Method for determining the degree of oxidative degradation of polysorbate 20 in aqueous formulations

Abstract

A method is provided for determining the degree of oxidative degradation of polysorbate (20) in an aqueous formulation or in several aqueous formulations, in particular biopharmaceutical formulations, each containing polysorbate (20), using a liquid chromatography-mass spectrometry-based method in which the relative peak area of ​​the compound polyethylene glycol-6-laurate, a marker substance for the oxidative degradation of polysorbate (20), is determined by comparing a reference sample with the sample being investigated. The relative peak area for the sample under investigation is proportional to the degree of oxidative degradation of polysorbate (20) in the sample. The method can also be performed without the use of a reference sample by comparing the peak areas of polyethylene glycol-6-laurate of two or more aqueous formulations with each other. Further provided is a process for producing a recombinant protein, comprising, inter alia, formulating the recombinant protein using polysorbate (20) into a pharma- ceutical acceptable aqueous formulation suitable for administration, obtaining therefrom at least one sample containing the recombinant protein and polysorbate (20), and detecting the degree of oxidative degradation of polysorbate (20).

Description

Technical Field

[0001] The present invention relates to a method for determining the degree of oxidative degradation of polysorbate 20 in aqueous formulations.

Background Art

[0002] Polysorbates (PS) constitute a group of nonionic surfactants and are widely used as wetting agents or emulsifiers, for example, in cosmetics, pharmaceuticals, foods, detergents, and cleaners. Due to their biocompatibility and low toxicity, polysorbates are the most widely used surfactants for biopharmaceutical formulations and function as excipients for biopharmaceutical formulations that solubilize substances such as proteins that are not water-soluble per se in water. In particular, they stabilize proteins against interfacial tension and prevent interface-induced aggregation or surface adsorption. In this way, they help to stabilize components, such as proteins, in aqueous formulations and thus maintain the activity and effectiveness of the components. Polysorbates are derived from a core structure of sorbitan or isosorbide linked to poly(oxy)ethylene (POE) chains of variable length. These can then be esterified with fatty acids. Exemplary representatives of polysorbates are polysorbate 20 (PS20) or polyoxyethylene (20) sorbitan monolaurate; polysorbate 40 (PS40) or polyoxyethylene (20) sorbitan monopalmitate; polysorbate 60 (PS60) or polyoxyethylene (20) sorbitan monostearate; and polysorbate 80 (PS80) or polyoxyethylene (20) sorbitan monooleate respectively.

[0003] The numerical values 20, 40, 60, and 80 following the term "polysorbate" represent the types of fatty acids associated with the polyoxyethylene sorbitan residues of the molecule. That is, monolaurate is indicated by 20, monopalmitate is indicated by 40, monostearate is indicated by 60, and monooleate is indicated by 80. The numerical value 20 following the term "polyoxyethylene" refers to the total number of oxyethylene-(CH2CH2O)-groups present in the molecule. Commercially available polysorbates usually consist of a mixture of structurally related molecules such as non-esterified sorbitan and / or isosorbitan-polyethylene glycol (PEG) species, mono, di, tri, tetra esters, fatty acids, etc. The two main types of polysorbates, namely polysorbate 20 (PS20) and polysorbate 80 (PS80), are the most relevant to biopharmaceutical formulations, but mainly differ in the distribution of the elements of the fatty acid and poly(oxy)ethylene chains. Polysorbate 40 and 60 are more commonly used in the food or cosmetic industries. Polysorbates exhibit several decomposition reactions, which result in the formation of many decomposition products, loss of functional properties, and potentially lead to the instability of the aqueous formulations used. Therefore, the decomposition of polysorbates can result in particle formation in aqueous formulations and can be a major quality issue and potential risk factor.

[0004] For the degradation of polysorbates observed in biopharmaceutical products, for example, there are two major degradation pathways: hydrolysis, typically catalyzed by host cell impurities, representing ester hydrolysis and resulting in the liberation of free fatty acids, and oxidative degradation, initiated by free radicals and also called auto-oxidation. As is generally known, auto-oxidation can occur at any location within a molecule as long as the kinetic characteristics of H abstraction at a given site are favorable. In this context, for complex molecules such as polysorbate 20, it must be considered that a very large number of degradation products that can be detected by several different analytical techniques can generally be present. In [1, 2], for example, about 60 different species are envisioned. The auto-oxidation of unsaturated fatty acids, particularly oleic acid, has been extensively studied for decades due to its importance in the oxidation of lipids in foods and oils, but little is known about the actual degradation pathways and stability of the individual components of polysorbates under oxidative stress.

[0005] The oxidative degradation of both polysorbate 20 and polysorbate 80 has already been described in the literature. These references aim to describe the mechanism of oxidative degradation that occurs either along the unsaturated fatty acid structure where double bonds are present [1] or within the poly(oxy)ethylene chain structure [2], and both factors are sensitive to free-radical-induced fragmentation.

[0006] For polysorbate 80, the first pathway is prevalent due to the presence of oleic acid containing its double bond. Thus, the specific individual components resulting from oxidative degradation, which appear during decomposition and can be detected by LC-MS (liquid chromatography-mass spectrometry) analysis, representing different substructures, are identified. Such individual components are also referred to as oxidation markers. For example, in [1], the major degradation products of polysorbate 20 and polysorbate 80 are evaluated by oxidative stress induced by 2,2’-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH). AAPH is a water-soluble azo compound used as a model oxidizing agent and free radical generator. The identified oxidation markers of polysorbate 80 include poly(oxy)ethylene sorbitan esterified with several structures such as hydroperoxy-oleate (hydroperoxy-C18:1), keto-oleate (keto-C18:1), hydroxyl-oleate (hydroxyl-C18:1), epoxy-stearate (epoxy-C18:0), and oxo-nonanoate (oxo-C9:0), and are described including the mass-to-charge ratio (m / z) of each ion used in a mass spectrometry method enabling its analysis by detection based on LC-MS.

[0007] For polysorbate 20, due to the loss of unsaturated fatty acids, the oxidative degradation of polysorbate mainly proceeds through the fragmentation of ether bonds within the poly(oxy)ethylene structure [2], which does not necessarily result in the emergence of new structures that can be used as marker structures. Thus, the current literature has not yet described any oxidation marker structures for analysis based on liquid chromatography-mass spectrometry (LC-MS) targeting the poly(oxy)ethylene substructure region.

[0008] According to Borisov et al. [1], it has been proposed that poly(oxy)ethylene esters containing 4 - 5 oxy-ethylene (OE) units are by-products of oxidative degradation. Kishore et al. [2] describe poly(oxy)ethylene esters containing 1 - 6 oxy-ethylene units. Zhang et al. [3] describe the formation of poly(oxy)ethylene esters (POE-laurates) containing 1 - 8 ethylene oxide (EO) units, where POE-laurate containing 6 EO units is the dominant mixture, which was under forced oxidation conditions where the solution was further saturated with O2 gas in the simultaneous presence of L-histidine and polysorbate 20. However, none of these studies provide the corresponding mass-to-charge ratios (m / z ratios) of the chemical structures and ions that enable detection and analysis via a method based on liquid chromatography-mass spectrometry (LC-MS). In the above-mentioned literature, it is demonstrated that all poly(oxy)ethylene-laurates, for example those having 1 - 8 oxy-ethylene units, originate from oxidative stress and from polysorbate 20 used as the stressed substance. This typically cannot be transferred to aqueous formulations under ambient conditions and is not applicable especially to biopharmaceutical formulations. In fact, this is not an effective approach for implementation.

[0009] More importantly, the degradation products were artificially generated using substances that generate free radicals, which are removed from the actual situation and the conditions of normal aqueous formulations. Furthermore, for example, the reality within natural biopharmaceutical formulations was not considered under everyday stabilization conditions. Additionally, the degradation products were not related to the oxidative degradation of polysorbate without using artificial radical initiators. Moreover, none of the degradation products are related to the mass-to-charge ratios (m / z ratios) of the ions that enable monitoring via a method based on liquid chromatography-mass spectrometry (LC-MS). In addition to label-free analysis based on liquid chromatography-mass spectrometry (LC-MS), the oxidative degradation of polysorbate has been investigated using other techniques. For example, the use of 2,4-dinitrophenylhydrazine for derivatization of free fatty acid esters resulting as by-products of oxidative stress applied to polysorbate 20 has been described [4]. On the other hand, charged aerosol detection coupled with reversed-phase chromatography is used [5]. However, these techniques do not reveal the individual degradation species of polysorbate under oxidative stress.

[0010] However, the degradation of polysorbate is a major issue, especially in the development of biopharmaceuticals, and can pose an important risk to successful product development, for example by deteriorating the quality of the product or increasing safety concerns. Therefore, understanding the causes and processes of polysorbate degradation is important for assembling suitable treatment strategies. According to current state-of-the-art techniques, it is not possible to differentiate between the hydrolysis and oxidative degradation of polysorbate by direct and clear analytical tools, and in particular, no substance is known to identify the presence of oxidative modification of polysorbate 20, that is, no marker substance is known to be derived from the oxidative degradation of polysorbate 20, which is widely used as a surfactant in aqueous formulations. Furthermore, to date, no analytical method has been described for detecting and quantifying such marker substances in aqueous formulations, especially in biopharmaceutical formulations.

[0011] Furthermore, the method of liquid chromatography-mass spectrometry (LC-MS) disclosed in, for example, [1] is not intended as a method for the absolute quantification of fatty acid esters in polysorbate and only allows semi-quantitative determination using this method. Furthermore, a method for studying the mechanism of polysorbate (PS) degradation is disclosed in

[13] . Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) 18 is used to characterize polysorbate 20 and its degradation products by measuring O incorporation, and the two main so-called degradation products were free lauric acid and polyoxyethylene (POE) laurate.

[0012]

[14] According to, a process for quantifying at least one polysorbate derivative of a sample, comprising: - performing LC-MS analysis of the sample based on the signal from the dioxolanilium ion; - performing internal calibration with an internal standard of polysorbate A process is provided. An internal standard refers to a chemical compound that is added in a fixed amount to a sample, blank, and calibration standard in chemical analysis. This is a compound that is generally very similar to, but not identical to, polysorbate in the sample. In one embodiment, the internal standard (IS) can be selected from compounds having a polyethylene glycol chain esterified with one or more carboxylic acids so as to have physicochemical properties similar to those of polysorbate.

[15] describes a method for reducing polysorbate degradation in an aqueous formulation containing polysorbate, which includes adding cyclodextrin to the formulation, wherein the resulting mass ratio of cyclodextrin to polysorbate is greater than about 37.5:1.

[0013] In fact, patents and academic literature as a whole do not fully address the use of specific marker substances, especially unlabeled oxidized marker species, that are specific to polysorbate 20 and are particularly related to naturally occurring oxidative polysorbate degradation. Furthermore, to date, no oxidation by-products have been identified as established markers for tracking the oxidation of polysorbate 20 in aqueous formulations containing polysorbate 20, especially in biopharmaceutical formulations containing biological APIs (pharmaceutical active ingredients) and common excipients. Accordingly, an object of the present invention is to provide a method that can overcome at least these prior art problems and identify and determine marker substances, especially unlabeled oxidized marker species, that are specific to polysorbate 20 and are particularly related to the oxidative degradation of polysorbate 20. This method should enable the monitoring and quantification of the oxidative marker substances in aqueous formulations. SUMMARY OF THE INVENTION

[0014] Surprisingly, a specific marker substance, namely polyethylene glycol-6-laurate (PEG-6-laurate), has been found to be associated with the oxidative degradation pathway of polysorbate 20, as this marker substance is formed during the oxidative degradation of polysorbate 20. This marker substance enables the determination of the degree of oxidative degradation of polysorbate 20 in an aqueous formulation by an analytical method, as the relative level of said marker substance indicates that oxidative degradation has occurred.

[0015] Accordingly, a (first) method (in a first alternative) for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20, comprising the following steps: Step 1 of preparing a first sample of the aqueous formulation to be tested, which functions as a reference sample; Step 2 of preparing a second sample and any further samples of the aqueous formulation to be tested; Separating polyethylene glycol-6-laurate and using a method based on liquid chromatography-mass spectrometry to determine the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, from the reference sample of step 1 and the second sample and any further samples of step 2, respectively; Step 3 of determining the relative peak area of polyethylene glycol-6-laurate based on the peak area of polyethylene glycol-6-laurate of the second sample and any further samples with respect to the peak area of polyethylene glycol-6-laurate of the reference sample, wherein the relative peak area is proportional to the degree of oxidative degradation of polysorbate 20; and Step 4 of optionally quantifying the amount of polyethylene glycol-6-laurate of the second sample and any further samples based on the relative peak area of each sample is provided.

[0016] According to one embodiment, the relative peak area of polyethylene glycol-6-laurate for the second sample of step 3 and each additional sample, if any, is - calculating the difference in the peak areas obtained by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak area of polyethylene glycol-6-laurate of the second sample and from the peak area of polyethylene glycol-6-laurate of each additional sample, if any; or - normalizing the peak areas obtained by setting the peak area of polyethylene glycol-6-laurate of the reference sample to 100% and calculating and normalizing the peak area of polyethylene glycol-6-laurate of the second sample and of each additional sample, if any, based thereon determined by.

