" ("biologics" whose drug
substance is made by a living organism or derived from a living
organism or through recombinant DNA or controlled gene expression
methodologies) that have been previously officially granted
marketing authorisation. Since biologics have a high degree of
molecular complexity, and are generally sensitive to changes in
manufacturing processes (e.g. if different cell lines are used in
their production), and since subsequent follow-on manufacturers
generally do not have access to the originator's molecular clone,
cell bank, know-how regarding the fermentation and purification
process, nor to the active drug substance itself (only the
innovator's commercialized drug product), any "biosimilar" is
unlikely to be exactly the same as the innovator drug product.
Herein, the term "buffer" or "buffer solution" refers to a
generally aqueous solution comprising a mixture of an acid (usually
a weak acid, e.g. acetic acid, citric acid, imidazolium form of
histidine) and its conjugate base (e.g. an acetate or citrate salt,
for example, sodium acetate, sodium citrate, or histidine) or
alternatively a mixture of a base (usually a weak base, e.g.
histidine) and its conjugate acid (e.g. protonated histidine salt).
The pH of a "buffer solution" will change very only slightly upon
addition of a small quantity of strong acid or base due to the
"buffering effect" imparted by the "buffering agent".
[0052] Herein, a "buffer system" comprises one or more buffering
agent(s) and/or an acid/base conjugate(s) thereof, and more
suitably comprises one or more buffering agent(s) and an acid/base
conjugate(s) thereof, and most suitably comprises one buffering
agent only and an acid/base conjugate thereof. Unless stated
otherwise, any concentrations stipulated herein in relation to a
"buffer system" (i.e. a buffer concentration) suitably refers to
the combined concentration of the buffering agent(s) and/or
acid/base conjugate(s) thereof. In other words, concentrations
stipulated herein in relation to a "buffer system" suitably refer
to the combined concentration of all the relevant buffering species
(i.e. the species in dynamic equilibrium with one another, e.g.
citrate/citric acid). As such, a given concentration of a histidine
buffer system generally relates to the combined concentration of
histidine and the imidazolium form of histidine. However, in the
case of histidine, such concentrations are usually straightforward
to calculate by reference to the input quantities of histidine or a
salt thereof. The overall pH of the composition comprising the
relevant buffer system is generally a reflection of the equilibrium
concentration of each of the relevant buffering species (i.e. the
balance of buffering agent(s) to acid/base conjugate(s)
thereof).
[0053] Herein, the term "buffering agent" refers to an acid or base
component (usually a weak acid or weak base) of a buffer or buffer
solution. A buffering agent helps maintain the pH of a given
solution at or near to a pre-determined value, and the buffering
agents are generally chosen to complement the pre-determined value.
A buffering agent is suitably a single compound which gives rise to
a desired buffering effect, especially when said buffering agent is
mixed with (and suitably capable of proton exchange with) an
appropriate amount (depending on the pre-determined pH desired) of
its corresponding "acid/base conjugate", or if the required amount
of its corresponding "acid/base conjugate" is formed in situ--this
may be achieved by adding strong acid or base until the required pH
is reached. For example in the sodium acetate buffer system, it is
possible to start out with a solution of sodium acetate (basic)
which is then acidified with, e.g., hydrochloric acid, or to a
solution of acetic acid (acidic), sodium hydroxide or sodium
acetate is added until the desired pH is reached.
[0054] Generally, a "stabiliser" refers to a component which
facilitates maintenance of the structural integrity of the
biopharmaceutical drug, particularly during freezing and/or
lyophilization and/or storage (especially when exposed to stress).
This stabilising effect may arise for a variety of reasons, though
typically such stabilisers may act as osmolytes which mitigate
against protein denaturation. As used herein, stabilisers are amino
acids (i.e. free amino acids not part of a peptide or protein--e.g.
glycine, arginine, histidine, aspartic acid, lysine) and sugar
stabilisers, such as a sugar polyol (e.g. mannitol, sorbitol),
and/or a disaccharide (e.g. trehalose, sucrose, maltose,
lactose).
[0055] Agents used as buffering agents, antioxidants or surfactants
according to the invention, are excluded from the meaning of the
term "stabilisers" as used herein, even if they may exhibit, i.a.
stabilising activity.
[0056] Herein, the term "surfactant" refers to a surface-active
agent, preferably a nonionic surfactant. Examples of surfactants
used herein include polysorbate (for example, polysorbate 20
(polyoxyethylene (20) sorbitan monolaurate) also known under the
tradename Tween 20); poloxamer (e.g. poloxamer 188, a non-ionic
triblock copolymers composed of a central hydrophobic chain of
polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic
chains of polyoxyethylene (poly(ethylene oxide)), also known under
the tradename Lutrol F 68).
[0057] Herein, the term "stable" generally refers to the physical
stability and/or chemical stability and/or biological stability of
a component, typically an active or composition thereof, during
preservation/storage.
[0058] Agents used as buffering agents, antioxidants or stabilisers
according to the invention, are excluded from the meaning of the
term "surfactants" as used herein, even if they may exhibit, i.a.
surfactant activity.
[0059] Herein, the term "antioxidant" refers to an agent capable of
preventing or decreasing oxidation of the biopharmaceutical drug to
be stabilized in the formulation.
[0060] Antioxidants include radical scavengers (e.g. ascorbic acid,
BHT, sodium sulfite, p-amino benzoic acid, glutathione or propyl
gallate), chelating agents (e.g. EDTA or citric acid) or chain
terminators (e.g. methionine or N-acetyl cysteine).
[0061] Agents used as buffering agents, stabilisers or surfactants
according to the invention, are excluded from the meaning of the
term "antioxidants" as used herein, even if they may exhibit, i.a.
antioxidative activity.
[0062] A "diluent" is an agent that constitutes the balance of
ingredients in any liquid pharmaceutical composition, for instance
so that the weight percentages total 100%. Herein, the liquid
pharmaceutical composition is an aqueous pharmaceutical
composition, so that a "diluent" as used herein is water,
preferably water for injection (WFI).
[0063] Herein, the term "particle size" or "pore size" refers
respectively to the length of the longest dimension of a given
particle or pore. Both sizes may be measured using a laser particle
size analyser and/or electron microscopes (e.g. tunneling electron
microscope, TEM, or scanning electron microscope, SEM). The
particle count (for any given size) can be obtained using the
protocols and equipment outlined in the Examples, which relates to
the particle count of sub-visible particles.
[0064] Herein, the term "about" refers to the usual error range for
the respective value readily known to the skilled person in this
technical field. Reference to "about" a value or parameter herein
includes (and describes) embodiments that are directed to that
value or parameter per se. In case of doubt, or should there be no
art recognized common understanding regarding the error range for a
certain value or parameter, "about" means .+-.5% of this value or
parameter.
[0065] Herein, the term "percent share" in connection with glycan
species refers directly to the number of different species. For
example the term "said FA2G1 has a share of 25%-41% of all glycan
species" means that in 50 antibody molecules analysed, having 100
heavy chains, 25-41 of the heavy chains will exhibit the FA2G1
glycosylation pattern.
[0066] It is to be appreciated that references to "treating" or
"treatment" include prophylaxis as well as the alleviation of
established symptoms of a condition. "Treating" or "treatment" of a
state, disorder or condition therefore includes: (1) preventing or
delaying the appearance of clinical symptoms of the state, disorder
or condition developing in a human that may be afflicted with or
predisposed to the state, disorder or condition but does not yet
experience or display clinical or subclinical symptoms of the
state, disorder or condition, (2) inhibiting the state, disorder or
condition, i.e., arresting, reducing or delaying the development of
the disease or a relapse thereof (in case of maintenance treatment)
or at least one clinical or subclinical symptom thereof, or (3)
relieving or attenuating the disease, i.e., causing regression of
the state, disorder or condition or at least one of its clinical or
subclinical symptoms.
[0067] Aqueous anti-PD-L1 Antibody Formulation
[0068] In a first aspect, the invention provides a novel aqueous
pharmaceutical antibody formulation, comprising:
[0069] (i) Avelumab in a concentration of 1 mg/mL to 30 mg/mL as
the antibody;
[0070] (ii) acetate or histidine in a concentration of 5 mM to 15
mM as the buffering agent;
[0071] (iii) D-mannitol or trehalose in a concentration of 240 mM
to 320 mM, or a combination of arginine HCl in a concentration of
50 to 150 mM and glutamic acid in a concentration of 25 mM to 75 mM
as a stabiliser;
[0072] (iv) Poloxamer 188 or Polysorbate 20 in a concentration of
0.25 mg/mL to 0.75 mg/mL, as surfactant, or no surfactant;
[0073] wherein the formulation does not comprise methionine,
and
[0074] further wherein the formulation has a pH of 5.0 to 6.0,
preferably, 5.0 to 5.6.
[0075] In a preferred embodiment the formulation does not comprise
any antioxidant.
[0076] In an embodiment the concentration of Avelumab in the said
formulation is about 10 mg/mL to about 20 mg/mL.
[0077] In another embodiment the concentration of acetate or
histidine in the said formulation is about 10 mM.
[0078] In yet another embodiment the concentration of D-mannitol or
trehalose in the said formulation is about 280 mM, or for the
combination of arginine HCl and glutamic acid, the concentration of
arginine HCl is about 150 mM and the concentration of glutamic acid
is about 50 mM.
[0079] In yet another embodiment the concentration of Poloxamer 188
or Polysorbate 20 in the said formulation is about 0.5 mg/mL.
[0080] In yet another embodiment the pH of the said formulation is
5.2 (.+-.0.1) to 5.5 (.+-.0.1).
[0081] In a preferred embodiment the said formulation comprises
acetate in a concentration of about 10 mM, and does not comprise
any other buffering agent.
[0082] In another preferred embodiment the said formulation
comprises D-mannitol or trehalose in a concentration of about 280
mM, and does not comprise any other stabiliser.
[0083] In yet another preferred embodiment the said formulation
comprises Polysorbate 20 or Poloxamer 188 in a concentration of
about 0.5 mg/mL, and does not comprise any other surfactant.
[0084] In an embodiment the said formulation comprises:
[0085] (i) Avelumab in a concentration of about 10 mg/mL as the
antibody;
[0086] (ii) acetate in a concentration of about 10 mM as the
buffering agent;
[0087] (iii) D-mannitol or trehalose in a concentration of about
280 mM as a stabiliser;
[0088] (iv) Polysorbate 20 or Poloxamer 188 in a concentration of
about 0.5 mg/mL as surfactant;
[0089] and does not comprise methionine, and has a pH of about
5.5.
[0090] In a preferred embodiment the said formulation
comprises:
[0091] (i) Avelumab in a concentration of 10 mg/mL;
[0092] (ii) acetate in a concentration of 10 mM;
[0093] (iii) D-mannitol or trehalose in a concentration of 280
mM;
[0094] (iv) Polysorbate 20 or Poloxamer 188 in a concentration of
0.5 mg/mL;
[0095] and has a pH of 5.5 (.+-.0.1).
[0096] In a preferred embodiment the said formulation consists
of:
[0097] (i) Avelumab in a concentration of 10 mg/mL;
[0098] (ii) sodium acetate trihydrate in a concentration of 10
mM;
[0099] (iii) D-mannitol or trehalose in a concentration of 280
mM;
[0100] (iv) Polysorbate 20 or Poloxamer 188 in a concentration of
0.5 mg/mL;
[0101] (v) HCl to adjust the pH;
[0102] (vi) water (for injection) as the solvent;
[0103] and has a pH of 5.5 (.+-.0.1).
[0104] In a preferred embodiment the said formulation consists
of:
[0105] (i) Avelumab in a concentration of 10 mg/mL;
[0106] (ii) sodium acetate trihydrate in a concentration of 10
mM;
[0107] (iii) trehalose dihydrate in a concentration of 280 mM;
[0108] (iv) Polysorbate 20 in a concentration of 0.5 mg/mL;
[0109] (v) HCl to adjust the pH;
[0110] (vi) water (for injection) as the diluent;
[0111] and has a pH of 5.5 (.+-.0.1).
[0112] In a more preferred embodiment the said formulation consists
of:
[0113] (i) Avelumab in a concentration of 10 mg/mL;
[0114] (ii) sodium acetate trihydrate in a concentration of 10
mM;
[0115] (iii) D-mannitol in a concentration of 280 mM;
[0116] (iv) Polysorbate 20 in a concentration of 0.5 mg/mL;
[0117] (v) HCl to adjust the pH;
[0118] (vi) water (for injection) as the diluent;
[0119] and has a pH of 5.5 (.+-.0.1).
