Method for detecting polysorbate

Abstract

The present invention relates to providing a method for detecting polysorbate in pharmaceutical products.

Description

Technology Field

[0001] The present invention relates to providing a method for detecting polysorbate in pharmaceutical products. Background Technology

[0002] Polysorbates are non-ionic surfactants commonly used in both food and biopharmaceutical products. In biopharmaceutical products, they can be used to prevent protein adsorption, aggregation, and particle formation on surfaces. However, there are concerns that degradation products of these polysorbates may cause problems such as injection site irritation when used parenterally. Due to these uses, there is significant interest in developing analytical methods to monitor the integrity of polysorbates, specifically polysorbate 80 (polyoxyethylene sorbitan mono-oleate). Commercially available PS80 is heterogeneous, and the most common process-related subspecies are polyoxyethylene (POE) groups, POE isosorbide mono-esters, and POE sorbitan / isosorbide di-, tri-, and tetra-esters; therefore, the development of analytical methods has been challenging. Several methods have been reported.

[0003] None of these provide the following: (1) Specificity—the immunogenicity of the PS80 mono-ester peak enables quantification in degraded samples; (2) Sensitivity, ≤ 20 ppm; (3) Accuracy, 95–105%; (4) Precision, ≤ 5%; (5) Mobility, including quality control (QC); (6) Ability to validate with similar time and nearly the same cost as traditional UV-visible high-performance liquid chromatography (HPLC) methods; (7) Ability to monitor intact and degraded PS80 and related subspecies; (8) Ability to function as a platform method for protein-containing biopharmaceutical formulations; (9) Linearity, R 2>0.99; (10) degradation of fatty acids; (11) convertible mass spectrometry; (12) not using derivatization or / or (13) not using quantification dependent on micelle encapsulation.

[0004] Therefore, there is a need to provide an improved polysorbate detection method capable of resolving the aforementioned defects and / or detecting intact polysorbate and / or degraded polysorbate products.

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

[0006] Therefore, in the first aspect of the present invention,

[0007] (i) a step of precipitating the protein by exposing a sample containing the protein to an organic protic polar solvent or an organic aprotic polar solvent,

[0008] (ii) a step of separating proteins from a precipitated sample by centrifuging the precipitated sample to pellet the proteins and obtaining a liquid supernatant,

[0009] (iii) a step of separating polysorbate by applying the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column containing fixed cyano groups and eluting the bound polysorbate using a mobile phase composition gradient, and

[0010] (iv) A step of detecting separated polysorbates using a chromophore-lack detector to identify polysorbates.

[0011] A method is provided for identifying polysorbate, e.g., intact polysorbate and / or degraded polysorbate products in a protein-containing sample, comprising applying the above sample to the sample.

[0012] In a second aspect, the present invention

[0013] (a) (i) a step of precipitating the protein by exposing the protein sample to an organic protic polar solvent or an organic aprotic polar solvent,

[0014] (ii) a step of separating proteins from precipitated samples by centrifuging the precipitated samples to pellet the proteins or peptides and obtaining a liquid supernatant,

[0015] (iii) a step of separating polysorbate by applying the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column containing fixed cyano groups and eluting the bound polysorbate using a mobile phase composition gradient, and

[0016] (iv) A step of detecting separated polysorbates using a chromophore-lack detector to identify polysorbates.

[0017] A step of measuring polysorbate in the protein sample comprising;

[0018] (b) a step of identifying protein sample(s) from (a) in which the level of intact polysorbate, such as PS80, is about 10 ppm to about 5000 ppm;

[0019] (c) Step of isolating and recovering the protein(s) identified in step (b).

[0020] A method for identifying protein sample(s) from a plurality of proteins, for example, comprising, wherein the identified protein sample(s) contain about 10 ppm to about 5000 ppm of intact polysorbate.

[0021] In addition, the use of said protein in the manufacture of a pharmaceutical preparation, for example, to be administered to a human subject, is also provided, which is obtained or is obtainable by the method of the second aspect of the present invention.

[0022] In embodiments of the first and second aspects of the present invention, the provided method is a method for identifying polysorbate 80 (e.g., intact and / or degraded PS80 polysorbate) in a protein-containing sample, e.g., an antibody sample such as an mAb.

[0023] The precipitation and separation steps combined with elution enable the separation of polysorbate products from the sample, and the detection step enables the detection and analysis of the intact and / or degraded polysorbate products, such as PS80 and / or PS60 and / or PS40 and / or PS20.

[0024] In one embodiment of the first and second aspects of the present invention, a method for identifying polysorbate in a sample containing a protein or peptide, such as an antibody such as the 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 said sample, comprising measuring the intact and / or degraded products, such as PS80 and / or PS60 and / or PS40 and / or PS20. Brief explanation of the drawing

[0025] Figure 1 shows the last two steps of the synthesis path for PS80. Figure 2 shows the degradation products of the two most common types of degradation in polysorbate. Figure 3 shows a chromatogram obtained using an HPLC-CAD method to quantify PS80 mono-esters and qualitatively / semi-quantitatively monitor four different subspecies groups. Figure 4 shows chromatograms for PS80 (solid line) and blank (dotted line) of various sources. Figure 5 shows chromatograms for various polysorbates (solid lines) and blanks (dotted lines). Figure 6 shows the chromatography profile obtained using the HPLC-CAD method of PS80 mono-ester. Figure 7 shows the kinetics of PS80 decomposition in samples from 5, 25, 40, or -70°C up to 21 days. Figure 8 shows the Arrhenius plot of the rate constants (at 5, 25, and 40°C) for the decomposition of PS80 mono-ester. Figure 9 shows an overlay of chromatograms (200 ppm standard solution (multi-compensated J.T. Baker PS80), an mAb sample with decomposed PS80, and a blank solution). Specific details for implementing the invention

[0026] In this specification, the invention has been described with reference to embodiments in a manner that enables a clear and concise specification. It should be understood that embodiments may be combined or separated in various ways without departing from the invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the relevant technical field (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques were used for quantitative analysis methods.

[0028] All publications cited herein, including but not limited to patents and patent applications, are incorporated herein by reference as fully presented.

[0029] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of amino acid residues. Polypeptides may be of natural (tissue-derived) origin, recombinants or natural expressions from prokaryotic or eukaryotic cell preparations, or may be chemically produced through synthetic methods. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. An amino acid mimic refers to a chemical compound that has a structure different from the general chemical structure of an amino acid but functions in a manner similar to that of a naturally occurring amino acid. Non-natural residues are well described in scientific and patent literature; and some exemplary non-natural compositions and guidelines useful as mimics of natural amino acid residues are described below. Mimics of aromatic amino acids include, for example, D- or L-nafilalanine; D- or L-phenylglycine; D- or L-2 thienylalanine; D- or L-1, -2,3- or 4-pyrenylalanine; 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-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine: Dp-fluoro-phenylalanine; D- or Lp-biphenylphenylalanine; K- or Lp-methoxy-biphenylphenylalanine: D- or L-2-indole(alkyl)alanine; and may be produced by substitution with D- or L-alkylalanine, wherein the alkyl may be a substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or non-acidic amino acid. Aromatic rings of non-natural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0030] As used herein, the term "antigen-binding polypeptide" refers to antibodies, antibody fragments, and other protein constructs capable of binding to an antigen.

