U.S. patent application number 15/743413 was filed with the patent office on 2019-07-18 for compositions comprising antibody-duocarmycin drug conjugates.
This patent application is currently assigned to Synthon Biopharmaceuticals B.V.. The applicant listed for this patent is Synthon Biopharmaceuticals B.V.. Invention is credited to Niels Jaap OSINGA, Ernst Johannes Bernardus VAN BOCKXMEER.
Application Number | 20190216942 15/743413 |
Document ID | / |
Family ID | 53673006 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190216942 |
Kind Code |
A9 |
OSINGA; Niels Jaap ; et
al. |
July 18, 2019 |
COMPOSITIONS COMPRISING ANTIBODY-DUOCARMYCIN DRUG CONJUGATES
Abstract
The present invention relates to lyophilized compositions
comprising antibody-duocarmycin drug conjugates.
Inventors: |
OSINGA; Niels Jaap;
(Nijmegen, NL) ; VAN BOCKXMEER; Ernst Johannes
Bernardus; (Nijmegen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synthon Biopharmaceuticals B.V. |
Nijmegen |
|
NL |
|
|
Assignee: |
Synthon Biopharmaceuticals
B.V.
Nijmegen
NL
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180200382 A1 |
July 19, 2018 |
|
|
Family ID: |
53673006 |
Appl. No.: |
15/743413 |
Filed: |
July 8, 2016 |
PCT Filed: |
July 8, 2016 |
PCT NO: |
PCT/EP2016/066347 PCKC 00 |
371 Date: |
January 10, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6803 20170801;
A61P 35/00 20180101; C07K 2317/24 20130101; C07K 16/32 20130101;
A61K 31/4184 20130101; A61K 47/183 20130101; A61K 47/6851 20170801;
C07K 16/2863 20130101; A61K 47/6809 20170801; C07K 16/30 20130101;
A61K 9/19 20130101; A61K 47/26 20130101; A61K 39/3955 20130101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; A61K 47/26 20060101 A61K047/26; A61K 47/18 20060101
A61K047/18; C07K 16/28 20060101 C07K016/28; C07K 16/30 20060101
C07K016/30; A61K 9/19 20060101 A61K009/19; A61K 31/4184 20060101
A61K031/4184; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 10, 2015 |
EP |
15176307.5 |
Claims
1. A lyophilized composition comprising an antibody-drug conjugate
of formula (I), ##STR00001## a buffering agent, a lyoprotectant,
and a surfactant wherein the molar ratio of lyoprotectant to
antibody-drug conjugate is 1,400-3,200 to 1, wherein mAb is a
monoclonal antibody.
2. The composition according to claim 1 wherein the lyoprotectant
is a non-reducing saccharide.
3. The composition according to claim 2, wherein the lyoprotectant
is sucrose, trehalose or a mixture thereof.
4. The composition according to claim 1, wherein the antibody-drug
conjugate is present in a concentration of 1-30 mg/ml when
reconstituted with water.
5. The composition according to claim 1, wherein the buffering
agent is histidine, citrate or succinate.
6. The composition according to claim 1, wherein the buffering
agent is present in a concentration of 2.5-25 mM when reconstituted
with water.
7. The composition according to claim 1, wherein the surfactant is
an alkyl glycoside, poloxamer or polysorbate.
8. The composition according to claim 1, wherein, when
reconstituted with water, the aqueous solution has a pH of
5.3-6.0.
9. The composition according to claim 1, wherein the monoclonal
antibody is an anti-HER2 antibody.
10. The composition according to claim 1, consisting essentially of
an antibody-drug conjugate of formula (I), a buffering agent, a
lyoprotectant, and a surfactant wherein the molar ratio of
lyoprotectant to antibody-drug conjugate is 1,400-3,200 to 1.
11. The composition according to claim 1, wherein the molar ratio
of lyoprotectant to antibody-drug conjugate is 1,400-2,000 to
1.
12. The composition according to claim 1, comprising an
antibody-drug conjugate of formula (II), ##STR00002## histidine,
trehalose, and polysorbate 20, wherein the molar ratio of trehalose
to antibody-drug conjugate is about 1,605 to 1, and, when
reconstituted with water, the antibody-drug conjugate is present in
an amount of about 10 mg/ml, the concentration of histidine is
about 5 mM, the amount of polysorbate 20 is about 0.01% (m/v), and
the pH is about 5.7.
13. The composition according to claim 12 consisting essentially of
an antibody-drug conjugate of formula (II), histidine, trehalose,
and polysorbate 20, wherein the molar ratio of trehalose to
antibody-drug conjugate is about 1,605 to 1, and, when
reconstituted with water, the ADC is present in an amount of about
10 mg/ml, the concentration of histidine is about 5 mM, the amount
of polysorbate 20 is about 0.01% (m/v), and the pH is about
5.7.
14. A process for the preparation of the composition according to
claim 1, comprising the steps of a) freezing a pre-lyophilization
aqueous solution of a composition of claim 1, b) primary drying at
a product temperature below the collapse temperature of the
composition at a pressure below atmospheric pressure, and c)
secondary drying at a product temperature above 0.degree. C. and
below the glass transition temperature of the composition at a
pressure below atmospheric pressure.
15. The process of claim 14 wherein the freezing step a) comprises
an annealing step, wherein the annealing step is performed for 0.5
to 6 hours at a shelf temperature in the range of -25.degree. C. to
-10.degree. C.
16. The composition according to claim 11, wherein the molar ratio
of lyoprotectant to antibody-drug conjugate is 1,400-1,800 to 1.
Description
FIELD OF THE PRESENT INVENTION
[0001] The present invention relates to lyophilized compositions
comprising antibody-duocarmycin drug conjugates and reconstituted
aqueous solutions thereof.
