Method for detecting polysorbates

Abstract

To provide a method for detection of intact polysorbate or degraded polysorbate products in a sample containing protein such as a pharmaceutical protein product.SOLUTION: The method comprises: precipitating protein by exposing a protein-containing sample to an organic protic polar solvent or an organic aprotic polar solvent; then centrifuging the sample; and subjecting the supernatant to chromatographic separation using a stationary phase column having immobilized cyano groups, where the chromatographic separation is performed by gradient elution and detected with a charged particle detector.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention provides a method for detecting polysorbates in pharmaceutical products. [Background technology]

[0002] Polysorbates are nonionic surfactants commonly used in both food and biopharmaceutical products. In biopharmaceutical products, they can be used to prevent protein adsorption to surfaces, aggregates, and particulate formulations. However, degradation products of these polysorbates can cause irritation, for example, at the injection site, when used parenterally. Because of this use, there has been great interest in developing analytical methods to monitor the integrity of polysorbates, particularly polysorbate 80 (polyoxyethylene sorbitan monooleate). Commercially available PS80 is heterogeneous, with the most common process-related subspecies being polyoxyethylene (POE) groups, POE isosorbide monoesters, and POE sorbitan / isosorbide di-, tri-, and tetraesters. Therefore, analytical method development has been challenging. Several methods have been reported.

[0003] The following have not been achieved: (1) specificity - immunity of the PS80 mono-ester peak enables quantification in degraded samples; (2) sensitivity, ≤ 20 ppm; (3) accuracy, 95-105%; (4) precision, ≤ 5%; (5) transferability, including quality control (QC); (6) ability to validate in a time and at approximately the same cost as conventional ultraviolet-visible high performance liquid chromatography (HPLC) methods; (7) ability to monitor intact and degraded PS80 and related subspecies; (8) ability to serve as a platform method for protein-containing biopharmaceutical formulations; (9) linearity, R 2>0.99; (10) fatty acid resolution; (11) interpretable by mass spectrometry; (12) not utilizing derivatization; and / or (13) not utilizing quantitation dependent on micellar encapsulation.

[0004] Therefore, there is a need to provide improved methods for detecting polysorbates that address the drawbacks detailed above and / or that are capable of detecting intact polysorbates and / or polysorbate degradation products. Summary of the Invention

[0005] Thus, the present invention provides methods for detecting polysorbate, e.g., intact polysorbate and / or polysorbate degradation products, in a sample containing a protein (e.g., of a pharmaceutical protein product such as an antigen-binding polypeptide (e.g., a monoclonal antibody (mAb))).

[0006] Therefore, in a first aspect of the present invention there is provided a method for identifying polysorbates, such as intact polysorbates and / or polysorbate degradation products, in a protein-containing sample, said sample being subjected to the following steps: (i) precipitating the protein by exposing the sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) separating the protein or peptide from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by subjecting the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column comprising immobilized cyano groups and eluting the bound polysorbate using a mobile phase composition gradient; and (iv) detecting the separated polysorbate using a chromophore-free detector and identifying the polysorbate. A method is provided, comprising subjecting the

[0007] In a second aspect, the present invention provides a method for identifying a protein sample, e.g., from a plurality of proteins, comprising: the identified protein sample contains about 10 ppm to about 5000 ppm of intact polysorbate; The method comprises the steps of: (a) measuring polysorbate in said sample, said step comprising the steps of: (i) precipitating the protein by exposing the sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) separating the protein or peptide from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by subjecting the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column comprising immobilized cyano groups and eluting the bound polysorbate using a mobile phase composition gradient; and (iv) detecting the separated polysorbate using a chromophore-free detector and identifying the polysorbate. a process comprising: (b) identifying the protein sample from step (a) having a concentration of intact polysorbate, e.g., PS80, of about 10 ppm to about 5000 ppm; and (c) isolating and recovering the protein identified in step (b). The present invention provides a method comprising:

[0008] Further provided is a protein obtainable or obtained by the method of the second aspect of the invention, and also the use of said protein in medicine, for example the use of said protein in the preparation of a pharmaceutical formulation for administration to a human subject.

[0009] In certain embodiments of the first and second aspects of the invention, methods are provided for identifying polysorbate 80 (e.g., intact and / or degraded PS80 polysorbate) in a protein-containing sample (e.g., an antibody sample such as a mAb).

[0010] The precipitation step and the separation step combined with elution allow for the separation of polysorbate products in the sample, and the detection step allows for the detection and analysis of intact polysorbate and / or polysorbate degradation products, such as PS80 and / or PS60 and / or PS40 and / or PS20.

[0011] In certain embodiments of the first and second aspects of the invention, the method for identifying polysorbates in a sample containing a protein or peptide (e.g., an antibody such as an mAb sample provided herein) is a quantitative method that can be used to measure the amount of polysorbate, such as PS80 and / or PS60 and / or PS40 and / or PS20, present in a sample, including measurement of intact polysorbate and / or polysorbate degradation products (e.g., of PS80 and / or PS60 and / or PS40 and / or PS20). [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the final two steps of the synthetic route for PS80. [Figure 2] FIG. 2 shows the degradation products of the two most common types of degradation in polysorbates. [Figure 3] FIG. 3 shows chromatograms obtained using the HPLC-CAD method to quantify PS80 monoesters and qualitatively / semi-quantitatively monitor the other four groups of subspecies. [Figure 4] FIG. 4 shows chromatograms for various sources of PS80 (solid lines) and a blank (dotted line). [Figure 5] FIG. 5 shows chromatograms for various polysorbates (solid lines) and a blank (dotted line). [Figure 6] FIG. 6 shows the chromatographic profile of PS80 monoester obtained using the HPLC-CAD method. [Figure 7] FIG. 7 shows the kinetics of PS80 degradation in samples at 5° C., 25° C., 40° C., or −70° C. for up to 21 days. [Figure 8] FIG. 8 shows an Arrhenius plot of the rate constants (at 5° C., 25° C., and 40° C.) for the decomposition of PS80 monoester. [Figure 9] FIG. 9 shows an overlay of chromatograms of a 200 ppm standard solution (PS80 from JT Baker, which is listed in multiple compendia), a mAb sample containing degraded PS80, and a blank solution. DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention has been described herein with reference to embodiments in a manner that makes it possible to write a clear and concise specification, which embodiments are intended, and should be so construed, to be capable of being variously combined or separated without departing from the invention.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). For quantitative analysis, standard techniques are used.

[0015] All publications (including but not limited to patents and patent applications) cited herein are hereby incorporated by reference as if fully set forth.

[0016] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of amino acid residues. Polypeptides can be of natural (tissue-derived) origin, recombinant or naturally expressed from prokaryotic or eukaryotic cell preparations, or chemically produced by synthetic methods. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid mimetic refers to a chemical compound that has a structure that differs from the general chemical structure of an amino acid but functions in a manner similar to a naturally occurring amino acid. Non-naturally occurring residues are well described in the scientific and patent literature, and some exemplary non-naturally occurring moieties useful as mimetics of natural amino acid residues and as guidelines are provided below. Aromatic amino acid mimetics include, for example, D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2 thienylalanine; D- or L-1, 2-, 3-, or 4-pyreneylalanine; D- or L-3 thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(triamino)-alanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylalanines, where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isotyl, isopentyl, or a non-acidic amino acid.Aromatic rings of non-neutral amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0017] The term "antigen-binding polypeptide" as used herein refers to antibodies, antibody fragments, and other protein constructs capable of binding to an antigen.