[0031] The term “antibody” is used herein in its broadest sense to refer to molecules having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE), including monoclonal, recombinant, polyclonal, chimeric, human, humanized, and multispecific antibodies including bispecific antibodies, and heterozygous antibodies, single variable domains (e.g., domain antibodies (DAB)), antigen-binding antibody fragments, Fab, F(ab')2, Fv, disulfide-bound Fv, single-stranded Fv, disulfide-bound scFv, diabodies, TANDABS, etc., and any modified versions of the foregoing (for a summary of alternative “antibody” formats, see [Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136]). Alternative antibody formats are also considered, including alternative scaffolds in which one or more CDRs of antigen-binding proteins can be arranged on a suitable non-immunoglobulin protein scaffold or backbone, such as apibodies, SpA scaffolds, LDL receptor class A domains, avimers, or EGF domains.

[0032] The terms "complete," "whole," or "defective antibody," used interchangeably herein, refer to a heterotemeric glycoprotein having a molecular weight of approximately 150,000 daltons. The defective antibody consists of two identical heavy chains (HC) and two identical light chains (LC) connected by covalent disulfide bonds. This H2L2 structure folds to form three functional domains comprising two antigen-binding fragments known as the "Fab" fragment and a crystallizable fragment called the "Fc" fragment. The Fab fragment consists of an amino-terminal variable domain, a variable heavy chain (VH) or variable light chain (VL), and a carboxyl-terminal constant domain, CH1 (heavy chain) and CL (light chain). The Fc fragment consists of two domains formed by the dimerization of paired CH2 and CH3 regions. Fc can derive effector function by binding to a receptor on an immune cell or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies—IgM, IgA, IgG, IgE, and IgD—are defined by distinct heavy chain amino acid sequences called μ, α, γ, ε, and δ, respectively, and each heavy chain can be paired with a κ or λ light chain. The majority of antibodies in serum belong to the IgG class, and there are four isoforms of human IgG: IgG1, IgG2, IgG3, and IgG4, whose sequences differ mainly in the hinge region.

[0033] As used herein, the “fragment” is a protein or polypeptide having the same amino acid sequence as a part, rather than the whole, of the amino acid sequence of a whole naturally occurring protein / polypeptide, when used in relation to a protein or polypeptide. The fragment may be “independent” or may be contained within a larger protein or polypeptide, and its part or region forms a single continuous region within a single larger protein / polypeptide.

[0034] The term “single variable domain” refers to a folded polypeptide domain comprising a characteristic sequence of an antibody variable domain. Thus, it includes a complete antibody variable domain, e.g., VH, VHH, and VL, and a modified antibody variable domain in which, e.g., one or more loops are replaced by sequences not characteristic of the antibody variable domain, or an antibody variable domain that is truncated or includes N- or C-terminal extensions, as well as a folded fragment of a variable domain that retains at least the binding activity and specificity of the full-length domain. A single variable domain as defined herein may bind to an antigen or epitope independently of other variable regions or domains. “Domain antibody” or “DAB” may be considered identical to a human “single variable domain.” The single variable domain may be a human single variable domain, but also includes a single variable domain from other species, such as rodents (e.g., as disclosed in WO 00 / 29004), and the nurse shark and camel VHH camel VHH is an immunoglobulin single variable domain polypeptide derived from species including camels, llamas, alpacas, dromedary camels, and guanacos that naturally produce heavy chain monoantibodies lacking light chains. The said VHH domain may be humanized according to standard techniques available in the art, and said domain is considered a “single variable domain.”

[0035] As used herein, the term "polysorbate" refers to any one (or all) of ordinary intact polysorbates selected from polysorbate 80 (PS80), polysorbate 60 (PS60), polysorbate 40 (PS40), and polysorbate 20 (PS20) and their degradation products. Intact polysorbate refers to polysorbate when it exists as a monoester. The degradation products of polysorbate 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 comprising POE esters of fatty acids, and short-chain fatty acids. FIG. 2 shows the degradation products of the two most common types of degradation in polysorbate.

[0036] Polysorbates are commonly used as non-ionic surfactants in both food and biopharmaceutical products. In biopharmaceutical products, they are used to prevent protein adsorption, aggregation, and particle formation on surfaces. However, it is known that the degradation of intact polysorbates is problematic, for example, as the degraded polysorbate products can cause irritation in injectable pharmaceutical products and also cause excessive turbidity in samples, for example, pharmaceutical samples. Generally, about 10 ppm to about 5000 ppm of intact polysorbate is considered a desirable amount in pharmaceutical products.

[0037] Therefore, polysorbate, in particular polysorbate 80 (polyoxyethylene sorbitan mono-oleate or Tween), which is the most commonly used polysorbate TMThere is great interest in developing analytical methods to monitor these polysorbates, including the integrity of 80). Commercially available PS80 is heterogeneous, and the most common process-related subspecies are polyoxyethylene (POE) groups, POE isosorbide mono-esters, and POE sorbitan / isosorbide di-, tri-, tetra-esters. Figure 1 shows the last two steps of the synthesis pathway for polysorbate 80 (PS80).

[0038] However, the development of analytical methods to detect intact polysorbates, such as PS80, as well as PS60, PS40, and PS20, and their degradation products has been a challenge, and there is a need for such methods, particularly those applicable to protein-containing biopharmaceutical preparations. Furthermore, the pharmaceutically acceptable release of protein materials by agencies, such as the FDA, depends on protein materials containing prescribed levels of polysorbates, including specific polysorbate products.

[0039] Accordingly, the present invention provides a method for enabling the identification of such polysorbates, e.g., intact polysorbates and / or degraded polysorbate products, in pharmaceutical formulations, e.g., biopharmaceutical formulations containing proteins.

[0040] The present invention also provides a method for enabling the measurement of polysorbates in biopharmaceutical formulations containing proteins or peptides. As used herein, the term "measurement" of polysorbates refers to the identification and also quantification of said polysorbates. The measured polysorbates may be intact and / or degraded polysorbate products. The method provided herein is advantageous because it is accurate, can be performed within a timeframe similar to traditional HPLC, and does not rely on the use of derivatization or micelle encapsulation, which can be problematic. Derivatization or micelle encapsulation can increase the complexity of sample preparation, be subject to equilibrium kinetics that can negatively affect precision, and use additional components in the matrix that negatively affect the signal-to-noise ratio.

[0041] Accordingly, the present invention in a first aspect

[0042] (i) a step of precipitating the protein by exposing a sample containing the protein to an organic protic polar solvent or an organic aprotic polar solvent,

[0043] (ii) a step of separating proteins from precipitated samples by centrifuging the precipitated samples to pellet the proteins or peptides and obtaining a liquid supernatant,

[0044] (iii) a step of separating polysorbate by applying the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column containing fixed cyano groups and eluting the bound polysorbate using a mobile phase composition gradient, and

[0045] (iv) A step of detecting separated polysorbates using a chromophore-lack detector to identify polysorbates.

[0046] A method is provided for identifying polysorbate, e.g., intact polysorbate and / or degraded polysorbate products in a protein-containing sample, e.g., an antibody, e.g., an mAb sample, comprising applying the above sample to the sample.