[0017] Thus, the method of the present invention provides for the identification of polyethylene glycol-6-laurate as a by-product of the oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20, separates, detects and determines this marker substance with respect to a reference by a method based on liquid chromatography-mass spectrometry (LC-MS-based method), and enables the quantification of this marker substance in the aqueous formulation by a specific analytical method. Although it is known that the peak area (integration) is proportional to the concentration of the analyte, the relative peak area enables the correlation of the relative peak area of polyethylene glycol-6-laurate with the degree of oxidative degradation of polysorbate 20.

[0018] In a first alternative of the above (first) method, the first sample of the aqueous formulation to be tested is taken as the reference sample. In an alternative embodiment, an externally generated or provided reference sample is used as the reference, i.e., the external reference sample is provided independently. Accordingly, according to another embodiment, a (first) method (in a second alternative) for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20, the following steps: Step 1': preparing a reference sample which is an aqueous solution containing polysorbate 20 and has the same polysorbate 20 content as the aqueous formulation to be tested; Step 2': preparing a first sample of the aqueous formulation to be tested and any additional samples; Separating polyethylene glycol-6-laurate, and respectively using a method based on liquid chromatography-mass spectrometry to determine the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, from the reference sample of step 1', and the first sample and any additional samples of step 2'; Step 3': determining the relative peak area of polyethylene glycol-6-laurate based on the peak area of polyethylene glycol-6-laurate of the first sample and any additional samples with respect to the peak area of polyethylene glycol-6-laurate of the reference sample, wherein the relative peak area is proportional to the degree of oxidative degradation of polysorbate 20; and Step 4': optionally quantifying the amount of polyethylene glycol-6-laurate in the first sample and any additional samples based on the relative peak area of each sample A method is provided that includes.

[0019] According to one embodiment, the relative peak area of polyethylene glycol-6-laurate for the first sample and any additional samples of step 3' is - calculating the difference in the obtained peak areas by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak area of polyethylene glycol-6-laurate of the first sample and the peak area of polyethylene glycol-6-laurate of each of any additional samples, respectively, to calculate; or - normalizing the obtained peak areas by setting the peak area of polyethylene glycol-6-laurate of the reference sample to 100% and calculating and normalizing the peak areas of polyethylene glycol-6-laurate of each of the first sample and any further samples based thereon is determined by

[0020] In one embodiment of the present invention, in step 2 or step 2', several samples of the aqueous formulation containing polysorbate 20 are taken at successive times T1, T2, T3...., and it is possible to determine not only random samples in the aqueous formulation over time but also possible oxidative degradation pathways of polysorbate 20. For example, in step 2, two, three or more further samples of the aqueous formulation containing polysorbate 20 can be taken at successive time intervals T1, T2, T3.... which can be of the same length or different lengths.

[0021] In the first method (first and second alternatives) described above, one aqueous formulation was tested for oxidative degradation of polysorbate 20. However, several aqueous formulations can also be examined. Thus, in a further embodiment of the present invention, the following steps: Step 1a of preparing a sample of each aqueous formulation to be examined; Separating polyethylene glycol-6-laurate and using a method based on liquid chromatography and mass spectrometry to determine the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, from each sample of step 1a Step 2a of comparing the peak areas of the samples with each other, wherein the difference in the peak areas of the samples (relative peak areas) is proportional to the degree of oxidative degradation of polysorbate 20 in each respective sample; and Step 3a of optionally quantifying the amount of polyethylene glycol-6-laurate in each sample based on the obtained peak areas of each sample Provided is a (second) method for determining the degree of oxidative degradation of polysorbate 20 in several aqueous formulations each containing polysorbate 20, in particular in several aqueous formulations, such as two, three, four or more aqueous formulations, where each aqueous formulation contains the same amount of polysorbate 20.

[0022] In this (second) method of the invention, the peak areas can be directly compared to each other, or the relative peak areas can be determined using a reference and then the samples can be compared to the reference. The reference can be one of the samples taken, and the selection of the sample that can be used as the reference depends on the individual case. The reference can also be an external reference sample as already described in connection with the first method.

[0023] In the (second) method, the relative peak area of polyethylene glycol-6-laurate of each sample can be determined relative to a reference sample, and this procedure is - calculating the difference in the peak areas obtained by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak area of polyethylene glycol-6-laurate of each sample in step 2a; or - normalizing the peak areas obtained by setting the peak area of polyethylene glycol-6-laurate of the reference sample to 100% and calculating and normalizing the peak area of polyethylene glycol-6-laurate of each sample in step 2a based thereon and is carried out thereby.

[0024] The separation of polyethylene glycol-6-laurate, identified as an oxidative marker substance for the oxidative degradation of polysorbate 20, is achieved by liquid chromatography as part of a method based on liquid chromatography-mass spectrometry by using its hydrophobicity. In subsequent mass spectrometry procedures, the molecules to be examined are transferred to the gas phase and ionized. The molecules can be fragmented by this method. Therefore, the detection and determination of the relative levels of polyethylene glycol-6-laurate in step 3, step 3' or step 2a are carried out using a method based on liquid chromatography-mass spectrometry. Any quantification in step 4, step 4' or step 3a can be carried out, for example, by external calibration using a standard solution of polyethylene glycol-6-laurate. According to one embodiment of the method based on liquid chromatography-mass spectrometry, the liquid chromatography is used as separation chromatography, especially in the form of column chromatography, more specifically as reverse-phase chromatography. In a further embodiment, the liquid chromatography as part of the method based on liquid chromatography-mass spectrometry is high-performance liquid chromatography (HPLC). Ultra-high performance liquid chromatography (UPLC) can also be used. According to one embodiment of the method based on liquid chromatography-mass spectrometry in liquid chromatography, gradient elution is used.

[0025] In another embodiment, a polar solvent is added to the polysorbate-containing sample to be examined before performing the liquid chromatography, especially the solvent used in the liquid chromatography. In a further embodiment, the mass spectrometry as part of the method based on liquid chromatography-mass spectrometry is selected to be high-resolution mass spectrometry (HR-MS). High-resolution mass spectrometry can be especially used for structure elucidation and is advantageous for this purpose. According to one embodiment, for data evaluation of a method based on the liquid chromatography mass spectrometry method of the present invention, an extracted ion chromatogram is created from a mass-to-charge ratio that includes a range of about 487 amu, particularly includes or consists of the range of 486.5 to 487.5 amu.

[0026] Any optional additional step 5 that may follow step 4 or step 3, or any optional additional step 5' that may follow step 4' or step 3', or any optional additional step 4a that may follow step 3a or step 2a includes estimating to what extent polysorbate 20 has already been oxidatively decomposed in the sample based on the inverse proportional relationship between the polyethylene glycol-6-laurate level and the polysorbate 20 level. A decrease in the content of polysorbate 20 in the sample and thus in the aqueous formulation was found to clearly correlate with an increase in the relative level of the oxidative marker substance polyethylene glycol-6-laurate, whereby polysorbate 20 was found to increase increasingly.

[0027] According to a further embodiment of the present invention, the aqueous formulation contains one or more proteins. The protein can be selected from monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, single-chain antibodies or antibody fragments thereof, such as Fv, scFv, Fab, Fab', scFab, F(ab')2, Fab2, Fc and Fc' fragments, immunoglobulin heavy and light chains and their constant, variable or hypervariable regions and Fv and Fd fragments, bispecific antibodies, trispecific antibodies, scFv-Fc, minibodies, or single-domain antibodies, or mixtures thereof. The protein can be selected from therapeutic proteins used for the prevention or treatment of diseases or disorders. According to another embodiment, the aqueous formulation can be an aqueous pharmaceutical formulation, particularly an aqueous biopharmaceutical formulation, containing polysorbate 20. For example, the aqueous biopharmaceutical formulation can contain pharmaceutically acceptable polysorbate 20, optionally an API such as one or more proteins, and any pharmaceutically acceptable excipients, and is used for the prevention or treatment of diseases or disorders. The present polyoxyethylene sorbitan monolaurate 20 may be intended for pharmaceutical use, especially for use in drugs. In particular, polyoxyethylene sorbitan monolaurate 20 may be pharmaceutically acceptable and may have a pharmaceutical grade quality, such as that of the European / US / Chinese pharmacopoeia. For example, polyoxyethylene sorbitan monolaurate 20 may be suitable for parenteral administration. The following commercial products of polyoxyethylene sorbitan monolaurate 20 are illustratively described: Super Refined™ PS20, Tween™ 20, Tween™ 20 HP, Tween™ 20 pharmaceutical grade or PS20 HP, which are available in particular from Croda, Edison, NJ, USA or Croda Europe Ltd., or PS20 China grade, which is available in particular from Nanjing Well Pharmaceutical Co., Ltd.

[0028] The present invention also relates to a process (third method) for producing a recombinant protein, comprising the following steps: Step a) of culturing eukaryotic cells expressing the recombinant protein in cell culture; Step b) of harvesting the recombinant protein, Step c) of purifying the recombinant protein using an aqueous formulation; and Step d) of formulating the recombinant protein into a pharmaceutically acceptable aqueous formulation suitable for administration using polyoxyethylene sorbitan monolaurate 20; and Step e) of obtaining at least one sample of the pharmaceutically acceptable aqueous formulation of step d); comprising The process (third method) further comprises performing the (first or second) method for determining the degree of oxidative degradation of polyoxyethylene sorbitan monolaurate 20 disclosed in the present invention on the sample obtained in step e).

[0029] According to a further embodiment, the process (third method) for producing a recombinant protein comprises the step of obtaining at least one sample containing the recombinant protein and polyoxyethylene sorbitan monolaurate 20 in step e) as described above, and the sample from step d) is a drug substance sample or a pharmaceutical product sample. The present invention also relates to a method for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation or several aqueous formulations each containing polysorbate 20, in particular to the use of polyethylene glycol-6-laurate as an oxidation marker in the (first or second) method disclosed herein. A further object of the present invention is the use of the (first or second) method disclosed herein as a development, production, shelf-life, or stability test during storage of an aqueous formulation or several aqueous formulations each containing polysorbate 20 for determining the degree of oxidative degradation of polysorbate 20 in the aqueous formulation.

[0030] Embodiments of the present invention are described by way of example with reference to the accompanying drawings. The drawings of the present disclosure are incorporated into and form part of this specification, and show embodiments of the present invention without limiting the specific embodiments described herein. The drawings, together with the general description and the detailed description, serve to explain the principles of the present disclosure.

Brief Description of the Drawings

[0031]

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Mode for Carrying Out the Invention

[0032] Definition of General Terms Terms not specifically defined herein should be given the meanings ascribed to them by those skilled in the art in light of the present disclosure and context.

[0033] The term "polysorbate 20" or "PS20" refers to polyoxyethylene (20) sorbitan monolaurate and is commercially available as a mixture of structurally related molecules containing the major sorbitan polyoxyethylene fatty acid esters and substantial amounts of polyoxyethylene, sorbitan polyoxyethylene, and isosorbide polyoxyethylene [1, 2, 4]. The molecular heterogeneity of polysorbates results from variability within its three functional groups, namely, polydispersity with respect to the length of the EO chains in the hydrophilic head, differences in chain length (number of carbon atoms), and degree of unsaturation of the fatty acids in the hydrophobic tail, as well as the presence of species containing a core consisting of sorbitol monoanhydride and dianhydride (sorbitan and isosorbide, respectively). Further, depending on the number of fatty acid esters per structure, polysorbates can contain mono-, di-, tri-, and tetra-esters. An overview of the compositions of various polysorbates is provided in Table 1 of [1].

[0034] According to the specifications of the European Pharmacopoeia (Ph.Eur.), with respect to the fatty acid content, lauric acid esters account for 40 - 60% of the esters in polysorbate 20, and the remaining esters are in the range of C8 - C18. However, for the sake of simplicity, it is customary to consider PS20 as the polyethoxylated (POE) sorbitan monoester of lauric acid. [6], The theoretical structure of polysorbate 20 shown in Figure 1 is as follows:

[0035]

Chemical Structure

[0036] The term "oxidation marker" or "oxidative marker" (also abbreviated as "oxmarker") as used herein means a compound related to the oxidative degradation of polysorbate 20, particularly a compound formed during its oxidative degradation. Thus, the presence of an oxidation marker at a higher level than in the case of its impurities means that the oxidative degradation of polysorbate 20 has occurred or is occurring. The oxidation marker herein is polyethylene glycol-6-laurate. The expression "oxidative degradation" of polysorbate 20 refers to a free-radical-initiated degradation, also known as autoxidation. Oxidative degradation is an ester hydrolysis that is typically catalyzed by impurities in the host cell and results in the release of free fatty acids, unlike the hydrolysis of polysorbate 20.