[0018] The term "antibody" is used herein in the broadest sense to refer to molecules having immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD, or IgE), and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies (including bispecific antibodies), and heteroconjugate antibodies; single variable domains (e.g., domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab'), Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., as well as modified forms of any of the foregoing (for a review of alternative "antibody" forms, see Holliger and Hudson, Nature Biotechnology, 2005, Vol. 23, No. 9, 1126-1136). Alternative antibody formats are also contemplated, including alternative scaffolds in which one or more CDRs of an antigen binding protein may be placed on a suitable non-immunoglobulin protein scaffold or framework (e.g., an affibody, an SpA scaffold, an LDL receptor class A domain, an avimer, or an EGF domain).

[0019] The terms full-length antibody, whole antibody, or intact antibody, used interchangeably herein, refer to a heterotetrameric glycoprotein with a molecular weight of approximately 150,000 daltons. Intact antibodies are composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This H2L2 structure folds to form three functional domains, including two antigen-binding fragments (known as 'Fab' fragments) and an 'Fc' crystallizable fragment. The Fab fragment consists of an amino-terminal variable domain (variable heavy (VH) or variable light (VL)) and a carboxyl-terminal constant domain (CH1 (heavy) and CL (light)). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. The Fc can trigger effector functions by binding to receptors on immune cells or by binding C1q (the first component of the classical complement pathway). The five classes of antibodies (IgM, IgA, IgG, IgE, and IgD) are defined by different heavy chain amino acid sequences, called μ, α, γ, ε, and δ, respectively, and each heavy chain can be paired with either a κ or λ light chain. The majority of antibodies in serum belong to the IgG class, and human IgG exists in four isotypes: IgG1, IgG2, IgG3, and IgG4, whose sequences differ mainly in the hinge region.

[0020] As used herein, a "fragment," when used in reference to a protein or polypeptide, is a protein or polypeptide having an amino acid sequence that is the same as some, but not all, of the amino acid sequence of the entire naturally occurring protein / polypeptide. A fragment may be "free-standing" or may be contained within a larger protein or polypeptide (forming as a single, contiguous region of that single, larger protein / polypeptide, part or region thereof).

[0021] The term "single variable domain" refers to a folded polypeptide domain comprising a sequence typical of an antibody variable domain. "Single variable domain" therefore includes complete antibody variable domains such as VH, VHH, and VL, as well as modified antibody variable domains (e.g., in which one or more loops are replaced by sequences not typical of antibody variable domains), or antibody variable domains that are truncated or contain N- or C-terminal extensions, and folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain. A single variable domain as defined herein is capable of binding to an antigen or epitope independently of the antigen or epitope of a different variable region or domain. A "domain antibody" or "DAB" may be considered the same as a human "single variable domain." Single variable domains can be human single variable domains, but also include single variable domains from other species, such as rodent (e.g., rodents as described in WO 00 / 29004), nurse shark, and camelid VHHs. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species including camels, llamas, alpacas, dromedaries, and guanacos, which produce heavy chain-only antibodies naturally devoid of light chains. Such VHH domains can be humanized by standard techniques available in the art, and such domains are considered to be "single variable domains."

[0022] As used herein, the term "polysorbate" refers to a common intact polysorbate selected from polysorbate 80 (PS80), polysorbate 60 (PS60), polysorbate 40 (PS40), and polysorbate 20 (PS20), as well as any one (or all) of their degradation products. Intact polysorbate refers to polysorbate when it exists as a monoester. Degradation products of polysorbates that can be detected by the method of the present invention include long-chain fatty acids (e.g., palmitic acid, linoleic acid, oleic acid), polyoxyethylene (POE) groups containing POE esters of fatty acids, and short-chain fatty acids. Figure 2 shows the degradation products of the two most common types of degradation in polysorbates.

[0023] Polysorbates are commonly used as nonionic surfactants in both food and biopharmaceutical products. In biopharmaceutical products, polysorbates are used to prevent protein adsorption to surfaces, aggregates, and particle formulations. However, it is known that intact polysorbates can be problematic when decomposed. For example, polysorbate degradation products can cause irritation in pharmaceutical product injections and also lead to excessive turbidity in samples (e.g., pharmaceutical samples). An amount of intact polysorbates of about 10 ppm to about 5000 ppm is generally considered to be the desired amount in pharmaceutical products.

[0024] There is considerable interest in developing methods to monitor polysorbates, including their integrity, particularly polysorbate 80 (polyoxyethylene sorbitan monooleate or Tween™ 80), which is the most commonly used polysorbate. Commercially available PS80 is heterogeneous, with the most common process-related subspecies being polyoxyethylene (POE) groups, POE isosorbide monoesters, and POE sorbitan / isosorbide di-, tri-, and tetraesters. Figure 1 shows the final two steps of the synthetic route for polysorbate 80 (PS80).

[0025] However, developing analytical methods to detect not only intact polysorbates such as PS80, but also their degradation products, such as PS60, PS40, and PS20, is challenging, and there is a need for such methods, particularly those applicable to protein-containing biopharmaceutical formulations. Furthermore, approval of a protein material as pharmaceutically acceptable by agencies such as the FDA depends on the protein material containing predetermined levels of polysorbates, including certain polysorbate products.

[0026] The present invention therefore provides methods that allow for the identification of such polysorbates (e.g., intact polysorbates and / or polysorbate degradation products) in pharmaceutical formulations, such as biopharmaceutical formulations, that contain proteins.

[0027] The present invention also provides a method for measuring polysorbate in biopharmaceutical preparations containing proteins or peptides. The term "measurement" of polysorbate, as used herein, refers to the identification and even quantification of such polysorbate. The measured polysorbate may be intact polysorbate and / or polysorbate degradation products. The method provided herein is accurate, can be performed in a similar time to conventional HPLC, and is advantageous because it does not rely on the use of derivatization or micellar encapsulation, which can be problematic. Derivatization or micellar encapsulation can increase the complexity of sample preparation, depend on equilibrium kinetics (which can adversely affect accuracy), and may utilize additional components that adversely affect the signal-to-noise ratio relative to the matrix.

[0028] Therefore, in a first aspect, the present invention provides a method for identifying polysorbates (e.g., intact polysorbates and / or polysorbate degradation products) in a protein-containing sample (e.g., an antibody, such as a mAb sample), comprising subjecting said sample to the following steps: (i) precipitating the protein by exposing the sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) separating the proteins or peptides from the precipitated sample by centrifuging the precipitated sample to pellet the proteins or peptides, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by subjecting the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column comprising immobilized cyano groups and eluting the bound polysorbate using a mobile phase composition gradient; and (iv) detecting the separated polysorbate using a chromophore-free detector and identifying the polysorbate. The method includes subjecting the

[0029] The precipitation and separation steps combined with elution allow for the separation of polysorbate products (e.g., intact polysorbate and polysorbate degradation products) in the sample, and the detection step allows for the detection, identification, and quantification of polysorbate products (e.g., intact polysorbate and polysorbate degradation products, e.g., intact PS80 and / or PS60 and / or PS40 and / or PS20 and their degradation products). In one embodiment, the method for measuring intact polysorbate in a protein-containing sample (e.g., an antibody, such as a mAb sample provided herein) is a quantitative method that allows for the determination of the amount of intact polysorbate and / or polysorbate degradation products, such as PS80 and / or PS60 and / or PS40 and / or PS20, present in the sample.