[0047] The precipitation and separation steps combined with elution allow for the separation of polysorbate products within a sample, e.g., intact and degraded polysorbate products, and the detection step allows for the detection, identification, and quantification of polysorbate products, e.g., intact and degraded polysorbate products, such as intact PS80 and / or PS60 and / or PS40 and / or PS20 and their degradation products. In one embodiment, a method for measuring intact polysorbate in a protein-containing sample, e.g., an antibody, e.g., an mAb sample provided herein is a quantitative method that allows for the measurement of the amount of intact and / or degraded polysorbate products, e.g., PS80 and / or PS60 and / or PS40 and / or PS20, present in said sample.

[0048] In one embodiment, the method of the present invention may be used to monitor the degradation of intact polysorbate over time in a sample, e.g., a protein-containing sample, e.g., an antibody, e.g., an mAb sample, or a cell or protein-containing vector expressing a heterologous therapeutic gene, for example, to evaluate the stability of such protein-containing samples.

[0049] The present invention also provides a method for measuring the amount of intact polysorbate in a protein-containing sample, for example, the amount of intact PS80 and / or intact PS60 and / or intact PS40 and / or intact PS20 present in such sample.

[0050] In a second aspect, the present invention

[0051] (a) (i) a step of precipitating the protein by exposing the protein sample to an organic protic polar solvent or an organic aprotic polar solvent,

[0052] (ii) a step of separating proteins from precipitated samples by centrifuging the precipitated samples to pellet the proteins or peptides and obtaining a liquid supernatant,

[0053] (iii) a step of separating polysorbate by applying the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column containing fixed cyano groups and eluting the bound polysorbate using a mobile phase composition gradient, and

[0054] (iv) A step of detecting separated polysorbates using a chromophore-lack detector to identify polysorbates.

[0055] A step of measuring polysorbate in the protein sample comprising;

[0056] (b) a step of identifying protein sample(s) from (a) having a level of intact polysorbate of about 10 ppm to about 5000 ppm;

[0057] (c) Step of isolating and recovering the protein(s) identified in step (b).

[0058] A method for identifying protein sample(s) from a plurality of proteins, for example, comprising, wherein the identified protein sample(s) contain about 10 ppm to about 5000 ppm of intact polysorbate.

[0059] In one embodiment of the second aspect of the present invention, the damage-free polysorbate is PS80 and / or PS60 and / or PS40 and / or PS20.

[0060] In addition, a protein obtained or obtainable by the method of the second aspect of the present invention, and also a use of said protein in the manufacture of medicines, for example, pharmaceutical formulations to be administered to human subjects.

[0061] The present invention also provides a protein (e.g., an antibody) obtainable or obtained from the method of the second aspect of the present invention, containing intact polysorbate present in an amount of about 10 ppm to about 4000 ppm or to about 3000 ppm or to about 2000 ppm or to about 800 ppm or to about 700 ppm, and also provides the use of said protein in medicine in the manufacture of pharmaceutical formulations for administration to human subjects, for example.

[0062] In one embodiment, where the protein is a protein-containing vector expressing an antibody or cell or a xenotherapy gene for use in cell therapy, 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 to which the method of the present invention can be applied may be about 5 mg / ml to about 300 mg / ml, about 5 to about 200 mg / ml, about 5 to about 50 mg / ml, about 5 to about 20 mg / ml, about 5 to about 10 mg / ml, about 10 to about 20 mg / ml, about 15 or about 20 mg / ml to about 50 mg / ml.

[0063] The method of the present invention may be applied to any natural or recombinant protein. Protein samples may include, for example, therapeutic proteins, preventive proteins, or diagnostic proteins. For example, the method may be applied to samples comprising antigen-binding constructs, such as antibodies or antibody fragments, such as biologically functional fragments of antibodies, and the method may also be applied to vaccine compositions, cells, or protein-containing vectors expressing heterologous therapeutic genes.

[0064] If the protein sample is an antibody, it may be, for example, a monoclonal antibody (mAb), or a bispecific or multispecific antibody or a fragment thereof. The antibody may be a chimeric, humanized, or human antibody. If the protein is an antibody fragment, it may be, for example, Fab, F(ab')2, Fv, disulfide-linked Fv, single-stranded Fv, disulfide-linked scFv, Diabadi, TANDABS TM , it may be a modified version of the antibody CDR and any of the aforementioned.

[0065] Antibody fragments also include a single variable domain (or dAb), e.g., a human VH or VL single variable domain, or non-human sources, e.g., llama or camel, e.g., camel VHH, e.g., nanobody TM It may be a single variable domain derived from (e.g., specifically described in WO 94 / 04678 and WO 95 / 04079). For example, the use of any of these antibodies or CDRs of a single variable domain as part of a protein scaffold is also considered.

[0066] Protein samples for use in the method of the present invention may be in the form of a liquid or suspension in an aqueous medium, or they may be reconstituted, for example, after being freeze-dried and then reconstituted in an aqueous medium. In addition to the protein and water, the protein samples may further comprise additional diluents, for example, pharmaceutically acceptable diluents. Examples of such pharmaceutically acceptable diluents include solvents, such as water, sodium chloride solution, sugars, buffers such as acetate, salts such as sodium chloride, and / or other excipients. In one embodiment, the buffers are acetate and citrate.

[0067] The method of the present invention is 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, e.g., liquid biopharmaceutical preparations, e.g., mAb preparations.

[0068] The method of the present invention can also be applied to gene therapy products such as samples containing oligonucleotides, engineered cells for cell therapy, and engineered vectors (e.g., viral vectors) containing therapeutic genes for administration to human subjects.

[0069] The method of the present invention can also be used to measure polysorbates in samples containing small molecules that are chemical substances (NCE), and if such NCE samples do not contain proteins, protein precipitation may be omitted and the amount of organic protic polar solvent or organic aprotic polar solvent may be adjusted.

[0070] Since the pH value is not critical to the performance of the method, the method of the present invention can be performed over a wide range of pH values, for example, about pH 5 to about pH 10, or about pH 6 to about pH 8. Protein samples analyzed according to the method of the present invention may have a pH of about pH 6.0 to about pH 8.0, for example, about 7.4 to about 6.8.

[0071] Organic protic polar solvents for use in the protein precipitation step are widely known in the relevant art, and the term as used herein refers to organic solvents containing unstable protons and capable of ionization. Examples of such solvents that can be used in the method of the present invention are widely known to those skilled in the art and include, for example, methanol, ethanol, and isopropyl alcohol (IPA). For example, if the protein is an antibody, methanol, IPA, or acetone may be used in the protein precipitation step.

[0072] Organic aprotic polar solvents for use in the protein precipitation step are also widely known in the relevant art, and the term used herein refers to organic solvents that do not contain unstable protons. Examples of such solvents that can be used in the method of the present invention are widely known to those skilled in the art and include, for example, acetone, tetrahydrofuran (THF), and acetonitrile.

[0073] The method can be performed over a wide range of solvent concentrations, and when the method is performed on a sample containing antibodies, the volume / volume dilution can be about 1 part sample to about 5, 9, or about 19 parts solvent.

[0074] The centrifugation step can be performed at a speed and time sufficient to obtain a protein pellet, for example, at least about 10,000 rpm for at least about 10 minutes.

[0075] The separation step can be performed using a column chromatography method, for example, reverse-phase medium or mixed-mode retention chromatography. Such mixed-mode chromatography includes the combination of two or more retention mechanisms, for example, normal phase, cation exchange, and anion exchange.