[0037] The term "degree of oxidative degradation" as used in the present invention indicates the extent of oxidative degradation of polysorbate 20, particularly how far the oxidative degradation has already progressed, so that it is possible to estimate how much or to what extent polysorbate 20 has already been oxidatively degraded. The degree of oxidative degradation is proportional to the relative or absolute amount or level of the oxidation marker polyethylene glycol-6-laurate in an aqueous formulation containing polysorbate 20. The relative amount or level in a sample means relative to a reference sample or to the amount or level of another sample. The absolute amount or level is the actual amount analytically determined by quantitative analysis.

[0038] The term "proportional" in the expression "[the relative peak area of polyethylene glycol-6-laurate] is proportional to the degree of oxidative degradation of polysorbate 20" and similar phrases should be understood to mean that there is a relationship or correlation between the relative peak area of the oxidation marker polyethylene glycol-6-laurate in a sample and the degree of oxidative degradation of polysorbate 20. In particular, a proportional relationship or more precisely an inverse proportional relationship exists, i.e., the (relative or absolute) amount or level of polyethylene glycol-6-laurate and the (relative or absolute) amount or level of polysorbate 20 are uncorrelated with each other, as will be explained in more detail later. The term "level" should be understood in its broadest sense and means the amount, portion, content, or concentration of a compound present in an aqueous liquid, particularly a reference sample or an aqueous formulation. "Relative level" means the amount, portion, content, or concentration of a compound expressed relative to or with respect to a reference.

[0039] The term "aqueous" is intended to mean that water is present in the formulation. For example, water can constitute part of the solvent, such as a portion or the major portion of the solvent, or can represent the only solvent present. In fact, any type of water can be used. Purified water may be preferred in some cases, but according to some embodiments, tap water can also be used. The type of water selected depends on the intended use of the aqueous formulation. The purified water used in the present invention is water that has been subjected to purification processes such as distillation, reverse osmosis, carbon filtration, volumetric or electro-deionization, microfiltration or ultrafiltration, ultraviolet oxidation, etc. to remove impurities so as to be suitable for use. Combinations of these processes can also be used to achieve high-purity water, such as ultrapure water, where the trace contaminants are measured in parts per billion (ppb) or parts per trillion (ppt). The water used can also be ultrapure water, such as type 1 ultrapure water (Milli-Q® water) according to ASTM D1193 or ISO3696. The water used can also be sterile water suitable for administration to a subject, such as water for injection (WFI). Also, distilled water, double-distilled water or deionized water can be used. The term "aqueous formulation" refers to a solution in which one of the solvents or the solvent is water. The solution includes true solutions, dispersions, suspensions, etc., unless otherwise described. The aqueous formulations according to the present invention include polysorbate and any one or more proteins and any excipients.

[0040] The term "protein" (used interchangeably with "polypeptide" and "amino acid residue sequence") refers to a polymer of any amino acids (natural or artificial) of any length. The term "protein" also includes proteins that are post-translationally modified through reactions including, but not limited to, glycosylation, glycation, acetylation, phosphorylation, oxidation, amidation or protein processing. Modifications and changes, such as fusions to other proteins, amino acid sequence substitutions, deletions or insertions, can preferably result in the structure of the polypeptide while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can result in a polypeptide or its underlying nucleic acid coding sequence and obtain a protein with similar properties. Amino acid modifications can be prepared, for example, by performing site-directed mutagenesis or mutagenesis via polymerase chain reaction in its underlying nucleic acid sequence. Thus, the term "protein" or "polypeptide" also includes, for example, fusion proteins consisting of immunoglobulin components, such as Fc components, and growth factors, such as interleukins.

[0041] The term "protein" includes proteins, polypeptides, fragments thereof, peptides, fusion proteins, all of which can be expressed in a host cell line or synthesized via chemical methods. The desired protein can be, for example, an antibody, enzyme, cytokine, lymphokine, adhesion molecule, receptor and derivatives or fragments thereof, and any other protein or polypeptide that functions as an agonist or antagonist and / or can have therapeutic or diagnostic uses.

[0042] Furthermore, the desired protein or polypeptide may be, for example, but not limited to: insulin, insulin-like growth factor, hGH, tPA, cytokines such as interleukins (IL) such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosis factor (TNF) such as TNF alpha and TNF beta, TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1, VEGF and single domain antibodies (e.g., from camelids). Also included is the production of erythropoietin or any other hormone growth factor and any other polypeptide that can function as an agonist or antagonist and / or have therapeutic or diagnostic use. Thus, the protein can be, for example, an antibody such as a monoclonal antibody, polyclonal antibody, monospecific antibody, multispecific antibody, single-chain antibody or an antibody fragment thereof such as Fv, scFv, Fab, Fab’, scFab, F(ab’)2, Fab2, Fc and Fc’ fragments, immunoglobulin heavy and light chains and their constant, variable or hypervariable regions and Fv and Fd fragments, bispecific antibody, trispecific antibody, scFv-Fc, minibody, or single domain antibody, or a mixture thereof.

[0043] As used herein, the term "antibody" generally refers to a monospecific antibody, although an antibody can also be multispecific or a fragment thereof. Exemplary antibodies within the scope of the present invention include, but are not limited to, antibodies against CD2, CD3, CD20, CD22, CD30, CD33, CD37, CD40, CD44, CD44v6, CD49d, CD52, EGFR1 (HER1), EGFR2 (HER2), GD3, IGF, VEGF, TNF alpha, IL2, IL-5R, IL-36R or IgE, preferably selected from the group consisting of antibodies against CD20, CD33, CD37, CD40, CD44, CD52, HER2 / neu (erbB2), EGFR, IGF, VEGF, TNF alpha, IL2, IL-36R and IgE.

[0044] As used herein, the terms "antibody", "antibodies", or "immunoglobulin" refer to proteins selected from immunoglobulins, which are naturally formed as a reaction of the host organism to foreign substances (= antigens) from differentiated B lymphocytes (plasma cells). There are various classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW. An antibody can be an IgG antibody such as an IgG1 antibody or an IgG4 antibody. The terms "immunoglobulin" and "antibody" are used interchangeably herein. Antibodies include monoclonal, monospecific and multispecific (e.g., bispecific or trispecific) antibodies, single-chain antibodies, antigen-binding fragments of antibodies (e.g., Fab or F(ab')2 fragments), disulfide-linked Fvs, etc. Antibodies can be of any species, including chimeric and humanized antibodies.

[0045] The term "sample" is understood as broadly as possible to mean a limited or smaller amount of an aqueous preparation taken for analysis. Sampling can be done manually or by an automated method. The term "liquid chromatography mass spectrometry" or "LC-MS method" refers to a method known in the prior art based on a specific analytical chemistry technique that combines physical separation by liquid chromatography (or HPLC) with mass spectrometry (MS) using mass spectrometry. This expression is intended to include any technique that combines liquid chromatography and mass spectrometry known to those skilled in the art. Coupled liquid chromatography-MS systems are known in chemical analysis. These typically show the advantage of synergistically improving the two methods in combination. LC-MS systems also contain certain types of interfaces that allow substances separated by the LC procedure to enter the MS ion source. The interface is necessary because the LC device and the MS device are not actually compatible with each other. In the LC system, the mobile phase is under pressure, while the MS analyzer is usually under high vacuum.

[0046] The expressions "method based on liquid chromatography mass spectrometry" or "method based on LC-MS" should be understood as the methods used in the present invention that include liquid chromatography and mass spectrometry, and by directly performing them in sequence, the methods known to those skilled in the prior art (the "LC-MS method" defined above) function as the basis. The method of the present invention is based on this, and is modified and adapted in the manner disclosed herein to meet the desired requirements, particularly for oxidation markers, and may be quantified.

[0047] Expressions such as "quantifying" or "quantification" should be understood in their broadest sense and represent determining the concentration or amount of the substance polyethylene glycol-6-laurate based on the relative levels (relative peak areas) found, and are present in the aqueous formulation from which the sample is taken and tested. Quantification is achieved by external calibration using a standard solution of polyethylene glycol-6-laurate. The term "recombinant protein", as used herein, refers to a protein produced by recombinant techniques such as molecular cloning. Such methods involve combining genetic material from multiple sources or creating sequences that do not occur naturally. Recombinant proteins typically are based on sequences from different species from different cells or organisms, or from the host cell of the recipient used to produce the protein, such as CHO cells or HEK293 cells, or are based on artificial sequences such as fusion proteins. In the context of the present invention, the recombinant protein preferably is a therapeutic protein, such as an antibody, an antibody fragment, a molecule derived from an antibody (e.g., scFv, bispecific or multispecific antibody), or a fusion protein (e.g., Fc fusion protein).

[0048] The term "expressing a recombinant protein", as used herein, refers to a cell containing a DNA sequence encoding the recombinant protein, which is transcribed and translated into a protein sequence including post-translational modifications, i.e., resulting in the production of the recombinant protein in cell culture. The term "eukaryotic cell", as used herein, refers to a cell having a nucleus within a nuclear membrane and includes animal cells, human cells, plant cells, and yeast cells. In the present invention, "eukaryotic cells" specifically include mammalian cells, such as cells derived from Chinese hamster ovary (CHO) cells or HEK293 cells, and yeast cells. The term "prodrug", as used herein, refers to a formulated pharmaceutical active ingredient (API) containing an excipient. The API has a therapeutic effect in the body as opposed to the excipient and aids in the delivery of the API. In the case of biological therapies, the formulated API containing an excipient typically means the API in the final formulation buffer at at least the maximum concentration used in the final dosage form and is also referred to as a pharmaceutical.

[0049] APIs may be present in aqueous formulations. According to the WHO guidelines, “Active Pharmaceutical Ingredients (APIs)” are any substances or combinations of substances intended to be used in a Finished Pharmaceutical Product (FPP) that possess pharmacological activity, or have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or in the restoration, correction or modification of physiological functions in humans. Additionally, excipients may be present in aqueous formulations. APIs can be, for example, one or more proteins. An aqueous formulation containing an API is considered an aqueous biopharmaceutical formulation. The term “pharmaceutical product” as used herein refers, in the case of a marketed final dosage form of a drug substance, e.g., a tablet or capsule, or in the case of a biologic, typically an injectable solution in a suitable enclosure such as a vial or syringe. A pharmaceutical product can also be in lyophilized form. The term “host cell protein” (HCP) refers to process-related protein impurities produced by the host organism during the manufacture and production of biopharmaceuticals. During the purification process, most of the HCPs produced, usually more than 99% of the impurities, are removed from the final product. However, the remaining HCPs remain in the final pharmaceutical product, so the removal of HCPs is one of the most significant challenges in the production of biopharmaceuticals. Therefore, all proteins present in the expression system or substrates near the protein in question are considered impurities and are typically summarized under the term host cell protein (HCP).

[0050] The expressions “comprising,” “comprises,” “comprised of,” “including,” “includes,” or “included” also include, unless otherwise stated or apparent from the context, the more specific term “consisting of.” In addition, it should be noted that in the present disclosure, the singular and plural forms are not used in a restricted manner. Thus, as used herein, the singular forms “a,” “an,” “one,” and “the” refer to both the singular and plural, unless otherwise stated or apparent from the context. The expressions "about" or "approximately" mean within 10% of the specific value shown or the value of the upper or lower limit range, particularly 5% or more, more specifically within 1% or within 0.1%. For mass-to-charge ratio, the term "about" has an average of ±0.5. This means that "about 227 amu" represents the range of 227 - 0.5 amu to 227 + 0.5 amu or 226.5 to 227.5 amu, and "about 487 amu" represents the range of 487 - 0.5 amu to 487 + 0.5 amu or 486.5 to 487.5 amu.

[0051] Embodiments of the present invention Subsequently, a multi-step method according to the present invention will be described. The optimal method conditions and parameters for each individual step can vary depending on the specific aqueous formulation present, any excipients, the selected solvent other than water, and any API. Unless otherwise specified, the method conditions and parameters for each method step can be easily selected by those skilled in the art. The experimental procedures are presented in the experimental section. Polyethylene glycol-6-laurate has been found to be a marker substance that provides an indication of the degree of oxidative degradation of polysorbate 20. Since polyethylene glycol-6-laurate is identified as a novel marker substance that enables specific monitoring of the oxidative degradation of polysorbate 20, it is also referred to as an oxidative marker or oxidation marker for the degradation of polysorbate 20. By using a method based on liquid chromatography mass spectrometry that is adapted and modified accordingly for the present invention, the relative level of the marker substance in a sample can be determined, and thus the oxidative degradation of polysorbate 20 can be inferred.

[0052] The general procedure of the method based on liquid chromatography mass spectrometry according to the present invention is as follows: First, a chromatography method is performed. This is a process of liquid chromatography, particularly an HPLC process. The chromatographic procedure is followed by mass spectrometry. In mass spectrometry, electrospray ionization (ESI) is commonly used to generate the ions that are used to determine the molecular weight. In this method, the polysorbate species are (partially) fragmented at the ESI source, separating in particular the fragments containing fatty acids. This means that 1,3-dioxolanium ions are generated. In this way, the total ions can be measured. Usually, the total ions are measured at a mass-to-charge ratio (m / z) in the range of about 100 to about 2000. For evaluation, it is possible to extract the ion traces of the detected species: these are the so-called extracted ion chromatograms or XIC (extracted ion chromatogram).