[0030] In certain embodiments, the methods of the present invention can be used to monitor the degradation of intact polysorbate in samples such as protein-containing samples (e.g., antibodies such as mAb samples, or cells, or protein containing vectors expressing heterologous therapeutic genes), e.g., over time to assess the stability of such protein-containing samples.

[0031] The present invention also provides the use of the method to measure the amount of intact polysorbate in a protein-containing sample (e.g., to measure the amount of intact PS80 and / or intact PS60 and / or intact PS40 and / or intact PS20 present in such a sample).

[0032] In a second aspect, the present invention provides a method for identifying a protein sample, e.g., from a plurality of proteins, comprising: the identified protein sample contains about 10 ppm to about 5000 ppm of intact polysorbate; The method comprises the steps of: (a) measuring polysorbates in said protein sample, said step comprising the steps of: (i) precipitating the protein by exposing the sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) separating the protein or peptide from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by subjecting the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column comprising immobilized cyano groups and eluting the bound polysorbate using a mobile phase composition gradient; and (iv) detecting the separated polysorbate using a chromophore-free detector and identifying the polysorbate; a process comprising: (b) identifying the protein sample from step (a) having an intact polysorbate concentration of about 10 ppm to about 5000 ppm; and (c) isolating and recovering the protein identified in step (b). The present invention provides a method comprising:

[0033] In one embodiment of the second aspect of the invention, the intact polysorbate is PS80 and / or PS60 and / or PS40 and / or PS20.

[0034] Further provided is a protein obtained or obtainable by the method of the second aspect of the invention, and also the use of said protein in medicine, for example the use of said protein in the preparation of a pharmaceutical formulation for administration to a human subject.

[0035] The present invention also provides a protein (e.g., an antibody) obtainable or obtained by the method of the second aspect of the present invention (said protein containing intact polysorbate present at about 10 ppm to about 4000 ppm, or about 10 ppm to about 3000 ppm, or about 10 ppm to about 2000 ppm, or about 10 ppm to about 800 ppm, or about 10 ppm to about 700 ppm), and further provides the use of said protein in medicine, for example in a pharmaceutical formulation for administration to a human subject.

[0036] In embodiments in which the protein is an antibody for use in cell therapy, or cells, or a protein containing a vector expressing a heterologous therapeutic gene, the amount of intact polysorbate present in the protein is about 10 ppm to about 700 ppm. The concentration of the protein present in the sample, and to which the method of the present invention can be applied, can be about 5 mg / mL to about 300 mg / mL, about 5 mg / mL to about 200 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 20 mg / mL, about 5 mg / mL to about 10 mg / mL, about 10 mg / mL to about 20 mg / mL, about 15 mg / mL to about 50 mg / mL, or about 20 mg / mL to about 50 mg / mL.

[0037] The methods of the present invention can be applied to any natural or recombinant protein. Protein samples can include, for example, therapeutic, prophylactic, or diagnostic proteins. For example, the methods can be applied to samples containing antigen-binding constructs (e.g., antibodies or antibody fragments (e.g., biologically functional fragments of antibodies)), and the methods can also be applied to vaccine compositions, cells, or protein-containing vectors expressing heterologous therapeutic genes.

[0038] If the protein sample is an antibody, the antibody can be, for example, a monoclonal antibody (mAb) or a bispecific or multispecific antibody, or a fragment thereof. The antibody can be a chimeric, humanized, or human antibody. If the protein is an antibody fragment, the antibody fragment can be, for example, Fab, F(ab'), Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabody, TANDABS™, antibody CDR, and modified forms of any of the foregoing.

[0039] Antibody fragments may also be single variable domains (or dABs), such as human VH or VL single variable domains, or single variable domains of non-human origin, such as llama or camelid (e.g., camelid VHHs, including Nanobodies™ (described, inter alia, in WO94 / 04678 and WO95 / 04079)). The use of the CDRs of any of these antibodies or single variable domains (e.g., as part of a protein scaffold) is also contemplated.

[0040] The protein sample for use in the method of the present invention can be in the form of a liquid or suspension in an aqueous medium, or can be, for example, lyophilized and then reconstituted in an aqueous medium.In addition to protein and water, the protein sample can further contain an additional diluent (e.g., a pharmaceutically acceptable diluent).Such pharmaceutically acceptable diluents include, for example, solvents such as water, sodium chloride solution, sugars, buffers such as acetic acid, salts such as sodium chloride, and / or other excipients.In one embodiment, the buffer can be an acetic acid or citrate buffer.

[0041] The methods of the invention are particularly useful for detecting polysorbates (e.g., intact polysorbates) and degraded species of polysorbates (e.g., PS80 and / or PS60 and / or PS40 and / or PS20) in protein containing liquid samples, such as liquid biopharmaceutical formulations (e.g., mAb formulations).

[0042] The methods of the present invention may also be applied to samples containing oligonucleotides, engineered cells for cell therapy, and also to gene therapy products such as engineered vectors (e.g., viral vectors) containing therapeutic genes for administration to human subjects.

[0043] The methods of the present invention can also be used to measure polysorbate in samples containing small molecules that are chemical entities (NCEs), where such NCE samples do not contain protein, thus protein precipitation can be omitted, and the amount of organic protic polar solvent or organic aprotic polar solvent can be adjusted.

[0044] The method of the invention can be carried out over a wide pH range, for example, from about pH 5 to about pH 10, or from about pH 6 to about pH 8, since the pH value is not critical to the practice of the method. Protein samples analyzed according to the method of the invention can have a pH of from about pH 6.0 to about pH 8.0, for example, a pH of from about 7.4 to about 6.8.

[0045] The organic protic polar solvent used in protein precipitation step is well known in the art, and this term as used herein refers to the organic solvent that contains protons that are susceptible to chemical change and can be ionized.The examples of such solvents that can be used in the method of the present invention are well known to those skilled in the art, and include, for example, methanol, ethanol, and isopropyl alcohol (IPA).For example, when protein is an antibody, methanol, IPA, or acetone can be used in protein precipitation step.

[0046] The organic aprotic polar solvents used in the protein precipitation process are well known in the art, and as used herein, this term refers to an organic solvent that does not contain a proton that is chemically labile. Examples of such solvents that can be used in the method of the present invention are well known to those skilled in the art, and include, for example, acetone, tetrahydrofuran (THF), and acetonitrile.

[0047] This method can be performed over a wide range of solvent concentrations; when the method is performed on a sample containing an antibody, the volume / volume dilution can be about 1 part sample to about 5 parts, 9 parts, or about 19 parts solvent.