BACKGROUND OF THE PRESENT INVENTION
[0002] Duocarmycins are toxins isolated from Streptomyces sp. in
1988. These DNA binding and alkylating agents exhibit potent
cytotoxicity in vitro. However, their application in cancer
treatment is limited because, in vivo, they have unfavourable side
effects resulting in a small therapeutic index.
[0003] The therapeutic index of anti-tumour agents can be improved
by incorporating them in an antibody-drug conjugate (ADC). An ADC
is obtained by conjugating a drug, via a cleavable or non-cleavable
linker (linker drug), to an antibody.
[0004] Currently, two ADCs are marketed, i.e., brentuximab vedotin
and trastuzumab emtansine, and over 30 ADCs are in various phases
of clinical development. The developments regarding ADCs prompted
renewed interest in developing duocarmycins and particularly
duocarmycin derivatives as drugs in ADCs. These ADCs are referred
to as antibody-duocarmycin drug conjugates or duocarmycin-derived
ADCs in the present application.
[0005] Two duocarmycin-derived ADCs, i.e., SYD985 (NCT02277717
(2014); Sponsor: Synthon Biopharmaceuticals) and MDX-1203
(NCT00944905 (2009); Sponsor: Bristol-Myers Squibb), are currently
in clinical development.
[0006] Compared to naked (monoclonal) antibodies, ADCs have
different physicochemical properties; hence, conventional
pharmaceutical formulations suitable for monoclonal antibodies are
not equally suitable for ADCs. Most linker drugs, in particular
those containing duocarmycin derivatives, have a low solubility in
water. When these linker drugs are conjugated to antibodies, the
resulting conjugate has an increased hydrophobicity as compared to
the naked antibody, decreasing the colloidal stability of the ADC
in an aqueous solution. The hydrophobicity increase upon
conjugation of a linker drug is pronounced as compared to the
variation in hydrophobicity between different antibodies. This
poses difficulties for pharmaceutical formulation development. Not
only the type of linker drug, but also the number of linker drugs
per antibody (Drug-to-Antibody Ratio, DAR) and the site(s) of
conjugation of the linker drug to the antibody influence the
physicochemical properties of the ADC. As compared to a naked
antibody, the corresponding ADC tends to aggregate more easily.
Furthermore, the higher the DAR, the higher is the tendency for
aggregation in solution.
[0007] Additionally, to ensure the chemical stability of the linker
drug, especially in the case of a cleavable linker, the ADC
formulation has to comply with requirements different from those
necessary for the stability of the naked antibody per se. As each
linker drug has a different chemical stability and hydrophobicity,
each ADC is unique and requires a dedicated, tailored
composition.
[0008] In order to have sufficient shelf life, protein-derived
drugs are often marketed as a lyophilized powder for reconstitution
with water. To obtain a lyophilized powder, a lyophilization or
freeze-drying process is employed. This lyophilisation process has
three stages, i.e., freezing, primary drying, and secondary drying.
Primary drying conditions are chosen in such a way that the product
temperature remains below the collapse temperature of the
composition to prevent physical collapse of the cake. The collapse
temperature of the composition can be determined using Freeze-Dry
Microscopy. Secondary drying is performed below the glass
transition temperature (Tg) of the composition, which temperature
is usually determined by differential scanning calorimetry
(DSC).
[0009] Before the lyophilisation process can commence, the active
pharmaceutical ingredient needs to be provided in a suitable
formulation. In the case of an ADC formulation, the purification
formulation typically is exchanged with a formulation suitable for
lyophilisation, i.e., to provide a pre-lyophilisation solution. The
pre-lyophilisation solution needs to fulfil several demands, i.e.,
it should solubilise the ADC completely; in the pre-lyophilisation
solution the ADC should be colloidally and chemically stable for a
certain period of time at various conditions, e.g. temperature,
generally occurring during lyophilisation; the process time should
be acceptable; and the ADC should be stable in the resulting
lyophilized composition, typically referred to as lyophilized
powder or cake. This cake should have an acceptable appearance.
Additionally, the lyophilized composition should be easily
reconstitutable.
[0010] Usually, lyophilized protein formulations contain a
buffering agent, a lyoprotectant and, optionally, a surfactant or a
bulking agent or both. The lyophilized powder typically is stored
in a vial and is reconstituted with bacteriostatic or sterile water
for injection.
[0011] The ADCs that are currently marketed are available as a
lyophilized powder. However, these ADCs contain toxins, viz. a
maytansinoid (e.g. DM1) or an auristatin (e.g. monomethyl
auristatin E), that are less hydrophobic than the
duocarmycin-derived toxins currently being investigated as drugs in
ADCs and these ADCs have different DARs than the
duocarmycin-derived ADCs used in the formulations of the present
invention. The physico-chemical properties of these and other
maytansinoid or auristatin ADCs are different from those of the
duocarmycin-derived ADCs used in the formulations of the present
invention and require different formulations.
[0012] For instance, the composition of T-DM1, i.e. trastuzumab
conjugated via a non-cleavable linker to emtansine, having a DAR of
3.5 (Kadcyla.RTM.) upon reconstitution, is 20 mg/ml T-DM1, 10 mM
sodium succinate, 6% (w/v) sucrose, 0.02% (w/v) polysorbate 20, pH
5.0.
[0013] WO2004/110498 relates to liquid and lyophilized compositions
of an antibody coupled to a maytansinoid and discloses inter alia
lyophilized compositions of huN901-DM1 ADCs (1-5 mg/ml), having a
DAR of about 4, of a solution comprising 10 mM sodium succinate,
0.01% (w/v) polysorbate 20, 0.5% (w/v) sucrose (i.e. over 10 times
less than the sucrose amount in the commercial formulation of
Kadcyla.RTM.), 250 mM glycine and pH 5.5. Example 6 of
WO2004/110498 teaches that compositions comprising 1 mg/ml
huN901:DM1 ADC, 10 mM sodium succinate in combination with 0.01%
polysorbate 20 and 250 mM glycine gives better stability than the
combination of 10 mM sodium citrate, 0.01% polysorbate 20 and 250
mM glycine. Formulations without polysorbate are not stable.