[0053] Such an extracted ion chromatogram or XIC is generated, for example, for the ion trace representing the 1,3-dioxolanium ion of lauric acid or for a typical fragment of polysorbate 20 at a mass-to-charge ratio of about 227 amu (from m / z 226.5 to m / z 227.5 amu). However, in the present invention, it has been found advantageous if the extracted ion chromatogram XIC is generated for data evaluation at a mass-to-charge ratio in the range that includes or consists of the ion trace or about 487 amu, in particular from m / z 486.5 to m / z 487.5 amu, and will be explained in more detail later.

[0054] The individual steps of the method according to the invention will now be explained in detail below: First method, 1. Alternative: In the first alternative of the first method for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20, a first sample of the aqueous formulation to be tested is taken as a reference sample.

[0055] - Process step 1 In process step 1, a first sample of the aqueous formulation to be tested, which functions as a reference sample, is provided. Since the aqueous formulation under investigation may already contain the oxidation marker as an impurity, the presence of the oxidation marker polyethylene glycol-6-laurate itself does not mean that oxidative degradation occurs, so a reference is needed. Oxidative degradation can also already occur in the aqueous formulation. When oxidative degradation of polysorbate 20 occurs, it is a continuous process that forms an increasing amount of polyethylene glycol-6-laurate. Oxidative degradation can be caused by various influences, especially environmental influences such as light, the presence of metal ions, etc. Accordingly, the first sample as a reference sample is used to determine the base value, and the polyethylene glycol-6-laurate in the sample is determined relative to the base value. In this context, it should be noted that since the raw material (solid) of commercially available polysorbate 20 is also present in the formulation containing polyethylene glycol-6-laurate in aqueous polysorbate 20, it usually easily contains polyethylene glycol-6-laurate. Contrary to expectations, if the reference sample does not contain polyethylene glycol-6-laurate, another reference sample is provided.

[0056] - Process step 2 In process step 2, a second sample and any further samples of the aqueous formulation to be inspected are provided. Accordingly, it is possible to collect two or more samples of the aqueous formulation to be inspected, for example 2, 3, 4, 5 or more further samples, in step 2. The number of samples depends on the individual case. According to one embodiment where the samples are collected at successive time intervals T1, T2, T3..., the times T1, T2, T3... can be of the same length or different lengths. For example, the samples can be collected sequentially at time intervals, for example of the same length. For example, one sample can be collected at one-month time intervals over a period of several months. In this way, it is possible to check whether oxidative degradation of polysorbate 20 occurs or to what extent oxidative degradation occurs over a certain period of time. Sampling in Steps 1 and 2 can be carried out by any procedure known to those skilled in the art, such as manually by a human or by an automated device or system. It goes without saying that sampling should be carried out in such a way that the following analytical procedures are not adversely affected or impaired. The amount of sample taken from the aqueous formulation is selected to be large enough to carry out a method based on liquid chromatography mass spectrometry. Those skilled in the art are familiar with the basis of this analytical technique.

[0057] - Process Step 3 In the subsequent Process Step 3, a method based on liquid chromatography mass spectrometry is used to separate polyethylene glycol-6-laurate and determine the relative peak area of polyethylene glycol-6-laurate, which is a marker substance for the oxidative degradation of polysorbate 20, in each of the reference sample of Step 1 and each sample of the aqueous formulation of Step 2. The determination of the "relative peak area" or "relative level" of polyethylene glycol-6-laurate are terms used synonymously and interchangeably herein, but in Step 3, it means that the polyethylene glycol-6-laurate level is determined in comparison with the reference sample. Specifically, polyethylene glycol-6-laurate is separated from the reference sample (the first sample) of Step 1, from the second sample, and from each additional sample of Step 2, and in each case, the peak area of polyethylene glycol-6-laurate in the reference sample, the second sample, and each additional sample is determined. Then, the peak area of polyethylene glycol-6-laurate in the second sample and each additional sample is determined relative to the peak area of polyethylene glycol-6-laurate in the reference sample to obtain the relative peak area of the second sample and each additional sample.

[0058] For the second sample in process step 3 and for each additional sample, the relative peak area of polyethylene glycol-6-laurate is determined by one of the following two possibilities: - Calculating the difference in the peak areas obtained by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak area of polyethylene glycol-6-laurate of the second sample and from the peak area of polyethylene glycol-6-laurate of each additional sample, respectively, to obtain and calculate the relative peak area for the second sample and for each additional sample; Or - Normalizing the peak areas obtained by setting the peak area of polyethylene glycol-6-laurate of the reference sample to 100% and calculating and normalizing the peak area of polyethylene glycol-6-laurate of each of the second sample and any additional samples based thereon can be determined by one of them.

[0059] If a difference is formed, the peak area of the reference sample is subtracted from the peak area of each sample. The reference peak area is determined appropriately only once, and then the obtained value (baseline) is subtracted from the peak area obtained for each sample. This is illustrated by an example.

[0060] The following peak areas are determined by a method based on liquid chromatography mass spectrometry: Peak area for the reference sample: 2000. Peak area for the first sample: 3000. Relative peak area for the first sample: 3000 - 2000 = 1000. Peak area for an additional sample: 4000. Relative peak area for the additional sample: 4000 - 2000 = 2000. Negative values cannot be obtained since the reference sample is the first sample taken from the aqueous formulation and thus usually has an equal or lower content of polyethylene glycol-6 laurate than the other sample(s) taken or any additional sample(s). When normalizing the peak areas obtained, the peak area of polyethylene glycol-6-laurate in the reference sample is set to 100% and the peak areas of polyethylene glycol-6-laurate in the second sample and each additional sample are calculated based thereon. This will be illustrated using another example.

[0061] The following peak areas are determined by a method based on liquid chromatography mass spectrometry: Peak area relative to the reference sample: 2000. Peak area relative to the other sample: 3000. The relative peak area relative to the reference sample is set to 100%. Relative peak area relative to the second sample: ((3000×100):2000 =) 150%. Peak area relative to an additional sample: 4000. The relative peak area relative to the reference sample remains set to 100%. Relative peak area relative to the second sample: ((4000×100):2000 =) 200%. Therefore, in order to determine whether oxidative degradation of polysorbate 20 occurs, it is only necessary to determine the relative level of polyethylene glycol-6-laurate in the sample of the aqueous formulation compared to the reference.

[0062] When taking and analyzing several samples, it goes without saying that these polyethylene glycol-6-laurate levels are always determined as relative levels compared to the reference sample (relative peak area). Thus, in step 3, one, two, three, four, five or more samples are each analyzed using a method based on liquid chromatography mass spectrometry: for this purpose, the polyethylene glycol-6-laurate contained in the other samples is separated, its peak area is determined, and the relative peak area is determined relative to the peak area of the reference sample (the first sample) using subtraction or normalization as already described to obtain the relative peak area of the other samples. The same procedure is then carried out for each additional sample, i.e., the polyethylene glycol-6-laurate contained in each additional sample is separated, its peak area is determined, and the relative peak area is determined relative to the peak area of the reference sample using subtraction or normalization as already described to obtain the relative peak area of each additional sample. Since this procedure is repeated for all samples, a series of relative peak areas can be determined. According to one embodiment, the relative peak area can be determined as a function of the sampling times T1, T2,....

[0063] Calculating the relative peak area relative to the reference sample is useful when it is carried out in the same way for all samples, i.e., if the difference is formed for one sample, it is useful if the difference is used for all samples. If normalization is used for one sample, it is appropriate to use normalization for all samples. This ensures the comparability of the determination of the relative peak area of the samples.

[0064] The relative peak area with respect to polyethylene glycol-6-laurate is proportional to the degree of oxidative degradation of polysorbate 20. That is, the degree of oxidative degradation is directly related to determining the relative level of the oxidation marker polyethylene glycol-6-laurate in the aqueous formulation being examined. The oxidation marker polyethylene glycol-6-laurate is formed to the extent that polysorbate 20 is oxidatively degraded. Therefore, based on the determined relative level of the oxidation marker, the degree of oxidative degradation of polysorbate 20 can be estimated, especially how much oxidative degradation has already progressed, i.e., how much or to what extent polysorbate 20 has already been oxidatively degraded. More precisely, there is an inverse relationship (non-correlation) between the amount or level of polyethylene glycol-6-laurate and the amount or level of polysorbate 20, which will be explained in more detail later. Therefore, the sample can be used to determine the possible oxidative degradation pathways. For example, a significantly high level of the oxidation marker present in the sample (e.g., a stability sample) relative to the corresponding reference sample represents a high degree of oxidative degradation of polysorbate 20.

[0065] However, in this context, it must be taken into account that the proportional relationship cannot be obtained throughout the degradation process of polysorbate 20 because polyethylene glycol-6-laurate itself degrades over time. Thus, one skilled in the art can understand that the teachings of the present invention can relate to the range in which polyethylene glycol-6-laurate has not yet been degraded and the described proportional relationship can be obtained. Therefore, the disclosed method based on liquid chromatography-mass spectrometry (LC-MS) functions as an analytical tool for determining the level of polyethylene glycol-6-laurate and corresponds to a certain degree of oxidative degradation of polysorbate 20 in the NBE (novel biological formulation) solution. Thus, the method of the present invention can also distinguish between oxidatively degraded and non-oxidatively degraded polysorbate 20 and can be explained in more detail later.

[0066] - Process Step 4 In process step 4, following step 3, polyethylene glycol-6-laurate may be quantified in the sample based on the relative peak area of the second sample and any further individual samples. This step may be optional as it may be sufficient to determine whether oxidative degradation of polysorbate 20 has already occurred based on the relative level of the found polyethylene glycol-6-laurate. However, if a quantitative reference to the degree of oxidative degradation actually occurring is required or desired, quantitative analysis may be performed in any case. Quantification is performed, in particular, via external calibration. For example, the quantification in step 4 may be carried out by external calibration using a standard solution of polyethylene glycol-6-laurate. This procedure is known to those skilled in the art and thus it is not necessary to describe the procedure in all details. Quantification of polyethylene glycol-6-laurate can be very useful, for example, when the absolute values obtained by quantification are used in an established array of limits and / or ranges.

[0067] - Process step 5 Process step 5 involves estimating to what extent polysorbate 20 has already been oxidatively degraded in the sample based on an (approximate) inverse proportional relationship between the polyethylene glycol-6-laurate level and the polysorbate 20 level. The degree of oxidative degradation of polysorbate 20 in the sample can be estimated based on the relative peak area of polyethylene glycol-6-laurate determined in step 3 or the amount of polyethylene glycol-6-laurate determined in step 4 based on the relative peak area of polyethylene glycol-6-laurate.

[0068] Therefore, not only is it possible to distinguish between non-oxidative and oxidative degradation of polysorbate 20 in an aqueous formulation, but also the pathway and progression of oxidative degradation can be determined using the method of the present invention. An (approximate) inverse relationship is shown by determining the (relative) level or content of polysorbate 20 in the aqueous formulation of the sample over time and determining the (relative) level or content of polyethylene glycol 6-laurate in the aqueous formulation of the sample over time. When the determined values are plotted on two curves and compared to each other, the two curves are (approximately) inversely proportional. Otherwise, the curves are not inversely proportional to each other, and oxidative degradation of polysorbate 20 does not occur.

[0069] As shown in Figure 2A, which will be described in detail later, there is a correlation between the decrease in the polysorbate 20 concentration due to oxidative degradation and the increase in the relative level of polyethylene glycol-6-laurate formed during oxidative degradation. For approximations, it can be assumed that the concentration of polysorbate 20 is inversely proportional to the relative level of polyethylene glycol-6-laurate. This indicates that oxidative degradation of polysorbate 20 occurs in Figure 2A. This enables the estimation of the oxidative degradation of polysorbate 20 based on the relative level of polyethylene glycol-6-laurate found in method step 3. Therefore, a high value for the relative level of polyethylene glycol-6-laurate means high degradation of polysorbate 20 in the sample investigated. A median value for the relative level of polyethylene glycol-6-laurate means moderate degradation of polysorbate 20 in the sample tested, and a low value for the relative level of polyethylene glycol-6-laurate means low degradation of polysorbate 20 in the sample tested.

[0070] When the estimated oxidative degradation based on the determined relative polyethylene glycol-6-laurate level correlates with time, very useful information about the oxidative degradation behavior of polysorbate 20 in aqueous formulations is obtained. This is of particular interest, for example, in the development, manufacture, shelf life, or stability testing of aqueous formulations during their storage period, or in stability testing for aqueous biopharmaceutical formulations, for example, to check their quality over time. Next, it is also possible to estimate how likely the oxidative degradation of polysorbate 20 will progress over time in an aqueous formulation containing polysorbate 20, particularly an aqueous biopharmaceutical formulation, or at what level the oxidative degradation of polysorbate 20 can be predicted at a specific point in time. This can be derived by estimating the level of oxidative degradation of polysorbate 20 in an aqueous formulation, such as a biopharmaceutical formulation, implemented using the method of the present invention. Identical or very similar biopharmaceutical formulations behave in the same or corresponding similar ways. Therefore, the estimation is possible and particularly useful for the development of aqueous formulations, particularly aqueous biopharmaceutical formulations. First method, 2. Alternative: In an alternative embodiment of the first method (2. Alternative) for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20, an external reference sample is used. Except for this difference, all the explanations for the first alternative apply to the second alternative in the same way.