[0048] The centrifugation step can be carried out at a speed and for a time sufficient to obtain a protein pellet, for example, at least about 10,000 rpm for at least about 10 minutes.

[0049] The separation step can be carried out using column chromatography, for example, using reversed-phase media or mixed-mode retention chromatography, which involves the combined use of two or more retention mechanisms, for example, normal phase, cation exchange, and anion exchange.

[0050] In certain embodiments, the separation methods of the present invention can be carried out on a reverse-phase chromatography column using methods known to those of skill in the art, where the column contains groups having carbon chains of C3 or longer, e.g., C4 to about C18.

[0051] In one embodiment, a column with immobilized cyano groups (e.g., a reverse-phase chromatography column with immobilized cyano groups on the stationary phase) is used. Cyano groups are well known in the art and are any chemicals containing the group -CN. Any cyano group can be used in the method of the present invention. Columns with CN groups that can be usefully used in accordance with the method of the present invention include Agilent Zorbax SB300-CN, Agilent Zorbax SB300-CN, Phenomenex Luna CN, or Agilent InfinityLab Poroshell 120 EC-CN.

[0052] The column can be a silica bead column (e.g., with a pore size of about 80 angstroms or greater). In certain embodiments, the pore size is about 120 to about 300 angstroms. For example, a pore size of about 300 angstroms can be used. Suitable silica columns include, for example, Agilent Zorbax SB300-CN, Phenomenex CN, or Agilent InfinityLab Poroshell 120 EC-CN. In one embodiment, the column used is Agilent Zorbax SB300-CN, 3.5 μm, 150 × 4.6 mm (available from Agilent Co., Santa Clara, CA, USA). The column can be heated; for example, the column temperature can be about 20°C to about 80°C, or about 40°C to about 60°C, or about 50°C.

[0053] In certain embodiments, the elution step is performed using a gradient separation mobile phase, which may be, for example, a gradient separation mobile phase of A and B. In one embodiment, a gradient separation mobile phase of A and B is utilized, where A is a 0.1% to about 10% H2O mixture of an acid or ammonium acetate, which may be selected from trifluoroacetic acid (TFA), formic acid, acetic acid, and difluoroacetic acid, and B may be methanol, isopropanol, or acetonitrile. In certain embodiments, a gradient separation mobile phase of A and B is utilized, which is a 0.1% TFA H2O mixture, and B is methanol or acetonitrile. The gradient separation mobile phase of A and B may be achieved as detailed in Table 1 below.

[0054] [Table 1]

[0055] The detectors used in the methods of the present invention are chromophore-free detectors, that is, detectors that function when the sample to be detected lacks a chromophore.

[0056] In certain embodiments, the detector used in the methods of the present invention may be an evaporative light scattering detector, or mass spectrometry may be used for detection.

[0057] In another embodiment, a charged particle detector (CAD) is used as a detector in the method of the present invention. This is a detector used, for example, in combination with high-performance liquid chromatography (HPLC). It functions by charging nonvolatile and semivolatile analytes with nitrogen gas charged with a high-voltage corona wire. The charged analyte particles then pass through an ion trap that removes high-mobility species (i.e., solvent) and subsequently move to a collector, where they are measured by a highly sensitive electrometer. CADs that can be used include Corona Veo (available from Thermo Waltham, MA, USA), Corona Veo RS (available from Thermo Waltham, MA, USA), Vanquish (available from Waltham, MA, USA), Corona Ultra and Ultra RS (available from Thermo Waltham, MA, USA), and Corona Plus (available from Thermo Waltham, MA, USA).

[0058] One feature that CAD offers is the ability to measure intact species by charging the surface of the analyte, unlike mass spectrometry, which produces charged fragments. Furthermore, responses are similar for analytes with similar surface areas and densities. Finally, when using highly volatile eluents, CAD methods can also be very sensitive (less than 1 nanogram).

[0059] Although CAD is easy to operate, there are additional considerations in developing a standard HPLC-UV / Vis (ultraviolet-visible spectroscopy) analytical method. These include (1) selecting a column that does not drop out; (2) using high-purity solvents in the mobile phase for a low, reproducible baseline; and (3) cleaning glassware and plastics because of the greater potential for interference from contaminants. Achieving high specificity is important when using CAD, since there is no way to distinguish peak purity when using diode array or mass spectrometer (MS) detection. It should also be noted that if specificity cannot be achieved, the observed signal is not simply the sum of responses, as is the case in UV-Vis spectrophotometry; differences in response are often complicated by differences in charge, surface area, density, and voltage of the analyte relative to the components of the mobile phase. For these reasons, CAD is well suited as a detector for the analysis of PS80.

[0060] In one embodiment, the charged particle detector (CAD) used in the methods described herein is a Corona Veo RS (available from Thermo, Waltham, MA, USA).

[0061] The detection step performed using CAD results in a chromatogram, the baseline of which is obtained using a selected blank solution, containing peak areas for intact polysorbate, degradation products, and proteins and excipients in the sample. Evaluation of the peak areas using area under the curve calculation allows for quantification of polysorbates such as PS80, and / or PS60, and / or PS40, and / or PS20, and their degradation products. In one embodiment, this method allows for the identification of PS80, for example, intact PS80 and degraded PS80.

[0062] When referring to separation using the method of the present invention, what is meant is that the intact polysorbate peak (i.e., monoester) must be separated from the oleic acid peak. The resolution between the oleic acid peak and the intact polysorbate monoester peak is 1.5 or greater. Other peaks must be simply distinguishable from one another. Furthermore, in certain embodiments, there is a specificity requirement that there be no interfering peaks at the retention time of the intact polysorbate (i.e., monoester) that account for more than about 3% by area. In certain embodiments, the present invention provides a method for identifying polysorbates in a sample containing a protein (e.g., an antibody sample), comprising the following steps: (i) precipitating the proteins by exposing the sample to methanol or IPA; (ii) separating the protein or peptide from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by applying the supernatant to reverse-phase HPLC on a silica column having a pore size of about 300 angstroms and having immobilized cyano groups, and eluting with a mobile phase composition gradient consisting of A and B, where A is a mixture of 0.1% trifluoroacetic acid (TFA) in HO, and B is methanol or acetonitrile; and (iv) detecting the separated polysorbate products using a charged particle detector (CAD) and identifying the polysorbates. The present invention provides a method comprising: [Example]

[0063] The present invention will be further described with reference to the following examples, which are intended to illustrate various aspects of the invention only and are not intended to limit the invention.

[0064] Example 1: Comparison of the determination of PS80 and its subspecies present in a mAb pharmaceutical product by either (i) a new HPLC-CAD analysis by the method of the present invention, capable of quantifying PS80 monoesters, and (ii) a modified HPLC method using evaporative light scattering detection—HPLC-ELSD method.