Example 7 of WO2004/110498 shows that a similar formulation
comprising 5 mg/ml huN901-DM1, resulting in a 5 times lower
ADC-to-sucrose ratio than the preferred formulation of Example 6,
is stable as well. WO2004/110498 solely discloses lyophilized
formulations of huN901:DM1 ADC that comprise 250 mM glycine, which
not only acts as a bulking agent, but has cryo- or lyoprotective
properties as well. Therefore the disclosed formulations have a
high lyoprotectant/ADC molar ratio.
[0014] The commercial formulation of Adcetris.RTM. has the
composition after reconstitution of 5 mg/ml brentuximab vedotin, a
monomethyl auristatin E (MMAE) conjugate to a chimeric monoclonal
antibody of the IgG1 type (DAR of about 8), 5.6 mg/ml sodium
citrate, 0.21 mg/ml citric acid monohydrate, 70 mg/ml trehalose
dihydrate, 0.2 mg/ml polysorbate 80 and has a pH of 6.6.
[0015] WO2015/075201 discloses surfactant-free lyophilized
formulations comprising an ADC wherein an anti-TF IgG1 antibody is
conjugated to MMAE via a valine-citrulline linker, similar to the
linker drug of Adcetris.RTM.. This ADC has a DAR of 4 (p. 35, l.
6). Example 7 on p. 55 teaches that compositions containing 10
mg/ml ADC, 30 mM histidine, and 150 mM or 250 mM sucrose show
severe shrinkage (inferior cake quality/appearance) starting from a
fill volume of 4 ml in a 10 ml vial, which makes these formulations
unsuitable for commercial production. It also teaches that the
presence of a bulking agent (mannitol is present in the
pre-lyophilisation solution in a relatively high amount of at least
3% w/v) is necessary for good cake quality. Example 8 discloses
that a ratio of sucrose to ADC of 1,337 to 1 is preferred (i.e.
Formulation B), whereas Example 12 shows there is no difference in
stability between formulations having a sucrose to ADC ratio of
2,676 to 1 and formulations having a sucrose to ADC ratio of 446 to
1.
[0016] Several liquid formulations in relation to
duocarmycin-derived ADCs have been disclosed, e.g. in WO
2012/162482 (p. 35, l. 16-17), WO 2011/133039 (p. 218, l. 24), and
on p. 2 of in the supplementary information of DOKTER, W et al.
Preclinical Profile of the HER2-Targeting ADC SYD983/SYD985 Mol.
Cancer Ther.; 13(11) November 2014; 2618-2629. The formulation of
WO2012/162482 is not suitable for lyophilisation, as it contains a
high amount of sodium chloride which can reduce the collapse
temperature of the composition to impractically low values. WO
2011/133039 discloses a series of novel analogues of the DNA
alkylating agent CC-1065 and HER2-targeting ADCs formulated in the
commercial Herceptin.RTM. (trastuzumab) formulation comprising 4.2
mM histidine, 19.1 mg/ml trehalose dihydrate, and a polysorbate,
having a pH of 6. In DOKTER, W et al., a formulation comprising
histidine, 30 mg/ml trehalose dihydrate, and a polysorbate, having
a pH of 6 was used for the HER2-targeting duocarmycin-derived ADC
SYD983.
[0017] However, no lyophilized formulations comprising
duocarmycin-derived ADCs have been disclosed in the prior art. The
liquid formulations disclosed in WO 2011/133039 and DOKTER, W et
al. are unsuitable for lyophilization, because the stability of a
duocarmycin-derived ADC in the formulation and the quality of the
lyophilized cake is inferior in formulations that have a relatively
low molar ratio of lyoprotectant to ADC. In view of the lack of
suitable pharmaceutical formulations for duocarmycin-derived ADCs
in the state of the art, there is a clear need for lyophilized
compositions comprising duocarmycin-derived ADCs having a
acceptable cake appearance and which can be prepared with a
lyophilization process having an acceptable lyophilization time,
and in which the duocarmycin-derived ADCs are acceptably
stable.
BRIEF DESCRIPTION OF THE PRESENT INVENTION
[0018] The present invention relates to lyophilized compositions
comprising antibody-duocarmycin drug conjugates of formulae (I) or
(II) and corresponding reconstituted aqueous solutions thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: Lyophilized cakes of A: placebo solution with 30
mg/ml trehalose dihydrate (similar to composition II, but without
SYD985), B: composition II with 30 mg/ml trehalose dihydrate, C:
composition III with 40 mg/ml trehalose dihydrate
[0020] FIG. 2: Stability results: Y axis: percentage SYD985 monomer
in lyophilized composition I (.DELTA.), III (x), IV (.diamond.), V
(.circle-solid.), and VI (.box-solid.) at 40.degree. C., X axis:
time in months
[0021] FIG. 3: Stability results: Y axis: percentage high molecular
weight (HMW) particulates in lyophilized composition I (.DELTA.),
III (x), IV (.diamond.), V (.circle-solid.), and VI (.box-solid.)
at 40.degree. C., X axis: time in months
[0022] FIG. 4: SEM pictures of the top and middle layers of
lyophilized cakes of composition V without annealing (left) and
with annealing (right), white bar is 200 .mu.m
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0023] The present invention provides a lyophilized composition
comprising an antibody-drug conjugate (ADC) of formula (I),
a buffering agent, a lyoprotectant and a surfactant wherein the
molar ratio of lyoprotectant to antibody-drug conjugate is
1,400-3,200 to 1.
[0024] In the ADCs of formula (I) and (II) (see below) the linker
drug is conjugated to two or more antibody cysteine residues to
give an ADC having an average drug to antibody ratio (DAR) of 2-3.
Preferably, the ADC has an average DAR of 2.5-3.0, more preferably
an average DAR of 2.6-2.9.