[0120] In another embodiment the said formulation comprises:
[0121] (i) Avelumab in a concentration of about 20 mg/mL as the
antibody;
[0122] (ii) acetate in a concentration of about 10 mM as the
buffering agent;
[0123] (iii) D-mannitol or trehalose in a concentration of about
280 mM as a stabiliser;
[0124] (iv) Polysorbate 20 or Poloxamer 188 in a concentration of
about 0.5 mg/mL as surfactant;
[0125] and does not comprise methionine, and has a pH of 5.2
(.+-.0.1).
[0126] In a preferred embodiment the said formulation
comprises:
[0127] (i) Avelumab in a concentration of 20 mg/mL;
[0128] (ii) acetate in a concentration of 10 mM;
[0129] (iii) D-mannitol or trehalose in a concentration of 280
mM;
[0130] (iv) Polysorbate 20 or Poloxamer 188 in a concentration of
0.5 mg/mL;
[0131] and has a pH of 5.5 (.+-.0.1).
[0132] In a preferred embodiment the said formulation
comprises:
[0133] (i) Avelumab in a concentration of 20 mg/mL;
[0134] (ii) acetic acid in a concentration of 10 mM;
[0135] (iii) D-mannitol or trehalose dihydrate in a concentration
of 280 mM;
[0136] (iv) Polysorbate 20 or Poloxamer 188 in a concentration of
0.5 mg/mL;
[0137] (v) sodium acetate to adjust the pH;
[0138] (vi) water (for injection) as the diluent;
[0139] and has a pH of 5.2 (.+-.0.1).
[0140] In a more preferred embodiment the said formulation consists
of:
[0141] (i) Avelumab in a concentration of 20 mg/mL;
[0142] (ii) acetic acid in a concentration of 10 mM;
[0143] (iii) D-mannitol in a concentration of 280 mM;
[0144] (iv) Polysorbate 20 in a concentration of 0.5 mg/mL;
[0145] (v) sodium acetate to adjust the pH;
[0146] (vi) water (for injection) as the diluent;
[0147] and has a pH of 5.2 (.+-.0.1).
[0148] In a more preferred embodiment the said formulation consists
of:
[0149] (i) Avelumab in a concentration of 20 mg/mL;
[0150] (ii) acetic acid in a concentration of 10 mM;
[0151] (iii) trehalose dihydrate in a concentration of 280 mM;
[0152] (iv) Polysorbate 20 in a concentration of 0.5 mg/mL;
[0153] (v) sodium acetate to adjust the pH;
[0154] (vi) water (for injection) as the diluent;
[0155] and has a pH of 5.2 (.+-.0.1).
[0156] In a more preferred embodiment the said formulation consists
of:
[0157] (i) Avelumab in a concentration of 20 mg/mL;
[0158] (ii) acetic acid in a concentration of 10 mM;
[0159] (iii) D-mannitol in a concentration of 280 mM;
[0160] (iv) Poloxamer 188 in a concentration of 0.5 mg/mL;
[0161] (v) sodium acetate to adjust the pH;
[0162] (vi) water (for injection) as the diluent;
[0163] and has a pH of 5.2 (.+-.0.1).
[0164] In a more preferred embodiment the said formulation consists
of:
[0165] (i) Avelumab in a concentration of 20 mg/mL;
[0166] (ii) acetic acid in a concentration of 10 mM;
[0167] (iii) trehalose dihydrate in a concentration of 280 mM;
[0168] (iv) Poloxamer 188 in a concentration of 0.5 mg/mL;
[0169] (v) sodium acetate to adjust the pH;
[0170] (vi) water (for injection) as the diluent;
[0171] and has a pH of 5.2 (.+-.0.1).
[0172] In a preferred embodiment, the said formulation consists
of:
[0173] (i) Avelumab in a concentration of 20 mg/mL;
[0174] (ii) acetic acid in a concentration of 10 mM (0.6
mg/mL);
[0175] (iii) D-mannitol in a concentration of 280 mM (51
mg/mL);
[0176] (iv) Polysorbate 20 in a concentration of 0.5 mg/mL;
[0177] (v) sodium hydroxide in a concentration of 7.5 mM (0.3
mg/mL);
[0178] (vi) water (for injection) as the diluent;
[0179] and has a pH of 5.0 to 5.6, preferably 5.2 (.+-.0.1).
[0180] In a preferred embodiment, the latter formulation is made by
combining:
[0181] (i) 20 mg/mL of Avelumab;
[0182] (ii) 0.6 mg/mL of glacial acetic acid;
[0183] (iii) 51 mg/mL of D-mannitol;
[0184] (iv) 0.5 mg/mL of Polysorbate 20;
[0185] (v) 0.3 mg/mL of sodium hydroxide;
[0186] (vi) water (for injection) as the diluent;
[0187] to obtain the desired volume of the formulation.
[0188] In a further embodiment, the invention concerns an aqueous
pharmaceutical antibody formulation, whose pH is adjusted with
sodium hydroxide. Therefore, the formulation consists of Avelumab
in a concentration of 20 mg/mL as the active ingredient; and
glacial acetic acid, D-mannitol, Polysorbate 20, sodium hydroxide
and water for injection as the excipients; wherein the formulation
has a pH of 5.0 to 5.6, preferably 5.2 (.+-.0.1).
[0189] In a preferred embodiment, the formulation has a osmolality
between 270 and 330 mOsm/kg.
[0190] In an embodiment said Avelumab in the formulations as
described above has the heavy chain sequence of either FIG. 1a (SEQ
ID NO:1) or FIG. 1b (SEQ ID NO:2), the light chain sequence of FIG.
2 (SEQ ID NO:3), and carries a glycosylation on Asn300 comprising
FA2 and FA2G1 as the main glycan species, having a joint share of
>70% of all glycan species.
[0191] In a preferred embodiment, in the Avelumab glycosylation the
said FA2 has a share of 44%-54% and said FA2G1 has a share of
25%-41% of all glycan species.
[0192] In a preferred embodiment, in the Avelumab glycosylation the
said FA2 has a share of 47%-52% and said FA2G1 has a share of
29%-37% of all glycan species.
[0193] In a preferred embodiment, in the Avelumab glycosylation the
said FA2 has a share of about 49% and said FA2G1 has a share of
about 30%-about 35% of all glycan species.
[0194] In a preferred embodiment the Avelumab glycosylation further
comprises as minor glycan species A2 with a share of <5%, A2G1
with a share of <5%, A2G2 with a share of <5% and FA2G2 with
a share of <7% of all glycan species.
[0195] In a preferred embodiment, in the Avelumab glycosylation
said A2 has a share of 3%-5%, said A2G1 has a share of <4%, said
A2G2 has a share of <3% and said FA2G2 has a share of 5%-6% of
all glycan species.
[0196] In a preferred embodiment, in the Avelumab glycosylation
said A2 has a share of about 3.5%-about 4.5%, said A2G1 has a share
of about 0.5%-about 3.5%, said A2G2 has a share of <2.5% and
said FA2G2 has a share of about 5.5% of all glycan species.
[0197] In an embodiment the said Avelumab in the formulation as
described above has the heavy chain sequence of FIG. 1b (SEQ ID
NO:2).
[0198] In an embodiment the Avelumab formulation as described above
is for intravenous (IV) administration.
[0199] Drug-delivery Device
[0200] In a second aspect the present invention provides a drug
delivery device comprising a liquid pharmaceutical composition as
defined herein. Suitably the drug delivery device comprises a
chamber within which the pharmaceutical composition resides.
Suitably the drug delivery device is sterile.
[0201] The drug delivery device may a vial, ampoule, syringe,
injection pen (e.g. essentially incorporating a syringe), or i.v.
(intravenous) bag.
[0202] The aqueous pharmaceutical formulations are parenterally
administered, preferably via sub-cutaneous injection, intramuscular
injection, i.v. injection or i.v. infusion. The most preferred way
of administration is i.v. infusion.
[0203] In a preferred embodiment, the drug delivery device is a
vial containing the formulation as described above.
[0204] In a more preferred embodiment the said vial contains 200 mg
avelumab in 10 mL of solution for a concentration of 20 mg/mL.
[0205] In an even more preferred embodiment the vial is a glass
vial.
[0206] Medical Treatment
[0207] In a third aspect, the invention provides a method of
treating cancer comprising administering the formulation as
described above to a patient.
[0208] In an embodiment the cancer to be treated is selected from
non-small cell lung cancer, urothelial carcinoma, bladder cancer,
mesothelioma, Merkel cell carcinoma, gastric or gastroesophageal
junction cancer, ovarian cancer, breast cancer, thymoma,
adenocarcinoma of the stomach, adrenocortical carcinoma, head and
neck squamous cell carcinoma, renal cell carcinoma, melanoma,
and/or classical Hodgkin's lymphoma.
ABBREVIATIONS
[0209] ANOVA Analysis of variance
[0210] CD Circular Dichroism
[0211] CE Capillary Electrophoresis
[0212] DoE Design of Experiments
[0213] DP Drug Product
[0214] DS Drug Substance
[0215] DSF Differential Scanning Fluorimetry
[0216] DTT Dithiothreitol
[0217] ESI Electrospray Ionization
[0218] HILIC Hydrophilic Interaction Liquid Chromatography
[0219] HMWs Higher Molecular Weights
[0220] HPLC High Performance Liquid Chromatography
[0221] iCE Capillary Isoelectric Focusing
[0222] LC Liquid Chromatography
[0223] LMWs Lower Molecular Weights
[0224] MALDI Matrix-Assisted Laser Desorption Ionization
[0225] MS Mass Spectrometry
[0226] NTU Nephelometry Turbidity Units
[0227] OD Optical density
[0228] PBS Poly Buffer Saline
[0229] PES Polyethersulfone
[0230] PVDF Polyvinylidene Fluoride
[0231] SDS-PAGE Sodium Dodecyl Sulphate-PolyAcrylamide Gel
Electrophoresis
[0232] SE Size Exclusion
[0233] TOF Time of Flight
[0234] UPLC Ultra Performance Liquid Chromatography
[0235] RH Residual Humidity
[0236] UV Ultraviolet
EXAMPLES
[0237] Methods Used to Determine Stability
[0238] In order to assess the stability of the antibody
formulations tested, and select the best candidates, thermal
stress, mechanical stress, light exposure, osmolality, turbidity,
protein content, total aggregates, fragmentation, pH, isoforms,
circular dichroism, sub-visible particles and biological activity
were determined as stability parameters according to the following
protocols:
[0239] Thermal Stress:
[0240] At 40.degree. C.: the samples in the original vial container
were incubated in a thermostatic cabinet at a temperature of
40.degree. C..+-.2.degree. C. (RH 75%.+-.5%) and withdrawn at
pre-determined time points.
[0241] At 25.degree. C.: the samples in the original vial container
were incubated in a thermostatic cabinet at a temperature of
25.degree. C..+-.2.degree. C. (RH 60%.+-.5%) and withdrawn at
pre-determined time points.
[0242] Mechanical Stress:
[0243] The samples in the original vial container were placed on an
orbital shaker maintained at 300 rpm for up to 24 hours (room
temperature).
[0244] Light Exposure:
[0245] The samples in the original vial container were exposed to a
light source for 7 hours adjusting the irradiance level in the
Suntest machine to 765 W/m.sup.2 (radiation wavelength between 320
nm and 800 nm).
[0246] Osmolality:
[0247] Normal human plasma has an osmolality of about 280 mOSm/kg
(Medical. Physiology--Principles for Clinical Medicine. Edited by
Rodney A. Rhoades PhD, David R. Bell PhD). In general, solutions
with osmolality close to 300 mOsm/kg are to be targeted when
developing parenteral formulations. Acceptable ranges (as per
product specifications) are 250-400 mOsm/kg.
[0248] Here, osmolality was determined by a cryoscopic method
determining the freezing point depression of water solutions after
addition of solutes. Amount of solutes, and hence the observed
osmolality value is proportional to the observed freezing point
depression of the compounded solution.
[0249] Turbidity:
[0250] The turbidity of the solutions were determined with a
nephelometer with the capability to measure scattered or attenuated
light (Hach Lange Model 2100AN). About 3 mL of solution in reduced
volume cuvettes were illuminated by an 870.+-.30 nm light emitting
diode (LED) assembly. A detector monitors the scattered light and
provided the turbidity (NTU) of the solution by comparison with a
series of standards of known turbidity.
