WO2024148187A1

WO2024148187A1 - Pharmaceutical composition comprising anti-hpa-1a antibody

Info

Publication number: WO2024148187A1
Application number: PCT/US2024/010348
Authority: WO
Inventors: Adrienne Rachael ALFORD; Adam W. LUCKA; Daniel KULLMANN; Johannes Pall MAGNUSSON; Claudia Mueller; Annette OSWALD
Original assignee: Rallybio Ipa, Llc
Priority date: 2023-01-04
Filing date: 2024-01-04
Publication date: 2024-07-11

Abstract

Provided is a stable pharmaceutical composition comprising an anti-human platelet antigen (HPA)-la antibody or antigen-binding fragment thereof.

Description

PHARMACEUTICAL COMPOSITION COMPRISING ANTI-HPA-la ANTIBODY

BACKGROUND

[0001] Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially life-threatening, rare disease that can cause uncontrolled bleeding in fetuses and newborns. Fetal -maternal incompatibility in the human platelet antigen (HPA)-l locus is the most common cause of FNAIT, accounting for 85% to 90% of severe FNAIT cases (Eksteen et al. 2015). There is currently no approved therapy for the prevention or treatment of FNAIT.

[0002] RLYB212 is a recombinant human immunoglobulin G1 monoclonal antibody developed from transformed memory B-cells isolated from a mother with severe FNAIT affected pregnancies. Formulation of RLYB212 in a pharmaceutical composition for parenteral administration has proved unexpectedly challenging, due to oxidation of its amino acid side chains at a low protein concentration. Accordingly, there is a need for a pharmaceutical composition comprising a low concentration of RLYB212, which is stable and resistant to oxidation.

SUMMARY OF THE INVENTION

[0003] Some of the main aspects of the present invention are summarized below.

Additional aspects are described in the Detailed Description of the Invention, Example, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various ways, and all such combinations are intended to fall within the scope of the present invention.

[0004] The disclosure provides a pharmaceutical composition comprising an antiHP A- la antibody, such as RLYB212, or an antigen-binding fragment thereof. One embodiment is a pharmaceutical composition comprising: (i) an anti-HPA-la antibody, or antigen-binding fragment thereof, which comprises the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2; and (ii) a stabilizing amount of tryptophan and/or methionine; and wherein the pharmaceutical composition is suitable for parenteral administration to a human subject. In certain embodiments, the composition is stable (i) at 25°C for 3 months or for 6 months or for 12 months; and/or (ii) at 40°C for 45 days or for 3 months. [0005] In one aspect, the composition is stable at 40°C for 3 months and/or at 25°C for 3 months or 6 months, each independently determined by at least two of (i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; (ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; (iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

[0006] In another aspect, the composition is stable at 40°C for 3 months, as determined by at least two of (i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; (ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; (iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC; and at 25°C for 3 months or 6 months, as determined by all of (i), (ii), and (iii).

[0007] In a further aspect, the composition is stable at 25°C for 6 months, as determined by (i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; (ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; and (iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

[0008] In some embodiments, the composition comprises at least about 0.05-2.0 mg/mL of the anti-HPA-la antibody or antigen-binding fragment thereof. In one embodiment, the composition comprises about 0.1 mg/mL of the anti-HPA-la antibody or antigen-binding fragment thereof. In another embodiment, the composition comprises about 0.35 mg/mL of the anti-HPA-la antibody or antigen-binding fragment thereof. In an additional embodiment, the composition comprises about 0.20 mg/mL of the anti- HPA-la antibody or antigen-binding fragment thereof.

[0009] In one embodiment, the composition comprises an anti-HPA-la antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment the composition comprises an anti-HPA-la antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0010] In some embodiments, the composition comprises at least about 1 mM tryptophan or at least about 5 mM methionine. In particular embodiments, the composition comprises at least about 2 mM tryptophan, or at least about 10 mM methionine, or at least about 2 mM tryptophan and at least about 10 mM methionine. [0011] In some embodiments, the composition comprises additional excipients. For example, in one embodiment, the composition comprises a buffer, such as a succinate buffer. In some embodiments, the composition comprises an amino acid, such as arginine. In some embodiments, the composition comprises a saccharide, such as sucrose. In certain embodiments, the composition comprises a surfactant, such as polysorbate 80. In a particular embodiment, the composition comprises succinate, arginine, sucrose, and polysorbate 80.

[0012] Pharmaceutical compositions of the invention have a pH that is suitable for parenteral administration. In one embodiment, the pH of the composition is about 6.0 to about 6.5. In one embodiment, the composition is suitable for subcutaneous administration. In one embodiment, the composition is suitable for intravenous administration.