[0065] The reagents and methods used are as follows. JT Baker's PS80, which is listed in multiple compendial publications, was purchased from Fisher Scientific (Atlanta, GA, USA, 02-003-654). Two sources of PS80 were purchased from Sigma-Aldrich (St. Louis, MO, USA): (1) PS80 stored in natural-colored plastic containers (part number P1754-25ML) and (2) PS80 stored in amber glass containers (part number 59925-100G). Highly purified PS80 was purchased from Croda Health Care (Edison, NJ, USA, SR48833). All-oleate ChP-compatible PS80 was purchased from NOF (White Plains, NY, USA) and was non-GMP PS80, POLO80(HX2) (19B803364). Polysorbate 60 was purchased from USP Reference Standard (Rockville, MD, USA, 154794). Polysorbate 40 was purchased from Fisher Scientific (Atlanta, GA, USA, AC334142500). Oleic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA, 75090-5ML). Linoleic acid was purchased from Fisher Scientific (Atlanta, GA, USA, AC215040250). Palmitic acid was purchased from MP Biomedicals (Santa Ana, CA, USA, 100905-10G). Palmitoleic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA, 76169). Chromatography (LC-MS or GC) grade methanol was purchased from Fisher Scientific (Atlanta, GA, USA, A456-4) or VWR (Honeywell / Burdick & Jackson, GC grade, purity ≥99.9%, BJGC 230-4). Ultrapure water (Milli-Q water) was produced using a Milli-Q water purification system (Millipore Corporation, Burlington, MA, USA). Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (St. Louis, MO, USA, 91707-10x1mL).Other precipitation solvents (isopropanol, acetone, tetrahydrofuran (THF)) were chromatographic grade and purchased from Sigma-Aldrich. For the HPLC-ELSD method, HPLC-grade methanol (646377-4L) and acetonitrile (439134-4L) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Honeywell Fluka formic acid (94318-250ML) was purchased from Fisher Scientific (Atlanta, GA, USA, AC334142500).

[0066] The instruments and analytical conditions used are as follows.

[0067] Protein precipitation and PS80 extraction The new HPLC-CAD analysis method was performed as follows. To remove the protein [i.e., mAb drug product] prior to injection, the protein was precipitated with a precipitation solvent (methanol, isopropanol, and / or acetone). Additionally, precipitation was utilized to disrupt any potential protein-PS80 interactions and to inhibit any degradation caused by lipases or esterases. Filtration was not a viable option because it would remove some polysorbate species. Therefore, 900 μL of the precipitation solvent was added to 100 μL of sample in a 1.5 mL Eppendorf safelock tube (Hauppauge, NY, USA, 022363204) that had been previously rinsed (with methanol or precipitation solvent). The sample preparation was then vortexed briefly (approximately 5 seconds) and centrifuged at 14,000 rpm for 10 minutes. PS80 species and fatty acids remained soluble in the supernatant. A minimum of 60 μL of the supernatant was transferred to an HPLC vial with a 300 μL insert.

[0068] A 1,000 ppm PS80 stock standard solution was prepared by weighing 100 ± 10 mg of multi-compendial JT Baker PS80 into a 100 mL Class A volumetric flask and diluting to volume with methanol. A 20 ppm PS80 working standard solution was prepared by vortexing 100 μL of MilliQ water, 20 μL of the 1,000 ppm PS80 stock standard, and 880 μL of methanol in a 1.5 mL Eppendorf tube previously rinsed (with precipitation solvent). Thus, the final diluent composition for the standard (90% precipitation solvent: 10% MilliQ HO / aqueous) was the same as that for the samples. PS60, PS40, and PS20 solutions were prepared similarly.

[0069] A resolution check solution containing 20 ppm PS80 and 5 ppm oleic acid stock standard solution was prepared. A sensitivity solution was prepared by mixing 898 μL of organic solvent, 100 μL of water, and 2 μL of the 1,000 ppm PS80 stock standard solution. The mAb formulation buffer was prepared in bulk, aliquoted, and stored at -70°C until the day of analysis. The 20 ppm PS80 formulation buffer preparation was made by dilution with LC-MS or GC-grade methanol. Because the sample preparations were protein-free, no centrifugation step was required.

[0070] For thermally stressed samples, multiple vials of mAb / protein-containing formulations were incubated at -70°C, 5°C, 25°C, and 40°C for 3 weeks, and at each time point (initiation, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, and 21 days), vials were removed from the oven and frozen at -70°C until the time of analysis.

[0071] The modified HPLC-ELSD analytical method was performed as follows. For suspected PS80-protein interactions and to stop any enzymatic degradation, proteins were precipitated with methanol instead of dilution with water. To precipitate proteins and extract PS80, 800 μL of organic solvent was added to 200 μL of sample in a 1.5 mL microcentrifuge tube and vortexed to mix. After mixing, the sample was centrifuged at 10,000 rpm at 5°C for 30 minutes. After centrifugation, 200 μL of supernatant was transferred to an HPLC vial with a 300 μL insert.

[0072] Quantification of the PS80 content in the samples was achieved by constructing a calibration curve. Due to the characteristics of the HPLC-ELSD detector, the matrix of the standard curve must be representative of the sample. To achieve a representative matrix, 500 mg of multi-compendial JT Baker PS80 was weighed into a 50.0 mL low-actinic Class A volumetric flask and diluted to volume with HPLC-grade methanol to prepare a 10,000 ppm PS80 stock solution. Additionally, 0.5 mL of the 10,000 ppm PS80 stock solution was then added to a 10.0 mL volumetric flask and brought to volume with HPLC-grade methanol to prepare a 500 ppm stock solution. The 500 ppm stock solution was used to prepare calibration standards in a methanol / water solution (80:20 (v / v)) with expected PS80 concentrations of 10 ppm, 25 ppm, 50 ppm, 100 ppm, 150 ppm, 200 ppm, and 250 ppm. A 20 ppm PS80 preparation was made by dilution with HPLC-grade methanol. Because the preparation was protein-free, no centrifugation step was performed. A sample chromatogram is shown in Figure 9. In summary, Figure 9 shows an overlay of chromatograms collected by the modified HPLC-ELSD method: a 200 ppm standard solution (PS80 from JT Baker, which is listed in multiple compendia), a mAb sample containing degraded PS80, and a blank solution. In Figure 9, the 200 ppm standard has the largest peak, and the -20°C sample has an intermediate, smaller peak.

[0073] New HPLC-CAD method: The Agilent HPLC 1260 system (Santa Clara, CA, USA) contained a binary solvent manager, a sample manager set to 23 °C, a column oven set to 50 °C, and a charged aerosol detector (CAD) Veo RS (Thermo, Waltham, MA, USA). The CAD was directly connected to the analytical column by 80 cm of tubing (Agilent, 01078-87305), and the analytical column was directly connected to a 3 μL Peltier element by 180 mm of tubing (Agilent, G1313-87305). The HPLC column heater was directly connected to the HPLC autosampler by standard tubing, and the ultraviolet-visible spectrophotometer (UV / Vis) and column switching valve were both bypassed.