[0076] In one embodiment, the separation step of the method of the present invention may be performed on a reverse-phase chromatography column using a method known to a person skilled in the art, wherein the column comprises a group having a carbon chain of C3 or more, for example, C4 to about C18 or less.

[0077] In one embodiment, the column comprises a fixed cyano group, and for example, a reverse-phase chromatography column comprising a cyano group fixed on a stationary phase is used. The cyano group is any chemical compound containing a -CN group that is widely known in the art. Any cyano group may be used in the method of the present invention. A column comprising a CN group that may be usefully used according to the method of the present invention is Agilent Zorbax SB300-CN, and includes Agilent Zorbax SB300-CN, Phenomenex Luna CN, or Agilent InfinityLab Poroshell 120 EC-CN.

[0078] The column may be a silica bead column having a pore size of, for example, about 80 angstroms or more. In one embodiment, the pore size is about 120 to about 300 angstroms. For example, a pore size of about 300 angstroms may be used. Examples of suitable silica columns include Agilent Zorbax SB300-CN, Phenomenex CN, or Agilent Infinity Lab Porroshell 120 EC-CN. In one embodiment, the column used is Agilent Zorbax SB300-CN, 3.5 µm, 150 x 4.6 mm (available from Agilent Co., Santa Clara, California, USA). The column may be heated, for example, the temperature of the column may be about 20°C to about 80°C, or about 40°C to about 60°C, or about 50°C.

[0079] In one embodiment, the elution step is performed using a gradient-separated mobile phase, which may be, for example, a gradient-separated mobile phase of A and B. In one embodiment, a gradient-separated mobile phase of A and B is used, wherein A is a mixture of 0.1% to about 10% of an acid or ammonium acetate in H2O, the acid may be selected from trifluoroacetic acid (TFA), formic acid, acetic acid, and difluoroacetic acid, and B may be methanol, isopropanol, or acetonitrile. In one embodiment, a gradient-separated mobile phase of A and B is used, which is a mixture of 0.1% trifluoroacetic acid (TFA) in H2O, and B is methanol or acetonitrile. The gradient-separated mobile phase of A and B may be performed as described in Table 1 below.

[0080]

[0081]

[0082] The detector used in the method of the present invention is a chromophore-deficient detector, and such a detector functions when the sample for detection lacks a chromophore.

[0083] In one embodiment, the detector used in the method of the present invention may be an evaporative light scattering detector, or mass spectrometry may be used for detection.

[0084] In another embodiment, a charged aerosol detector (CAD) is used as a detector in the method of the present invention, which is used, for example, with high-performance liquid chromatography (HPLC) and is a detector that operates by charging nitrogen gas charged by a high-voltage corona wire into non-volatile and semi-volatile analytes. Subsequently, the charged analyte particles pass through an ion trap that removes high-mobility species (i.e., solvent) and continue to a collector, where they are measured by a sensitive potentiometer. Examples of CADs available for use include Corona Veo (available from Thermo in Waltham, Massachusetts), Corona Veo RS (available from Thermo in Waltham, Massachusetts), Vanquish (available from Waltham, Massachusetts), Corona Ultra and Ultra RS (available from Thermo in Waltham, Massachusetts), and Corona Plus (available from Thermo in Waltham, Massachusetts).

[0085] One of the features provided by CAD is the ability to measure intact species by charging the surface of the analyte, unlike mass spectrometry which generates charged fragments. Additionally, for analytes with similar surface area and density, the response is similar. Finally, when a highly volatile eluent is used, the CAD method can also be very sensitive (sub-nanogram).

[0086] Although CAD is easy to operate, there are additional considerations in the development of standard HPLC-UV / Vis analysis methods. These include: (1) the selection of a non-efferent column; (2) the use of high-purity solvents in the mobile phase for a low-reproducible baseline; and (3) the cleaning of glassware and plastics due to the greater possibility of interference from contaminants. Since there is no way to identify peak purity as is possible when using diode array or mass spectrometer (MS) detection, it is important to achieve specificity when using CAD. Furthermore, it should be noted that if specificity is not achieved, the observed signal is not simply the sum of reactions as in UV-Vis spectrophotometry, and differences in reactions are often complicated by differences in the charge, surface area, density, and volatility of the analyte relative to the components of the mobile phase. For this reason, CAD is highly suitable as a detector for the analysis of the PS80.

[0087] In one embodiment, the charged aerosol detector (CAD) used in the method described herein is a Corona Veo RS (available from Thermo, Waltham, Massachusetts, USA).

[0088] A detection step performed using CAD involves obtaining a baseline using selected blank solution(s) and obtaining a chromatogram containing peak areas for intact polysorbates, degradation products, as well as proteins and excipients in the sample. Evaluation of peak areas using area under the curve calculation enables the quantification of polysorbates, e.g., PS80, and / or PS60, and / or PS40 and / or PS20, and their degradation products. In one embodiment, the method enables the identification of PS80, e.g., intact and degraded PS80.

[0089] When the inventors refer to separation using the method of the present invention, this implies that the intact polysorbate peak (i.e., monoester) must be decomposed 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 simply need to be distinguishable from one another. Additionally, in one embodiment, there is a specificity requirement that there are no interference peaks at a residence time of intact polysorbate (i.e., monoester) exceeding about 3 area percent. In one embodiment, the present invention

[0090] (i) a step of precipitating the protein by exposing a sample containing the protein to methanol or IPA,

[0091] (ii) a step of separating proteins from precipitated samples by centrifuging the precipitated samples to pellet the proteins or peptides and obtaining a liquid supernatant,

[0092] (iii) a step of separating polysorbate by applying the supernatant to a reverse-phase HPLC on a silica column having a pore size of about 300 angstroms containing fixed cyano groups and eluting using a gradient of mobile phase compositions consisting of A and B, wherein A is a mixture of 0.1% trifluoroacetic acid (TFA) with H2O and B is methanol or acetonitrile,

[0093] (iv) A step of detecting separated polysorbates using a charged aerosol detector (CAD) to identify polysorbate products.

[0094] A method is provided to identify polysorbate in a protein-containing sample (e.g., antibody sample) including

[0095] Example:

[0096] The present invention is further described with reference to the following examples. These examples are merely for illustrating various aspects of the invention and are not intended to limit the invention.

[0097] Example 1: (i) a novel HPLC-CAD analysis according to the method of the present invention having the ability to quantify PS80 mono-esters, and (ii) a comparison of the measurement of PS80 and its subspecies present in mAb drug products through a modified HPLC method using evaporative light scattering detection—the HPLC-ELSD method.