[0013] Further aspects, features, and advantages of the present invention will be better appreciated upon a reading of the following detailed description of the invention and claims.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1A-1F show size exclusion high performance liquid chromatography (SE-HPLC) analysis of RLYB212 first-generation formulations initially (TO) and after storage at 25°C for up to 12 months. Quantification is shown for the main peak (FIG.

1A, ID), the high molecular weight species (HMWS) peak (FIG. IB, IE), and the low molecular weight species (LMWS) peak (FIG. 1C, IF).

[0015] FIG. 2A-2F show SE-HPLC analysis of RLYB212 first-generation formulations at TO and after storage at 40°C for 1.5 or 3 months. Quantification is shown for the main peak (FIG. 2A, 2D), the HMWS peak (FIG. 2B, 2E), and the LMWS peak (FIG. 2C, 2F)

[0016] FIG. 3A-3D show tailing factor of RLYB212 first-generation formulations at TO and after storage at 25 °C (FIG. 3A, 3C) or 40°C (FIG. 3B, 3D) for up to 12 months.

[0017] FIG. 4A-4C show cation exchange chromatography (CEX) analysis of RLYB212 first-generation formulations at TO and after storage at 25 °C for up to 12 months. Quantification is shown for the main peak (FIG. 4A), the basic regions (FIG. 4B), and the acidic regions (FIG. 4C). [0018] FIG. 5A-5C show CEX analysis of RLYB212 first-generation formulations at TO and after storage at 40°C for 1.5 or 3 months. Quantification is shown for the main peak (FIG. 5A), the basic regions (FIG. 5B), and the acidic regions (FIG. 5C).

[0019] FIG. 6A-6B show protein concentration of RLYB212 first-generation formulations at TO and after storage at 25°C (FIG. 6A) or 40°C (FIG. 6B) for up to 12 months.

[0020] FIG. 7A-7B show surfactant concentration in RLYB212 first-generation formulations at TO and after storage at 25 °C (FIG. 7A) or 40°C (FIG. 7B) for up to 12 months.

[0021] FIG. 8A-8C show SE-HPLC analysis of RLYB212 second-generation formulations at TO and after storage at 25°C for up to 12 months. Quantification is shown for the main peak (FIG. 8A), the HMWS peak (FIG. 8B), and the LMWS peak (FIG.

8C).

[0022] FIG. 9A-9C show SE-HPLC analysis of RLYB212 second-generation formulations at TO and after storage at 40°C for 1.5 or 3 months. Quantification is shown for the main peak (FIG. 9A), the HMWS peak (FIG. 9B), and the LMWS peak (FIG.

9C)

[0023] FIG. 10A-10B show tailing factor of RLYB212 second-generation formulations at TO and after storage at 25°C (FIG. 10A) or 40°C (FIG. 10B) for up to 12 months.

[0024] FIG. 11A-11C show CEX analysis of RLYB212 second-generation formulations at TO and after storage at 25°C for up to 12 months. Quantification is shown for the main peak (FIG. 11 A), the basic regions (FIG. 11B), and the acidic regions (FIG.

11C)

[0025] FIG. 12A-12C show CEX analysis of RLYB212 second-generation formulations at TO and after storage at 40°C for 1.5 or 3 months. Quantification is shown for the main peak (FIG. 12A), the basic regions (FIG. 12B), and the acidic regions (FIG.

12C)

[0026] FIG. 13A-13B show surfactant concentration in RLYB212 second-generation formulations at TO and after storage at 25°C (FIG. 13A) or 40°C (FIG. 13B) for up to 12 months.

[0027] FIG. 14A-14B show protein concentration of RLYB212 second-generation formulations at TO and after storage at 25°C (FIG. 14A) or 40°C (FIG. 14B) for up to 12 months. Values are in mg/mL, measured by A280/SE-HPLC. DETAILED DESCRIPTION OF THE INVENTION

[0028] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, immunology, hematology, cell biology, molecular biology, clinical pharmacology, and clinical practice, which are within the skill of the art.

[0029] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0030] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0031] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers' instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.

Definitions

[0032] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0033] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0034] Furthermore, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0035] Wherever embodiments are described with the language “comprising” or “having,” otherwise analogous embodiments described in terms of “consisting of’ and/or “consisting essentially of’ are included.

[0036] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10. Where a numeric term is preceded by “about,” the term includes the stated number and values ±10% of the stated number.

[0037] The term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The terms “antibody” or “immunoglobulin” are used interchangeably herein.