[0025] The ADCs of formulae (I) and (II) to be used in the
composition in accordance with the present invention have the
linker drug conjugated to the antibody through the S-atom of a
cysteine residue, i.e., they are cysteine-linked antibody-drug
conjugates. Typically, the cysteine residue is a natural cysteine
residue which is present in the heavy and/or light chain of the
(monoclonal) antibody (mAb) and forms interchain disulfide bonds.
Antibodies of different antibody classes contain different numbers
of interchain disulfide bonds. For example, IgG1 antibodies
typically have four interchain disulfide bonds--all four located in
the hinge region--and after (partial) reduction of the disulfide
bonds the linker drug is randomly attached to the free thiol
groups.
[0026] Antibody-drug conjugates of formulae (I) and (II) for use in
accordance with the present invention can be obtained according to
methods and procedures that are well known to a person skilled in
the art. Conjugation through interchain disulfide bonds can occur
after complete or partial reduction of said disulfide bonds.
Suitable methods for preparing such compounds can be found in the
description and examples of WO 2011/133039 as well as in the
supplementary information of DOKTER, W et al. in Mol. Cancer Ther.;
13(11) November 2014; 2618-2629 (disclosing HIC purification of the
ADC SYD983 to give SYD985). In particular, Example 15 of WO
2011/133039 describes the partial reduction of trastuzumab to
generate two free thiol groups per mAb and conjugation with a
number of linker drugs to ADCs having an average DAR of
approximately 2. It is easily understood by those skilled in the
art how to obtain ADCs having an average DAR of 2-3 in accordance
with the present invention.
[0027] In a typical embodiment of the composition of the present
invention, the antibody-drug conjugate (ADC) is present in the
composition in an amount providing a concentration of 1-30 mg/ml
when reconstituted with water. Preferably, the amount is providing
a concentration of 2-20 mg/ml, more preferably 5-15 mg/ml, most
preferably about 10 mg/ml when reconstituted with water.
[0028] The monoclonal antibody (mAb) used for conjugation with a
linker drug and for inclusion into a composition in accordance with
the present invention can be an IgA, IgD, IgE, IgG or IgM antibody.
The antibody can have .kappa. (kappa) light chains or .lamda.
(lambda) light chains. The IgG antibody can be an IgG1, IgG2, IgG3
or IgG4 antibody. Preferably, the antibody binds to a(n) antigen
target that is expressed in or on the cell membrane (e.g., on the
cell surface) of a tumour cell, more preferably, the antibody is
internalised by the cell after binding to the (antigen) target,
after which the duocarmycin drug is released intracellularly.
Preferably, the antibody is an IgG antibody, more preferably an
IgG1 antibody, most preferably an IgG1 antibody having .kappa.
light chains. Preferably, the IgG antibody carries a native
glycoside/carbohydrate moiety attached at N297 of the heavy chain
of the antibody.
[0029] Suitable antibodies include an anti-annexin A1 antibody, an
anti-CD19 antibody, an anti-CD22 antibody, an anti-CD30 antibody,
an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD38
antibody, an anti-CD44 antibody, an anti-CD47 antibody, an
anti-CD56 antibody, an anti-CD70 antibody, an anti-CD74 antibody,
an anti-CD79 antibody, an anti-CD115 antibody, an anti-CD123
antibody, an anti-CD138 antibody, an anti-CD203c antibody, an
anti-CD303 antibody, an anti-CEACAM antibody, an anti-CLL-1
antibody, an anti-c-MET (or anti-HGFR) antibody, an anti-Cripto
antibody, an anti-DLL3 antibody, an anti-EGFR antibody, an
anti-EPCAM antibody, an anti-EphA2 antibody, an anti-EphB3
antibody, an anti-ETBR antibody, an anti-FcRL5 antibody, an
anti-FOLR1 antibody, an anti-GCC antibody, an anti-GPNMB antibody,
an anti-HER2 antibody, an anti-HMW-MAA antibody, an anti-integrin
antibody, an anti-Lewis A like carbohydrate antibody, an anti-Lewis
Y antibody, an anti-LIV1 antibody, an anti-mesothelin antibody, an
anti-MN antibody, an anti-MUC1 antibody, an anti-MUC16 antibody, an
anti-NaPi2b antibody, an anti-Nectin-4 antibody, an anti-PSMA
antibody, an anti-SIRP.alpha. antibody, an anti-SLC44A4 antibody,
an anti-STEAP-1 antibody, an anti-5T4 (or anti-TPBG, trophoblast
glycoprotein) antibody, an anti-Tag72 antibody, an anti-TF (or
anti-tissue factor) antibody, an anti-TROP2 antibody, and an
anti-VLA antibody.
[0030] Preferably, the antibody is an anti-annexin A1 antibody, an
anti-HER2 antibody, an anti-CD115 antibody, an anti-CD123 antibody,
an anti-CLL-1 antibody, an anti-c-MET antibody, an anti-MUC1
antibody, an anti-PSMA antibody, an anti-5T4 antibody or an anti-TF
antibody. More preferably, the antibody is an anti-HER2 antibody,
an anti-PSMA antibody or an anti-5T4 antibody. Even more preferred
is an anti-HER2 antibody, in particular trastuzumab or a biosimilar
thereof.
[0031] The antibody to be used in accordance with the present
invention is a monoclonal antibody (mAb) and can be a chimeric,
humanized or human mAb. Preferably, in accordance with the present
invention a humanized or human mAb is used, more preferably a
humanized or human IgG antibody, most preferably a humanized or
human IgG1 mAb. Preferably, said antibody has .kappa. (kappa) light
chains, i.e., a humanized or human IgG1-.kappa. antibody.
[0032] The lyoprotectant to be used in the composition in
accordance with the present invention can be any excipient which,
when combined with the ADC, significantly prevents or reduces
chemical and/or physical instability of the ADC upon lyophilization
and subsequent storage.