[0251] Protein Content:
[0252] Protein content was determined via the optical density of
solutions (diluted to .about.0.5 mg/mL protein concentration with
relevant buffer) at 280 nm and 320 nm in 1 cm path length quartz
cuvettes. Assuming a molar extinction coefficient of 1.46
cm.sup.2/mg, protein concentration was obtained by applying the
formula: (A280-A320)/(1.46 cm.sup.2/mg.times.1 cm).
[0253] Total Aggregates:
[0254] The amount of aggregates was determined by the SE-HPLC
method. A sample volume of 20 .mu.L (sample diluted to about 0.5 mL
with PBS) was injected in a TSK gel Super SW3000 4.6 mm.times.30 cm
(cod. 18675) kept at a temperature of 22.+-.5.degree. C. at a flow
rate of 0.35 mL/min (mobile phase was 50 mM sodium phosphate+0.4
sodium perchlorate at pH 6.3.+-.0.1). UV detection at 214 nm.
[0255] Fragmentation:
[0256] Low molecular species (or fragments) were determined by
Bioanalyzer. Samples are analyzed at a concentration ranging
between 1.25-3.75 mg/mL (dilutions made with purified water). 34 of
each diluted sample were merged with 2 .mu.L of the corresponding
sample buffer (with the addition of DTT when tests were conducted
under reducing conditions) and 1 .mu.L of a 60 mM maleimide
solution. The samples were heated for 5 minutes at 70.degree. C.,
then 84 .mu.L of purified water were added and the solutions
vortexed and spun down. 6 .mu.L were loaded onto the chip
(0.25-0.75 .mu.g of protein). The chip was placed into the Agilent
2100 Bioanalyzer and the analysis started within the following five
minutes.
[0257] Isoforms:
[0258] Isoforms distribution was determined by iCE. An Fc coated
capillary cartridge (100 mm internal diameter and 50 mm length) was
used. The separation is conducted using a 100 mM NaOH solution in
0.1% methylcellulose as a cathodic solution and a 80 mM
o-phosphoric acid in 0.1% methylcellulose as an anodic solution.
The samples were prepared starting from 80 .mu.L of master mix
solution (obtained mixing 700 .mu.L of 0.1% methylcellulose, 10
.mu.L of Pharmalyte 5-8, 70 .mu.L of Pharmalyte 8-10.5, 10 .mu.L of
a 7.65 pl marker and 104 of a 9.77 pl marker), to which the
suitable volume of washed Avelumab sample (corresponding to 200
.mu.g of protein after washing to remove formulation components)
was added. An amount of purified water corresponding to (120
.mu.L--volume of washed Avelumab sample added at the previous step)
is added. The separation is conducted at a detection wavelength of
280 nm setting pre-focusing and focusing times of 1 and 15 minutes
respectively and pre-focusing and focusing voltages of 1500 V and
3000 V respectively. Samples were injected at a pressure of 1000
mBar.
[0259] pH: was determined by conventional potentiometry.
[0260] Circular Dichroism (CD):
[0261] Investigations on tertiary structure of Avelumab were
carried out using a CD spectropolarimeter by Jasco (mod. J810) in
the near UV range (320-250 nm). Samples were diluted to 1.5 mg/mL
protein concentration with purified water and, once filled in 1
cm-path length quartz cuvettes, analyzed at room temperature, at a
scanning speed of 20 nm/min, with a data pitch of 0.5 nm,
integration time of 8 s and standard sensitivity.
[0262] Sub-visible Particles:
[0263] Sub-visible particles were counted through the technique of
light obscuration method using a Pamas SVSS-C particle counter.
Samples were diluted 5-fold with purified water to obtain a final
volume of at least 25 mL to be tested.
[0264] Biological Activity:
[0265] For the long term stability studies described in Example 5
biological activity was measured as an additional stability
parameter.
[0266] The method used is based on the ability of Avelumab,
absorbed on an ELISA plate, to bind in a dose-dependent manner its
antigen PD-L1 present on the cell line HEK-293 (hPDL1, permanently
transfected with PD-L1). Dosages used were 400, 200, 100, 50, 25,
12.5, 6.25 and 3.12 ng/mL. From the data obtained EC.sub.50 values
were calculated. The biological activity (potency) of the samples
is expressed as the percentage of bioactivity of the sample against
the standard and is calculated as follows: Potency (sample)
[%]=(EC.sub.50 (sample)/EC.sub.50 (standard))*100.7.
[0267] Methods of Manufacturing
[0268] The present invention also provides a method of
manufacturing an aqueous pharmaceutical formulation as defined
herein. The method suitably comprises mixing together, in any
particular order deemed appropriate, any relevant components
required to form the aqueous pharmaceutical formulation. The
skilled person may refer to the examples or techniques well known
in the art for forming aqueous pharmaceutical formulations
(especially those for injection via syringe, or i.v. infusion).
[0269] The method may involve first preparing a pre-mixture (or
pre-solution) of some or all components (optionally with some or
all of the diluent) excluding Avelumab, and Avelumab may then
itself (optionally with or pre-dissolved in some of the diluent) be
mixed with the pre-mixture (or pre-solution) to afford the aqueous
pharmaceutical formulation, or a composition to which final
components are then added to furnish the final aqueous
pharmaceutical formulation. Preferably, the method involves forming
a buffer system, suitably a buffer system comprising a buffering
agent as defined herein. The buffer system is suitably formed in a
pre-mixture prior to the addition of Avelumab. The buffer system
may be formed through simply mixing the buffering agent (supplied
ready-made) with its acid/base conjugate (suitably in appropriate
relative quantities to provide the desired pH--this can be
determined by the skilled person either theoretically or
experimentally). In the case of an acetate buffer system, this
means e.g. mixing sodium acetate with HCl, or mixing acetic acid
with NaOH or acetate. The pH of either the pre-mixture of final
aqueous pharmaceutical formulation may be judiciously adjusted by
adding the required quantity of base or acid, or a quantity of
buffering agent or acid/base conjugate.
[0270] In certain embodiments, the buffering agent and/or buffer
system is pre-formed as a separate mixture, and the buffer system
is transferred to a precursor of the aqueous pharmaceutical
formulation (comprising some or all components save for the
buffering agent and/or buffer system, suitably comprising Avelumab
and potentially only Avelumab) via buffer exchange (e.g. using
diafiltration until the relevant concentrations or osmolality is
reached). Additional excipients may be added thereafter if
necessary in order to produce the final liquid pharmaceutical
composition. The pH may be adjusted once or before all the
components are present.
[0271] Any, some, or all components may be pre-dissolved or
pre-mixed with a diluent prior to mixing with other components.
[0272] The final aqueous pharmaceutical formulation may be
filtered, suitably to remove particulate matter. Suitably
filtration is through filters sized at or below 1 .mu.m, suitably
at 0.22 .mu.m. Suitably, filtration is through either PES filters
or PVDF filters, suitably with 0.22 .mu.m PES filters.
[0273] The person of skill in the art is well aware how an aqueous
pharmaceutical formulation can be used to prepare an IV solution,
so that the antibody drug substance can be administered
intravenously.
[0274] The preparation of the IV solution typically consists of a
certain amount of solution being withdrawn from saline bags (e.g.
0.9% or 0.45% saline) with a plastic syringe (PP) and a needle and
replaced with aqueous pharmaceutical formulation. The amount of
solution replaced will depend on the body weight of the
patients.
Example 1
Structure of Avelumab
[0275] 1.1 Primary Structure
[0276] Avelumab is an IgG with two heavy and two light chain
molecules. The amino acid sequences of the two chains are shown in
FIGS. 1a (SEQ ID NO:1)/1b (SEQ ID NO:2) and 2 (SEQ ID NO:3),
respectively.
[0277] 1.2 Secondary Structure
[0278] LC-MS and MS/MS methods were used to confirm the intact
chains of the molecule and the presence of post-translational
modifications to the proteins. The secondary structure of the
Avelumab molecule subunits are shown in FIG. 3.
[0279] As confirmed by UPLC-Q-TOF mass spectrometry of peptides
obtained by trypsin digestion, the disulfide bonds
Cys21-Cys96,Cys21-Cys90, Cys147-Cys203, Cys138-Cys197,
Cys215-Cys223, Cys229-Cys229, Cys232-Cys232, Cys264-Cys324 and
Cys370-Cys428 are forming the nine typical IgG bonding pattern.
[0280] 1.3 Glycosylation
[0281] The molecule contains one N-glycosylation site on Asn300 of
the heavy chain. As determined by peptide mapping, the main
structure identified by MALDI-TOF was a complex, biantennary type
core fucosylated oligosaccaride with zero (G0F), one (G1F), or two
galactose (G2F) residues. The main species are G0F and G1F.
[0282] Avelumab glycans fluorescence labeled by 2-aminobenzamide
have been analysed by HILIC-UPLC-ESI-Q-TOF. FIG. 4 shows the UPLC
profile of the glycan species found.
TABLE-US-00001 TABLE 1 Peak identification of 2AB HILIC-UPLC
chromatogram RT Measured Expected Oxford Peak (min) MW MW
Identification nomenclature Identification by 1a 5.99 1380.52 (M +
H) 1380.54 (M + H) ##STR00001## FA1 Manually identified by MS 2
6.01 1437.54 1437.56 ##STR00002## A2 Manually identified by MS 3
7.02 1583.74 (M + H) 1583.62 (M + H) ##STR00003## FA2 MS in source
fragmentation by GlycoworkBench 4 7.77 1355.57 (M + H) 1355.51 (M +
H) ##STR00004## M5 Manually identified by MS 5 8.16 1599.77 (M + H)
1599.62 (M + H) ##STR00005## A2G1 Manually identified by MS 6 9.82
1744.79 1744.67 ##STR00006## FA2G1 MS in source fragmentation by
GlycoworkBench 1462.90 1462.54 ##STR00007## FA2 freeEnd
GlycoworkBench identified by MS 7 10.07 1744.80 1744.67
##STR00008## FA2G1 MS in source fragmentation by GlycoworkBench
1462.91 1462.54 ##STR00009## FA2 freeEnd GlycoworkBench identified
by MS 8 10.44 1462.90 1462.54 ##STR00010## FA2 freeEnd
GlycoworkBench identified by MS 1744.79 1744.67 ##STR00011## FA2G1
Manually identified by MS 9 12.15 1177.50 (M + H) 1177.46 (M + H)
##STR00012## FM3 GlycoworkBench identified by MS 10 16.66 No No
ionization ionization 11 13.42 1906.33 1906.72 ##STR00013## FA2G2
MS in source fragmentation by GlycoworkBench 1624.71 1624.59
##STR00014## FA2G1 freeEnd GlycoworkBench identified by MS 12 13.71
954.40 (M + 2H)/2 954.36 (M + 2H)/2 ##STR00015## FA2G2 Manually
identified by MS 1626.69 1626.61 ##STR00016## FA2G1 redEnd
GlycoworkBench identified by MS 13 17.46 1099.97 (M + 2H)/2 1099.91
(M + 2H)/2 ##STR00017## FA2G2S MS in source fragmentation by
GlycoworkBench 14 18.54 1079.91 (M + 2H)/2 1079.86 (M + 2H)/2
##STR00018## FA2G2S freeEnd + S (probable- small traces) Manually
identified by MS 15 21.04 2489.05 2488.91 ##STR00019## FA2G2S2
Manually identified by MS
[0283] The geometric shapes representing the glycan building blocks
correspond to the following molecular entities:
[0284] Man .DELTA. Fuc .largecircle. Gal .quadrature. GalNAc GlcNAc
.diamond. NANA NGNA
[0285] Man: mannose, Fuc: fucose, Gal: galactose, GalNAc:
N-Acetylgalactosamine, NANA: sialic acid, NGNA:
N-glycolylneuraminic acid
[0286] The glycan nomenclature used follows the Oxford Notation as
proposed by Harvey et al. (Proteomics 2009, 9, 3796-3801). In
species containing fucose (FA2, FA2G1, FA2G2), the Fuc-GlcNAc
connectivity is a1-6. In species having a terminal GlcNAc, the
GlcNAc-Man connectivity is 81-2. In species containing galactose,
the Gal-GlcNAc connectivity is .beta.1-4.
[0287] The reported chromatographic profile has been integrated and
yielded the Glycan Species Distribution of Avelumab as shown in
Table 2a.