[0038] An antibody is typically composed of two identical pairs of polypeptide chains, each pair having one “heavy” chain and one “light” chain. Each chain is comprised of a variable region, which forms the antibody binding site, and a constant region, which may mediate the binding of the antibody to host tissues or factors. Immunoglobulin molecules can be divided into subclasses depending on the constant region of the heavy chain. The classes are immunoglobulin gamma (IgG), immunoglobulin mu (IgM), immunoglobulin delta (IgD), immunoglobulin epsilon (IgE), and immunoglobulin alpha (IgA). The heavy chain constant regions differ structurally and antigenically among the classes. IgG is the main type of antibody found in blood and extracellular fluid, and it plays a central role in the humoral immune response.

[0039] A “monoclonal antibody” (mAb) refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigen binding site (epitope).

[0040] The antigen-binding site can be defined using various terms and numbering schemes, including the following: (i) Kabat. In the Kabat scheme, CDRs are based on sequence variability (Wu and Kabat 1970). Generally, the antigen-binding site has three CDRs in each variable region (e.g., HCDR1, HCDR2 and HCDR3 in the heavy chain variable region (VH) and LCDR1, LCDR2 and LCDR3 in the light chain variable region (VL));

(ii) Chothia. In the Chothia scheme, the term “hypervariable region” refers to the regions of an antibody variable domain that are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk 1987). Generally, the antigenbinding site has three hypervariable regions in each VH (Hl, H2, H3) and VL (LI, L2, L3). Numbering systems as well as annotation of CDRs and HVs have been revised by Abhinandan and Martin (Abhinandan and Martin 2008).

(iii) IMGT. The IMGT scheme is based on the comparison of variable domains from immunoglobulins and T-cell receptors. The International ImMunoGeneTics (IMGT) database provides a standardized numbering and definition of these regions. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al. (2003).

(iv) AbM. The AbM scheme is a compromise between the Kabat and Chothia numbering schemes and is described by Martin (2010).

(v) SDRU. The antigen-binding site can also be delineated based on “Specificity Determining Residue Usage” (SDRU) (Almagro 2004), where SDR refers to amino acid residues of an immunoglobulin that are directly involved in antigen contact.

[0041] Glycoprotein Hb/IIIa (GPIIb/IIIa), also known as integrin allbp3, is a platelet membrane glycoprotein that binds fibrinogen and von Willebrand factor, and plays a role in platelet activation. Human platelet antigen (HPA)-l is a polymorphism at position 33 on the mature P3 chain of GPIIb/IIIa. In particular, individuals who have a Leu at position 33 in one or more copies of ITGB3 (z.e., the gene that encodes integrin P3) or in any of their integrin P3 are “HPA-la positive,” “positive for HPA-la,” or “HPA-la,” while individuals who do not have a Leu at position 33 (e.g., have a Pro at position 33) in all copies of ITGB3 or in all of their integrin P3 are “HPA-la negative” or “negative for HPA-la.”

[0042] An antibody that is “specific for HPA-la” or that “specifically binds to HPA- la,” is one that does not display detectable binding to HPA-lb. [0043] “Binding” generally refers to the non-covalent interaction between a single binding site of a molecule and its binding partner (e.g., a receptor and its ligand, an antibody and its antigen, two monomers that form a dimer, etc.). In the case of binding between an antibody and its antigen, the interaction can, for example, prevent other molecules from binding to or recognizing the antigen, can initiate the destruction of the antigen, or can alter the structure or functionality of the antigen.

[0044] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.

[0045] A “subject” or “individual” or “patient” is any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, and rodents, including rats and mice, rabbits, etc.

[0046] The invention relates to a pharmaceutical composition comprising an antiHP A- la antibody or antigen-binding fragment thereof, for example, comprising a VH having the amino acid sequence of SEQ ID NO: 1, and a VL having the amino acid sequence of SEQ ID NO: 2. Examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, and Fv fragments, diabodies, single chain antibodies (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0047] RLYB212 is a monoclonal antibody that specifically binds to anti-HPA-la. RLYB212 comprises a VH having the amino acid sequence of SEQ ID NO: 1, a VL having the amino acid sequence of SEQ ID NO: 2, a heavy chain (HC) having the amino acid sequence of SEQ ID NO: 9, and a light chain (LC) having the amino acid sequence of SEQ ID NO: 10. The CDRs of RLYB212, designated by the IMGT system, are as follows:

(a) VH CDR1 has the amino acid sequence of SEQ ID NO: 3,

(b) VH CDR2 has the amino acid sequence of SEQ ID NO: 4,

(c) VH CDR3 has the amino acid sequence of SEQ ID NO: 5,

(d) VL CDR1 has the amino acid sequence of SEQ ID NO: 6,

(e) VL CDR2 has the amino acid sequence of SEQ ID NO: 7, and

(f) VL CDR3 has the amino acid sequence of SEQ ID NO: 8. [0048] In some embodiments, the pharmaceutical composition of the invention comprises the anti-HPA-la antibody or antigen-binding fragment thereof at a concentration of about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.20, 0.22, 0.24, 0.26, 0.28, 0.30, 0.32, 0.34, 0.36, 0.38, 0.40, 0.42, 0.46, 0.48, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.9,0 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg/mL. These amounts can also serve as endpoints for a range of concentrations of the anti-HPA-la antibody or antigenbinding fragment thereof in the composition, for example, about 0.06 mg/mL to about 0.48 mg/mL, or about 0.1 mg/mL to about 0.35 mg/mL, etc. In a particular embodiment, the concentration of the anti-HPA-la antibody is about 0.20 mg/mL

[0049] The anti-HPA-la antibody or antigen-binding fragment thereof comprised in pharmaceutical compositions of the invention is particularly susceptible to oxidation. Accordingly, the composition comprises a stabilizing amount of tryptophan or methionine. Without wishing to be bound by theory, tryptophan and methionine act as an antioxidant to reduce oxidation of amino acid side chains in the antibody. In some embodiments, the composition comprises at least about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, or 2 mM tryptophan. In some embodiments, the composition comprises at least about 1, 2, 4, 6, 8, or 10 mM methionine. In certain embodiments, the composition comprises tryptophan and methionine.

[0050] The pharmaceutical composition comprising the anti-HPA-la antibody or antigen-binding fragment thereof is suitable for parenteral administration. Parenteral routes of administration include intravenous, intramuscular, intraperitoneal, intrathecal, and subcutaneous. In a preferred embodiment, the pharmaceutical composition is suitable for subcutaneous administration.

[0051] Pharmaceutical compositions of the invention preferably have a pH of between about 6.0 and about 6.5. Accordingly, in certain embodiments, the pharmaceutical composition comprises a buffer, such as an acetate, citrate, histidine, phosphate, or succinate buffer. In some embodiments, the concentration of buffer is 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM.

[0052] In some embodiments, the pharmaceutical composition comprises an amino acid, such as glycine, histidine, lysine, or arginine, to reduce viscosity and/or improve stability of the composition. Exemplary concentrations of amino acids include 50, 75, 100, 125, 150, 175, and 200 mM. [0053] Pharmaceutical compositions of the invention can comprise a sugar, such as glucose, maltose, sucrose, or trehalose. In some embodiments, the concentration of sugar is 60, 90, 120, 150, 180, or 200 mM.

[0054] Some pharmaceutical compositions of the invention comprise a surfactant, such as polysorbate 20 or polysorbate 80. The concentration of surfactant in the composition can be, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1% w/w.

[0055] In one embodiment, the provided pharmaceutical compositions are stable at 5°C for at least 3 months. In one embodiment, the provided pharmaceutical compositions are stable at 25°C for at least 3 months. In one embodiment, the provided pharmaceutical compositions are stable at 40°C for at least 45 days. In some embodiments, the pharmaceutical compositions are stable at 5 °C for at least 3 months, 6 months, 9 months, or 12 months. In some embodiments, the pharmaceutical compositions are stable at 25°C for at least 3 months, 6 months, 9 months, or 12 months. In some embodiments, the pharmaceutical compositions are stable at 40°C for at least 3 months, 6 months, 9 months, or 12 months.

[0056] Stability can be assessed, for example, by measuring the main protein peak, HMWS, LMWS, acidic regions, basic regions, main peak tailing factor, protein concentration, surfactant content, etc., at a given time after TO. For purposes of the present disclosure, “stable” at a stated temperature and duration means (i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; or (ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; or (iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC; or a combination of any two or more of (i), (ii), and (iii).

[0057] In one embodiment, the pharmaceutical composition is stable at 40°C for 3 months, as measured by at least two of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 3 months, as measured by all of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 6 months, as measured by at least two of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 6 months, as measured by all of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 3 months, as measured by at least two of (i), (ii), and (iii), and is stable at 40°C for 3 months, as measured by at least two of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 3 months, as measured by all of (i), (ii), and (iii), and is stable at 40°C for 3 months, as measured by at least two of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 6 months, as measured by at least two of (i), (ii), and (iii), and is stable at 40°C for 3 months, as measured by at least two of (i), (ii), and (iii). In one embodiment, the pharmaceutical composition is stable at 25°C for 6 months, as measured by all of (i), (ii), and (iii), and is stable at 40°C for 3 months, as measured by at least two of (i), (ii), and (iii).