[0033] Exemplary lyoprotectants include sugars such as reducing or
non-reducing saccharides; amino acids, such as glycine, arginine,
proline, lysine, alanine; a methylamine; a lyotropic salt; a polyol
such as trihydric or higher sugar alcohols, e.g. glycerin,
erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol;
propylene glycol; polyethylene glycol, polymers such as polyvinyl
pyrrolidine, polyvinyl alcohol, or polydextran, The preferred
lyoprotectant is a non-reducing saccharide, e.g. disaccharides such
as trehalose, isotrehaloses, sucrose and isosucroses;
trisaccharides, such as melezitose, gentianose, raffinose, erlose
and umbelli-ferose; tetrasaccharides, such as stachyose and
lychnose; and pentasaccharides, such as verbascose. The
lyoprotectant to be used in accordance with the composition of the
present invention preferably is trehalose or sucrose, or a mixture
thereof, most preferably trehalose. Typically, trehalose dihydrate
is used for preparing a composition in accordance with the present
invention.
[0034] The molar ratio of lyoprotectant to ADC to be used in the
composition in accordance with the present invention is 1,400-3,200
to 1. Preferably, a molar ratio of 1,400-3,000 to 1, more
preferably 1,400-2,500 to 1, even more preferably 1,400-2,000 to 1,
still more preferably 1,400-1,800 to 1, is used. Most preferably, a
lyoprotectant to ADC molar ratio of 1,500-1,700 to 1 is used.
[0035] The present inventors found that the stability of
duocarmycin-derived ADCs was optimal above a molar ratio of
lyoprotectant to ADC of 1,400 to 1. The stability and cake
appearance of such lyophilized compositions was found to be
improved compared to compositions having a molar ratio below 1,400
to 1. The lyoprotectant to ADC molar ratios of the prior art
compositions disclosed in WO 2012/162482, i.e., about 350 to 1, and
in WO 2011/133039 and DOKTER, W et al. in Mol. Cancer Ther.; 13(11)
November 2014; 2618-2629, i.e., 800 to 1, are well below 1,400 to
1. Compositions comprising duocarmycin-derived ADCs having a molar
ratio of lyoprotectant to ADC of lower than 1,400 to 1 showed an
inferior cake appearance, while placebo compositions, i.e.
compositions without a duocarmycin-derived ADC, with a similar
ratio showed a acceptable cake appearance. Moreover, the stability
of compositions comprising duocarmycin-derived ADCs having a molar
ratio of lyoprotectant to ADC of lower than 1,400 to 1 was inferior
as well. However, the molar ratio of lyoprotectant to ADC should
not be overly high, as the lyophilisation process of compositions
having a molar ratio of lyoprotectant to ADC of over 3,200 to 1 was
found to be unacceptably time consuming. A long lyophilisation
process time causes operational risks, such as degradation of the
ADC during the process, and makes the process unsuitable for
commercial production.
[0036] The buffering agent to be used in the composition in
accordance with the present invention may be any buffering agent
that does not have a major pH change during freezing. Suitable
buffering agents include tris(hydroxymethyl)methylamine,
4-2-hydroxyethyl-1-piperazine-ethanesulfonic acid, succinate,
citrate, and histidine. Preferred buffering agents are histidine,
citrate, and succinate. More preferred buffering agents are
histidine and succinate. The most preferred buffering agent is
histidine.
[0037] In the composition of the present invention, the buffering
agent is present in a concentration of 2.5-25 mM when reconstituted
with water, preferably in a concentration of 3.0-10 mM. The most
preferred concentration of the buffering agent is about 5 mM, as it
was observed that the stability of duocarmycin-derived ADCs in
aqueous solution was optimal at that concentration.
[0038] The composition according to the present invention typically
provides an aqueous solution having a pH of 5.3-6.0 when
reconstituted with water. Preferably, the pH is 5.5-5.8. Most
preferably, the pH is about 5.7. In the preferred pH ranges, a
compromise is reached between the stability of the antibody and the
chemical stability of the linker drug, resulting in an optimal
stability of the duocarmycin-derived ADC.
[0039] The surfactant to be used in the composition in accordance
with the present invention preferably is a non-ionic surfactant.
Suitable surfactants include alkyl glycosides, poloxamers, and
polysorbates. Preferred surfactants are polysorbates, such as
polysorbate 20 and polysorbate 80. The most preferred surfactant is
polysorbate 20.
[0040] The amount of surfactant to be present in the composition of
the present invention is such that it reduces aggregation of the
ADC in aqueous solution and minimizes the formation of (high
molecular weight) particulates when reconstituted with water. The
surfactant may be present in an amount providing a concentration of
0.001-0.5% mass/volume (m/v) when reconstituted with water,
preferably 0.005-0.05% (m/v), more preferably 0.005-0.02% (m/v).
Most preferred is an amount of about 0.01% (m/v).
[0041] In a preferred embodiment of the present invention, the
lyophilized composition comprises an antibody-drug conjugate of
formula (II),
histidine, trehalose, and polysorbate wherein the molar ratio of
trehalose to ADC is 1,500-1,700 to 1, and, when reconstituted with
water, the ADC of formula (II) is present in an amount of 5-15
mg/ml, the histidine concentration is 3.0-10 mM, the amount of
polysorbate is 0.005-0.02% (m/v), and the pH is 5.5-5.8.
[0042] In a more preferred embodiment of the present invention, the
lyophilized composition comprises an ADC of formula (II),
histidine, trehalose, and polysorbate 20, wherein the molar ratio
of trehalose to ADC is about 1,605 to 1, and when reconstituted
with water, the ADC of formula (II) is present in an amount of
about 10 mg/ml, the concentration of histidine is about 5 mM, the
amount of polysorbate 20 is about 0.01% (m/v), and the pH is
5.5-5.8.