[0071] - Process step 1’ In process step 1’, a reference sample is provided that is an aqueous solution containing polysorbate 20 and has the same polysorbate content as the aqueous formulation to be examined. For example, commercially available polysorbate 20 can be used. Since polysorbate 20 usually already contains polyethylene glycol-6-laurate, the aqueous solution of polysorbate 20 also contains this. In Process Step 1’, an external reference sample used to determine the base value is provided, and polyethylene glycol-6-laurate in the sample is determined relative to the base value. In other words, the description for the reference sample in Step 1 is applied to this specification with the necessary changes made.

[0072] - Process Step 2’ In Process Step 2’, a first sample and any additional samples of the aqueous formulation to be inspected are provided. Thus, it is possible to take two or more samples of the aqueous formulation to be inspected, for example, 2, 3, 4, 5 or more additional samples, in Step 2’. The number of samples depends on the individual case. According to one embodiment where samples are taken at successive time intervals T1, T2, T3..., the times T1, T2, T3... can be of the same length or different lengths. Sampling in Steps 1’ and 2’ can be carried out by any procedure known to an expert, such as manually by a human or by an automated device or system. It goes without saying that sampling should be carried out in such a way that the following analysis procedures are not adversely affected or impaired. The amount of sample taken from the aqueous formulation is selected to be large enough to carry out a method based on liquid chromatography mass spectrometry on the sample. Those skilled in the art are familiar with the basis of this analytical technique.

[0073] - Process Step 3’ In the following Process Step 3’, a method based on liquid chromatography mass spectrometry is used to separate polyethylene glycol-6-laurate, determine the relative peak area of polyethylene glycol-6-laurate in each sample, and thereby use an external reference instead of the first sample.

[0074] Specifically, using a method based on the liquid chromatography mass spectrometry method of the present invention, polyethylene glycol-6-laurate is separated from the reference sample in step 1' and from the first sample and any additional samples in step 2', and in each case, the peak area of polyethylene glycol-6-laurate in the reference sample, the first sample, and any additional samples is determined. Subsequently, the peak area of polyethylene glycol-6-laurate in the first sample and any additional samples is determined relative to the peak area of polyethylene glycol-6-laurate in the reference sample to obtain the relative peak area of the first sample and any additional samples. For each sample, it is convenient not to determine the peak area of polyethylene glycol-6-laurate in the reference sample, but once the peak area is determined, it is convenient to use this value (baseline) as a reference. Then, for each sample taken, its peak area is determined relative to this value (baseline) of the reference peak area.

[0075] The relative peak area in process step 3' has two possibilities: - Calculating the difference in the obtained peak areas by subtracting the peak area of polyethylene glycol-6-laurate in the reference sample from the peak area of polyethylene glycol-6-laurate in the first sample and the peak area of polyethylene glycol-6-laurate in any additional samples respectively to obtain and calculate the relative peak area for the first sample and any additional samples; Or - Normalizing the obtained peak areas by setting the peak area of polyethylene glycol-6-laurate in the reference sample to 100% and calculating and normalizing the peak areas of polyethylene glycol-6-laurate in the first sample and any additional samples based on this respectively can be determined by one of them. Furthermore, the further description of step 3 also applies herein.

[0076] - Process steps 4' and 5' In process step 4', the compound polyethylene glycol-6-laurate may be quantified, whereby the relative peak areas of the first sample and any further respective samples are obtained on a basis. In process step 5', it is possible to estimate to what extent polysorbate 20 has already been oxidatively decomposed in the sample based on the inverse proportional relationship between the polyethylene glycol-6-laurate level and the polysorbate 20 level. The description for process steps 4 and 5 applies herein in a manner corresponding to process steps 4' and 5'. Second method: In the present invention, a (second) method for determining the degree of oxidative decomposition of polysorbate 20 in some aqueous formulations is also provided, in particular each aqueous formulation contains the same amount of polysorbate 20. The number of aqueous formulations is not limited, for example 2, 3, 4, 5 or more aqueous formulations can be used, in particular up to 10, up to 20, up to 50 or even more aqueous formulations can be used. The aqueous formulations can differ from each other in terms of their compositions, but are selected in particular such that only the content of polysorbate 20 is the same.

[0077] - Process step 1a In process step 1a, samples of each aqueous formulation to be tested, each containing polysorbate 20, are provided. Sampling can be carried out by any procedure known to the expert, for example manually by a human or by an automated device or system. It goes without saying that sampling should be carried out in such a way that the following analysis procedures are not adversely affected or impaired. The amount of sample taken from the aqueous formulation is selected to be large enough to carry out a method based on liquid chromatography mass spectrometry on the sample. A person skilled in the art is familiar with the basis of this analytical technique. Since the peak areas of the samples in this second method can be compared with each other, there is no need to use a reference in this specification. This is particularly useful when conducting continuous tests. For example, different pharmaceutical compositions can be tested for their stability. According to one embodiment, it is also possible to select a reference sample from the collected samples and determine the relative peak area of each sample with respect to this reference sample. Which sample is selected as the reference sample may vary depending on the case.

[0078] - Process step 2a In process step 2a, a method based on liquid chromatography and mass spectrometry is used to separate polyethylene glycol-6-laurate from each sample in step 1a and determine the peak area of polyethylene glycol-6-laurate in each sample. Polyethylene glycol-6-laurate is a marker substance for the oxidative degradation of polysorbate 20. Then, the peak areas of the samples are compared with each other, whereby the change in the peak area of the sample is proportional to the degree of oxidative degradation of polysorbate 20. The relative peak area of polyethylene glycol-6-laurate does not need to be determined in this specification since the samples can be compared indirectly with each other. However, in another embodiment, the relative peak area of each sample can be determined with respect to a reference sample, which is one of the samples. Therefore, the descriptions for the first and second methods also apply to this specification.

[0079] - Process step 3a In process step 3a, any quantification of the compound polyethylene glycol-6-laurate is performed based on the obtained peak areas of the respective samples. For this purpose, an external calibration curve using a standard solution of polyethylene glycol-6-laurate is provided. Such a procedure has already been described in connection with process steps 4 and 4'.

[0080] - Process step 4a In process step 4a following step 3a, as detailed herein, it is possible to estimate to what extent polysorbate 20 has already been oxidatively decomposed in the sample based on the inverse proportional relationship between the polyethylene glycol-6-laurate level and the polysorbate 20 level. Therefore, since several aqueous formulations are examined for this purpose, a second method is also used to determine the degree of oxidative decomposition of polysorbate 20, to distinguish between non-oxidative decomposition and oxidative decomposition, and to determine the pathway of oxidative decomposition over time. The second method can be used, for example, for quality control. Thus, for example, it enables the identification of particularly stable compositions from several different aqueous formulations. Subsequently, some aspects of the present invention will be detailed.

[0081] Polyethylene glycol-6-laurate (PEG-6-laurate) as a polysorbate 20-specific oxidation marker In fact, polyethylene glycol-6-laurate is a commonly used and well-characterized substance, which has been described extensively in the scientific literature and various patent applications, for example, for its use as a detergent or as a liberator for various esterified compounds [7], and is also known as an additive substance in cosmetic compositions, such as hair conditioner / shampoo compositions [8, 9]. The molecular structure of polyethylene glycol-6-laurate (C 24 H 48 O8) is as follows:

[0082]

Chemical formula

[0083] The mixture of chemical compounds separated herein by liquid chromatography are various degradation products of polysorbate 20, which, as already explained, is itself already a mixture of different substances. Since the oxidation marker polyethylene glycol-6-laurate is separated from this mixture herein, its presence can be detected. For this purpose, the method based on liquid chromatography-mass spectrometry (LC-MS) is modified and adapted, inter alia, as follows: Liquid chromatography is used, inter alia, as separation chromatography, especially in the form of column chromatography. In column chromatography, a mixture of chemical compounds is separated using a column containing a solid stationary phase eluted with one or more mobile phases.

[0084] According to the present invention, in one embodiment, reverse-phase chromatography is used in liquid chromatography. In reverse-phase chromatography, in contrast to normal-phase chromatography, a stationary phase with non-polar surface properties and a mobile phase with polar properties are used. Known polar solvents for reverse-phase chromatography are, for example, water, methanol, and acetonitrile. Other solvents are also possible. According to one embodiment of the method based on liquid chromatography mass spectrometry in liquid chromatography, gradient elution is used. For example, two mobile phases containing a polar solvent are used. For example, water can be used as mobile phase A, and acetonitrile, which preferably contains formic acid, for example 1% formic acid, can be used as mobile phase B. The gradient can start at a few percent of one of the mobile phases, especially mobile phase B, for example 1-10%, especially 1-5% of mobile phase B, and end at 100% of mobile phase B. Then, mobile phase A represents the remaining percentage of 100%. A linear gradient of the gradient can be used. A person skilled in the art can easily find the suitable composition of one or more mobile phases and the appropriate gradient that can be used.

[0085] It is advantageous to add a polar solvent to the sample containing polysorbate 20 to be tested before performing liquid chromatography. The polar solvent is especially a solvent used in liquid chromatography. In one embodiment of the present invention, liquid chromatography as part of the method based on liquid chromatography mass spectrometry is high performance liquid chromatography HPLC, and ultra high performance liquid chromatography can be used. UPLC is more effective compared to HPLC because it uses a higher pump pressure. The pump pressure of HPLC is usually 40 MPa, and for UPLC it is approximately 100 MPa. UPLC uses a stationary phase with a smaller particle size compared to HPLC, so UPLC enables the separation of smaller particles. According to one embodiment, liquid chromatography is performed by high performance liquid chromatography using reverse phase chromatography, whereby the separation of the different polysorbate species present in polysorbate 20 is performed based on high hydrophobicity, so that polyethylene glycol-6-laurate can be separated by liquid chromatography using its hydrophobicity. The procedure itself is well known to experts. Modified and adapted features of methods based on liquid chromatography mass spectrometry are disclosed herein, and those skilled in the art can modify and adapt this procedure according to the disclosure to separate polyethylene glycol-6-laurate using the well-known procedures of liquid chromatography mass spectrometry. Therefore, it is not necessary to explain the procedure in all details. Thus, those skilled in the art can easily perform and optimize liquid chromatography as part of a method based on liquid chromatography mass spectrometry (LC-MS) based on the disclosed details and the given explanations.

[0086] After liquid chromatography is performed, the separated polyethylene glycol-6-laurate can then be identified by mass spectrometry coupled with liquid chromatography. In one embodiment, the mass spectrometry is performed with a heated electrospray ionization (HESI) source. In a further embodiment, the mass spectrometry is selected to be high resolution mass spectrometry (HR-MS). For example, high resolution mass spectrometry is particularly used for structure elucidation. According to another embodiment, for the data evaluation of the method based on liquid chromatography mass spectrometry of the present invention, an extracted ion chromatogram is created from mass-to-charge ratios that include or consist of a range including about 487 amu, particularly 486.5 - 487.5 amu. Polyethylene glycol-6-laurate surprisingly exhibits different m / z ratios in the mass spectrum that enable highly specific monitoring by analysis based on liquid chromatography mass spectrometry. This makes it possible to clearly identify polyethylene glycol-6-laurate if present.

[0087] A person skilled in the art can easily perform mass spectrometry based on the disclosed details and the given explanations. Referring to the spectra shown in FIGS. 1A - 1D to demonstrate the reason that a mass-to-charge ratio m / z of about 487 amu, particularly including or consisting of a mass-to-charge ratio m / z of 486.5 - 487.5 amu, is advantageous and especially suitable for detecting polyethylene glycol-6-laurate. FIGS. 1A - 1D are useful for showing the auto-oxidation of polysorbate 20 and illustrate how polyethylene glycol-6-laurate can be determined, particularly identified, in a sample by separation and detection using a method based on the liquid chromatography-mass spectrometry (LC-MS) of the present invention.

[0088] A sample consisting of polysorbate 20 in an aqueous solution is prepared, and an oxidation catalyst is added to the sample in the form of 2,2'-azobis-2-methyl-propaneimidamide dihydrochloride (also known in the art as 2,2-azobis(2-methylpropionamidine) dihydrochloride or 2,2'-azobis(2-amidinopropane) dihydrochloride) (AAPH) to induce the oxidative degradation of polysorbate 20, that is, to artificially induce the auto-oxidation of polysorbate 20. In particular, an oxidative stress is applied to the polysorbate 20 aqueous solution using 1.5 mM 2,2'-azobis-2-methyl-propaneimidamide dihydrochloride (AAPH). Details of the sample preparation are described in the experimental section, chapter "1.1 Materials", point "c) Sample preparation of artificially stressed polysorbate 20".

[0089] The oxidation catalyst AAPH is a water-soluble azo compound known as a model oxidant and a free radical generator. As already described, polysorbate 20 has a tendency to generate peroxides in situ as a result of auto-oxidation. Therefore, AAPH is used to stimulate the auto-oxidation of polysorbate 20. Furthermore, prepare another sample consisting of polysorbate 20 in an aqueous solution. The oxidation catalyst is not added to the other samples.