[0058] Parameters used to assess stability at different temperatures and timepoints can be chosen independently of one another. For instance, a composition can be stable at 40°C for 3 months as assessed by tailing factor and protein concentration, and at 25°C for 6 months as assessed by main peak/HMWS and protein concentration.

EXAMPLES

[0059] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

Example 1: Stability of First-Generation Formulations

[0060] Initial formulation studies revealed that RLYB212 was unusually susceptible to degradation at low concentrations. The observation that the formulation underwent more oxidation at lower protein concentrations was unexpected.

Methods

[0061] Formulations of RLYB212 were prepared in 20 mM succinate buffer with 0.02% w/v polysorbate 80, as shown in Table 1. Formulations were stored in a 1 mL prefilled syringe (PFS) at 0.5 mL fill.

Table 1. First-Generation RLYB212 Formulations

N/A: not applicable

[0062] Each formulation is evaluated initially (TO), after storage at 5°C, 25°C, and 40°C for 1.5, 3, 6, or 12 months, and after shaking for 5 days at 25°C. Table 2 shows evaluations performed on each formulation.

Table 2. Analytical Assays

CE-SDS: capillary electrophoresis-sodium dodecyl sulfate; FMA: fluorescence micelle assay;

LO: light obscuration; N/A: assay not applicable; X: assay performed

[0063] Protein content is assessed by SolvoVPE® for control formulation (F Ctrl), or by SE-HPLC for test formulations.

[0064] SE-HPLC is used for the separation and quantitative determination of high molecular weight species (HMWS), main peak and low molecular weight species (LMWS) present in tested formulations of RLYB212. HMWS are RLYB212 peaks with an earlier retention time/larger hydrodynamic radii, relative to the main peak. LMWS are all product-related peaks with a later retention time/ smaller hydrodynamic radii, relative to main RLYB212 peak.

[0065] Samples are injected onto the column and separated by differential permeation through a porous chromatography matrix. Species with a larger hydrodynamic radius are less likely to penetrate the pores of the stationary phase, resulting in faster elution. Conversely, analytes with relatively smaller hydrodynamic radii enter the pores of the stationary phase more frequently, resulting in later migration. The correlation of hydrodynamic radius to molecular weight allows the separation and quantification of high and low molecular weight species in each formulation through detection of eluted peaks by FLD excitation at 295 nm and emission at 348 nm. Quantitative determination of the RLYB212 main peak and all present HMWS and LMWS is achieved by relative area % evaluation.

[0066] CEX is used for the quantitative monitoring of charge heterogeneity and identity determination of RLYB212 in the tested formulations. Ion exchange chromatography is based on the interaction of differently charged proteins with the column. CEX retains positively charged proteins because the stationary phase of the column displays negatively charged functional groups. The proteins can be eluted from the column by changing the eluent pH (resulting in a change of the protein charge). The protein elution is monitored by a fluorescence detector (FLD) and excitation at 295nm and emission at 348nm. Quantitative determination of the RLYB212 main peak and all present acidic and basic variants was achieved by relative area % evaluation.

Results

[0067] Selected results of first-generation formulation analyses after 1.5 or 3 months at the indicated temperature are summarized in Table 3. Table 3. Results Summary of First-Generation Formulations

[0068] After 1.5 months at 25°C, SE-HPLC analysis showed a minimal decrease in purity of all formulations compared to TO (FIG. 1A-1C). The amount of LMWS ranged between below the level of quantification and 0.2% (FIG. 1C). Differences in purity between individual formulations were not significant. No significant differences in tailing factor were observed compared to TO, except that Fl showed a slight change in peak symmetry (FIG. 3A). All formulations were stable by CEX analysis (FIG. 4A-4C). Protein content (FIG. 6A) and surfactant content (FIG. 7A) were comparable and stable for all formulations.

[0069] After 3 months at 25 °C, all formulations showed minor degradation and minor loss of purity, with lowest overall purity loss displayed by F7 and F5 (FIG. 1A-1C). All formulations showed an increase in peak fronting except F7, F9, and F Ctrl (FIG. 3A). CEX analysis showed a transition to more acidic species for all formulations (FIG. 4A- 4C). The protein concentration of most formulations remained stable, except for Fl and F8, which displayed loss of protein content (FIG. 6A). In terms of surfactant content, formulations with sucrose and arginine generally performed better, while F7 showed the best overall performance (FIG. 7A).