TABLE-US-00002 TABLE 2a A2 FA2 A2G1 FA2G1 A2G2 FA2G2 M5** 3.6 48.7
3.4 35.6 2.3 5.4 1.0 **Probably Mannose 5, coelution with
biantennary mono-galactosylated species
[0288] The glycan mapping analysis confirmed the identification
carried out by peptide mapping (that allowed to identify the two
main glycan species), in addition secondary and minor species were
also characterized by this method, specific for glycan
analysis.
[0289] In another measurement the following Glycan Species
Distribution was observed.
TABLE-US-00003 TABLE 2b A2 FA2 A2G1 FA2G1 A2G2 FA2G2 4.0 50.2 1.0
30.0 0.1 5.6
Example 2
DoE1 Screening
[0290] A first Design of Experiment screening DoE1 at 10 mg/mL
Avelumab assessed the impact of several factors such as varying
buffer type/pH, excipients, surfactant type and relevant
concentration. The study led to the selection of the optimal
conditions which can maximize protein stability.
[0291] In DoE1 the following factors were taken into account for
investigation: [0292] Buffer type and pH: acetate, citrate and
histidine buffers to be evaluated in the pH range 5.0-6.0. [0293]
Excipients: 3 different excipients were considered in order to give
indications as to whether sugars/polyols or amino acids are to be
preferred for compounding in the formula, [0294] Surfactant type
and concentration: two alternative surfactants (Tween 20 and
Poloxamer 188) to be evaluated at varying concentrations (0-1
mg/mL).
[0295] The study was conducted in DINER vials (Schott) at a protein
concentration of 10 mg/mL with filling volumes of 8 mL (80
mg/vial).
[0296] Table 3 illustrates the selection of DoE1 formulas
investigated.
[0297] DoE1 allowed a selection of suitable buffer/pH, excipient
type and surfactant type to be made, that were used for the
subsequent DoE2 study described in Example 3.
TABLE-US-00004 TABLE 3 DoE1 screening formulations Surfactant
Avelumab Buffer concentration ID (mg/mL) pH (10 mM) Excipient
Surfactant (mg/mL) DoE1-1 10 5.00 Acetate Mannitol (51 mg/mL.sup.1)
Poloxamer 188 0.5 DoE1-2 10 5.00 Acetate Trehalose dihydrate (106
Tween 20 0.5 mg/mL.sup.1) DoE1-3 10 5.00 Citrate Mannitol (51
mg/mL.sup.1) Poloxamer 188 0.2 DoE1-4 10 5.25 Acetate Trehalose
dihydrate (106 Tween 20 0.2 mg/mL.sup.1) DoE1-5 10 5.25 Acetate
Arginine HCl (21.1 mg/mL.sup.2) + Poloxamer 188 0.2 Glutamic acid
(7.4 mg/mL.sup.3) DoE1-6 10 5.25 Citrate Arginine HCl (21.1
mg/mL.sup.2) + -- -- Glutamic acid (7.4 mg/mL.sup.3) DoE1-7 10 5.25
Citrate Mannitol (51 mg/mL.sup.1) Tween 20 0.2 DoE1-8 10 5.50
Acetate Mannitol (51 mg/mL.sup.1) Tween 20 0.5 DoE1-9 10 5.50
Acetate Trehalose dihydrate (106 -- -- mg/mL.sup.1) DoE1-10 10 5.50
Citrate Trehalose dihydrate (106 Poloxamer 188 1 mg/mL.sup.1)
DoE1-11 10 5.50 Citrate Arginine HCl (21.1 mg/mL.sup.2) + Tween 20
0.2 Glutamic acid (7.4 mg/mL.sup.3) DoE1-12 10 5.75 Citrate
Trehalose dihydrate (106 Tween 20 1 mg/mL) DoE1-13 10 5.75 Citrate
Mannitol (51 mg/mL) -- -- DoE1-14 10 5.75 Histidine Arginine HCl
(21.1 mg/mL) + Poloxamer 188 0.5 Glutamic acid (7.4 mg/mL) DoE1-15
10 5.75 Histidine Mannitol (51 mg/mL) Tween 20 1 DoE1-16 10 6.00
Citrate Arginine HCl (21.1 mg/mL) + Tween 20 1 Glutamic acid (7.4
mg/mL) DoE1-17 10 6.00 Citrate Trehalose dihydrate (106 Poloxamer
188 0.2 mg/mL) DoE1-18 10 6.00 Histidine Arginine HCl (21.1 mg/mL)
+ Poloxamer 188 1 Glutamic acid (7.4 mg/mL) DoE1-19 10 6.00
Histidine Trehalose dihydrate (106 Poloxamer 188 0.5 mg/mL)
Reference .sup.4 10 5.50 Acetate Mannitol (51 mg/mL)/L- Tween 20
0.5 Methionine (0.21 mg/mL) .sup.1Corresponds to 280 mM
.sup.2Corresponds to 150 mM .sup.3Corresponds to 50 mM .sup.4
Formulation disclosed in WO2013079174
[0298] 2.1 Manufacturing
[0299] The pre-formulated drug substance (DS) (10 (.+-.1) mg/mL
Avelumab, 1.36 mg/mL Sodium acetate trihydrate, 51 mg/mL
D-Mannitol, 0.21 mg/mL L-Methionine, hydrochloric acid q.b. to pH
5.5) was buffer exchanged by tangential flow filtration (using
Pellicon XL Biomax Cassettes with a 10 kDa cut-off) in the three
buffers: 10 mM sodium acetate pH 5.0, 10 mM sodium citrate pH 5.0
and 10 mM histidine pH 5.75 until a three-fold volume exchange was
achieved. At each step the DS solution was diluted 5-fold with
relevant buffer. Final target protein concentration in the
exchanged DS material was >10 mg/mL. The required excipients
were then added to the relevant buffer-exchanged DS material, pH
and final solution weight adjusted to the target so as to obtain
the DP compositions listed in Table 3.
[0300] The sequence of addition of ingredients to the exchanged DS
solutions was as follows:
[0301] Add D-Mannitol or Trehalose dihydrate or Arginine
HCl+Glutamic acid to the exchanged DS solution, stir until complete
dissolution, add L-Methionine and stir until complete dissolution
(only for Reference), add Poloxamer 188 or Polysorbate 20 (50 mg/mL
stock solution), stir until complete dissolution, check pH and
adjust to target with sodium hydroxide.
[0302] Drug product (DP) solutions were filled (8 mL) in DINER
vials (Schott).
[0303] Visual inspection during the DS diafiltration process
revealed that sodium citrate buffer caused generally higher
opalescence, whilst remarkably clearer solutions were obtained when
exchanges were made in sodium acetate and in histidine buffers.
[0304] In Table 4, the results of the experiments carried out to
determine protein recovery, osmolality (Osmomat 030/D, Gonotec) and
turbidity of the three DS materials upon buffer exchange are shown.
Satisfactory protein recoveries (>89%) and final osmolality
values (<61 mOsm/kg) were obtained. Turbidity analyses confirmed
the higher opalescence of the DS exchanged in sodium citrate.
TABLE-US-00005 TABLE 4 Results of recovery (by OD), osmolality and
turbidity experiments conducted on DS materials after buffer
exchange. Recovery Osmolality Turbidity Buffer (%) (mOsm/kg) (NTU)
Acetate 96 29 3 Citrate 89 38 30 Histidine 93 61 6
[0305] 2.2. Osmolality
[0306] The osmolality values of the DP formulations relevant to the
DoE1 screening were comprised in the range 299-396 mOsm/kg, with
most formulations having osmolalities below around 360 mOsm/kg.
[0307] The measurements were carried out at time 0, upon
manufacturing completion.
[0308] The values obtained were in line with target (acceptable
range 250-400 mOsm/Kg). Solutions containing Trehalose dihydrate
showing higher values (close to 400 mOsm/kg) due to effect of this
ingredient on freezing point and subsequent (apparent) increase in
osmolality.
[0309] 2.3 Thermal Stress
[0310] 2.3.1 Protein Content
[0311] As determined by OD measurements, the time 0 content values
were in line with theoretical values (10 mg/mL). No significant
changes were observed after 1 month at 40.degree. C.
[0312] 2.3.2 Total Aggregates
[0313] Total aggregates DoE1 formulations were determined by
SE-HPLC at time 0 and after 2 and 4 weeks of storage at 40.degree.
C.
[0314] No statistically significant variations in terms of
aggregates upon thermal stress at 40.degree. C. could be
highlighted, thus indicating that the different matrices tested led
to invariant/negligible changes in the aggregation pattern.
[0315] 2.3.3 Fragmentation
[0316] Fragmentation by Bioanalyzer (2100 Bioanalyzer, Agilent) in
DoE1 formulations was determined at time 0 and after 2 and 4 weeks
of storage at 40.degree. C.
[0317] The data indicated that: [0318] pH is a critical factor to
protein fragmentation at 40.degree. C. At pH>5.75, fragmentation
tends to significantly increase (most typically in formulations
from DoE1-13 to DoE1-19, in citrate and histidine buffers). [0319]
The formulations presenting the lowest variations in fragmentation
are those in a pH range of 5.0-5.75 preferably in presence of
either D-Mannitol or Trehalose dihydrate (DoE1-2-8-9-10-12). [0320]
Formulation DoE1-7 (citrate buffer at pH 5.25, in presence of
D-Mannitol and Tween 20) presented abnormal profiles with
consistent peak doubling (some issues might be related to usage of
citrate as a buffering agent in terms of fragmentation, in addition
to those already highlighted during manufacturing with the increase
in turbidity/opalescence).
[0321] 2.3.4 Turbidity
[0322] Turbidity by nephelometry in DoE1 formulations was
determined at time 0 and after 2 and 4 weeks of storage at
40.degree. C.
[0323] Opalescence/strong opalescence consistently observed in all
DP formulations containing citrate as a pH buffering agent.
[0324] All formulations in sodium acetate and histidine were found
to be clear/slightly opalescent with no significant changes
observed over 1 month of storage at 40.degree. C.
[0325] 2.3.5 pH
[0326] No pH changes were observed.
[0327] 2.4 Mechanical Stress
[0328] The DoE1 formulations were subjected to 24-hour orbital
shaking in vials at 300 rpm (room temperature). Upon stress
termination the samples were tested for aggregates and
opalescence.
[0329] 2.4.1 Total Aggregates
[0330] Total aggregates were determined by SE-HPLC after mechanical
stress and compared to time 0 results. Negligible changes were
observed.
[0331] 2.4.2 Turbidity
[0332] Turbidity of DoE1 formulations was determined by
nephelometry (2100AN IS, Hach Lange) after mechanical stress and
compared to time 0 results. The data were evaluated by ANOVA and a
moderately significant impact deriving from surfactant presence
(0.01<p-value<0.05) was observed. Either Tween 20 or
Poloxamer 188 can help minimize turbidity changes after mechanical
stress.
[0333] 2.5 Light Exposure
[0334] The DoE1 formulations were subjected to 7-hour irradiation
at 765 W/m.sup.2 (Suntest CPS, Atlas). Upon light stress
termination the samples were tested for aggregates, opalescence, pH
and isoforms profile.
[0335] 2.5.1 Total Aggregates
[0336] Using SE-HPLC (Alliance, Waters) slight variations were
observed, most frequently when sodium citrate buffer is used
(p-value<0.01).
[0337] Sodium acetate and histidine are the buffers to be preferred
in order to minimize aggregation changes.
[0338] 2.5.2 Turbidity
[0339] As determined by nephelometry the most evident turbidity
increases were typically found in citrate buffer at pH
values>5.75 (DoE1-13 and DoE1-16 and DoE1-17).
[0340] 2.5.3 pH
[0341] No changes were observed.
[0342] 2.6 DoE1: Outcome
[0343] The data obtained in the frame of the thermal, mechanical
and light stress were evaluated in order to determine conditions
that provide maximal protein resistance against stresses.
[0344] The results of the analysis are reported in Table 5.
TABLE-US-00006 TABLE 5 Components of highly stabilized Avelumab
formulations at 10 mg/mL protein concentration ID# Buffer pH
Excipient Surfactant Extrapolated 10 mM Acetate 5.20 Trehalose
Tween 20 dihydrate (0.5 mg/mL) (280 mM) DoE1-4 10 mM Acetate 5.25
Trehalose Tween 20 dihydrate (0.2 mg/mL) (280 mM)
[0345] The extrapolated formulation is highlighted in green
(ID#=Extrapolated), whilst the most similar formula in the set of
those tested is the DoE1-4, also reported.