[0090] Using a method based on the liquid chromatography mass spectrometry method of the present invention, investigate a sample, one sample in which an oxidation catalyst is present, and one sample that does not contain an oxidation catalyst. The mass spectrometry method used as part of the method based on the liquid chromatography mass spectrometry method is used in the full scan mode, and the spectra obtained are compared. Details of the method based on the liquid chromatography mass spectrometry method used are described in the experimental section, chapter "1.2 Method", point "c) Quantification of oxidation marker PEG-6 laurate (P6L) via liquid chromatography mass spectrometry (LCMS P6L)", but the final step in the described procedure, namely the quantification of polyethylene glycol-6-laurate, is not performed. The results are shown in Figures 1A to 1D.

[0091] In Figure 1A, on the left side, the extraction of the total ion chromatogram (TIC) of two full scans of polysorbate 20 in an aqueous solution obtained from an embodiment of the method based on the liquid chromatography mass spectrometry (LC-MS) of the present invention is shown at a mass-to-charge ratio m / z of about 227 amu (226.5 - 227.5 amu). The m / z ratio of about 227 amu is used to correspond to the compound esterified with laurate, which is known to be the most prominent in polysorbate 20, namely the 1,3-dioxolanilium ion of lauric acid in particular. In Figure 1A, on the left side, the upper panel shows a sample of a stressed polysorbate 20 solution, namely polysorbate 20 in an aqueous solution, to which the oxidation catalyst AAPH is added to artificially initiate the oxidative degradation of polysorbate 20. The lower panel on the left side of Figure 1A shows an unstressed sample of a polysorbate 20 solution, namely polysorbate 20 in an aqueous solution, to which the oxidation catalyst AAPH is not added. The table shown on the left side of Figure 1A outlines the observed peaks and peak areas of the spectra shown on the left side with (referred to as "1.5 mM AAPH" in Figure 1A) and without (referred to as "without AAPH" in Figure 1A) the oxidation catalyst AAPH across the upper panel.

[0092] Comparing the sample not under stress (Figure 1A, left, lower panel) with the sample stressed with AAPH (Figure 1A, left, upper panel), a structure eluting at a retention time of approximately 18.4 minutes, approximately 3.4 times higher, i.e., an increase in polyethylene glycol-6-laurate, is observed in the stressed sample. In Figure 1A, on the right side, the extraction of the total ion chromatograms (TIC) of two full scans of polysorbate 20 in an aqueous solution obtained from an embodiment of the method based on the liquid chromatography-mass spectrometry (LC-MS) of the present invention is shown. Both show a mass-to-charge ratio m / z of approximately 487 amu (486.5 - 487.5 amu). The upper chromatogram shows the addition of the oxidation catalyst AAPH, and the lower chromatogram shows no inclusion of the oxidation catalyst AAPH. However, both chromatograms in Figure 1A show on the right side at a mass-to-charge ratio m / z of approximately 487 amu (486.5 - 487.5 amu). In these chromatograms, it can be seen that the peak at approximately 18.4 minutes is surprisingly 20 times higher (Figure 1A, right side) than the peak at approximately 18.4 minutes in the chromatogram at a mass-to-charge ratio m / z of approximately 227 amu (Figure 1A, left side). As a result, it can be concluded that the chromatogram has high selectivity, low interference, high sensitivity, and the signal increases approximately 20 times at a retention time of approximately 18.4 minutes after AAPH stress at an m / z ratio including or consisting of approximately 487 amu.

[0093] To show the difference between chromatograms based on data evaluation, refer to Figure 1B. In Figure 1B, ion chromatograms obtained from an embodiment of a method based on liquid chromatography mass spectrometry (LC-MS) of the present invention in an aqueous solution of oxidized polyoxyethylene (20) sorbitan, i.e., total ion chromatogram (TIC; upper chromatogram), extracted ion chromatogram generated at a mass-to-charge ratio m / z of about 227 amu (from m / z 226.5× to m / z 227.5 amu; middle chromatogram), and extracted ion chromatogram generated at a mass-to-charge ratio m / z of about 487 amu (from m / z 486.5× to m / z 487.5 amu; lower chromatogram) are shown. In the upper chromatogram, several peaks overlap. In the middle extracted ion chromatogram generated at an m / z ratio of about 227 amu (226.5 - 227.5 amu), there is likely interference from two peaks at a retention time of about 18.4 minutes, while the peak of the oxidation marker polyethylene glycol-6-laurate (referred to as "PEG-6-laurate") is included. In the lower extracted ion chromatogram generated at an m / z ratio including about 487 amu (486.5 - 487.5 amu), since the mass spectrum is at an m / z ratio including about 487 amu, there is a single peak of the oxidation marker polyethylene glycol-6-laurate with excellent selectivity and sensitivity. Therefore, an m / z ratio including or consisting of about 487 amu exhibits excellent selectivity and sensitivity compared to an m / z including or consisting of about 227 amu. Thus, it can be understood that an m / z ratio including or consisting of about 227 amu with fragmentation of free fatty acids at the source is significantly less suitable than an m / z ratio including or consisting of about 487 amu without fragmentation at the source. Therefore, data evaluation of a method based on liquid chromatography mass spectrometry (LC-MS) using a mass-to-charge ratio m / z including or consisting of about 487 amu is particularly advantageous and is particularly suitable for detecting polyethylene glycol-6-laurate in a method based on liquid chromatography mass spectrometry (LC-MS).

[0094] In this regard, it should be noted that the value for a mass-to-charge ratio of 487 amu rather represents a value of about 487 amu. Thus, according to the present invention, the mass-to-charge ratio is, in particular, in the range of 486.5 to 487.5 amu corresponding to about 487 amu.

[0095] To demonstrate that the structure underlying the peak eluting at about 18.4 minutes was previously identified and actually represents polyethylene glycol-6-laurate, a method based on liquid chromatography mass spectrometry using high-resolution mass spectrometry is implemented. Details of the method based on liquid chromatography mass spectrometry using high-resolution mass spectrometry are described in the experimental section, chapter "1.2 Method", point "b) High-resolution mass spectrometry (HR-MS)".

[0096] The deconvoluted mass spectra are shown in Figure 1C and show a series of structures having about 443 Da, about 487 Da and about 531 Da. These structures could be identified via the exact determination of the monoisotopic peaks as sodium adducts of POE (polyoxyethylene) chain fragments esterified with laurate containing 5, 6 and 7 oxy-ethylene units, respectively. The corresponding molecular structures are as follows: Peak at about 443 Da: C 22 H 44 Sodium adduct of O7(PEG-5-laurate), Peak at about 487 Da: C 24 H 48 Sodium adduct of O8(PEG-6-laurate), and Peak at about 531 Da: C 26 H 52 Sodium adduct of O9(PEG-7-laurate).

[0097] Polyethylene glycol-6-laurate (also referred to herein as PEG-6-laurate or P6L) exhibits a maximum signal intensity at approximately 487 Da. It is particularly important that these three structures identified herein correspond to the concepts [1, 2] in the art that propose reduction of the POE chain as a potential oxidation mechanism. Therefore, from these structures, evidence is provided that the oxidation of polysorbate 20 occurs via this pathway.

[0098] To clearly provide the identification and structure of polyethylene glycol-6-laurate by an orthogonal approach (verification of the chemical structure of polyethylene glycol-6-laurate by synthesis and reanalysis), a commercially available polyethylene glycol-6-laurate synthesized by ChiroBlock GmbH, Bitterfeld-Wolfen, Germany was purchased and analyzed by a method based on the liquid chromatography-mass spectrometry of the present invention, and directly compared with a sample of stressed polysorbate 20 solution (polysorbate 20 in an aqueous solution added with AAPH). Figure 1D shows the upper mass spectrum (upper panel) and the lower mass spectrum of commercially available polyethylene glycol-6-laurate obtained from an embodiment of the method based on the liquid chromatography-mass spectrometry (LC-MS) of the present invention, and a sample of polyethylene glycol-6-laurate from polysorbate 20 stressed with AAPH (lower panel). As can be obtained from Figure 1D, the polyethylene glycol-6-laurate in the upper spectrum and the lower spectrum at 487.32497 Da and 487.32493 Da respectively are in very good agreement with the experimentally determined values, indicating that the commercially available polyethylene glycol-6-laurate corresponds to the experimentally determined oxidation marker polyethylene glycol-6-laurate, and confirming the identification of the oxidation marker structure of polyethylene glycol-6-laurate. The different numerical values in the last decimal place are due to measurement errors but are within the variability of the measurements.

[0099] Correlation between the degradation of polysorbate 20 and the concentration of polyethylene glycol-6-laurate The oxidation marker of polysorbate 20 is increasingly formed as the oxidative degradation of polysorbate 20 progresses. Therefore, it is desirable that the decrease in the content of polysorbate 20 is accompanied by an increase in the content of polyethylene glycol-6-laurate. In fact, it can be confirmed that there is a direct correlation between the oxidative degradation of polysorbate 20 and the detection of the relative levels of the oxidation marker substances, which will be described below. To investigate the correlation between the relative polyethylene glycol-6-laurate level and the artificially induced degradation of polysorbate 20, the polysorbate 20 sample is artificially degraded at a suitable temperature selected to stimulate the auto-oxidation of polysorbate 20 by adding 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). For the preparation of the sample, refer to the experimental section, chapter "1.1 Materials", point "c) Preparation of samples of artificially stressed polysorbate 20".

[0100] To evaluate the progress of the oxidative degradation of polysorbate 20, samples are taken at different time points. The progress of the oxidative degradation of polysorbate 20 is also referred to herein as the stress period. Specifically, the polysorbate 20 concentration, the relative level of polyethylene glycol-6-laurate, and the relative level of the non-treated polysorbate 20 species of the artificially oxidized polysorbate 20 are determined at different sampling points. The polysorbate 20 concentration is analyzed in the sample via HPLC-CAD, and the levels of the oxidation marker polyethylene glycol-6-laurate and the non-treated polysorbate 20 species present in the sample are evaluated via an embodiment of a method based on the liquid chromatography mass spectrometry (LC-MS) of the present invention. For a detailed description of the analysis of the polysorbate 20 concentration performed by HPLC-CAD, refer to the experimental section, chapter "1.2 Methods", point "d) Quantification of polysorbate 20 via high performance liquid chromatography charged aerosol detection (HPLC-CAD)".

[0101] For a detailed description of the analysis of the relative levels of the oxidation marker polyethylene glycol-6-laurate via embodiments of the method based on the liquid chromatography mass spectrometry (LC-MS) of the present invention, refer to the experimental section, chapter "1.2 Methods", point "c) Quantification of the oxidation marker PEG-6 laurate (P6L) (LCMS P6L) via liquid chromatography mass spectrometry". For a detailed description of the analysis of the relative levels of the species of untreated polysorbate 20 present in the sample via embodiments of the method based on the liquid chromatography mass spectrometry (LC-MS) of the present invention, refer to the experimental section, chapter "1.2 Methods", point "a) Screening of the species of untreated polysorbate 20 (LC-MS POE) via liquid chromatography mass spectrometry".

[0102] The results of the analysis are shown in Figure 2A, where the relative peak areas of the polysorbate 20 concentration [mg / mL] (left y-axis) and the oxidative marker (referred to as "Oxmarker") polyethylene glycol-6-laurate (right y-axis) are plotted against time [hours]. The polysorbate 20 concentration measured by HPLC-CAD is shown by the curve with triangles, while the relative levels of the oxidation marker polyethylene glycol-6-laurate measured in embodiments of the method based on the liquid chromatography mass spectrometry (LC-MS) of the present invention are shown by the curve with circles.

[0103] In Figure 2A, the concentration of polysorbate 20 represented by the curve with triangles shows a continuous decrease from 0.043 mg / mL (0 hours) to 0.009 mg / mL (35 hours), which corresponds to a 79% loss of polysorbate 20 concentration from sampling time T0 (0 hours) to sampling time T6 (35 hours). This means that through the progression of the decomposition of polysorbate 20, a continuous decrease in the polysorbate 20 concentration was observed. In addition, the HPLC-CAD method was confirmed as an HPLC method showing stability (data not shown), and it was found that the decomposition of polysorbate 20 using AAPH was reliably and accurately reproduced and demonstrated. As a result, the measured curve (curve with triangles) representing the progression of the decomposition of polysorbate 20 reliably and precisely shows values.

[0104] Furthermore, the relative level of the oxidation marker polyethylene glycol-6-laurate was evaluated via an embodiment of a method based on the liquid chromatography mass spectrometry (LC-MS) method of the present invention. The data of the liquid chromatography mass spectrometry (LC-MS) analysis are also shown in Figure 2A. The reference sample is taken at time T0. Consistent with and correlating with the detected decomposition of polysorbate 20, the relative peak area of polyethylene glycol-6-laurate represented by the curve with circles shows an increase up to a maximum at sampling time T4 (12 hours) (see Figure 2A), and then a slight decrease. After 12 hours, the relative level of polyethylene glycol-6-laurate decreases but remains higher than the initial level. Then, the relative level of polyethylene glycol-6-laurate slightly decreases again after 24 hours, which is consistent with the further progression of the decomposition of polysorbate 20 by this time. In this regard, it should be noted that polyethylene glycol-6-laurate is only an intermediate product of the oxidative decomposition of polysorbate 20, which is further decomposed, resulting in the observed decrease in the polyethylene glycol-6-laurate level after 24 hours. Thus, it is actually shown that the decrease in the polysorbate 20 concentration correlates with the increase in the relative level of polyethylene glycol-6-laurate.