[0070] F5 and F7 were analyzed after 6 and 12 months at 25°C. The two formulations are identical except that F7 contains a combination of two potential antioxidant sinks, 2 mM tryptophan and 10 mM methionine, while F5 is a base control without the antioxidant amino acids. F7 displayed substantially better protection of purity (FIG. 1D-1F), protein content (FIG. 6A), and surfactant content (FIG. 7A), and resulted in minimized peak fronting (FIG. 3C), after 6 or 12 months at 25°C, compared to F5. Preservation of surfactant is an important factor in long-term stability. The surfactant protects the protein (active agent) from adsorption and aggregation, enabling a longer shelf life for the pharmaceutical composition.

[0071] After 1.5 months at 40°C, all formulations showed decreased purity by SE- HPLC (FIG. 2A-2F) and CEX analysis (FIG. 5A-5C), compared to TO. Formulation F7, containing tryptophan and methionine, had the best stability profile by SE-HPLC analysis, while F3 showed the highest loss in purity. Formulations with a higher RLYB212 concentration (F8, F9) showed higher aggregation, but lower fragmentation. All test formulations except F7 showed an increase in peak fronting (FIG. 3B, 3D) and a loss of protein content (FIG. 6B). Surfactant content was stable for all formulations (FIG. 7B)

[0072] SE-HPLC purity analysis of all formulations after 3 months at 40°C showed increased HMWS (FIG. 2B) and LMWS (FIG. 2C) in many formulations. Notably, increased HMWS were not observed in F7 (FIG. 2E). Tailing factor was not suitable for formulations without methionine and tryptophan, while F7 displayed only a small change in degraded species (FIG. 3B, 3D). CEX analysis showed a strong loss in main peak area and transition to acidic species for all formulations (FIG. 5A-5C). Protein concentration decreased substantially for all formulations, except F7 (FIG. 6B). Formulations with a higher RLYB212 concentration (F8, F9) showed an equal loss of protein content to the lower-concentration formulations (F1-F6). All formulations showed substantial degradation of polysorbate 80, except F7 (FIG. 7B). Example 2: Stability of Second-Generation Formulations

Methods

[0073] Based on the results observed for the first-generation formulations, five new formulations of RLYB212, F10-F14, were prepared in 20 mM succinate buffer with 0.02% w/v polysorbate 80, as shown in Table 4. Formulations were stored in a 1 mL prefilled syringe (PFS) at 0.5 mL fill. Each formulation is evaluated as described in Example 1.

Table 4. Second-Generation RLYB212 Formulations

Results

[0074] Selected results of second-generation formulation analyses after storage at 25°C for the indicated time periods are summarized in Table 4. TO data was collected at 5°C.

Table 4. Stability Results of Second-Generation Formulations After Storage at

25°C

[0075] After storage at 25 °C for up to 12 months, all test formulations were practically free from visible particles. No differences were observed among the formulations by visual inspection. No significant differences in color, clarity/opalescence, or pH were observed between different formulations over the observation period. pH remained at target and stable for all formulations, within method variability. Sub-visible particles (>25 pm and >10 pm) were low for all formulations and within USP <T&1> limits.

[0076] SE-HPLC analysis after 1.5 or 3 months at 25°C showed good stability of all formulations and a low degradation profile, with a loss in main peak purity of -0.3-0.4% and a comparable increase in LMWS at 3 months (FIG. 8A-8C; Table 4). After 1.5 or 3 months at 25°C, all formulations containing methionine and/or tryptophan maintained peak symmetry equivalent to the reference standard. No decrease in the tailing factor was observed for any of the tested formulations (FIG. 10A; Table 4).

[0077] The stability of Fl 1 and Fl 3, which are identical but for their initial protein concentration, was also assessed by SE-HPLC after 6 and 12 months at 25°C. Both formulations remained stable over the observation period (FIG. 8A-8C, 10A, 13A, 14A; Table 4) After 6 months, Fl 1 and F13 maintained a main peak of at least 98%, with no increase in HMWS, and minimal changes in tailing factor. After 12 months, the main peak of Fl 1 and F13 showed a slight decrease of -1.5-2% compared with the 6-month data, with no increase in HMWS, and -3-4% increase in LMWS (FIG. 8A-8C; Table 4). The tailing factor for formulation F13 remained stable over 12 months, but was reduced by -0.3 for formulation Fl 1 (FIG. 10A; Table 4).

[0078] Likewise, all formulations displayed good stability and only minor changes in CEX profile for at least 6 months at 25°C (FIG. 11A-11C). A slight reduction in main peak purity to -44-46% was observed after 3 months; basic variants decreased to -31- 32% and acidic variants increased to -22-25%. This trend continued for Fl 1 and F13 after 12 months, resulting in a comparable content of -32-33% main peak, -20-21% basic variants, and -47% acidic variants. No differences between second-generation formulations were observed.