[0346] These data demonstrate that acetate buffer pH 5.0-5.5
provides improved protein stability, and that surfactant presence,
such as either Tween 20 or Poloxamer 188, at concentrations higher
than 0.2 mg/mL, is also important for improved protein stability in
the formulation.
Example 3
[0347] A second DoE screening "DoE2" aimed at fine-tuning the
formulations selected upon DoE1 completion and concurrently
increasing protein concentration to 20 mg/mL.
[0348] With this second formulation screening, six formulations at
20 mg/mL protein concentration varying in excipients (D-Mannitol,
Trehalose dihydrate) and surfactant (no surfactant, Poloxamer 188
or Polysorbate 20 at 0.5 mg/mL) in presence of 10 mM sodium acetate
buffer pH 5.2 were tested after thermal stress (1 month at
40.degree. C., 8 weeks at 25.degree. C. and 2-8.degree. C.) and
mechanical shaking (24 hours at 300 rpm, room temperature). The
relevant compositions are listed in Table 6.
TABLE-US-00007 TABLE 6 DoE2 screening formulations (protein
concentration = 20 mg/mL) Avelumab ID (mg/mL) Buffer Excipient
Surfactant DoE2-1 20 10 mM Mannitol -- acetate (51 mg/mL.sup.1) pH
5.2 DoE2-2 20 10 mM Trehalose dihydrate -- acetate (106
mg/mL.sup.1) pH 5.2 DoE2-3 20 10 mM Mannitol Tween 20 acetate (51
mg/mL.sup.1) (0.5 mg/mL) pH 5.2 DoE2-4 20 10 mM Trehalose dihydrate
Tween 20 acetate (106 mg/mL.sup.1) (0.5 mg/mL) pH 5.2 DoE2-5 20 10
mM Mannitol Poloxamer 188 acetate (51 mg/mL.sup.1) (0.5 mg/mL) pH
5.2 DoE2-6 20 10 mM Trehalose dihydrate Poloxamer 188 acetate (106
mg/mL.sup.1) (0.5 mg/mL) pH 5.2 DoE1-8 20 10 mM Mannitol Tween 20
acetate (51 mg/mL.sup.1) (0.5 mg/mL) pH 5.5 Reference 20 10 mM
Mannitol Tween 20 acetate (51 mg/mL)/L- (0.5 mg/mL) pH 5.5
Methionine (0.21 mg/mL)
[0349] The DoE2 study was conducted to comparatively evaluate the
effect of D-Mannitol vs. Trehalose dihydrate, and the impact of
surfactant (either Tween 20 or Poloxamer 188, or no surfactant) in
sodium acetate buffer at pH 5.2, at the increased protein
concentration of 20 mg/mL. Two pH 5.5 reference samples have been
included in the design: "Reference" with L-Methionine, and a
reference formulation without L-Methionine, corresponding to
DoE1-8.
[0350] 3.1 Manufacturing
[0351] The pre-formulated drug substance (DS) (27.1 mg/mL Avelumab
in 10 mM sodium acetate pH 5.5) was used. The required excipients
were then added to the DS material.
[0352] The sequence of addition of ingredients to the DS solution
was as follows:
[0353] Add D-Mannitol or Trehalose dihydrate, stir until complete
dissolution, add Poloxamer 188 or Polysorbate 20 (20 mg/mL stock
solution), stir until complete dissolution, add L-Methionine and
stir until complete dissolution (only for Reference), stir until
complete dissolution, check pH and adjust to target with sodium
hydroxide or diluted acetic acid.
[0354] The solutions were weight adjusted to the target with
relevant buffer so as to obtain the DP compositions listed in Table
7.
[0355] DP solutions were filled (8 mL) in DIN6R vials.
[0356] 3.2 Thermal Stress
[0357] 3.2.1 Protein Content
[0358] No protein content (OD, Lambda 35, Perkin Elmer) changes
observed over 4 weeks at 40.degree. C. (Table 7) and 8 weeks at
25.degree. C. (Table 8).
TABLE-US-00008 TABLE 7 Protein content (mg/mL) by OD of DoE2
formulations (thermal stress at 40.degree. C.) Protein conc
Excipient Time 2 weeks 4 weeks # ID (mg/mL) Buffer (280 mM)
Surfactant 0 (40.degree. C.) (40.degree. C.) 1 DoE2 - 1 20 10 mM
acetate D-Mannitol No 22.3 20.0 20.9 pH 5.2 2 DoE2 - 2 20 10 mM
acetate Trehalose No 22.0 20.6 21.6 pH 5.2 dihydrate 3 DoE2 - 3 20
10 mM acetate D-Mannitol Tween 20 (0.5 21.9 20.5 21.6 pH 5.2 mg/mL)
4 DoE2 - 4 20 10 mM acetate Trehalose Tween 20 (0.5 22.1 20.5 22.3
pH 5.2 dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate D-Mannitol
Lutrol F-68 21.7 20.7 22.8 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20 10 mM
acetate Trehalose Lutrol F-68 22.7 21.3 22.5 pH 5.2 dihydrate (0.5
mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween 20 (0.5 21.5
20.5 23.5 pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol +
Tween 20 (0.5 21.5 20.4 23.3 pH 5.5 L-Methionine mg/mL)
TABLE-US-00009 TABLE 8 Protein content (mg/mL) by OD of DoE2
formulations (thermal stress at 25.degree. C.) Protein conc
Excipient Time 8 weeks # ID (mg/mL) Buffer (280 mM) Surfactant 0
(25.degree. C.) 1 DoE2 - 1 20 10 mM acetate D-Mannitol No 22.3 20.6
pH 5.2 2 DoE2 - 2 20 10 mM acetate Trehalose No 22.0 21.0 pH 5.2
dihydrate 3 DoE2 - 3 20 10 mM acetate D-Mannitol Tween 20 (0.5 21.9
21.3 pH 5.2 mg/mL) 4 DoE2 - 4 20 10 mM acetate Trehalose Tween 20
(0.5 22.1 21.5 pH 5.2 dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate
D-Mannitol Lutrol F-68 21.7 20.5 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20
10 mM acetate Trehalose Lutrol F-68 22.7 21.0 pH 5.2 dihydrate (0.5
mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween 20 (0.5 21.5
21.1 pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol + Tween
20 (0.5 21.5 21.2 pH 5.5 L-Methionine mg/mL)
[0359] 3.2.2 Total Aggregates
[0360] Total aggregates determined by SE-HPLC over stability at
40.degree. C. and 25.degree. C. are represented in FIGS. 6 and 7
respectively. Only minor, non-significant changes in aggregation
were observed.
[0361] 3.2.3 Fragmentation by Bioanalyzer
[0362] Fragments were evaluated over 1 month at 40.degree. C. and
after 2 months at 25.degree. C. The relevant results are shown in
FIG. 8 and FIG. 9 respectively.
[0363] At 40.degree. C., aside from formulation DoE2-1, which
presented an amount of fragments higher than 7% after 1 month, the
other formulas were observed to have similar behavior (4-6% in
fragments after 1 month) with slightly better performances of
formulations DoE2-4, DoE2-5 and DoE2-6 (4.0-4.5% in fragments after
1 month at 40.degree. C.).
[0364] At 25.degree. C., similar fragmentation percentages were
found after 2 months (4.6-6.1%) 3.2.4 Isoforms profile
[0365] The isoforms profile by iCE280 (Fast IEF Analyzer,
Convergent Bioscience) in DoE2 formulations was determined at time
0 and after 4 weeks of storage at 40.degree. C. Upon storage at
40.degree. C. typically increases in the acidic cluster can be
determined, while a concurrent decrease in the basic isoforms is
observed.
[0366] The isoforms profiles were evaluated over 1 month at
40.degree. C. (Table 9) and after 8 weeks at 25.degree. C. (FIG.
10).
[0367] Comparable variations were observed in all samples at both
stressing conditions.
TABLE-US-00010 TABLE 9 iCE280 results for DoE2 formulations after 4
weeks at 40.degree. C. Time 0 4 weeks at 40.degree. C. Acidic Main
Basic Acidic Main Basic forms peak forms forms peak forms (%) (%)
(%) (%) (%) (%) DoE2 - 1 32.3 36.0 31.7 40.3 31.9 27.8 DoE2 - 2
32.0 37.7 30.4 38.0 33.6 28.5 DoE2 - 3 32.2 36.7 31.1 39.9 32.7
27.5 DoE2 - 4 32.7 36.7 30.6 39.7 33.0 27.3 DoE2 - 5 32.5 37.4 30.2
38.1 33.4 28.5 DoE2 - 6 32.4 37.0 30.7 38.3 33.7 28.0 DoE1 - 8 33.2
36.9 30.0 38.8 33.5 27.7 Reference 32.2 36.2 31.7 37.7 33.4
28.9
[0368] 3.2.6 Turbidity
[0369] No variations observed after 1 month at 40.degree. C. (Table
8) and 2 months at 25.degree. C. (Table 9).
TABLE-US-00011 TABLE 10 Turbidity of DoE2 formulations after 1
month at 40.degree. C. Protein conc Excipient Time 2 weeks 4 weeks
# ID (mg/mL) Buffer (280 mM) Surfactant 0 (40.degree. C.)
(40.degree. C.) 1 DoE2 - 1 20 10 mM acetate D-Mannitol No 2 2 2 pH
5.2 2 DoE2 - 2 20 10 mM acetate Trehalose No 2 2 2 pH 5.2 dihydrate
3 DoE2 - 3 20 10 mM acetate D-Mannitol Tween 20 (0.5 2 2 2 pH 5.2
mg/mL) 4 DoE2 - 4 20 10 mM acetate Trehalose Tween 20 (0.5 2 2 2 pH
5.2 dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate D-Mannitol Lutrol
F-68 2 2 2 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20 10 mM acetate Trehalose
Lutrol F-68 2 2 2 pH 5.2 dihydrate (0.5 mg/mL) 7 DoE1 - 8 20 10 mM
acetate D-Mannitol Tween 20 (0.5 2 2 2 pH 5.5 mg/mL) 8 Reference 20
10 mM acetate D-Mannitol + Tween 20 (0.5 3 3 2 pH 5.5 L-Methionine
mg/mL)
TABLE-US-00012 TABLE 11 Turbidity of DoE2 formulations after 2
months at 25.degree. C. Protein conc Excipient Time 8 weeks # ID
(mg/mL) Buffer (280 mM) Surfactant 0 (25.degree. C.) 1 DoE2 - 1 20
10 mM acetate D-Mannitol No 2 2 pH 5.2 2 DoE2 - 2 20 10 mM acetate
Trehalose No 2 2 pH 5.2 dihydrate 3 DoE2 - 3 20 10 mM acetate
D-Mannitol Tween 20 (0.5 2 2 pH 5.2 mg/mL) 4 DoE2 - 4 20 10 mM
acetate Trehalose Tween 20 (0.5 2 2 pH 5.2 dihydrate mg/mL) 5 DoE2
- 5 20 10 mM acetate D-Mannitol Lutrol F-68 2 2 pH 5.2 (0.5 mg/mL)
6 DoE2 - 6 20 10 mM acetate Trehalose Lutrol F-68 2 2 pH 5.2
dihydrate (0.5 mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween
20 (0.5 2 3 pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol +
Tween 20 (0.5 3 3 pH 5.5 L-Methionine mg/mL)
[0370] 3.2.7 pH
[0371] No variations observed after 1 month at 40.degree. C. (Table
12) and 2 months at 25.degree. C. (Table 13).
TABLE-US-00013 TABLE 12 pH of DoE2 formulations after 1 month at
40.degree. C. Protein conc Excipient Time 2 weeks 4 weeks # ID
(mg/mL) Buffer (280 mM) Surfactant 0 (40.degree. C.) (40.degree.