[0098] The reagents and methods used are as follows:

[0099] Multi-compendial J.T. Baker PS80 was purchased from Fisher Scientific (Atlanta, Georgia, USA, 02-003-654). Two sources of PS80 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA): (1) PS80 stored in natural-colored plastic containers (Part # P1754 - 25ML), and (2) PS80 stored in amber glass containers (Part # 59925 - 100G). Highly purified PS80 was purchased from Croda Health Care (Edison, New Jersey, USA, SR48833). All-oleate ChP-compliant PS80 was purchased from NOF (White Plains, New York, USA), and non-GMP PS80 was purchased from POLO80(HX2) 19B803364. Polysorbate 60 was purchased from USP reference standard (Rockville, Maryland, USA, 154794). Polysorbate 40 was purchased from Fisher Scientific (Atlanta, Georgia, USA, AC334142500). Oleic acid was purchased from Sigma-Aldrich (St. Louis, Missouri, USA, 75090-5ML). Linoleic acid was purchased from Fisher Scientific (Atlanta, Georgia, USA, AC215040250). Palmitic acid was purchased from MP Biomedicals (Santa Ana, California, USA, 100905-10G). Palmitoleic acid was purchased from Sigma-Aldrich (76169, St. Louis, Missouri, USA). Chromatography (LC-MS or GC) grade methanol was purchased from Fisher Scientific (A456-4, Atlanta, Georgia, USA) or VWR (Honeywell / Burdick and Jackson, GC grade, ≥ 99.9% purity, BJGC 230-4).Ultrapure water (Milli-Q water) was produced using a Milli-Q water purification system (Millipore Corporation, Burlington, Massachusetts, USA). Trifluoroacetic acid (TFA) was purchased from Sigma-Aldrich (91707-10x1 mL, St. Louis, Missouri, USA). Other precipitation solvents (isopropanol, acetone, tetrahydrofuran (THF)) were chromatography grade and were purchased from Sigma-Aldrich. For the HPLC-ELSD method, HPLC-grade methanol (646377-4 L) and acetonitrile (439134-4 L) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Honeywell Flucaformic Acid (94318-250ML) was purchased from Fisher Scientific (Atlanta, Georgia, USA, AC334142500).

[0100] The instruments and analysis conditions used are as follows:

[0101] Protein Precipitation and PS80 Extraction:

[0102] For the novel HPLC-CAD analysis method, this was performed as follows:

[0103] To remove proteins [i.e., mAb drug products] prior to injection, the proteins were precipitated using a precipitation solvent (methanol, isopropanol, and / or acetone). Additionally, precipitation was used to disrupt any potential protein-PS80 interactions and inhibit any degradation caused by lipase or esterase. Filtration was not a viable option due to the removal of some polysorbate species. Therefore, 900 μL of precipitation solvent (using methanol or precipitation solvent) was added to 100 μL of the sample in a 1.5 mL Eppendorf safety-lock tube (Harpuji, NY, 022363204). Subsequently, the sample formulation was briefly mixed (~5 seconds) by vortexing and centrifuged at 14,000 rpm for 10 minutes. PS80 species and fatty acids remained soluble in the supernatant. At least 60 μL of supernatant was transferred to an HPLC vial equipped with 300 μL of insert.

[0104] A 1,000 ppm PS80 stock standard solution was prepared by weighing 100 ± 10 mg of multi-compensated J.T. Baker PS80 into a 100 mL Class A volumetric flask and diluting it by volume with methanol. A 20 ppm PS80 working standard solution was prepared by mixing 100 μL of MilliQ water, 20 μL of the 1,000 ppm PS80 stock standard solution, and 880 μL of methanol by vortexing in a pre-rinsed (using precipitation solvent) 1.5 mL Eppendorf tube. Thus, the final diluent composition for the standard (90% precipitation solvent: 10% MilliQ H2O / aqueous) was identical to that of the sample. PS60, PS40, and PS20 solutions were prepared in the same manner.

[0105] A resolution check solution containing 20 ppm PS80 and 5 ppm oleic acid stock standard solutions was prepared. A sensitivity solution was prepared by mixing 898 μL of organic solvent, 100 μL of water, and 2 μL of 1,000 ppm PS80 stock standard solution. The mAb buffer was prepared in bulk, aliquoted, and stored at -70°C until the date of analysis. The 20 ppm PS80 buffer was prepared by diluting it with LC-MS or GC-grade methanol. A centrifugation step was not required for protein-deficient sample preparations.

[0106] For thermally stressed samples, multiple vials of mAb / protein-containing preparations were incubated at -70, 5, 25, and 40°C for 3 weeks, the vials were removed from the oven at each time point (initial, 1, 2, 3, 4, 7, 14, and 21 days), and frozen at -70°C until the time of analysis.

[0107] For the modified HPLC-ELSD analysis method, it was performed as follows:

[0108] Due to suspected PS80-protein interactions and the cessation of any enzymatic degradation, the protein was precipitated with methanol instead of diluting it with water. To precipitate the protein and extract PS80, 800 μL of organic solvent was added to 200 μL of the sample in a 1.5 mL microcentrifuge tube and mixed by vortexing. After mixing, the sample was centrifuged at 10,000 rpm for 30 minutes at 5°C. After centrifugation, 200 μL of the supernatant was transferred to an HPLC vial fitted with a 300 μL insert.

[0109] Quantification of the PS80 content in the samples was achieved through the preparation of a calibration curve. Due to the properties of the HPLC-ELSD detector, the matrix of the standard curve must be representative of the samples. To obtain a representative matrix, 500 mg of multi-compensatory J.T. Baker PS80 was weighed into a 50.0 mL chemical line Class A volumetric flask and diluted by volume with HPLC-grade methanol to prepare a 10,000 ppm PS80 stock solution. Subsequently, 0.5 mL of the 10,000 ppm PS80 stock solution was added to a 10.0 mL volumetric flask and diluted by volume with HPLC-grade methanol to prepare a 500 ppm stock solution. Calibration standards in a methanol / water solution (80:20 v / v) were prepared using a 500 ppm stock solution at expected PS80 concentrations of 10, 25, 50, 100, 150, 200, and 250 ppm. A 20 ppm PS80 formulation was prepared by diluting with HPLC-grade methanol. A centrifugation step was not performed on the protein-deficient formulation. Sample chromatograms are provided in Fig. 9. In summary, Fig. 9 shows an overlay of chromatograms collected by the modified HPLC-ELSD method (200 ppm standard solution (Multi-Compendial J.T. Baker PS80), mAb samples containing decomposed PS80, and blank solutions). In Fig. 9, the 200 ppm standard has the largest peak, and the -20 sample has a smaller peak in the middle.

[0110] Novel HPLC-CAD Method:

[0111] The Agilent HPLC 1260 system (Santa Clara, California, USA) included a binary solvent manager, a sample manager set to 23°C, a column oven set to 50°C, and a charge aerosol detector (CAD) Veo RS (Thermo, Waltham, Massachusetts, USA). The CAD was connected directly to the analytical column via 80 cm of tubing (Agilent, 01078-87305) and directly connected to a 3 μL Peltier via 180 mm of tubing (Agilent, G1313-87305). The HPLC column heater was connected to the HPLC autosampler via standard tubing, bypassing both the UV-VIS and the column switching valve.

[0112] The analytical column was a Zorbax SB300-CN (150 mm x 4.6 mm, 3.5 μm 300 Å, 863973-905) from Agilent Technologies (Wilmington, Delaware, USA). A volatile mobile phase (MP) consisting of 0.1% v / v TFA (MP A) and 100% LC-MS or GC grade methanol (MP B) in MilliQ water was used. Additionally, the mobile phase was pre-screened for cleanliness by flowing at 1.2 mL / min in a 35% MP A: 65% MP B ratio and ensuring that the CAD had a baseline level of less than 10 mV with the parameters listed above. Separation of the PS80 subspecies was achieved by gradient elution (0.1% TFA in MilliQ water: 0 min–100%; 1 min–100%; 3 min–50%; 8 min–50%; 27 min–5%; 30 min–5%; and 30.1 min–100%) at a flow rate of 1.2 mL / min. The total run time of the method was 40 minutes. The injection volume was 30.0 μL. The ThermoVeo RS CAD was operated with the following settings: evaporation temperature, 60°C; power function, 1.00; output offset, 0%; filter, 5.0 sec; range 100 pA. An in-house nitrogen supply was used. The CAD analog signals were converted into digital signals using an e-SAT / IN module (Waters, Milford, Massachusetts, USA, 668000230).