[0079] Selected results of second-generation formulation analyses after storage at

40°C for the indicated time periods are summarized in Table 5. TO data is shown in

Table 4

Table 5. Stability Results of Second-Generation Formulations After Storage at 40°C

[0080] After 1.5 or 3 months at 40°C, all formulations were practically free of visible particles. The pH and turbidity remained stable within method variability and unchanged from TO. pH remained at target and stable for all formulations, within method variability. Sub-visible particles (>25 pm and >10 pm) were low for all formulations and within USP <T&1> limits.

[0081] SE-HPLC analysis after 1.5 months at 40°C showed high stability (>98%) of all second-generation formulations (FIG. 9A-9C; Table 5). Some degradation was observed after 3 months at 40°C (FIG. 9A).

[0082] After 1.5 or 3 months at 40°C, all formulations containing methionine and/or tryptophan maintained peak symmetry equivalent to the reference standard (FIG. 10B). All formulations displayed moderate changes in CEX profile after 1.5 or 3 months at 40°C, relative to TO (FIG. 12A-12C).

[0083] Surfactant stability was maintained in formulations containing methionine and/or tryptophan after 1.5 months at 25°C (FIG. 13A) or 40°C (FIG. 13B), which was not the case for first-generation formulations, except for F7, which contained methionine and tryptophan. Formulations containing methionine displayed better surfactant stability than those without methionine.

[0084] Protein concentration was maintained for up to 12 months at 25°C (FIG. 14A;

Table 4) and for up to 3 months at 40°C (FIG. 14B; Table 5).

SEQUENCES

SEQ ID NO: 1 - RLYB212 Heavy Chain Variable (Vn) Region

Gin Vai Gin Leu Gin Gin Ser Gly Pro Gly Leu Vai Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Ala He Ser Gly Asp Ser Vai Ser Ser Asn Ser Ala Ala Trp Asn Trp He Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Phe Arg Ser Asn Trp Tyr Asn Asp Tyr Ala Ala Ser Vai Lys Ser Arg He Thr He Asn Gin Asp Thr Ser Lys Asn Gin Leu Ser Leu Gin Leu Asn Ser Vai Thr Pro Glu Asp Thr Ala Vai Tyr Tyr Cys Ala Arg Asp Gly Ala Trp Gly Gly Ser Ser Trp Trp Pro Gly Leu Pro His His Tyr Tyr Ser Gly Met Asp Vai Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

SEQ ID NO: 2 - RLYB212 Light Chain Variable (VL) Region

Glu lie Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu He Tyr Asp Ala Ser Lys Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr He Arg Ser Leu Glu Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asp Trp Gin Gly Leu Thr Phe Gly Gly Gly Thr Lys Vai Glu lie Lys

SEQ ID NO: 3 - RLYB212 VH Complementarity Determining Region (CDR) 1 Gly Asp Ser Vai Ser Ser Asn Ser Ala Ala

SEQ ID NO: 4 - RLYB212 VH CDR 2

Thr Tyr Phe Arg Ser Asn Trp Tyr Asn

SEQ ID NO: 5 - RLYB212 VH CDR 3

Ala Arg Asp Gly Ala Trp Gly Gly Ser Ser Trp Trp Pro Gly Leu Pro His His Tyr Tyr Ser Gly Met Asp Vai

SEQ ID NO: 6 - RLYB212 VL CDR 1

Gin Ser Vai Ser Ser Tyr

SEQ ID NO: 7 - RLYB212 VL CDR 2

Asp Ala Ser

SEQ ID NO: 8 - RLYB212 VL CDR 3

Gin Gin Arg Ser Asp Trp Gin Gly Leu Thr

SEQ ID NO: 9 - RLYB212 Heavy Chain

Gin Vai Gin Leu Gin Gin Ser Gly Pro Gly Leu Vai Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Ala He Ser Gly Asp Ser Vai Ser Ser Asn Ser Ala Ala Trp Asn Trp He Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Phe Arg Ser Asn Trp Tyr Asn Asp Tyr Ala Ala Ser Vai Lys Ser Arg He Thr He Asn Gin Asp Thr Ser Lys Asn Gin Leu Ser Leu Gin Leu Asn Ser Vai Thr Pro Glu Asp Thr Ala Vai Tyr Tyr Cys Ala Arg Asp Gly Ala Trp Gly Gly Ser Ser Trp Trp Pro Gly Leu Pro His His Tyr Tyr Ser Gly Met Asp Vai Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys SEQ ID NO: 10 - RLYB212 Light Chain

Glu He Vai Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Vai Ser Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu He Tyr Asp Ala Ser Lys Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr He Arg Ser Leu Glu Pro Glu Asp Phe Ala Vai Tyr Tyr Cys Gin Gin Arg Ser Asp Trp Gin Gly Leu Thr Phe Gly Gly Gly Thr Lys Vai Glu lie Lys Arg Thr Vai Ala Ala Pro Ser Vai Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser Phe Asn Arg Gly Glu Cys

REFERENCES

Abhinandan KR and Martin ACR. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol. Immunol. 45: 3832-3839 (2008).