[0105] This means that there is an obvious non-correlation between polyethylene glycol-6-laurate and polysorbate 20 within the range that can be easily determined by those skilled in the art, and demonstrates the suitability of polyethylene glycol-6-laurate as a direct marker for the artificial oxidative degradation of polysorbate 20. As already described, polysorbate 20 does not represent a single substance, but rather a mixture of a series of different substances and species. The auto-oxidation of polysorbate 20 affects all the compounds and species present in polysorbate 20. Therefore, it was checked whether the artificially induced auto-oxidation and decomposition of polysorbate 20 actually occurred and covered other compounds. Thus, the relative levels of some untreated polysorbate 20 species present in the polysorbate 20 starting material, i.e., the polyoxyethylene esters present in polysorbate 20, were analyzed before the decomposition of polysorbate 20 (reference, time T0 = 0 h) and during the decomposition of polysorbate 20 by an embodiment of the method based on liquid chromatography-mass spectrometry of the present invention. The results are shown in the block diagram of Figure 2B where the relative peak areas of the found untreated polysorbate 20 species of polysorbate 20 are plotted against time [h]. The untreated polysorbate 20 species to be analyzed are POE-sorbitan mono C12, POE-isosorbide mono C12, POE mono C12, and POE-sorbitan di C12, and their chemical structures are shown in Figure 3 (see

[10] ).

[0106] Figure 2B shows a continuous decrease in the relative levels (relative peak areas) for the species POE-sorbitan mono C12, POE-isosorbide mono C12, and POE-sorbitan di C12, all of which decrease with increasing duration of oxidative stress. The largest decrease is observed for the species POE-sorbitan di C12, which was found to be sensitive to oxidative degradation in polysorbate 20 during the experiment (data not shown). In other words, the degradation of polysorbate 20 in solution is confirmed by the relative levels of the species of untreated polysorbate 20 found in embodiments of the method based on liquid chromatography mass spectrometry (LC-MS) as shown in Figure 2B. Therefore, there is an apparent lack of correlation between the polyethylene glycol-6-laurate level and the level of untreated polysorbate 20, demonstrating the suitability of polyethylene glycol-6-laurate as a direct marker for the artificial oxidative degradation of polysorbate 20.

[0107] An aqueous formulation comprising an excipient and any other components It is found that polyethylene glycol-6-laurate functions as a reliable oxidative marker for samples artificially subjected to oxidative stress. To be used as an oxidative marker, it should be possible to determine that it is reliable and accurate under ambient conditions, especially in aqueous biopharmaceutical formulations, without the artificial influence of added catalysts, so as to distinguish different degradation pathways of polysorbate 20. In this context, it is also important to confirm that, for example, the excipients of the formulations typically present in aqueous biopharmaceutical formulations do not interfere with the determination of polyethylene glycol-6-laurate. In fact, the presence of polyethylene glycol-6-laurate as an oxidative marker in the oxidative degradation of polysorbate 20 is found to occur not only in artificially generated oxidative degradation, but also during the naturally occurring oxidative degradation of polysorbate 20 in aqueous formulations. It has also been found that the presence of the excipients of various normal formulations in aqueous formulations does not interfere with the determination of polyethylene glycol-6-laurate and does not adversely affect the method of the present invention in any way. Furthermore, the presence of other components, such as one or more proteins, also does not interfere with the determination of polyethylene glycol-6-laurate.

[0108] Proteins that can be advantageously used are selected, for example, from monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, single-chain antibodies or antibody fragments thereof, such as Fv, scFv, Fab, Fab’, scFab, F(ab’)2, Fab2, Fc and Fc’ fragments, immunoglobulin heavy and light chains and their constant, variable or hypervariable regions and Fv and Fd fragments, bispecific antibodies, trispecific antibodies, scFv-Fc, minibodies, or single-domain antibodies, or mixtures thereof. Thus, the aqueous formulation can be an aqueous formulation containing conventional formulation excipients. This can be an aqueous formulation that can be used in the field of biopharmaceuticals and contains one or more excipients and any one or more other components such as proteins.

[0109] Aqueous formulation as a biopharmaceutical formulation According to one embodiment, the aqueous formulation can be an aqueous biopharmaceutical formulation. The aqueous biopharmaceutical formulation can contain pharmaceutically acceptable polysorbate 20, one or more pharmaceutically acceptable excipients, and any one or more pharmaceutical active ingredients (APIs). Examples of pharmaceutically acceptable polysorbate 20 have already been described. Pharmaceutically acceptable excipients that can be used in biopharmaceutical formulations are well known to those skilled in the art. One or more pharmaceutical active ingredients (APIs) can be, for example, one or more proteins selected from therapeutic proteins used for the prevention or treatment of diseases or disorders. Thus, the oxidation marker can also be used in relation to an aqueous biopharmaceutical formulation to determine whether oxidative degradation of polysorbate 20 has already occurred. That the method of the present invention also functions in an aqueous biopharmaceutical formulation is demonstrated and confirmed from the following investigations.

[0110] The oxidative degradation of polysorbate 20 is investigated in several aqueous biopharmaceutical formulations. For this purpose, two different monoclonal antibody formulations called mAb-1 formulation and mAb-2 formulation, as well as a placebo for the mAb-1 formulation were used. The placebo for the mAb-1 formulation is an aqueous biopharmaceutical formulation that is the same as the mAb-1 formulation but without the monoclonal antibody mAb-1. Both the mAb formulations as well as the placebo formulation are aqueous formulations and contain a pharmaceutically acceptable polysorbate as well as pharmaceutically acceptable excipients. Detailed information on the mAb-1 and mAb-2 monoclonal antibodies, as well as the composition of the aqueous biopharmaceutical formulations, namely the mAb-1 formulation and the mAb-2 formulation as well as the placebo for the mAb-1 formulation, can be found in the experimental section, chapter "1.1 Materials", point "a) Monoclonal antibodies". In addition, the mAb-1 used in the mAb-1 formulation was prepared by different variants, namely variant A and variant B, whereby the downstream process of variant B was optimized for the removal of host cell impurities. Thus, the mAb-1 formulation was examined in two alternatives, by variant A and B.

[0111] Also, the mAb-2 used in the mAb-2 formulation was prepared by different variants, namely variant A and variant B. Variant A was carried out on a laboratory scale and variant B was carried out in a biopharmaceutical plant. Thus, the mAb-2 formulation was also examined in two alternatives, by variant A and B. Both monoclonal antibodies mAb-1 and mAb-2 are therapeutic proteins used for the prevention or treatment of a disease or disorder. To investigate the oxidative degradation of polysorbate 20 over time, the mAb-1 formulation described above was stored at room temperature (25 °C) for 3 months without the addition of any artificial oxidation catalyst such as AAPH; the mAb-2 formulation was stored at room temperature (25 °C) for 6 months without the addition of any artificial oxidation catalyst such as AAPH.

[0112] First, the polysorbate 20 concentration and relative levels of polyethylene glycol-6-laurate oxidation markers for the mAB-1 formulation and the corresponding placebo formulation were determined at different sampling times. The results are shown in Figures 4A and 4B. Figure 4A shows the polysorbate 20 concentration [mg / mL] plotted against time [months] determined by HPLC-CAD analysis. Details of the HPLC-CAD analysis can be found in the Experimental section, chapter "1.2 Methods", point "d) Quantification of polysorbate 20 via high performance liquid chromatography charged aerosol detection (HPLC-CAD)". Figure 4B shows the relative levels of polyethylene glycol-6-laurate oxidation markers based on relative peak area [%] plotted against time [months] determined by a method based on liquid chromatography-mass spectrometry (LC-MS) of the present invention. For details of the method, refer to the Experimental section, chapter "1.2 Methods", point "c) Quantification of oxidation marker PEG-6 laurate (P6L) (LCMS P6L) via liquid chromatography-mass spectrometry".

[0113] In Figure 4A, both mAb1 formulations show a decrease in the polysorbate 20 level, respectively, although mAb-1 was generated using two different variants A and B and different reaction kinetics. Also, the placebo formulation shows a decrease in the polysorbate 20 level (curve with triangles). Specifically, in Figure 4A, the mAb-1 formulation resulting from variant A (curve with circles: mAb-1 (variant A)) shows a significant loss of total polysorbate 20 during storage as measured by HPLC-CAD. In contrast to variant A, in variant B (curve with squares: mAb-1 (variant B)), the downstream process was optimized for the removal of host cell impurities, resulting in an mAb-1 formulation that is substantially stable during storage and shows minimal degradation of polysorbate 20.

[0114] In Figure 4B, only the placebo formulation (curve with triangles: placebo against mAb-1 formulation) shows an increase in the relative level of polyethylene glycol-6-laurate oxidization marker. Further, as can be seen in Figure 4B, both Variant A and B of the mAb-1 formulation are equal and do not show an increase in the relative level of polyethylene glycol-6-laurate. However, when compared to the corresponding placebo formulation, a significant loss of polysorbate 20 in the placebo formulation is observed in conjunction with a simultaneous increase in the relative level of polyethylene glycol-6-laurate (Figure 4B: curve with triangles also) (Figure 4A: curve with triangles). Since degradation caused by host cell protein (HCP) can be excluded in the placebo formulation, the loss of polysorbate 20 should be due to oxidative degradation. Furthermore, since both Variant A and B of the mAb-1 formulation do not show an increase in the relative level of polyethylene glycol-6-laurate shown in Figure 4B, oxidative degradation of polysorbate 20 does not occur in both mAb-1 formulations. Further, it can be assumed from Figures 4A and 4B that Variant A of polysorbate 20 in the mAB-1 formulation is subject to hydrolysis rather than oxidative degradation. As a result, Figures 4A and 4B clearly demonstrate that it is possible to distinguish between non-oxidative degradation and oxidative degradation in the degradation of polysorbate 20 based on the relative levels of polyethylene glycol-6-laurate found.

[0115] In fact, the stability study confirmed the above conclusion. For this reason, the stability study was carried out. The placebo and the mAb-1 formulation prepared by Variant Method A were analyzed using the method of liquid chromatography mass spectrometry (LC-MS). The data found are shown in the bar graphs of FIGS. 5A and 5B. FIGS. 5A and 5B show the stability study of the formulation for 3 months, and the relative peak areas plotted against time [months] were analyzed by LC-MS-POE according to an embodiment of the method based on the liquid chromatography mass spectrometry (LC-MS) of the present invention and LC-MS-FFA by the method of liquid chromatography mass spectrometry (LC-MS). Details of the LC-MS-POE and LC-MS-FFA methods are described in the experimental section (Chapter "1.2 Methods", Point "a) Screening of species of untreated polysorbate 20 via liquid chromatography mass spectrometry (LC-MS POE)" and Chapter "1.2 Methods", Point "e) Determination of free fatty acids (FFA) via liquid chromatography mass spectrometry (LC-MS FFA)"). Data for LC-MS-FFA are not shown. FIG. 5A shows a block diagram of the content levels of species of untreated polysorbate 20 as relative peak areas [%] for a 3-month stability study of the placebo against the mAb-1 formulation. Also, the species of untreated polysorbate 20 are compounds present in polysorbate 20 before the decomposition of oxidized polysorbate 20 occurs (reference, start) and during the oxidative decomposition of polysorbate 20 observed up to 3 months. The species of untreated polysorbate 20 to be analyzed are again POE-sorbitan mono C12, POE-isosorbide mono C12, POE mono C12 and POE-sorbitan di C12, and their chemical structures are shown in FIG. 3 (see

[10] ).

[0116] In FIGS. 5A and 5B, the first sample of the aqueous formulation is not taken as a reference sample, but another reference sample is used. The other reference sample is polysorbate 20 (a commercially available raw material) in an aqueous solution (not shown in the figure). For the determination of the relative peak area, the described normalization procedure is used. Since another reference sample is used, the relative peak area of the starting sample is not 100%. FIG. 5B shows a block diagram of the relative levels of the species of untreated polysorbate 20 as the relative peak area [%] over a 3-month stability study for the mAb-1 formulation (variant A). The comparison of the profiles of the species of untreated polysorbate 20 over the stability storage period shows a decrease in all species for the mAb-1 formulation (variant A) in FIG. 5B, and only a clear decrease in POE-sorbitan diC12 for the corresponding placebo formulation. The large decrease in POE-sorbitan diC12 in polysorbate 20 of the placebo formulation (FIG. 5A) compared to the mAb-1 formulation (FIG. 5B) can be explained by the fact that species of esterified polysorbate 20 such as POE-sorbitan diC12 are hardly sensitive to enzymatic degradation due to steric hindrance, while typically decreasing rapidly during oxidative degradation (see FIG. 5A)[1].