[0043] In an even more preferred embodiment of the present
invention, the lyophilized composition comprises an ADC of formula
(II), histidine, trehalose, and polysorbate 20, wherein the molar
ratio of trehalose to ADC is about 1,605 to 1, and when
reconstituted with water, the ADC of formula (II) is present in an
amount of about 10 mg/ml, the concentration of histidine is about 5
mM, the amount of polysorbate 20 is about 0.01% (m/v), and the pH
is about 5.7.
[0044] In a most preferred embodiment of the present invention, the
lyophilized composition consists of or consists essentially of an
ADC of formula (II), histidine, trehalose, and polysorbate 20,
wherein the molar ratio of trehalose to ADC is about 1,605 to 1,
and when reconstituted with water, the ADC of formula (II) is
present in an amount of about 10 mg/ml, the concentration of
histidine is about 5 mM, the amount of polysorbate 20 is about
0.01% (m/v), and the pH is about 5.7.
[0045] The compound of formula (II) referred to as SYD985 in the
present specification has an average DAR in the range of
2.6-2.9.
[0046] In one embodiment, SYD985, also known as trastuzumab
vc-seco-DUBA, is an ADC of formula (II) having an average DAR of
about 2.8. This SYD985 is a mixture consisting of about 65% DAR2
species, about 30% DAR4 species, and about 5% DAR6 species. The
DAR2 species consist of ADCs wherein two linker drugs are
conjugated to the cysteine residues of one interchain disulfide
bridge between the heavy- and light chains and ADCs wherein two
linker drugs are conjugated to the cysteine residues of one
interchain disulfide bridge between the heavy chains. The DAR4
species consist of ADCs wherein four linker drugs are conjugated to
the cysteine residues, predominantly in two isomers, in one isomer
the four linker drugs are conjugated to cystein residues of the two
interchain disulfide bridges between the heavy and light chains, in
the other isomer the four linker drugs are conjugated to the
cysteine residues of the two interchain disulfide bridges between
the heavy chains. The DAR6 species consist of ADCs wherein six
linker drugs are conjugated, the predominant isomer is an ADC
wherein two linker drugs are conjugated to the cysteine residues of
one interchain disulfide bridge between the heavy and light chains
and four linker drugs are conjugated to the cysteine residues of
the two interchain disulfide bridges between the heavy chains.
[0047] The compositions of the present invention additionally may
comprise a bulking agent. Typical bulking agents include dextran,
polyvinylpyrrolidone, serine, glycine, mannitol, inositol,
sorbitol, and hydroxyethyl starch. Preferred bulking agents are
mannitol and sorbitol. The most preferred bulking agent is
mannitol. The bulking agent is present in an amount sufficient to
decrease the lyophilization process time and/or to improve cake
appearance. Preferably, no bulking agent is included in the
composition in accordance with the present invention. Most
preferably, the composition of the present invention consists of or
consists essentially of an ADC of formula (I) or (II), a buffering
agent, a lyoprotectant, and a surfactant as described herein
above.
[0048] The present invention further provides a process for the
lyophilization of a composition comprising an ADC of formula (I) or
(II) in accordance with the present invention, the process
comprising the steps of a) freezing a pre-lyophilization aqueous
solution of a composition comprising an ADC of formula (I) or (II),
a buffering agent, a lyoprotectant, and a surfactant wherein the
molar ratio of lyoprotectant to ADC is 1,400-3,200 to 1, b) primary
drying at a product temperature below the collapse temperature of
the composition at a pressure below atmospheric pressure, and c)
secondary drying at a product temperature above 0.degree. C. and
below the glass transition temperature of the composition at a
pressure below atmospheric pressure. Preferably, the composition is
frozen using a shelf temperature of -45.degree. C. to -30.degree.
C., the primary drying is performed at a shelf temperature of
-25.degree. C. to -5.degree. C. at a pressure below 0.2 mbar, and
the secondary drying is performed at a shelf temperature of
15.degree. C. to 40.degree. C., 20.degree. C. to 40.degree. C. or
25.degree. C. to 40.degree. C. at a pressure below 0.2 mbar.
Preferably, secondary drying is performed at a shelf temperature of
about 20.degree. C.
[0049] In an alternative embodiment, the process comprises the
steps of a) freezing a pre-lyophilization aqueous solution of a
composition in accordance with the present invention, b) primary
drying at a product temperature below the collapse temperature of
the composition at a pressure below atmospheric pressure, and c)
secondary drying at a product temperature below the glass
transition temperature of the composition at a pressure below
atmospheric pressure, wherein the freezing step a) comprises an
annealing step.
[0050] Preferably, the annealing step is performed at a shelf
temperature of -25.degree. C. to -10.degree. C. for 0.5 to 6 hours
or 1 to 6 hrs. More preferred is an annealing step at a shelf
temperature of -25.degree. C. to -15.degree. C. for 1 to 5 hrs or 2
to 5 hrs. Even more preferred, step a) comprises lowering the shelf
temperature of the apparatus at a pressure of 1,000 mbar at a rate
of 0.2-1.degree. C./min, to a shelf temperature of -50.degree. C.
to -30.degree. C., subsequently maintaining this temperature for 30
min to 1.5 hrs, then raising the shelf temperature at a rate of
0.2-1.degree. C./min to a shelf temperature of -25.degree. C. to
-10.degree. C., and maintaining this temperature for 0.5 to 3 hours
or 1.5 to 3 hrs, followed by cooling at a rate of 0.2-1.0.degree.
C./min to a shelf temperature of -50.degree. C. to -40.degree. C.,
followed by maintaining that temperature for 1-2 hrs.
[0051] The present inventors found that inclusion of an annealing
step surprisingly decreased the lyophilisation process time
drastically. When an annealing step of no more than 4 hours is
included, the total process time decreases by 40 hours or more. As
well, the morphology of the lyophilisate is optimal if an annealing
step is included. The porosity of the cake increases, leading to
faster and more homogeneous drying, and residual moisture after
primary drying is low.