[0074] The analytical column was a Zorbax SB300-CN (150 mm × 4.6 mm, 3.5 μm 300 Å, 863973-905) from Agilent Technologies (Wilmington, DE, USA). A volatile mobile phase (MP) consisting of 0.1% (v / v) TFA in MilliQ water (MP A) and 100% LC-MS or GC-grade methanol (MP B) was used. To further ensure cleanliness and baseline CAD levels below 10 mV at the parameters listed above, the mobile phase was prescreened by running 35% MP A:65% MP B at 1.2 mL / min. Separation of the PS80 subspecies was achieved by gradient elution at a flow rate of 1.2 mL / min (0.1% TFA in MilliQ water: 0 min - 100%; 1 min - 100%; 3 min - 50%; 8 min - 5%; 27 min - 5%; 30 min - 5%; and 30.1 min - 100%). The overall run time of the method was 40 min. The injection volume was 30.0 μL. The Thermo Veo RS CAD was operated with the following settings: evaporation temperature, 60 °C; power function, 1.00; output offset, 0%; filter, 5.0 s; and range, 100 pA. An in-house nitrogen supply was used. The CAD analog signal was converted to a digital signal using an e-SAT / IN module (Waters, Milford, MA, USA, 668000230).

[0075] Modified HPLC-ELSD method: The present method was modified from the method of Hewitt and Koppolu. The Agilent HPLC 1100 system (Santa Clara, CA, USA) contained a binary solvent manager, a sample manager set at 25°C, a column oven set at 30°C, and an Agilent 1260 Infinity G4260B evaporative light scattering detector (ELSD, Agilent Technologies, Wilmington, DE, USA). The ELSD was directly connected to the analytical column, which was directly connected to a 3 μL Peltier element. The HPLC column heater was directly connected to the HPLC autosampler via standard plumbing, and the ultraviolet-visible spectrophotometer (UV / Vis) and column switching valve were both bypassed.

[0076] The analytical column was an Oasis® MAX (20 mm × 2.1 mm, 30 μm 80 Å, part number 186002052) from Waters Corporation (Milford, MA, USA). A volatile mobile phase consisting of 2% (v / v) formic acid in MilliQ water and 2% (v / v) formic acid in isopropanol was used. Flow bypassed the ELSD for the first 4 minutes of the run, and separation was achieved by gradient elution at a flow rate of 1.0 mL / min (2% formic acid in MilliQ water: 0 min - 90%; 1 min - 80%; 3.4 min - 80%; 3.5 min - 0%; 4.5 min - 0%; 4.6 min - 90%; and 10 min - 90%). The injection volume was 50.0 μL. The Agilent 1260 Infinity G4260B ELSD was operated with the following settings: LED, 10; gain (PMT), 2; smooth (Smth), 1; data output, 80 Hz; vapor temperature, 80 °C; nebulizer temperature, 50 °C; gas flow (SLM), 1. An in-house nitrogen supply was used. CAD analog signals were converted to digital signals using an e-SAT / IN module (Waters, Milford, MA, USA, 668000230).

[0077] For the new HPLC-CAD analytical data analysis, integration and calculations were performed as follows: The average PS80 monoester concentration of triplicate preparations was reported. To quantify all esters (monoester and multiester) for comparison with the modified HPLC-ELSD method, a calibration curve for all esters was generated by grouping the areas of the PS80 monoester and multiester in a linearity setup. Thus, the total ester area in the HPLC-CAD method resembles a single peak in the modified HPLC-ELSD method; the POE group is not included in the single peak because it elutes during the first 4 minutes of each injection when the valve switches to waste.

[0078] Arrhenius kinetic modeling was used to assess the rate of PS80 decomposition and to estimate the stability or activation energy (E a ) was used to estimate the rate constants. The hydrolytic degradation of the PS80 monoester was assumed to be a pseudo-first-order reaction, as previously described. Rate constants were determined from the slope of the natural logarithm of the concentration versus time plot, assuming no influential changes due to changes in dynamic viscosity. For all linear plots, a relative error analysis of the slope was performed as previously described.

[0079]

number

[0080] where n is the number of data points, a is the slope, and b is the y-intercept.

[0081] For the modified HPLC-ELSD analytical method data analysis, integration and calculations were performed using Empower3, allowing for batch data processing to obtain retention times, peak areas, and other chromatographic performance indices. Similarly, for the new HPLC-CAD method data analysis, integration and calculations were performed using Empower3, allowing for batch data processing to obtain retention times, peak areas, resolution, S / N, and other chromatographic performance indices.

[0082] result: The new HPLC-CAD method described above was found to accurately and precisely quantify PS80 monoesters and qualitatively / semiquantitatively monitor the other four subgroups. For simplicity, we selected a concentration range where the CAD response is linear, even though it is nonlinear. The calibration curve can also be linearized by applying a power function algorithm; however, without baseline reproducibility, such an algorithm may not always be applicable.

[0083] Matrix interference was assessed by evaluating the recovery of spiked PS80 in (1) a PS80-free IgG drug product; and (2) a mAb sample containing a PS80 monoester that was completely degraded (<limit of quantitation (LOQ)) (see Table 2 and discussion below). The degraded sample contained protein in an aqueous buffer containing trehalose, methionine, arginine, histidine, mM EDTA, and PS80. The other sample contained protein in an aqueous buffer containing trehalose, citric acid, EDTA, and PS80. To assess specificity in the degraded samples, fatty acids (linoleic acid, palmitic acid, oleic acid, and palmitoleic acid) were also spiked at 5 ppm into a 20 ppm PS80 working standard solution (see Figure 3). In summary, Figure 3 shows a chromatogram obtained using the detailed HPLC-CAD method to quantify PS80 monoesters and qualitatively / semiquantitatively monitor the other four subgroups. The chromatogram shows an overlay of a blank (dashed line) and a 20 ppm PS80 (JT Baker, listed in multiple compendia) standard solution spiked with 10 ppm fatty acids (solid line). Peaks: 1 = unretained formulation buffer components (trehalose, amino acids, EDTA, salt impurities in solvents, and / or sample residues); 2 = POE groups; 3 = palmitoleic acid; 4 = linoleic acid; 5 = palmitic acid; 6 = oleic acid; 7 = PS80 monoesters (sorbitan and isosorbide); 8 = diesters; 9 = triesters; 10 = tetraesters; * (if present), contaminants from the Eppendorf tube. We believe that the identification of the PS80 peak is consistent with previously reported LC-MS results [17, 30], with the elution order of the analytes expected based on their relative hydrophobicity.

[0084] Validation of the method with mAb drug products has been completed.

[0085] The assay consisted of precision, linearity, accuracy, specificity, and LOQ (Table 2). The CAD response for each injection was used to calculate the mean concentration, standard deviation, and relative standard deviation. Precision was assessed by analysis of the average of triplicate preparations on two to three occasions. Precision was also assessed by reproducibility analysis of duplicate injections on five assay occasions, between two analyses, using formulation buffer (or assay control). Intermediate precision was determined by one analyst performing two independent assay occasions on one system and a second analyst performing three independent assay occasions on a second system.

[0086] [Table 2]

[0087] The mAb drug products were tested in triplicate and the results were statistically analyzed to determine the mean concentration, standard deviation, and relative standard deviation.