[0113] Modified HPLC-ELSD Method:

[0114] This method is a modified version of the Hewitt and Copolu method. The Agilent HPLC 1100 system (Santa Clara, California, USA) included a binary solvent manager, a sample manager set to 25°C, a column oven set to 30°C, and a 1260 Infinity G4260B Evaporative Light Scattering Detector (ELSD, Agilent Technologies, Wilmington, Delaware, USA). The ELSD was connected directly to the analytical column and directly to a 3 μL Peltier. The HPLC column heater was connected to the HPLC autosampler via standard tubing, bypassing both the UV-VIS and the column switching valve.

[0115] The analytical column was an Oasis® MAX (20 mm x 2.1 mm, 30 μm 80 Å, Part #186002052) from Waters Corporation (Milford, Massachusetts, USA). A volatile mobile phase consisting of 2% v / v formic acid in water and 2% v / v formic acid in isopropanol was used. Separation was achieved by gradient elution at a flow rate of 1.0 mL / min (2% formic acid in water was 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%), and the flow was diverted from the ELSD at the first 4 minutes of the run. The injection volume was 50.0 μL. The Agilent 1260 Infinity G4260B ELSD was operated with the following settings: LED, 10; Gain (PMT), 2; Smoothing (Smth), 1; Data Output, 80 Hz; Evaporation Temperature, 80°C; Nebulizer Temperature, 50°C; Gas Flow (SLM), 1. An in-house nitrogen supply was used. The CAD analog signal was converted into a digital signal using an e-SAT / IN module (Waters, Milford, Massachusetts, USA, 668000230).

[0116] For the novel HPLC-CAD analysis method, data analysis, integration, and calculations were performed as follows:

[0117] The average concentration of PS80 mono-ester in the triple formulation was reported. To quantify total esters (mono-esters and multi-esters) for comparison with the modified HPLC-ELSD method, a calibration curve for total esters was constructed by classifying the areas of PS80 mono-esters and multi-esters in the linear formulation. Thus, the total ester area in the HPLC-CAD method is similar to the single peak in the modified HPLC-ELSD method; the POE group is not included in the single peak, as they are eluted when the valve switch is switched to waste during the first 4 minutes of each injection.

[0118] The degradation rate of PS80 was evaluated using Arrhenius kinetics modeling, and the stability or activation energy (Ea) was estimated. The hydrolytic degradation of PS80 mono-esters was assumed to be pseudo-first order, as previously described. Rate constants were determined from the slopes of the natural logarithm of the concentration versus time plot, under the assumption that there are no significant effects due to changes in dynamic viscosity. For all linear plots, a relative error analysis of the slopes was performed as previously described:

[0119] Equation 1

[0120] Here, n is the number of data points, a is the slope, and b is the y-intercept.

[0121] For the modified HPLC-ELSD analysis method, data analysis, integration, and calculations were performed using Empower 3 to enable batch data processing to obtain retention time, peak area, and other chromatographic performance indices. Similarly, for the novel HPLC-CAD method, data analysis, integration, and calculations were performed using Empower 3 to enable batch data processing to obtain retention time, peak area, resolution, S / N, and other chromatographic performance indices.

[0122] result:

[0123] The novel HPLC-CAD method described above has been found to accurately and clearly quantify PS80 mono-esters and to qualitatively / semi-quantitatively monitor four different groups of subspecies. For simplicity, the inventors selected a concentration range that is linear even though the CAD response is non-linear. The calibration curve can also be linearized by applying a power-function algorithm; however, without baseline reproducibility, such an algorithm may not always remain true.

[0124] Matrix interference was evaluated by assessing the recovery of spiked PS80 in (1) PS80-free IgG drug products; and (2) mAb samples containing fully degraded (<limit of quantification (LOQ)) PS80 mono-esters (see Table 2 and discussion below). The degraded samples contained proteins in an aqueous buffer containing trehalose, methionine, arginine, histidine, mM EDTA, and PS80. The other samples contained proteins in an aqueous buffer containing trehalose, citrate, EDTA, and PS80. To evaluate specificity in the degraded samples, fatty acids (linoleic acid, palmitic acid, oleic acid, and palmitoleic acid) were also spiked at a concentration of 5 ppm in a 20 ppm PS80 working standard solution (see Fig. 3). In summary, Figure 3 illustrates a chromatogram obtained using the HPLC-CAD method to quantify PS80 mono-esters and qualitatively / semi-quantitatively monitor four different subspecies groups. It shows a blank (dotted line) and an overlay of a 20 ppm PS80 (Multi-Compendial J.T. Baker) standard solution spiked with 10 ppm fatty acids (solid line). Peaks: 1 = Non-retaining agent buffer components (trehalose, amino acids, EDTA, salt impurities in solvent, and / or sample residues); 2 = POE group; 3 = Palmitoleic acid; 4 = Linoleic acid; 5 = Palmitic acid; 6 = Oleic acid; 7 = PS80 mono-ester (sorbitan and isosorbide); 8 = Di-ester; 9 = Tri-ester; 10 = Tetra-ester; * If present, contaminants from the Eppendorf tube. The identification of the PS80 peak is consistent with previously reported LC-MS results [17, 30] and is assumed to be due to the expected elution order of the analytes based on the relative hydrophobicity of the analytes.

[0125] The method was standardized into an mAb drug product.

[0126] This consisted of precision, linearity, accuracy, specificity, and LOQ (Table 2). Using the CAD response for each injection, the mean concentration, standard deviation, and relative standard deviation were calculated. Precision was evaluated through the average analysis of 2 to 3 replicate preparations. Precision was also evaluated using a formulation buffer (or analysis control) through the repeatability analysis of duplicate injections for 5 analysis cases between 2 analysts. Intermediate precision was determined by one analyst performing 2 independent analysis cases for one system and a second analyst performing 3 independent analysis cases for the second system.

[0127] Table 2 Accuracy, repeatability, intermediate precision, and linearity of PS80 mono-ester in formulation buffers and mAb formulations.

[0128]

[0129] mAb drug products were triple-tested, and the results were statistically analyzed to determine the mean concentration, standard deviation, and relative standard deviation.

[0130] Linearity was evaluated by two analysts using five replicates of independent analyses. The coefficient of determination (R²) for each curve. 2...was determined by linear regression. Accuracy was determined using the spike recovery approach. Two analysts tripled the introduction of 20 ppm PS80 into samples without PS80 in the formulation for five analysis cases. Specificity was also verified using these formulations. Specificity was also evaluated by the resolution of PS80 mono-ester and oleic acid peaks in the resolution check solution and by ensuring the absence of interference peaks within the elution window (± 0.5 min) of PS80 mono-ester during the injection of a 90% organic solvent / 10% water blank. Since CAD is a universal detector and sometimes absorbs small trace contaminants, peak specificity was demonstrated by the absence of peaks exceeding 2% of the standard area. The signal-to-noise ratio (S / N) was estimated to be ≥ 10 for the 2 ppm PS80 solution.