Examples
[0052] Preparation of Pre-Lyophilisation Solutions of SYD985
[0053] The ADC SYD 985 (i.e. trastuzumab vc-seco-DUBA) was prepared
and purified according to methods and procedures published in the
supplement of DOKTER, W et al. in Mol. Cancer Ther.; 13(11)
November 2014, 2618-2629. After purification, the SYD985 solution
was concentrated and diafiltrated using the aqueous solutions as
depicted in Table 1 without surfactant using a single-use
tangential flow cassette having a polyethersulfone membrane with a
30 kDa cut-off (Sius.TM.). After diafiltration, surfactant was
added and the solution was diluted to 10 mg/ml of SYD985. Then the
final solution was immediately frozen and stored at -70.degree. C.
The SYD985 concentration was determined using UV-VIS and is
expressed in mg/ml. Molar amounts were calculated using a molecular
weight of 151.8 kDa for SYD 985 (average DAR of 2.7).
TABLE-US-00001 TABLE 1 Composition of pre-lyophilization solution*
I II VI Comparative Comparative III IV V Comparative SYD985 10
mg/ml 10 mg/ml 10 mg/ml 10 mg/ml 10 mg/ml 10 mg/ml Histidine 10 mM
10 mM 10 mM 5 mM 5 mM 10 mM Trehalose. 20 mg/ml 30 mg/ml 40 mg/ml
40 mg/ml 40 mg/ml 82.5 mg/ml 2H.sub.2O Mannitol 50 mg/ml 0 mg/ml 0
mg/ml 0 mg/ml 0 mg/ml 0 mg/ml Polysorbate 0.01% 0.01% 0.01% 0.01%
0.01% 0.01% 20 (m/v) pH 5.7 6.0 6.0 6.0 5.7 6.0 Molar ratio
Trehalose: 800:1 1,200:1 1,605:1 1,605:1 1,605:1 3,310:1 SYD985
*The composition of the pre-lyophilization solution is the same as
the composition of the solution obtained by reconstituting the
lyophilized cake.
[0054] Lyophilization Process Used to Determine R&D Stability
of Solutions I-VI
[0055] The frozen solutions I-VI were thawed at room temperature
(RT) and, if free of precipitates, filtered over a 0.22 .mu.m
filter and filled into vials. The vials were loaded into a small
scale freeze drying apparatus. The lyophilization was performed by
lowering the shelf temperature to -35.degree. C., followed by
primary drying at 0.075 mbar at a shelf temperature of -10.degree.
C., and secondary drying at a shelf temperature of 40.degree. C. In
composition I containing mannitol, an annealing step of 4 hrs at a
shelf temperature of -20.degree. C. was introduced between the
freezing and primary drying step. In composition VI having over 80
mg/ml trehalose, an annealing step at -12.degree. C. for 5 hrs was
introduced after freezing to -40.degree. C., primary drying was
performed at 0.075 mbar and -21.degree. C., required to avoid
collapse of the lyophilisate. An intermediate drying step at
-5.degree. C. was introduced between primary and secondary drying
to prevent melting. The secondary drying step was performed at
20.degree. C.
[0056] Lyophilisation Process (General Method)
[0057] The following process is a general procedure to obtain a
lyophilized composition in accordance with the present
invention.
[0058] For lyophilization of SYD985, a frozen solution of SYD985
(10 mg/ml) containing 5 mM histidine, 40 mg/ml trehalose dihydrate,
and 0.01% (m/v) polysorbate 20 was thawed and filtered, and 20 ml
vials were filled with 8.3 ml of SYD985 solution. The vials were
loaded into an Epsilon 2-6D (MartinChrist) R&D freeze dry
apparatus. The shelf temperature of the apparatus was lowered at a
pressure of 1,000 mbar at a rate of 0.2-1.degree. C./min to a shelf
temperature of -50.degree. C. to -30.degree. C., subsequently
maintaining this temperature for 1 to 2 hrs, then the shelf
temperature was raised at a rate of 0.2-1.degree. C./min to a
temperature of -25.degree. C. to -10.degree. C., this temperature
was maintained for 1.5 to 3 hrs. The next step was a cooling step
at a rate of 0.2-1.0.degree. C./min to a shelf temperature of
-50.degree. C. to -40.degree. C., followed by maintaining that
temperature for 1-2 hrs. Subsequently, the pressure was decreased
to 0.05-0.3 mbar, and the shelf temperature was raised again at a
rate of 0.2-1.degree. C./min to -15.degree. C. to -5.degree. C.,
and that temperature was maintained for 25 to 35 hrs at a pressure
of 0.05-0.3 mbar. Then the shelf temperature was raised to
20.degree. C. to 40.degree. C. at a rate of 5-20.degree. C./hour,
and this temperature was maintained for 1-10 hrs.
[0059] Comparative Example without Annealing Step
[0060] The frozen solution of composition V was thawed at room
temperature and, if free of precipitates, filtered over a 0.22
.mu.m filter and filled into vials. The vials were loaded into a
Epsilon 2-6D (MartinChrist) R&D freeze dry apparatus. The
lyophilization was performed by lowering the shelf temperature to
-40.degree. C. at a rate of 0.2-1.degree. C./minute, followed by
primary drying at 0.075 mbar at a shelf temperature of -10.degree.
C. for 50 hrs. After primary drying, the shelf temperature was
raised to 40.degree. C. at a rate of 3.degree. C./hr and kept at
that temperature for 10 hrs. Total process time was about 92
hrs.
[0061] Lyophilization Process Results
[0062] Appearance/Quality of the Lyophilized Cake
[0063] The appearance of the lyophilized cakes of the various
formulations is summarized in Table 4, row 3. Formulations I, III,
IV and V of Table 1 had a good appearance after lyophilization. An
amount of lyoprotectant of less than 1,400 times the molar amount
of ADC gives unsatisfactorily results for the final lyophilized
product, except in the presence of a considerable amount of
mannitol (Formulation I). Lyophilisation of solution II resulted in
a lyophilized cake of inferior quality (see FIG. 1B). Upon visual
inspection, cracks and crumbles were visible. The inferior cake
quality was not expected as a similar solution, without SYD985, the
placebo, had a acceptable cake appearance as lyophilisate (see FIG.