[0088] Linearity was assessed by two analysts on five independent assay replicates. The coefficient of determination (R 2 ) was determined by linear regression. Precision was determined using a spike-and-recovery approach. Two analysts introduced 20 ppm PS80 to samples without PS80 in the formulation in triplicate on five assay occasions. This preparation was also used to confirm specificity. Specificity was also assessed by the resolution of the PS80 monoester and oleic acid peaks in a resolution check solution and by ensuring the absence of interfering peaks within the elution window (±0.5 min) of the PS80 monoester in a 90% organic solvent / 10% water blank injection. Because the CAD is a versatile detector and may detect traces of contaminants, peak specificity is indicated by the absence of peaks with an area greater than 2% of that observed for the standard. The signal-to-noise ratio (S / N) was estimated to be ≥10 for the 2 ppm PS80 solution.

[0089] [Table 3]

[0090] Various origins and varieties of PS80 were tested with this method (Table 3). As shown in Figure 4, the chromatograms for each PS80 source were visually comparable. In summary, Figure 4 shows chromatograms for various origins of PS80 (solid lines) and a blank (dotted line): (A) all-oleate ChP-adapted PS80; (B) Sigma-Aldrich PS80 stored in amber glass containers; (C) Croda super-refined PS80; (D) Sigma-Aldrich PS80 stored in natural-colored plastic containers; and (E) multi-compendial JT Baker PS80.

[0091] Assuming that the PS80 monoesters in all-oleate PS80 and the multi-compendial JT Baker PS80 are equal, the peak area for all-oleate PS80 would be larger as a result of its slightly higher molecular weight. Some variation in subspecies between batches has been observed as a result of inconsistent synthetic routes for polysorbates. Physicochemical properties have been shown to vary from batch to batch. As evidenced by the alternate storage container for Sigma-Aldrich PS80, it is recommended that PS80 standard solutions be prepared from material derived from the same source and lot as the PS80 used in biopharmaceutical formulations.

[0092] Polysorbate 40 (PS40, polyoxyethylene (20) sorbitan monopalmitate) and polysorbate 60 (PS60, polyoxyethylene (20) sorbitan monostearate) were also evaluated (Figure 5), and in each chromatographic profile, the monoesters were separated from the multiesters. In summary, Figure 5 shows chromatograms for various polysorbates (solid lines) and a blank (dotted line): (A) polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate); (B) polysorbate 40 (PS40, polyoxyethylene (20) sorbitan monopalmitate); (C) polysorbate 60 (PS60, polyoxyethylene (20) sorbitan monostearate); and (D) polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In each chromatographic profile, the monoesters were separated from the multiesters. PS60 appears to contain two major monoester forms, or a significant amount of POE isosorbide monoester, and further experiments with mass spectrometry may be required to identify the identity of these peaks. Polysorbate 20 (PS20, polyoxyethylene (20) sorbitan monolaurate), which is listed in multiple compendials, was also included, but a complex chromatogram was obtained (SM Figure 7). Complex chromatograms with PS20 have been reported previously, and a simpler chromatogram was obtained by using total laurate PS20. PS60 appears to contain two major monoester forms, or a significant amount of POE isosorbide monoester, and further experiments with mass spectrometry may be required to identify the identity of these peaks.

[0093] Thus, this method has useful applications for PS20, PS40, and PS60.

[0094] To evaluate the accuracy and specificity of the method for the PS80 monoester, a complete monoester degradation sample was added (spiked) and analyzed using mAb. PS80 monoester degradation was achieved by incubating the sample at 5 °C for 36 months (Figure 6). Briefly, Figure 6 shows the chromatographic profile of the PS80 monoester (samples containing added (spiked) complete monoester degradation and analyzed using mAb) obtained using the HPLC-CAD method. PS80 monoester degradation was achieved by incubating the sample at 5 °C for 36 months. This figure shows an overlay of chromatograms collected by the new CAD method, indicating the broadening of the multi-ester peaks due to oxidative degradation in the mAb products stored at 5 °C and -20 °C for 36 months. Furthermore, nearly complete degradation of the monoester corresponded to an increase in the POE groups. The standard was PS80 listed in multiple official documents of J.T.Baker. The peak at 17.5 minutes was a variable contaminant from un-rinsed Eppendorf tubes. Analysis of the degraded samples confirmed that they contained no quantifiable amount of PS80 monoester (<LOQ). As expected, significant increases in the POE groups occurred due to the degradation of the PS80 monoester. To confirm whether subsequent degradation interfered with monoester quantification, a spike recovery approach was employed. 20 ppm PS80 was added (spiked) to the degraded sample preparation (this corresponds to a 200 ppm PS80 sample concentration). The PS80 monoester was recovered at a value of 93%, and it was confirmed that the significant increase in degradation products did not adversely affect the detection of the monoester. Furthermore, the multi-ester peak decreased slightly and showed peak broadening. The most likely cause could be the decomposition and reformation of oxidative degradation products with slightly different hydrophobicity and / or size after being involved in radically induced degradation.

[0095] Comparison of ELSD and CAD methods A series of samples was prepared and tested using both methods. Samples were formulation buffer and mAb product stored at 5°C and -20°C for 36 months, with or without one freeze-thaw (FT) cycle. The modified ELSD method incorporates a bypass during the first 4 minutes, so that POE groups and proteins do not pass through the detector, as expected from previously reported methods. A direct comparison of these two methods is summarized in Table 4 and shows good agreement.

[0096] [Table 4]

[0097] This method has been validated with IgG1, IgG2, and IgG4 mAbs (Table 5). During this experiment, it was found that some precipitation solvents (e.g., acetone, THF) resulted in low or insufficient recovery of PS80 monoester or subspecies in the formulation buffer (data not shown).

[0098] [Table 5]

[0099] Example 2: PS80 Kinetic Study: Concentration-time data were obtained by quantifying the amount of PS80 monoester and subspecies at each time point (initial, 1 day, 2 days, 4 days, 7 days, 14 days, and 21 days) using the new HPLC-CAD method (Figure 7). In summary, Figure 7 shows the kinetics of PS80 degradation in samples at 5°C, 25°C, 40°C, or -70°C for up to 21 days. (A) Monoester, (B) Multiester (di-, tri-, and tetraester), (C) POE groups, and (D) Total Mass Balance were quantified. Here, the PS80 monoester peak was truly quantitative, while the subspecies were semi-quantitative. Concentration-time data were obtained by quantifying the amount of PS80 monoester and subspecies at each time point (initial, 1 day, 2 days, 4 days, 7 days, 14 days, and 21 days) using the new HPLC-CAD method. For truly quantifiable PS80 monoester, an Arrhenius plot was compiled for the decomposition of PS80 monoester with rate constants for 5°C, 25°C, and 40°C data, resulting in an activation energy for PS80 monoester of 35.8 ± 7.2 kJ / mol; a linear least-squares fit yielded y = 4311.5x - 0.1696 (R 2 = 0.961) (Figure 8). The observed activation energy is similar to previously published observations of PS80 hydrolysis, as Kishore reported an activation energy of approximately 35 kJ / mol for the first 30% degradation of PS80. However, the analytical method employed did not have the specificity to distinguish between the monoester and multiester forms. In our study, degradation was most likely hydrolytic degradation due to the presence of lipase; therefore, in this study, the broadening of the multiester peak was very small; therefore, POE groups and multiesters were also quantified (Figure 7). Mass balance was calculated by summing the concentrations (ppm) of POE groups, PS80 monoesters, and multiesters, with an accuracy of <10% for all mass balance data. Negligible amounts of oleic acid were observed at 40°C.