Almagro JC. Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires. Mol. Recognit. 17: 132-143 (2004).

Chothia C and Lesk AM. Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196: 901-917 (1987).

Eksteen M, et al. Characterization of a human platelet antigen- la-specific monoclonal antibody derived from a B cell from a woman alloimmunized in pregnancy. J. Immunol. 194: 5751-5760 (2015).

Lefranc M-P, et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev. Comp. Immunol. 27: 55-77 (2003).

Martin ACR. Antibody Engineering. Eds. Kontermann R, Dubel S (Springer-Verlag, Berlin). 2: 33-51 (2010).

Wu TT and Kabat EA, An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132: 211-250 (1970).

***

The invention is further described by the following claims.

Claims

1. A pharmaceutical composition comprising: a) about 0.05-2.0 mg/mL of an anti-human platelet antigen (HPA)-la antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and b) at least about 1 mM tryptophan or at least about 5 mM methionine; wherein the pharmaceutical composition is stable at 40°C for 3 months; and wherein the pharmaceutical composition is suitable for parenteral administration to a human subject.

2. The pharmaceutical composition of claim 1, comprising at least about 2 mM tryptophan.

3. The pharmaceutical composition of claim 1, comprising at least about 10 mM methionine.

4. The pharmaceutical composition of claim 1, comprising about 2 mM tryptophan and about 10 mM methionine.

5. The pharmaceutical composition of claim 1, comprising an anti-HPA-la monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

6. The pharmaceutical composition of claim 1, comprising an anti-HPA-la monoclonal antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

7. The pharmaceutical composition of any one of claims 1-6, comprising about 0.1 mg/mL of the anti-HPA-la antibody or antigen-binding fragment thereof.

8. The pharmaceutical composition of any one of claims 1-6, comprising about 0.35 mg/mL of the anti-HPA-la antibody or antigen-binding fragment thereof.

9. The pharmaceutical composition of any one of claims 1-6, comprising about 0.20 mg/mL of the anti-HPA-la antibody or antigen-binding fragment thereof.

10. The pharmaceutical composition of claim 1, comprising succinate.

11. The pharmaceutical composition of claim 1, comprising arginine.

12. The pharmaceutical composition of claim 1, comprising sucrose.

13. The pharmaceutical composition of claim 1, comprising polysorbate 80.

14. The pharmaceutical composition of claim 1, comprising succinate, arginine, sucrose, and polysorbate 80.

15. The pharmaceutical composition of claim 1, having a pH of about 6.0 to about 6.5.

16. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is stable at 25°C for 3 months.

17. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is stable at 25°C for 6 months.

18. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is stable at 25°C for 12 months.

19. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for subcutaneous administration.

20. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is suitable for intravenous administration.

21. The pharmaceutical composition of claim 1, which is stable at 40°C for 3 months, as determined by at least two of: i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

22. The pharmaceutical composition of claim 21, which is stable at 25°C for 3 months, as determined by at least two of: i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

23. The pharmaceutical composition of claim 21, which is stable at 25°C for 3 months, as determined by: i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; and iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

24. The pharmaceutical composition of claim 21, which is stable at 25°C for 6 months, as determined by at least two of: i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

25. The pharmaceutical composition of claim 21, which is stable at 25°C for 6 months, as determined by: i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; and iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC.

26. A pharmaceutical composition comprising: a) about 0.05-2.0 mg/mL of an anti-human platelet antigen (HPA)-la antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2; and b) at least about 1 mM tryptophan or at least about 5 mM methionine; wherein the pharmaceutical composition is stable at 25°C for 6 months, as determined by at least two of: i) a main peak of at least 97% and no more than an increase of 0.1% in HMWS, relative to TO, as measured by SE-HPLC; ii) a change in tailing factor of no more than ± 0.2, relative to TO, as measured by SE-HPLC; iii) a decrease in protein concentration of less than 10%, relative to TO, as measured by A280/SE-HPLC. and wherein the pharmaceutical composition is suitable for parenteral administration to a human subject.

27. The pharmaceutical composition of claim 26, which is stable at 25°C for 6 months, as determined by i), ii), and iii).