1A). Lyophilization of solutions III, IV and V of Table 1 with a
trehalose molar excess of 1,605 results in a cake of the desired
quality (cake of solution III is shown in FIG. 1C).
[0064] Duration of the Lyophilization Process
[0065] The duration for the various lyophilization processes is
summarized in Table 2.
TABLE-US-00002 TABLE 2 Summary Lyophilization time I II IV V VI
R&D process no annealing ~92 hrs ~92 hrs ~92 hrs ~92 hrs ~138
hrs with annealing ND ND ND ~44 hrs ~122 hrs Scale-up process no
annealing ND ND ND ND ~13 days* with annealing ND ND ND ~63 hrs ~8
days ND = not determined *cake collapsed
[0066] The R&D lyophilization process for solution VI of Table
1 required at least 5 days (about 122 hrs), with an annealing step
at -12.degree. C. for 5 hrs, whereas lyophilization of solutions I,
II, IV and V took less than 4 days (about 92 hrs without
annealing).
[0067] The lyophilization process with annealing step has a total
process time of about 44 hrs for formulation V. FIG. 4 shows a SEM
picture of a lyophilized cake of solution V obtained by the
comparative process which is without an annealing step (left) and a
picture of a lyophilized cake with annealing (right). The porosity
of the cake is clearly increased. Scale-up of solution V resulted
in a process having a process time of about 63 hours.
[0068] Scale-up of solution VI to a commercial lyophilization
process was unsuccessful, as the shortest commercial scale process
obtained after optimization of the conditions still took over 8
days, with an annealing step. Without the annealing step the
process took about 13 days and resulted in a collapsed cake. A high
trehalose content in a SYD985 liquid pre-lyophilization solution is
disadvantageous for the lyophilisation process.
[0069] Stability Measurements
[0070] a) Stability in Solution
[0071] The frozen solution was thawed at RT and, if free of
precipitates, filtered over a 0.22 .mu.m filter and filled into
vials. Sub-visible particulate matter analysis was determined by
the light obscuration technique (LO) using a PAMAS CVSS particle
counter (HCB-LD-25/25 sensor, Partikelmess- and Analyse Systeme
GmbH). The procedure is performed in accordance with the Ph. Eur.
<2.9.19>, particulate contamination; sub-visible particles. A
sample volume of 300 .mu.l is measured (prerun volume: 0.8 mL,
rinse volume: 5 mL, fill and rinse rate: 10 mL/min). At least 3
successive LO measurements were performed per sample. Results are
analyzed using PMA software.
[0072] Z.sub.average, an indication of particle size, to determine
aggregation in solution, was measured using dynamic light
scattering (DLS). Analysis was performed on a Zetasizer APS
(Malvern Instruments, .lamda.0=830 nm, .theta.=90.degree.). A
sample volume of 100 .mu.l is measured. Per sample at least 3
successive DLS measurements were performed with 2-minutes time
intervals to allow solutions to be at rest. During all
measurements, temperature was kept constant at 25.degree. C. and
scattering results were compensated for viscosity.
[0073] b) Stability of the Lyophilized Compositions
[0074] Lyophilized samples were reconstituted using water for
injection. All measured lyophilized compositions were easily
reconstitutable.
[0075] The percentage of high molecular weight material (HMW) and
monomer of SYD985 in the reconstituted compositions I, III, IV, V
and VI was determined by High Performance Liquid Chromatography
(HPLC) in Size Exclusion Chromatography (SEC) on a Shimadzu UFLC
system with a: TSKgel G3000SWxl, 5 .mu.m, 7.8 mm.times.30 cm
analytical column of Joint Analytical Systems (PN 08541) at a
column temperature of 25.degree. C., using a 50 mM phosphate buffer
with 300 mM NaCl of pH 7.5 as mobile phase at a flow rate of 0.3
ml/minute. Typically, 50 .mu.l of the reconstituted formulation was
diluted to a concentration of approximately 1.0 mg SYD985/mL.
[0076] Stability Results
[0077] a) Stability in Solution
[0078] Table 3 provides a summary of the stability results for the
pre-lyophilization solutions of Table 1. The stability of solution
II was not determined as the appearance after lyophilization was
not acceptable. All five measured solutions measured showed
comparable stability, except for solution I. Solution I of Table 1
with a trehalose molar excess of about 800 resulted in decreased
stability in solution (see Table 3). After 1 month at RT, a
protein-aceous precipitate was observed. For solution I decreased
colloidal stability was observed as well with LO and DLS
measurements after 1 month at room temperature (RT). Solution I has
a large amount of particles >10 .mu.m at t=1 month as observed
with LO, whereas the other solutions had low amounts of particles
of this size. Z.sub.average measured with DLS for the solutions
kept for 1 month is as well significantly increased in solution I
as compared to the other solutions.
[0079] Both solutions IV and V with a histidine concentration of 5
mM (IV and V) have lower Z.sub.average values than the values
measured for III and VI, having a histidine concentration of 10
mM.
[0080] b) Stability of the Lyophilized Compositions
[0081] FIGS. 2 and 3 show that when solution I of Table 1 is
lyophilized, in the lyophilized cake the % monomer decreases,
whereas the % HMW increases over time at 40.degree. C., indicative
of decreased stability. FIGS. 2 and 3 show that for the
formulations V and VI the decrease in % monomer as well as the
increase in % HMW over time is significantly lower, as compared to
formulation I. The amount of monomer in reconstituted solutions III
and IV at t=1 month is higher than the amount of monomer in
solution I. The amounts of HMW at t=1 month are similar for
solutions I, III and IV.
* * * * *