[0131] Table 3 Weight-adjusted peak area of ​​PS80 mono-ester.

[0132]

[0133] Various sources and types of PS80 were tested using this method (Table 3). As demonstrated by Figure 4, the chromatograms of each PS80 source were visually equivalent. In summary, Figure 4 shows the chromatograms for various sources of PS80 (solid line) and blank (dotted line): (A) All-oleate ChP-compliant PS80; (B) Sigma-Aldrich PS80 stored in an amber glass container; (C) Croda Super-Refined PS80; (D) Sigma-Aldrich PS80 stored in a natural-colored plastic container; and (E) Multi-Compendial J.T. Baker PS80.

[0134] Assuming the same PS80 mono-ester is used in all-oleate PS80 and multi-compensated J.T. Baker PS80, the peak area of ​​all-oleate PS80 would be higher as a result of its slightly larger molecular weight. The discrepancy in the synthesis pathway for polysorbate has led to the observation of some variability in subspecies between batches. Physicochemical properties have been proven to vary from batch to batch. As demonstrated by alternative storage containers for Sigma-Aldrich PS80, it is recommended that PS80 standard solutions be prepared from material from the same source and lot as the PS80 used in biopharmaceutical formulations.

[0135] Polysorbate 40 (PS40, polyoxyethylene (20) sorbitan mono-palmitate) and polysorbate 60 (PS60, polyoxyethylene (20) sorbitan mono-stearate) were also evaluated (Fig. 5), and their respective chromatographic profiles separated the mono-ester from the multi-ester. In summary, Fig. 5 shows chromatograms for various polysorbates (solid lines) and blanks (dotted lines): (A) Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate); (B) Polysorbate 40 (PS40, polyoxyethylene (20) sorbitan mono-palmitate); (C) Polysorbate 60 (PS60, polyoxyethylene (20) sorbitan mono-stearate); and (D) polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). Each chromatographic profile separated the mono-ester from the multi-ester. PS60 appears to contain two major mono-ester forms or a significant amount of POE isosorbide mono-ester; further investigation via mass spectrometry may be necessary to identify the identity of these peaks. Multi-compensated polysorbate 20 (PS20, polyoxyethylene (20) sorbitan mono-laurate) was also included, but a complex chromatogram was generated (SM Fig. 7). A complex chromatogram with PS20 has been previously reported, and a simpler chromatogram was achieved by using all-laurate PS20. PS60 appears to contain two major mono-ester forms or a significant amount of POE isosorbide mono-ester; Further investigation using mass spectrometry may be necessary to identify the nature of these peaks.

[0136] Therefore, this method has a useful application for the PS20, PS40, and PS60.

[0137] To evaluate the accuracy and specificity of the method in terms of PS80 monoesters, samples with complete monoester degradation were spiked with mAb and analyzed. PS80 monoester degradation was achieved by incubating the samples at 5°C for 36 months (Fig. 6). In summary, Fig. 6 shows the chromatographic profile of PS80 monoesters obtained using the HPLC-CAD method, which is a sample with complete monoester degradation spiked with mAb and analyzed. PS80 monoester degradation was achieved by incubating the samples at 5°C for 36 months. The figure shows an overlay of chromatograms collected via the novel CAD method demonstrating peak broadening of the multiester due to oxidative degradation in the mAb products stored at 5°C and -20°C for 36 months. Additionally, the near-complete degradation of the monoester corresponds to an increase in POE groups. The standard is multi-compensated J.T. It was Baker PS80. The peak at 17.5 min is variable contaminant from the pre-rinsed Eppendorf tube. Analysis of the decomposed sample revealed the absence of a quantifiable amount of PS80 mono-ester ( <LOQ)를 확인하였다. 예상된 바와 같이, PS80 모노-에스테르의 분해는 POE 기의 현저한 증가를 생성하였다. 후속 분해가 모노-에스테르 정량화를 방해하는지를 결정하기 위해, 스파이킹된 회수 접근법을 사용하였다. 분해된 샘플 제제를 200 ppm PS80 샘플 농도와 상관관계가 있는 20 ppm PS80으로 스파이킹하였다. PS80 모노-에스테르는 93%의 값으로 회수되었으며, 이는 분해 산물의 유의한 증가가 모노-에스테르의 검출에 불리한 영향을 미치지 않는다는 것을 확인시켜 준다. 추가로, 다중-에스테르 피크는 약간 감소하였고, 피크 확장을 입증하였다. 이는 아마도 라디칼-유도된 분해에 참여한 후 약간 상이한 소수성 및 / 또는 크기를 갖는 산화된 분해물의 분해 및 재형성에 기인할 수 있다.

[0138] Comparison of ELSD and CAD Methods:

[0139] A series of samples were prepared and tested using two methods. The samples were formulation buffers and mAb products stored for 36 months at 5°C and -20°C with or without a single freeze-thaw (FT) cycle. Since the modified ELSD method involves orientation during the first 4 minutes, it is stated that POE groups and proteins do not pass through the detector, as inferred from previously reported methods. A direct comparison of total esters in the two methods is summarized in Table 4 and demonstrated excellent agreement.

[0140] Table 4 Comparison of the modified HPLC-ELSD method and the novel CAD method using a 36 M mAb sample at 5℃. Percent match was calculated as % match = [C2 / C1] * 100%, where C1 and C2 are the total PS80 ester concentrations determined by the HPLC-ELSD and HPLC-CAD methods, respectively.

[0141]

[0142] This method was validated with IgG1, IgG2, and IgG4 mAbs (Table 5). During this investigation, some precipitation solvents (e.g., acetone, THF) were found to have low or poor recovery of PS80 mono-esters or subspecies in the formulation buffer (data not shown).

[0143] Various mAb formulations with proven novel CAD methods.

[0144]

[0145] Example 2:PS80 Kinetics Study: Concentration-time data were obtained by quantifying the amounts of PS80 monoesters and subspecies at each time point (initial, 1, 2, 4, 7, 14, and 21 days) using a novel HPLC-CAD method (Fig. 7). In summary, Fig. 7 shows the kinetics of PS80 degradation in samples at 5, 25, 40, or -70°C up to 21 days. (A) monoesters, (B) multiesters (di-, tri-, and tetra-esters), (C) POE groups, and (D) total mass balance were quantified. Here, the PS80 monoester peaks were truly quantitative, while the subspecies were semi-quantitative. Concentration-time data were obtained by quantifying the amounts of PS80 monoesters and subspecies at each time point (initial, 1, 2, 4, 7, 14, and 21 days) using a novel HPLC-CAD method. For the actually quantifiable PS80 monoester, an Arrhenius plot was compiled for the degradation of PS80 monoester with rate constants for the 5, 25, and 40°C data to generate an activation energy of 35.8 ± 7.2 kJ / mol for PS80 monoester; a linear least-squares fitting yielded y = 4311.5x - 0.1696, where R 2= 0.961 (Fig. 8). Since Kishore reports an activation energy of ~35 kJ / mol for the first ~30% degradation of PS80, the observed activation energy is similar to previously published observations regarding PS80 hydrolysis. However, the analytical method used did not possess the specificity to distinguish between mono- and multi-ester forms. In the inventors' study, there was almost no peak broadening of the multi-ester because degradation was likely mostly hydrolytic due to the presence of lipase. Therefore, the POE group and multi-ester were also quantified (Fig. 7). The mass balance was calculated by summing the concentrations (ppm) of the POE group, PS80 mono-ester, and multi-ester; the precision for all mass-balance data was <10%. A negligible amount of oleic acid was observed at 40°C.