[0100] The PS80 monoester in JT Baker's multi-compendial PS80 was significantly less stable than the multiester. This is consistent with previously published data. Although the monoester is significantly degraded, the remaining large amount of multiester may still provide protection from protein aggregation or colloidal stability.

[0101] Previously, suspected protein-PS80 interactions were thought to reduce the initial amount of PS80 measured in protein-containing pharmaceutical products. Interestingly, this method can also determine whether rapid degradation is occurring, as there will be a decrease in PS80 monoester concentration and a corresponding increase in POE groups. If there is no increase in POE groups, there is most likely no interaction with the protein or container.

[0102] Conclusion: A new, sensitive, and specific platform analytical method was developed for PS80 in biopharmaceutical formulations using HPLC-CAD. The method utilizes protein precipitation to mitigate potential interferences that would hinder specificity and to inhibit any active degradative enzymes (e.g., lipases and esterases). Specificity was demonstrated using PS40, PS60, and various strains of PS80. Application of the present invention with multiple strains of IgG mAb provides further support for the specificity that can be achieved by this method with fresh, significantly degraded pharmaceutical products.

[0103] The method was validated to demonstrate chromatographic specificity to monitor PS80 monoester, POE sorbitan / isosorbide, fatty acid, and multiester subspecies for degradation. Validation studies demonstrated that the method exhibited excellent reproducibility (2.2% RSD), intermediate precision (6.5% RSD), accuracy (101% recovery), linearity (average R 2 ≥ 0.999), specificity (no interfering peaks observed in the matrix and R sIt was concluded that the method performed satisfactorily with respect to the quantitation limit (≥ 1.5 oleic acids / PS80 monoester) and the limit of quantitation (approximately 20 ppm for the sample and 2 ppm for the protein-free sample). Studies of a severely degraded mAb drug product showed that the PS80 monoester was degraded below the LOQ, and acceptable recoveries (93%) could be achieved. It is noteworthy that the decrease in PS80 monoester was coupled with an increase in POE groups. The study of the severely degraded product also allowed for comparison of the specific method with established methods by quantifying all esterified PS species.

[0104] In conclusion, the analytical CAD method described above was shown to provide selective, sensitive, and specific quantitative and qualitative information about PS80 in biopharmaceutical products. The potential for this method to be used as a platform method for purposeful validation was demonstrated by utilizing modifications to the precipitation solvent using multiple subtypes of IgG mAbs (IgG1, IgG2, and IgG4). Therefore, this method is a useful tool for supporting stability testing for these mAbs and other biopharmaceutical products.

Claims

1. 1. A method for identifying polysorbates in a sample containing protein, comprising the steps of: (i) precipitating the protein by exposing the sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) separating the protein or peptide from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by subjecting the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column comprising immobilized cyano groups and eluting the bound polysorbate using a mobile phase composition gradient; and (iv) detecting the separated polysorbates using a chromophore-free detector and identifying the polysorbates; A method comprising:

2. 10. The method of claim 1, wherein the method identifies intact polysorbate and / or polysorbate degradation products.

3. 3. The method of claim 1 or 2, further comprising quantifying polysorbate.

4. The method of any one of claims 1 to 3, wherein the protein sample comprises an antibody.

5. The method according to any one of claims 1 to 4, wherein the protein is a monoclonal antibody or a fragment thereof.

6. 6. The method of any one of claims 1 to 5, wherein the method detects a polysorbate selected from any one of PS80, PS60, PS40 and PS20.

7. The method of any one of claims 1 to 6, wherein the chromophore-free detector is a charged particle detector (CAD).

8. The method of any one of claims 1 to 7, wherein the protein sample comprises an acetate or citrate buffer.

9. 9. The method of claim 1, wherein the protein in the sample is present at a concentration of about 5 mg / mL to about 300 mg / mL.

10. 10. The method of any one of claims 1 to 9, wherein the protein precipitation is carried out using a solvent selected from methanol, isopropyl alcohol (IPA), THF or acetone.

11. 11. The method according to any one of claims 1 to 10, wherein the separation of the polysorbates is carried out using a reversed-phase HPLC column equipped with immobilized cyano groups.

12. 12. The method of claim 11, wherein the column is a silica column having a pore size of ≥ 80 Angstroms.

13. The elution was carried out using a gradient separation mobile phase consisting of buffer A and buffer B, where buffer A was a 0.1% trifluoroacetic acid (TFA) solution. ２ 13. The method according to claim 1, wherein the buffer is a 0 mixture and buffer B is methanol or acetonitrile.

14. 14. The method of any one of claims 1 to 13, wherein the separation of the polysorbate is carried out using a heated column having a temperature of about 20°C to about 80°C.

15. The following steps: (i) precipitating the proteins by exposing the sample to methanol or IPA; (ii) separating the protein from the precipitated sample by centrifuging the precipitated sample to pellet the protein and obtain a liquid supernatant; (iii) separating the polysorbate by applying the supernatant to reverse-phase HPLC on a silica column having a pore size of about 300 angstroms and containing immobilized cyano groups, and eluting with a mobile phase gradient consisting of A and B, where A is H of 0.1% trifluoroacetic acid (TFA); ２ O mixture, and B is methanol or acetonitrile; and (iv) detecting the separated polysorbates using a charged particle detector (CAD) and identifying the polysorbates; The method of claim 1 , comprising:

16. 1. A method for identifying a protein sample, comprising: the protein sample being identified contains from about 10 ppm to about 5000 ppm intact polysorbate; The method comprises the steps of: (a) measuring polysorbate in said sample, said step comprising the steps of: (i) precipitating the protein by exposing the sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) separating the protein or peptide from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide, thereby obtaining a liquid supernatant; (iii) separating the polysorbate by subjecting the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column comprising immobilized cyano groups and eluting the bound polysorbate using a mobile phase composition gradient; and (iv) detecting the separated polysorbates using a chromophore-free detector and identifying the polysorbates; a process comprising: (b) identifying the protein sample from step (a) having a concentration of intact polysorbate of about 10 ppm to about 5000 ppm; and (c) isolating and recovering the protein identified in step (b). A method comprising:

17. 17. The method of claim 16, wherein the protein sample isolated in step (c) contains from about 10 ppm to about 700 ppm intact polysorbate.

18. 18. The method of claim 16 or 17, wherein the polysorbate is PS80.

19. The method of any one of claims 16 to 18, wherein the chromophore-free detector is a charged particle detector (CAD).

20. A protein or peptide polysorbate obtainable or obtained by the method according to any one of claims 16 to 19.

21. 21. The protein of claim 20, which is a monoclonal antibody or a fragment thereof.

22. 22. A protein according to claim 20 or 21 for use in medicine.

23. 22. Use of a protein according to claim 20 or 21 for the treatment or diagnosis of a human subject.

24. 22. Use of a protein according to claim 20 or 21 in the manufacture of a medicament for treating a disease in a human subject.

25. 22. A pharmaceutical formulation comprising a protein according to claim 20 or 21 in combination with one or more pharmaceutically acceptable excipients.

Images