C.) 1 DoE2 - 1 20 10 mM acetate D-Mannitol No 5.2 5.2 5.2 pH 5.2 2
DoE2 - 2 20 10 mM acetate Trehalose No 5.2 5.2 5.2 pH 5.2 dihydrate
3 DoE2 - 3 20 10 mM acetate D-Mannitol Tween 20 (0.5 5.2 5.2 5.2 pH
5.2 mg/mL) 4 DoE2 - 4 20 10 mM acetate Trehalose Tween 20 (0.5 5.2
5.2 5.2 pH 5.2 dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate
D-Mannitol Lutrol F-68 5.2 5.2 5.2 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20
10 mM acetate Trehalose Lutrol F-68 5.2 5.2 5.2 pH 5.2 dihydrate
(0.5 mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween 20 (0.5
5.5 5.5 5.5 pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol +
Tween 20 (0.5 5.5 5.5 5.5 pH 5.5 L-Methionine mg/mL)
TABLE-US-00014 TABLE 13 pH of DoE2 formulations after 2 months at
25.degree. C. Protein conc Excipient Time 8 weeks # ID (mg/mL)
Buffer (280 mM) Surfactant 0 (25.degree. C.) 1 DoE2 - 1 20 10 mM
acetate D-Mannitol No 5.2 5.2 pH 5.2 2 DoE2 - 2 20 10 mM acetate
Trehalose No 5.2 5.2 pH 5.2 dihydrate 3 DoE2 - 3 20 10 mM acetate
D-Mannitol Tween 20 (0.5 5.2 5.3 pH 5.2 mg/mL) 4 DoE2 - 4 20 10 mM
acetate Trehalose Tween 20 (0.5 5.2 5.2 pH 5.2 dihydrate mg/mL) 5
DoE2 - 5 20 10 mM acetate D-Mannitol Lutrol F-68 5.2 5.2 pH 5.2
(0.5 mg/mL) 6 DoE2 - 6 20 10 mM acetate Trehalose Lutrol F-68 5.2
5.2 pH 5.2 dihydrate (0.5 mg/mL) 7 DoE1 - 8 20 10 mM acetate
D-Mannitol Tween 20 (0.5 5.5 5.5 pH 5.5 mg/mL) 8 Reference 20 10 mM
acetate D-Mannitol + Tween 20 (0.5 5.5 5.6 pH 5.5 L-Methionine
mg/mL)
[0372] 3.2.8 Circular Dichroism
[0373] CD spectra (J-810 Spectropolarimeter, Jasco) of DoE2
formulations were collected at time 0 and after 4 weeks at
40.degree. C. and 8 weeks at 25.degree. C. in the near UV
range.
[0374] Protein in all formulation generally retains its tertiary
structure after 4 weeks at 40.degree. C. and 8 weeks at 25.degree.
C.
[0375] 3.2.9 Sub-visible Particles
[0376] The sub-visible particles of the DoE2 formulations after 8
weeks of storage at 2-8.degree. C. were determined. The results are
shown in Table 14. The values were found within European
Pharmacopoeia limits (for solutions supplied in containers with a
nominal content of less than 100 mL).
TABLE-US-00015 TABLE 14 Sub-visible particles of DoE2 formulations
after 8 weeks at 2-8.degree. C. Sub-visible Sub-visible particles
>10 particles >25 Protein conc Excipient .mu.m (per .mu.m
(per # ID (mg/mL) Buffer (280 mM) Surfactant container) container)
1 DoE2 - 1 20 10 mM acetate D-Mannitol No 754 33 pH 5.2 2 DoE2 - 2
20 10 mM acetate Trehalose No 716 14 pH 5.2 dihydrate 3 DoE2 - 3 20
10 mM acetate D-Mannitol Tween 20 (0.5 597 24 pH 5.2 mg/mL) 4 DoE2
- 4 20 10 mM acetate Trehalose Tween 20 (0.5 1839 100 pH 5.2
dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate D-Mannitol Lutrol F-68
431 38 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20 10 mM acetate Trehalose
Lutrol F-68 521 28 pH 5.2 dihydrate (0.5 mg/mL) 7 DoE1 - 8 20 10 mM
acetate D-Mannitol Tween 20 (0.5 915 14 pH 5.5 mg/mL) 8 Reference
20 10 mM acetate D-Mannitol + Tween 20 (0.5 1873 52 pH 5.5
L-Methionine mg/mL)
[0377] 3.3 Mechanical Stress
[0378] 3.3.1 Fragmentation by Bioanalyzer
[0379] After 24 hours at 300 rpm, slight variations in fragments
(Table 15) were observed in all samples (up to 5.0-6.5%) with no
specific relation to the specific compositions tested.
TABLE-US-00016 TABLE 15 Fragments (%) by Bioanalyzer of DoE2
formulations after 24-hour shaking (300 rpm; room temperature)
Protein conc Excipient Time 24 H # ID (mg/mL) Buffer (280 mM)
Surfactant 0 300 RPM 1 DoE2 - 1 20 10 mM acetate D-Mannitol No 4.9
5.5 pH 5.2 2 DoE2 - 2 20 10 mM acetate Trehalose No 4.6 5.0 pH 5.2
dihydrate 3 DoE2 - 3 20 10 mM acetate D-Mannitol Tween 20 (0.5 4.7
5.7 pH 5.2 mg/mL) 4 DoE2 - 4 20 10 mM acetate Trehalose Tween 20
(0.5 4.6 6.5 pH 5.2 dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate
D-Mannitol Lutrol F-68 5.1 6.2 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20 10
mM acetate Trehalose Lutrol F-68 5.2 5.3 pH 5.2 dihydrate (0.5
mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween 20 (0.5 3.5 5.4
pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol + Tween 20
(0.5 3.4 5.4 pH 5.5 L-Methionine mg/mL)
[0380] 3.3.2 Aggregates
[0381] No changes were observed after mechanical shaking (Table
16).
TABLE-US-00017 TABLE 16 Aggregates (%) by SE-HPLC of DoE2
formulations after 24-hour shaking (300 rpm; room temperature)
Protein conc Excipient Time 24 H # ID (mg/mL) Buffer (280 mM)
Surfactant 0 300 RPM 1 DoE2 - 1 20 10 mM acetate D-Mannitol No 1.5
1.5 pH 5.2 2 DoE2 - 2 20 10 mM acetate Trehalose No 1.5 1.5 pH 5.2
dihydrate 3 DoE2 - 3 20 10 mM acetate D-Mannitol Tween 20 (0.5 1.6
1.5 pH 5.2 mg/mL) 4 DoE2 - 4 20 10 mM acetate Trehalose Tween 20
(0.5 1.6 1.5 pH 5.2 dihydrate mg/mL) 5 DoE2 - 5 20 10 mM acetate
D-Mannitol Lutrol F-68 1.6 1.5 pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20 10
mM acetate Trehalose Lutrol F-68 1.6 1.6 pH 5.2 dihydrate (0.5
mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween 20 (0.5 1.6 1.6
pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol + Tween 20
(0.5 1.6 1.6 pH 5.5 L-Methionine mg/mL)
[0382] 3.3.3 pH
[0383] No changes were observed after mechanical shaking (Table
17).
TABLE-US-00018 TABLE 17 pH of DoE2 formulations after 24-hour
shaking (300 rpm; room temperature) Protein conc Excipient Time 24
H # ID (mg/mL) Buffer (280 mM) Surfactant 0 300 RPM 1 DoE2 - 1 20
10 mM acetate D-Mannitol No 5.2 5.2 pH 5.2 2 DoE2 - 2 20 10 mM
acetate Trehalose No 5.2 5.2 pH 5.2 dihydrate 3 DoE2 - 3 20 10 mM
acetate D-Mannitol Tween 20 (0.5 5.2 5.2 pH 5.2 mg/mL) 4 DoE2 - 4
20 10 mM acetate Trehalose Tween 20 (0.5 5.2 5.2 pH 5.2 dihydrate
mg/mL) 5 DoE2 - 5 20 10 mM acetate D-Mannitol Lutrol F-68 5.2 5.2
pH 5.2 (0.5 mg/mL) 6 DoE2 - 6 20 10 mM acetate Trehalose Lutrol
F-68 5.2 5.2 pH 5.2 dihydrate (0.5 mg/mL) 7 DoE1 - 8 20 10 mM
acetate D-Mannitol Tween 20 (0.5 5.5 5.5 pH 5.5 mg/mL) 8 Referenee
20 10 mM acetate D-Mannitol + Tween 20 (0.5 5.5 5.5 pH 5.5
L-Methionine mg/mL)
[0384] 3.4 Turbidity
[0385] No changes were observed after mechanical shaking (Table
18).
TABLE-US-00019 TABLE 18 Turbidity (NTU) of DoE2 formulations after
24-hour shaking (300 rpm; room temperature) Protein conc Excipient
Time 24 h # ID (mg/mL) Buffer (280 mM) Surfactant 0 300 rpm 1 DoE2
- 1 20 10 mM acetate D-Mannitol No 2 2 pH 5.2 2 DoE2 - 2 20 10 mM
acetate Trehalose No 2 2 pH 5.2 dihydrate 3 DoE2 - 3 20 10 mM
acetate D-Mannitol Tween 20 (0.5 2 2 pH 5.2 mg/mL) 4 DoE2 - 4 20 10
mM acetate Trehalose Tween 20 (0.5 2 2 pH 5.2 dihydrate mg/mL) 5
DoE2 - 5 20 10 mM acetate D-Mannitol Lutrol F-68 2 2 pH 5.2 (0.5
mg/mL) 6 DoE2 - 6 20 10 mM acetate Trehalose Lutrol F-68 2 2 pH 5.2
dihydrate (0.5 mg/mL) 7 DoE1 - 8 20 10 mM acetate D-Mannitol Tween
20 (0.5 2 2 pH 5.5 mg/mL) 8 Reference 20 10 mM acetate D-Mannitol +
Tween 20 (0.5 3 2 pH 5.5 L-Methionine mg/mL)
[0386] 3.5 DoE2: Outcome
[0387] These results demonstrate that pH 5.2 (extrapolated from
DoE1) does not impact fragmentation and therefore is suitable for
use in a stable formulation. Optimal pH for preserving protein
stability was demonstrated to be in the range 5.0-5.5 (DoE1). In
contrast, pH values of 5.6-5.7 could result in higher
fragmentation.
[0388] Mannitol and Trehalose dihydrate resulted in similar
behavior.
[0389] No superiority of Poloxamer 188 over Tween 20 was found.
[0390] These results also demonstrate that higher protein
concentration (20 mg/mL) in the DoE2 formulation is feasible with
no observed or anticipated stability issues.
[0391] DoE2: Formulation 3 (the formula most preferred and finally
selected for further use at 20 mg/mL) was compared in terms of
isoforms profiles to the reference formula at time 0, after 4 weeks
at 40.degree. C. and 8 weeks at 25.degree. C. in order to evaluate
whether different behavior between the two formulas are present
over stability time at different conditions. The results are
presented in Table 19.
TABLE-US-00020 TABLE 19 Isoforms profiles by iCE280 of DoE2
Formulation 3 and Reference formulation at time 0, after 4 weeks
(40.degree. C.) and 8 weeks (25.degree. C.) Time 4 weeks 8 weeks
ID# Buffer pH Excipient Surfactant Cluster 0 (40.degree. C.)
(25.degree. C.) DoE2 - 3 10 mM 5.20 D-Mannitol Tween 20 1 1.6 3.1
2.9 Acetate (0.5 mg/mL) 2 4.5 5.3 7.1 3 9.9 10.8 9.1 4 16.2 20.7
18.0 5 36.7 32.7 34.9 6 22.2 19.7 20.3 7 8.9 7.8 7.7 Reference 10
mM 5.50 Mannitol + Tween 20 1 1.8 2.3 3.4 Acetate L-Methionine (0.5
mg/mL) 2 4.3 5.0 8.1 3 9.8 10.6 10.0 4 16.3 19.8 16.9 5 36.2 33.4
34.7 6 22.6 21.1 19.6 7 9.1 7.8 7.4
[0392] Also the additional timepoint (8 weeks) at 25.degree. C.
highlighted no major issues deriving from the reduced pH with
respect to the reference formulation.
Example 4
Effect of Antioxidant (L-Methionine)
[0393] As methionine was used in the formulation disclosed in
WO2013079174, the present Avelumab formulation development aimed to
also clarify the impact of this compound as an antioxidant.
[0394] The 10 mg/mL samples (from the DoE1 set) were 2-fold diluted
with 200 .mu.L of 6% H.sub.2O.sub.2, obtaining a final protein
concentration of about 5 mg/mL and 3% H.sub.2O.sub.2, and then
incubated 3 h at 5.degree. C. At the end of the incubation the
sample was washed versus water by ultracentrifugation using an
Amicon Ultra (Millipore) 4 mL 10 kDa (4 washes 1 mL each step). The
final protein concentration after Amicon treatment was about 10
mg/mL.
[0395] DoE1: Formulation 8 is identical to Reference formula of
DoE2, except for the presence of L-Methionine: the forced oxidation
with H.sub.2O.sub.2 (3 hours at 2-8.degree. C.) of the two formulas
and following testing by iCE280 (oxidation generally leads to
increase in more acidic species in electropherograms) and
Bioanalyzer aimed to determine whether any differences arise in the
two formulations due to the presence of the antioxidant agent. The
results are presented in Tables 20 and 21.