[0146] In the multi-compensated J.T. Baker PS80, the PS80 monoester was significantly less stable than the multiester. This is consistent with previously published data. While the monoester is highly degradable, protection from protein aggregation or colloidal stability can still be achieved by the large amount of multiester remaining.

[0147] In the past, suspected protein-PS80 interactions were thought to lower the initial amount of PS80 determined in protein-containing drug products. Interestingly, this method can also determine whether rapid degradation occurs, as the PS80 mono-ester concentration will decrease and a corresponding increase in POE groups will be present. In the absence of an increase in POE groups, this is most likely due to an interaction with the protein or the container.

[0148] conclusion:

[0149] A novel, sensitive, and specific platform assay method for PS80 in biopharmaceutical formulations using HPLC-CAD has been developed. The method utilizes protein precipitation to prevent specificity and mitigate potential interference that terminates any active degrading enzymes (e.g., lipases and esterases). Specificity was demonstrated using PS40, PS60, and various types of PS80. The application of the method using multiple types of IgG mAbs provided further support for the specificity achievable by this method using fresh and heavily degraded drug products.

[0150] The above method was suitable for demonstrating the specificity of chromatography to monitor the degradation of PS80 mono-esters, POE sorbitan / isosorbide, fatty acids, and multiple ester subspecies. The qualification study evaluated the method for repeatability (2.2% RSD), medium precision (6.5% RSD), accuracy (101% recovery), and linearity (mean R). 2 ≥ 0.999), specificity (matrix and R s It was concluded that adequate performance was demonstrated in relation to ≥1.5 oleic acid / PS80 mono-ester (no interference peaks observed), and the limit of quantification (~20 ppm for samples and 2 ppm for protein-deficient samples). An investigation of heavily degraded mAb drug products demonstrated that PS80 mono-ester degraded below LOQ and acceptable recovery was achievable (93%). It should be noted that the reduction of PS80 mono-ester was associated with an increase in POE groups. The heavily degraded study also enabled a comparison of the specific method with established methods having low specificity by quantifying all esterified PS species.

[0151] Therefore, in conclusion, the analytical CAD method described above has been proven to provide selective, sensitive, and specific quantitative and qualitative information regarding PS80 in biopharmaceutical products. Its potential for use as a platform method for purposeful validation has been demonstrated using multiple sub-types of IgG mAbs (IgG1, IgG2, and IgG4) by employing variations in the precipitation solvent. Thus, this method is a valuable tool to support stability studies for these mAbs and other biopharmaceutical drug products.

Claims

Claim 1 A method for identifying polysorbates in a sample containing proteins, comprising: (i) a step of precipitating the protein by exposing the sample containing the protein to an organic protic polar solvent or an organic aprotic polar solvent; (ii) a step of separating the protein from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide and obtaining a liquid supernatant; (iii) a step of separating polysorbates by applying the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column containing a fixed cyano group and eluting the bound polysorbates using a mobile phase composition gradient; and (iv) a step of detecting the separated polysorbates using a chromophore-lack detector to identify the polysorbates. Claim 2 A method for identifying intact polysorbate and / or degraded polysorbate products in claim 1. Claim 3 A method according to claim 1 or 2, further comprising quantifying polysorbate. Claim 4 A method according to claim 1 or 2, wherein the protein sample comprises an antibody. Claim 5 A method according to claim 1 or 2, wherein the protein is a monoclonal antibody or a fragment thereof. Claim 6 A method for detecting a polysorbate selected from any one of PS80, PS60, PS40 and PS20 in claim 1 or 2. Claim 7 A method according to claim 1 or 2, wherein the chromophore-lack detector is a charged aerosol detector (CAD). Claim 8 A method according to claim 1 or 2, wherein the protein sample comprises an acetate or citrate buffer. Claim 9 A method according to claim 1 or 2, wherein the protein in the sample is present at a concentration of 5 mg / ml to 300 mg / ml. Claim 10 A method according to claim 1 or 2, wherein the protein precipitation is performed using a solvent selected from methanol, isopropyl alcohol (IPA), THF, or acetone. Claim 11 A method according to claim 1 or 2, wherein the separation of the polysorbate is performed using a reverse-phase HPLC column containing immobilized cyano groups. Claim 12 In claim 11, the method wherein the column is a silica column having a pore size of ≥ 80 Angstroms. Claim 13 A method according to claim 1 or 2, wherein the elution is performed using a gradient-separated mobile phase composed of buffer A and buffer B, wherein buffer A is a mixture of 0.1% trifluoroacetic acid (TFA) in H2O and buffer B is methanol or acetonitrile. Claim 14 A method according to claim 1 or 2, wherein the separation of the polysorbate is performed using a heated column having a temperature of 20°C to 80°C. Claim 15 A method according to claim 1, comprising: (i) precipitating a protein 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 obtaining a liquid supernatant; (iii) separating polysorbate by applying the supernatant to a reverse-phase HPLC on a silica column having a pore size of 120 Angstroms to 300 Angstroms containing fixed cyano groups and eluting using a mobile phase composition gradient consisting of A and B, wherein A is a mixture of 0.1% trifluoroacetic acid (TFA) in H2O and B is methanol or acetonitrile; and (iv) detecting the separated polysorbate using a charged aerosol detector (CAD) to identify the polysorbate. Claim 16 (a) (i) a step of precipitating a protein by exposing a protein sample to an organic protic polar solvent or an organic aprotic polar solvent; (ii) a step of separating the protein from the precipitated sample by centrifuging the precipitated sample to pellet the protein or peptide and obtaining a liquid supernatant; (iii) a step of separating polysorbates by applying the supernatant to chromatography, wherein the chromatography comprises applying the supernatant to a stationary phase column containing fixed cyano groups and eluting the bound polysorbates using a mobile phase composition gradient; and (iv) a step of measuring polysorbates in the sample, comprising detecting the separated polysorbates using a chromophore-lack detector to identify the polysorbates; (b) a step of identifying protein sample(s) from (a) having a level of intact polysorbate of 10 ppm to 5000 ppm; (c) a step of separating and recovering said protein(s) identified in step (b), a method for identifying a protein sample containing intact polysorbate of 10 ppm to 5000 ppm. Claim 17 A method according to claim 16, wherein the isolated protein sample in step (c) contains 10 ppm to 700 ppm of intact polysorbate. Claim 18 A method according to claim 16 or 17 in which the polysorbate is PS80. Claim 19 A method according to claim 16 or 17, wherein the chromophore-lack detector is a charged aerosol detector (CAD). Claim 20 Protein obtained or obtainable according to the method of paragraph 16 or 17. Claim 21 In paragraph 20, a protein that is a monoclonal antibody or a fragment thereof. Claim 22 A medicine for the therapy or diagnosis of a human subject comprising a protein according to paragraph 20. Claim 23 A pharmaceutical preparation comprising a protein according to paragraph 20 in combination with one or more pharmaceutically acceptable excipients. Claim 24 delete Claim 25 delete

Images