TABLE-US-00021 TABLE 20 Isoforms profiles by iCE280 of DoE1
Formulation 8 and Reference formulation after forced oxidation
treatment. Upper table: samples stored at 2-8.degree. C. Bottom
table: sample stored 4 weeks at 40.degree. C. + 6 weeks at
2-8.degree. C. Oxidised with 3% H.sub.2O.sub.2 Anti PD-L1 (after 10
week ID# Buffer pH (mg/mL) Excipient Surfactant Cluster storage at
2-8.degree. C.) DoE1 - 8 10 mM 5.50 10 Mannitol Tween 20 1 2.2
Acetate (280 mM) (0.5 mg/mL) 2 4.5 3 9.8 4 23.2 5 39.0 5 16.6 7 4.8
Reference 10 mM 5.50 10 Mannitol Tween 20 1 2.2 Acetate (280 mM) +
(0.5 mg/mL) 2 4.5 L-Methionine 3 10.1 (1.4 mM) 4 23.7 5 37.9 6 16.8
7 4.8 Oxidised with 3% H.sub.2O.sub.2 (after 4 week Anti PD-L1
storage at 40.degree. C. + ID# Buffer pH (mg/mL) Excipient
Surfactant Cluster 6 weeks at 2-8.degree. C.) DoE1 - 8 10 mM 5.50
10 Mannitol Tween 20 1 2.5 Acetate (280 mM) (0.5 mg/mL) 2 5.9 3
11.4 4 27.2 5 34.5 6 14.6 7 4.0 Reference 10 mM 5.50 10 Mannitol
Tween 20 1 2.7 Acetate (280 mM) + (0.5 mg/mL) 2 5.9 L-Methionine 3
11.1 (1.4 mM) 4 26.7 5 35.8 6 14.4 7 3.4
[0396] Comparable acidic clusters abundances were observed for the
two formulations (with or w/o methionine).
[0397] Fragments by Bioanalyzer were also tested for these samples
(Table 21):
[0398] comparable levels of fragmentation were observed for the two
formulations (with or w/o methionine).
TABLE-US-00022 TABLE 21 Fragments by Bioanalyzer of DoE1
Formulation 8 and Reference formulation after forced oxidation
treatment. Upper table: samples stored at 2-8.degree. C. Bottom
table: sample stored 4 weeks at 40.degree. C. + 6 weeks at
2-8.degree. C. Oxidized with 3% H.sub.2O.sub.2 Anti PD-L1 (after 10
week ID# Buffer pH (mg/mL) Excipient Surfactant storage at
2-8.degree. C.) DoE1 - 8 10 mM 5.50 10 Mannitol Tween 20 2.4
Acetate (280 mM) (0.5 mg/mL) Reference 10 mM 5.50 10 Mannitol Tween
20 2.5 Acetate (280 mM) + (0.5 mg/mL) L-Methionine (1.4 mM)
Oxidised with 3% H.sub.2O.sub.2 (after 4 week AntiPD-L1 storage at
40.degree. C. + ID# Buffer pH (mg/mL) Excipient Surfactant 6 weeks
at 2-8.degree. C.) DoE1 - 8 10 mM 5.50 10 Mannitol Tween 20 2.5
Acetate (280 mM) (0.5 mg/mL) Reference 10 mM 5.50 10 Mannitol Tween
20 3.0 Acetate (280 mM) + (0.5 mg/mL) L-Methionine (1.4 mM)
[0399] These results suggest that an antioxidant is not needed to
stabilize Avelumab and can, therefore, be omitted from the
formulation.
Example 5
Long Term Stability Studies 5.1 Drug Product Compositions and
Strengths
[0400] The Avelumab formulations 1, 2, 3, 4 and 5 listed in Table
22 were manufactured and used for a long term stability study. The
manufacturing process included a compounding followed by a
sterilizing double-filtration step through a 0.22 .mu.m membrane
(PES and PVDF filters were tested) before the final filling in
vials. Formulation 5 corresponds to the Reference used also in the
DoE-1 and -2 studies as described in Example 2 and 3.
TABLE-US-00023 TABLE 22 DP compositions DP Compositions Formu-
Formu- Formu- Formu- Refer- lation 1 lation 2 lation 3 lation 4
ence (DP 01- (DP 02- (DP 03- (DP 04- (DP 05- Ingredient(s) 190214)
190214) 180214) 180214) 190214) Avelumab 20 20 10 10 10 mg/mL mg/mL
mg/mL mg/mL mg/mL Sodium 10 mM 10 mM 10 mM 10 mM 10 mM acetate
buffer Mannitol 51 0 51 0 51 mg/mL mg/mL mg/mL mg/mL mg/mL
Trehalose 0 106 0 106 0 Dihydrate mg/mL mg/mL mg/mL mg/mL mg/mL
Polysorbate 20 0.5 0.5 0.5 0.5 0.5 mg/mL mg/mL mg/mL mg/mL mg/mL
L-Methionine 0 0 0 0 1.4 mM sodium q.s to pH q.s to pH q.s to pH
q.s to pH q.s to pH hydroxide or 5.2 .+-. 0.1 5.2 .+-. 0.1 5.2 .+-.
0.1 5.2 .+-. 0.1 5.5 .+-. 0.1 hydrochloric acid Filling volume 10
mL 10 mL 20 mL 20 mL 8 mL (in Type I glass vials)
[0401] Upon manufacturing (time 0), the osmolality was determined
and found in line with expected value (range: 320-350 mOsm/kg).
[0402] 5.2 Stability Study Plan and Duration
[0403] Concerning the stability of the formulations, the study
schedule, the storage conditions and the tests to be applied are
summarized in Table 23. For each time point the table indicates the
storage condition to be tested.
[0404] The storage of the samples has been carried out with the
vials in the upright position. The study is to be conducted over 1
month at 40.degree. C., 6 months at accelerated conditions (at
25.degree. C.) and 12 months at long term conditions (2-8.degree.
C.).
TABLE-US-00024 TABLE 23 Stability Plan T = 0.5 M 1 M 2 M 3 M 6 M 9
M 12 M Test 0 (2 wk) (4 wk) (8 wk) (13 wk) (26 wk) (39 wk) (52 wk)
Colour X 40.degree. C. 25.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. 2-8.degree. C. 40.degree. C.
25.degree. C. 25.degree. C. 25.degree. C. Turbidity X 40.degree. C.
25.degree. C. 2-8.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. 40.degree. C. 25.degree. C.
25.degree. C. 25.degree. C. pH X 40.degree. C. 25.degree. C.
2-8.degree. C. 2-8.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 40.degree. C. 25.degree. C. 25.degree. C. 25.degree.
C. Content A280- X 40.degree. C. 25.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. 2-8.degree. C. 2-8.degree. C. A320
40.degree. C. 25.degree. C. 25.degree. C. 25.degree. C. SE-HPLC X
40.degree. C. 25.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. 2-8.degree. C. 40.degree. C.
25.degree. C. 25.degree. C. 25.degree. C. SDS-page red X 40.degree.
C. 25.degree. C. 2-8.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. 40.degree. C. 25.degree. C.
25.degree. C. 25.degree. C. SDS-page non- X 40.degree. C.
25.degree. C. 2-8.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. red 40.degree. C. 25.degree. C.
25.degree. C. 25.degree. C. iCE-280 X 40.degree. C. 25.degree. C.
2-8.degree. C. 2-8.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 40.degree. C. 25.degree. C. 25.degree. C. 25.degree.
C. Osmolarity X 40.degree. C. N/A N/A N/A N/A N/A N/A Subvisible X
40.degree. C. 25.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 2-8.degree. C. 2-8.degree. C. particles 40.degree.
C. 25.degree. C. 25.degree. C. 25.degree. C. Potency X 40.degree.
C. 25.degree. C. N/A 2-8.degree. C. 2-8.degree. C. 2-8.degree. C.
2-8.degree. C. 40.degree. C. 25.degree. C. 25.degree. C.
[0405] Data were collected at 40.degree. C. (up to 1 month),
25.degree. C. (up to 6 months) and 2-8.degree. C. (up to 12
months).
[0406] 5.3 Stability at 2-8.degree. C.
[0407] 5.3.1 Degree of Coloration by Visual Inspection
[0408] No changes observed over stability. All solutions remain
clearer than clearest standard solution (<Y7). Values within
specifications.
[0409] 5.3.2 Degree of Opalescence by Nephelometry
[0410] All solutions show turbidity comprised in the range of clear
solutions (1-3 NTU). Values within specifications.
[0411] 5.3.3 pH
[0412] No changes observed over stability. All solutions show pH
values in line with target (5.2.+-.0.1 for formulations 1 to 4 and
5.5.+-.0.1 for Reference DP). Values within specifications.
[0413] 5.3.4 Protein Content by OD
[0414] Concentration of formulations 1 and 2 (target
concentration=20 mg/mL) was found in the range 18.7-19.8 mg/mL
(within .+-.10% limits with respect to target) during the study,
with no significant changes over time.
[0415] Concentration of formulations 3 and 4 and Reference DP
(target concentration=10 mg/mL) was found in the range 9.3-10.2
mg/mL during the study; no significant changes found.
[0416] Protein content remains therefore unaltered over 12 month
stability at 2-8 .degree. C. (Values within specifications).
[0417] 5.3.5 Dimers and HMWs by SE-HPLC
[0418] No changes in aggregation over 12 months at 2-8 .degree. C.
with respect to time 0. Values within specifications.
[0419] 5.3.6 Fragments (LMWs) by SDS-PAGE N-Red
[0420] As shown in FIG. 11 the samples showed a time 0 value of
LMWs by SDS-PAGE N-RED in the range 11.9-16.2%, followed by a +5-7%
increase at the next point (8 weeks) and by minor changes over the
rest of stability, up to six months.
[0421] 5.3.7 Sub-visible Particles
[0422] As for sub-visible particles per container, the counts were
below the limits set by United States, European and Japanese
Pharmacopoeia for solutions for infusion or injection with nominal
content of less than 100 mL (6000 per container equal to or greater
than 10 .mu.m and 600 per container equal to or greater than 25
.mu.m). The relevant bar charts for the two particle size ranges
are shown in FIG. 12 and FIG. 13 respectively for sub-visible
particles 10 .mu.m and sub-visible particles 25 .mu.m.
[0423] No changes in sub-visible particles upon storage were
highlighted.
[0424] 5.3.8 Biological Activity
[0425] Bioactivity values were typically in the range 89-110% for
all time points tested in the course of the stability study. No
decrease observed upon storage.
[0426] 5.3.9 Isoforms Pattern
[0427] The results from iCE280 experiments are reported in FIG. 14
(acidic cluster, sum of peaks 1-2-3-4), FIG. 15 (main peak) and
FIG. 16 (basic cluster, sum of peaks 6-7). Isoforms profile is
retained throughout the 12 month stability period. At refrigerated
conditions, no impact of pH on antibody's isoforms is observed.
[0428] 5.3.10 2-8.degree. C. Stability Outcome
[0429] None of the physico-chemical properties of the five
formulations tested was found to undergo significant changes over
the 12 month stability at 2-8.degree. C. This is surprising
especially for the isoforms patterns, as in formulations 1 to 4 no
methionine is present.
[0430] 5.4 Stability at 25.degree. C.
[0431] 5.4.1 Degree of Coloration by Visual Inspection
[0432] No changes observed over stability. All solutions remain
clearer than clearest standard solution (<Y7). Values within
specifications.
[0433] 5.4.2 Degree of Opalescence by Nephelometry
[0434] All solutions show turbidity comprised between 1-3 NTU
(clear solutions range). Values within specifications.
[0435] 5.4.3 pH
[0436] No changes observed over stability. All solutions show pH
values in line with target (5.2.+-.0.1 for formulations 1-2-3-4 and
5.5.+-.0.1 for Reference DP). Values within specifications.
[0437] 5.4.4 Protein Content by OD
[0438] Concentration of formulations 1 and 2 (target
concentration=20 mg/mL) was found in the range 18.5-20.0 mg/mL
(within .+-.10% limits with respect to target) during the study,
with no significant changes over time.
[0439] Concentration of formulations 3 and 4 and Reference DP
(target concentration=10 mg/mL) was found in the range 9.5-10.0
mg/mL during the study; no significant changes found.
[0440] Protein content remains therefore unaltered over six-month
stability at 25.degree. C. Values within specifications.
[0441] 5.4.5 Dimers and HMWs by SE-HPLC
[0442] No changes in aggregation over six months at 25.degree. C.
with respect to time 0.
[0443] Aggregation lower than specification limit (not more than
5%) was found throughout the study.
[0444] 5.4.6 Fragments (LMWs) by SDS-PAGE N-Red
[0445] The samples showed a time 0 value of LMWs by SDS-PAGE N-RED
in the range 11.9-16.2%, followed by step-wise increase at the next
point (4 weeks) followed by minor changes over the rest of
stability, up to six months (FIG. 17).
[0446] 5.4.7 Sub-visible Particles
[0447] As for sub-visible particles per container, the counts were
below the limits set by United States, European and Japanese
Pharmacopoeia for solutions for infusion or injection with nominal
content of less than 100 mL (6000 per container equal to or greater
than 10 .mu.m and 600 per container equal to or greater than 25
.mu.m). The relevant bar-charts are shown in FIG. 18 and FIG.
19.
[0448] No changes in sub-visible particles upon stability at
25.degree. C. were highlighted.
[0449] 5.4.8 Biological Activity
[0450] Bioactivity values were typically in the range 90-110% for
all time points tested in the course of the stability study. No
decreases observed upon stability at 25.degree. C.
[0451] 5.4.9 Isoforms Pattern
[0452] The results from iCE280 experiments are reported in FIG. 20
(acidic cluster, sum of peaks 1-2-3-4), FIG. 21 (main peak) and
FIG. 22 (basic cluster, sum of peaks 6-7).
[0453] Acidic cluster tends to increase over storage at 25.degree.
C. All samples show acidic cluster increase of about +10% after six
months at 25.degree. C. and concurrent decrease in main peak (-5%
after 6 months) and basic cluster (-5% after 6 months).
[0454] 5.4.10 25.degree. C. Stability Outcome
[0455] Over 6-month stability at 25.degree. C., the five
formulations tested showed no changes in terms of protein content,
appearance, clarity, pH, aggregates, sub-visible particles and
bioactivity with respect to time 0.
[0456] Fragments were found to increase by +5 percentage points
according to SDS-PAGE N-RED after six-months at 25.degree. C.,
while no statistically significant changes were highlighted by
Bioanalyzer.
[0457] Similar behavior in isoforms profile by iCE280: acidic
cluster of all formulations tend to increase by +10% over the six
month-study, with concurrent decreases in main peak and basic
cluster.
[0458] 5.5 Stability at 40.degree. C.
[0459] 5.5.1 Degree of Coloration by Visual Inspection
[0460] No changes observed over stability. All solutions remain
clearer than clearest standard solution (<Y7).
[0461] 5.5.2 Degree of Opalescence by Nephelometry
[0462] No changes observed over stability. All solutions show
turbidity comprised of 2 NTU (clear solutions range). Values within
specifications.
[0463] 5.5.3 pH
[0464] No changes observed over stability. All solutions show pH
values in line with target (5.2.+-.0.1 for formulations 1-2-3-4 and
5.5.+-.0.1 for Reference DP). Values within specifications.
[0465] 5.5.4 Protein Content by OD
[0466] Concentration of formulations 1 and 2 (target
concentration=20 mg/mL) was found in the range 18.0-19.0 mg/mL
(within .+-.10% limits with respect to target) during the study,
with no tendency towards loss in protein over time.
[0467] Concentration of formulations 3 and 4 and Reference DP
(target concentration=10 mg/mL) was found in the range 9.5-10.0
mg/mL during the study, with no tendency towards loss in protein
over time. Values within specifications.
[0468] Heat stress is, in conclusion, not detrimental to protein
content at the conditions tested (up to 1 month at 40.degree.
C.).
[0469] 5.5.5 Dimers and HMWs by SE-HPLC
[0470] No major changes in aggregation were highlighted after 1
month. All values below 1% total aggregates after 1 month (lower
than specification limits, that is not more than 5%). 5.5.6
Fragments (LMWs) by SDS-PAGE N-Red, Bioanalyzer
[0471] Given the variability of the SDS-PAGE N-RED method (for
instance, time 0 values of 11.9 and 14.5 were determined for DP
01-190214 and DP 02-190214 respectively) it can be concluded that
no major changes occur during the study at 40 .degree. C. (FIG.
23).
[0472] 5.5.7 Sub-visible Particles
[0473] As for sub-visible particles per container, the counts were
abundantly below the limits set by United States, European and JP
Pharmacopoeia for solutions for infusion or injection with nominal
content of less than 100 mL (6000 per container equal to or greater
than 10 .mu.m and 600 per container equal to or greater than 25
.mu.m). Relevant bar charts shown in FIG. 24 and FIG. 25.
[0474] No changes in sub-visible particles upon thermal stress were
highlighted.
[0475] 5.5.8 Biological Activity
[0476] Bioactivity values were typically in the range 99-120% for
all time points tested in the course of the stability study. No
decrease observed upon thermal stress in the samples.
[0477] 5.5.9 Isoforms Pattern
[0478] The results from iCE280 experiments are reported in FIG. 26
(acidic cluster, sum of peaks 1-2-3-4), FIG. 27 (main peak) and
FIG. 28 (basic cluster, sum of peaks 6-7).
[0479] Acidic cluster tends to increase over storage at 40.degree.
C.
[0480] Main peak variations (Fehler! Verweisquelle konnte nicht
gefunden werden.) confirmed a slightly better stability of new
formulas at 10 mg/mL and identical behavior of the remaining
compositions.
[0481] Results obtained with basic cluster determination confirmed
the above described results.
[0482] Up to two weeks, similar behavior was observed in the five
compositions. At higher stability times, slight differentiation
arises between the 20 mg/mL and the 10 mg/mL Avelumab DP (slightly
better resistance in formulas at 10 mg/mL).
[0483] 5.5.10 40.degree. C. Stability Outcome
[0484] At 40.degree. C. (1 month), the five formulations tested
showed no changes in terms of protein content, appearance, clarity,
pH, aggregates, sub-visible particles and bioactivity with respect
to time 0.
[0485] Small differences between 10 mg/mL and 20 mg/mL DP
formulations highlighted by iCE280 (acidic cluster tends to undergo
some increase upon storage, slightly more evident in 20 mg/mL than
in 10 mg/mL DP formulations).
[0486] 5.6 Conclusions
[0487] 5.6.1 Stability at 2-8.degree. C. (12 Months)
[0488] All formulations were found to be stable: no significant
changes observed in terms of appearance, turbidity (by
nephelometry), sub-visible particles, pH, protein content (by OD),
aggregation (by SE-HPLC), fragments (by SDS-PAGE N-RED and
Bioanalyzer), isoforms profile (by iCE280) and biological activity
(by bioassay) with respect to time 0.
[0489] 5.6.2 Stability at 25.degree. C. (6 Months)
[0490] No changes in terms of protein content, appearance, clarity,
pH, aggregates, sub-visible particles and bioactivity with respect
to time 0.
[0491] Fragments were found to increase by +5% according to
SDS-PAGE N-RED after six-months at 25.degree. C., while no
statistically significant changes were highlighted by Bioanalyzer
(a method used as an additional tool to add robustness to
conclusions on fragmentation occurrence).
[0492] A similar behavior was observed in isoforms profile by
iCE280: acidic cluster of all formulations tend to increase by +10%
over the six month-study, with concurrent decreases in main peak
and basic cluster.
[0493] 5.6.3 Stability at 40.degree. C. (1 Month)
[0494] No changes in terms of protein content, appearance, clarity,
pH, aggregates, sub-visible particles and bioactivity with respect
to time 0,
[0495] Small differences between 10 mg/mL and 20 mg/mL DP
formulations highlighted by iCE280 (acidic cluster tends to undergo
some increase upon storage, slightly more evident in 20 mg/mL than
in 10 mg/mL DP formulations) 5.7 Stability over 24 Months 5.7.1
Manufacturing of DP Compositions
[0496] The following DP compositions were manufactured and their
stability studied over a period of 24 months:
TABLE-US-00025 TABLE 24 DP compositions DP Compositions
Ingredient(s) DP 01-160414 DP 02-160414 avelumab 20 mg/mL 20 mg/mL
Acetate Acid Glacial (100%) 0.60 mg/mL * 0.60 mg/mL * Mannitol 51
mg/mL 51 mg/mL Polysorbate 20 0.5 mg/mL 0.5 mg/mL sodium hydroxide
0.30 mg/mL ** 0.30 mg/mL ** Filling volume 10 mL 30 mL Strength 200
mg/vial 600 mg/vial * Corresponding to 10 mM Sodium Acetate **
Final pH: 5.2
[0497] Both formulations correspond to formulation DP 01-190214 as
shown in Table 22. The only difference is that a fixed amount of
0.3 mg/mL (7.5 mM) of sodium hydroxide was used to yield a pH of
5.2 when combined with 0.6 mg/mL glacial acetic acid. The sole
difference between formulations DP 01-160414 and DP 02-160414 is
that the latter formulation has a volume of 30 mL per vial, while
the former has 10 mL per vial.
[0498] Both formulations were double-filtered through a 0.22 .mu.m
PVDF membrane, followed by the manual filling in vials. Protein
content was tested before and after filtration; the relevant
results indicate that no loss of protein occurs upon double aseptic
filtration.
[0499] Stability data up to 24 months (at +5.+-.3.degree. C.) and
up to 6 months at +25.degree. C..+-.2.degree. C. (RH 60%.+-.5%)
have been collected on the two formulations in the respective final
containers (vials).
[0500] 5.7.2 Stability up to 24 Months (at +5.+-.3.degree. C.)
[0501] At +5.+-.3.degree. C., up to 24 months, no changes in
protein content (by OD), HMWs (by SE-HPLC), turbidity (by
nephelometry), particles formation (by light obscuration), degree
of coloration (by visual inspection), and biopotency were observed.
Slight increase in acidic isoforms (+5% observed for all
compositions after 2 years).
[0502] No statistically significant changes were observed in terms
of fragmentation by SDS-PAGE N-RED, Bioanalyzer and CE-SDS
N-RED.
[0503] 5.7.3 Stability up to 6 Months at +25.degree.
C..+-.2.degree. C. (RH 60%.+-.5%)
[0504] At +25.degree. C..+-.2.degree. C. (RH 60%.+-.5%), up to 6
months, no changes in protein content (by OD), HMWs (by SE-HPLC),
turbidity (by nephelometry), particles formation (by light
obscuration), isoforms profile (by iCE280), degree of coloration
(by visual inspection), electrophoretic purity (by SDS/-PAGE RED)
and biopotency were observed. Similarly to stability at 5 .degree.
C., no statistically significant increase in fragmentation was
observed at +25.degree. C..+-.2.degree. C. (RH 60%.+-.5%) (results
confirmed by Bioanalyzer).
[0505] 5.7.4 Holding Time
[0506] Holding time before filtration (in bags, up to 24 hours at
room temperatures), holding time after filtration (in bags, up to
72 hours at room temperature) and shaking (up to 24 hours at 200
rpm at room temperature) showed no significant changes in protein
content, particles formation, aggregates and turbidity, thus
indicating no major issues that may arise during standard times of
operations typically considered during manufacturing process.
[0507] 5.7.5 Freeze/Thaw Study
[0508] A freeze/thaw study evidenced that the tested formulations
can safely be frozen at -80.degree. C. and then allowed to warm up
to +5.+-.3.degree. C., or +25.degree. C., with no major changes
occurring to the protein.
Sequence CWU 1
1
31450PRTArtificial SequenceHeavy chain sequence of Avelumab 1Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp
Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Lys Leu Gly Thr Val Thr Thr
Val Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445Gly Lys 4502449PRTArtificial SequenceHeavy chain
sequence of Avelumab, lacking the C-terminal K 2Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ile Met Met
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser
Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly3216PRTArtificial SequenceLight chain sequence of Avelumab
3Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1 5
10 15Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly
Tyr 20 25 30Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser
Asn Arg Phe 50 55 60Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu65 70 75 80Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Ser Ser Tyr Thr Ser Ser 85 90 95Ser Thr Arg Val Phe Gly Thr Gly Thr
Lys Val Thr Val Leu Gly Gln 100 105 110Pro Lys Ala Asn Pro Thr Val
Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140Pro Gly Ala
Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys145 150 155
160Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr
165 170 175Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
Ser His 180 185 190Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser
Thr Val Glu Lys 195 200 205Thr Val Ala Pro Thr Glu Cys Ser 210
215
* * * * *