[0117] The increase in the relative level of polyethylene glycol-6-laurate in the placebo formulation as shown in FIG. 4B clearly indicates the oxidative degradation of polysorbate 20 in the placebo formulation. Therefore, polyethylene glycol-6-laurate can also function as a marker substance for the presence of the oxidative degradation pathway in biopharmaceutical formulations. Therefore, the relative level of polyethylene glycol-6-laurate can be detected in these aqueous biopharmaceutical formulations under appropriate storage conditions without artificial oxidation catalysts, indicating that polyethylene glycol-6-laurate is also a reliable oxidation marker in natural biopharmaceutical formulations.

[0118] Many other experiments were conducted, and in all of them, polyethylene glycol-6-laurate was confirmed to be a highly reliable oxidation marker for the oxidative degradation of polysorbate 20 and could be determined in aqueous biopharmaceutical formulations regardless of the presence of normal excipients and / or pharmaceutical active ingredients (APIs). To demonstrate that polyethylene glycol-6-laurate is an effective and highly reliable oxidation marker for natural aqueous biopharmaceutical formulations, the results for another monoclonal antibody containing an aqueous biopharmaceutical formulation, i.e., the mAb-2 formulation, are shown below (detailed information on the monoclonal antibody mAb-2 and the composition of the aqueous biopharmaceutical formulation, i.e., the mAb-2 formulation, is shown in the experimental section, chapter 1.1, point a)). In this case, the mAb-2 formulation was investigated, and mAb-2 was prepared by two different processes, i.e., variant A and variant B. Variant A was carried out on a laboratory scale, and variant B was carried out in a biopharmaceutical plant. The data are shown in Figures 6A and 6B.

[0119] Figure 6A shows the polysorbate 20 concentration [mg / mL] plotted against the time [months] determined by HPLC-CAD analysis (see the experimental section, chapter 1.2, point d) for details). Figure 6B shows the relative level of the polyethylene glycol-6-laurate oxidation marker based on the relative peak area [%] plotted against the time [months] determined by a method based on liquid chromatography-mass spectrometry (LC-MS) (see the experimental section, chapter 1.2, point c) for details). As can be seen in Figure 6A, the mAb-2 formulations prepared by variant A and variant B show the degradation of all polysorbate 20 at the same rate, respectively, despite different reaction kinetics. However, as shown in Figure 6B, the increase in the relative level of polyethylene glycol-6-laurate was detected only in variant A (the curve with triangles) of the mAb-2 formulation, indicating that both variants are different in their degradation pathways, and thus only variant A shows an increase in the relative level of polyethylene glycol-6-laurate during stability storage, indicating the oxidative degradation of polysorbate 20.

[0120] Thus, FIGS. 6A and 6B also demonstrate that it is possible to distinguish non-oxidative degradation from oxidative degradation in the degradation of polysorbate 20 in aqueous biopharmaceutical formulations based on the relative levels of polyethylene glycol-6-laurate found. The determination of the relative levels of polyethylene glycol-6-laurate by its separation and detection is possible regardless of the presence of one or more excipients and regardless of the presence of one or more proteins, such as therapeutic proteins. Also, this correlates well with the LC-MS characterization data for the stability studies of variants A and B of the mAb-2 formulation found by LC-MS-POE and LC-MS-FFA (details of the LC-MS-POE and LC-MS-FFA methods are described in the experimental section, chapter 1.2, point a) and chapter 1.2, point e)). Data for LC-MS-FFA are not shown.

[0121] The results are shown in FIGS. 7A (variant B) and 7B (variant A). In FIGS. 7A and 7B, unlike FIGS. 5A and 5B, the first sample of the aqueous formulation under investigation is used as a reference sample. The described normalization procedure is used for the determination of the relative peak areas. Since the first sample functions as a reference sample, the relative peak area of the starting sample is 100%. In FIGS. 7A and 7B, both variants of the mAb-2 formulation show a decrease in the species of untreated polysorbate 20. However, only variant A (FIG. 7B) shows an increase in the relative levels of polyethylene glycol-6-laurate during stability storage showing oxidative degradation of polysorbate 20 as shown in FIG. 6B (curve with triangles). Oxidative degradation is confirmed by the decrease in the diesterified species POE-sorbitan diC12 for variant A of the mAb-2 formulation since the diesterified species of polysorbate 20 are less susceptible to enzymatic degradation due to steric hindrance (FIG. 7B), but typically show a decrease during oxidative degradation [1].

[0122] As further evidence that polyethylene glycol-6-laurate is indeed a highly reliable oxidation marker in natural aqueous biopharmaceutical formulations, Figures 8A and 8B show the results for an aqueous biopharmaceutical formulation, namely the mAb-4 formulation, and another monoclonal antibody containing a placebo for the mAb-4 formulation (detailed information on the monoclonal antibody mAb-4, as well as the compositions of the aqueous biopharmaceutical and placebo formulations, and the stability conditions are shown in the experimental section, Chapter 1.1, Points a) and b)). Figure 8A shows the polysorbate 20 concentration [mg / mL] plotted against the time [months] determined by HPLC-CAD analysis (see the experimental section, Chapter 1.2, Point d) for details). Figure 8B shows the relative levels of the polyethylene glycol-6-laurate oxidation marker based on the relative peak area [%] plotted against the time [months] determined by a method based on liquid chromatography-mass spectrometry (LC-MS) (see the experimental section, Chapter 1.2, Point c) for details). As can be seen in Figure 8A, the mAb-4 formulation (curve with triangles) shows substantially no decrease in the concentration of the untreated polysorbate 20 species, while the placebo for the mAb-4 formulation without antibody (curve with dots) shows a significant decrease in the concentration of the untreated polysorbate 20 species during stable storage. Thus, the two curves show that there is a clear degradation of polysorbate 20 only in the placebo formulation, and that polysorbate 20 is substantially completely degraded after 6 months.

[0123] Figure 8B shows that, according to the LC-MS-based method of the present invention, polyethylene glycol-6-laurate is formed to a large extent as a degradation product of polysorbate 20 in the placebo formulation, while the mAb formulation shows little increase in the PEG-6-laurate level. Thus, the curves in Figures 8A and 8B are temporarily uncorrelated with each other. Therefore, polyethylene glycol-6-laurate functions as a true oxidation marker not only under artificial conditions but also in natural aqueous formulations, such as in stability tests of biopharmaceutical formulations and samples. The marker substance, particularly its relative level, can indicate oxidative degradation both in the presence and absence of one or more active pharmaceutical ingredients (APIs), such as proteins, especially monoclonal antibodies. The oxidation marker polyethylene glycol-6-laurate can also distinguish between non-oxidative and oxidative degradation in the degradation of polysorbate 20 in aqueous formulations, particularly aqueous biopharmaceutical formulations, with or without one or more active pharmaceutical ingredients (APIs) and with or without one or more excipients.

[0124] Method implementation Therefore, the method based on liquid chromatography-mass spectrometry (LC-MS) of the present invention is adapted to be used for the separation, detection, and identification of polyethylene glycol-6-laurate, particularly from the perspective of relative peak area or its relative level and any quantification of polyethylene glycol-6-laurate. To verify that the method based on liquid chromatography-mass spectrometry (LC-MS) is a feasible and reliable test method, the method was investigated for linearity, specificity, authenticity, robustness, and accuracy using data from the known art in this field [11, 12]. A suitable method has been found that provides linear, precise, accurate, specific, and reliable results regarding the relative level of polyethylene glycol-6-laurate present. Details are shown in the experimental section (Chapter "2. Characterization of the LC-MS P6L method implementation" and Figure 8).

[0125] A further object of the present invention According to the present invention, a process (the third method) for producing a recombinant protein, comprising the following steps: Step a) culturing eukaryotic cells expressing the recombinant protein in cell culture; Step b) harvesting the recombinant protein; Step c) of purifying the recombinant protein using an aqueous formulation; Step d) of formulating the recombinant protein into a pharmaceutically acceptable aqueous formulation suitable for administration using polysorbate 20; and Step e) of obtaining at least one sample of the pharmaceutically acceptable aqueous formulation of step d); comprising The process (third method) further comprises performing a method (first or second) for determining the degree of oxidative degradation of polysorbate 20 disclosed in the present invention on the sample obtained in step e). A process is also provided.

[0126] According to one embodiment, the above process (third method) for producing a recombinant protein comprises obtaining at least one sample containing the recombinant protein and polysorbate 20 in step e) as described above, and the sample from step d) is a drug substance sample or a pharmaceutical sample. The recombinant protein of the above process of the present invention is produced in eukaryotic cells following expression, and the recombinant protein is harvested and further purified. The recombinant protein is recovered from the culture medium as a secreted protein in the harvested cell culture fluid (HCCF), or from the cell lysate (i.e., the fluid containing the contents of the cells lysed by any means including, but not limited to, enzymatic, chemical, osmotic, mechanical, and / or physical disruption of the cell membrane and any cell wall), and can be purified using techniques well known in the art. The recombinant protein is then formulated into a pharmaceutically acceptable aqueous formulation suitable for administration using polysorbate 20, and at least one sample containing the recombinant protein and the polysorbate 20 of the formulated recombinant protein is obtained.

[0127] The above process (third method of the present invention) further comprises performing a method (first or second method of the present invention) for determining the degree of oxidative degradation of polysorbate 20 disclosed in the present invention on the sample obtained in step e). The present invention also relates to a method associated with the oxidative degradation of polysorbate 20 in an aqueous formulation or several aqueous formulations each containing polysorbate 20, in particular to the use of polyethylene glycol-6-laurate as an oxidation marker in the (first or second) method disclosed herein. The subject of the present invention also relates to the development, manufacture, shelf life, or stability testing during storage of an aqueous formulation or several aqueous formulations each containing polysorbate 20 as a method for determining the oxidative degradation of polysorbate 20 in an aqueous formulation, as disclosed herein (first or second method).

[0128] The advantages of the present invention are diverse. Polysorbate 20 is particularly important due to its known function of stabilizing the components present in aqueous formulations, so its known auto-oxidation results in oxidative degradation of the aqueous formulation and the resulting disorders. Monitoring the oxidative degradation of polysorbate 20 becomes possible here for the first time using a compound that indicates degradation. This is polyethylene glycol-6-laurate, an oxidation marker for the oxidative degradation of polysorbate 20. In the art, oxidation markers for polysorbate 20 have been hypothesized based on various proposed degradation mechanisms, but these have not yet been experimentally identified as true components of the oxidative degradation process of polysorbate 20. It can be demonstrated that polyethylene glycol-6-laurate is an oxidation marker for a solution containing polysorbate 20 when the auto-oxidation of polysorbate 20 is artificially caused by the addition of an oxidation catalyst. However, polyethylene glycol-6-laurate specific to oxidative degradation also occurs during storage of an aqueous formulation containing polysorbate 20 without the addition of an oxidation catalyst, i.e., when the oxidative degradation of polysorbate 20 occurs naturally. Therefore, polyethylene glycol-6-laurate not only functions as a reliable oxidation marker for artificially stressed samples, but is also a highly reliable and accurate oxidation marker in aqueous formulations, such as aqueous biopharmaceutical formulations.

[0129] According to the present invention, a method based on liquid chromatography-mass spectrometry (LC-MS) provides for a reliable determination by separation and detection of the relative levels of the oxidation marker polyethylene glycol-6-laurate in aqueous formulations. The method based on liquid chromatography-mass spectrometry (LC-MS) is based on existing methods and is modified and adapted for the oxidation marker polyethylene glycol-6-laurate according to the present invention. A method based on liquid chromatography-mass spectrometry (LC-MS) for determining the relative levels of polyethylene glycol-6-laurate functions in simple aqueous solutions containing only polysorbate 20, as well as in very complex aqueous formulations and biopharmaceutical formulations containing excipients, with and without a pharmaceutical active ingredient (API). Biopharmaceutical formulations can also contain typical excipients such as buffers, sugars, stabilizers, etc., which do not interfere with the determination of the relative levels of the oxidation marker.

[0130] Regardless of the presence of the oxidation marker, the occurrence of oxidative degradation in polysorbate 20 has been confirmed in stability studies independent of the method of the present invention. In such stability studies, a decrease was observed in all untreated polysorbate 20 species, with a significant decrease in one diester species. These diester species are known to be little sensitive to enzymatic degradation due to steric hindrance, but typically decrease rapidly during oxidative degradation, thus confirming the oxidative degradation. Therefore, an analytical method based on liquid chromatography-mass spectrometry (LC-MS) enables monitoring of the relative levels of polyethylene glycol-6-laurate even in complex aqueous formulations, and monitoring of the relative levels of polyethylene glycol-6-laurate as an oxidation marker enables distinction between hydrolysis and oxidative degradation of polysorbate 20 in aqueous formulations. In fact, the method developed according to the present invention, based on liquid chromatography mass spectrometry (LC-MS), enables the relative levels of polyethylene glycol-6-laurate to be measured linearly, precisely, and accurately even in complex aqueous formulations.

[0131] An increase in the relative level of the marker substance polyethylene glycol-6-laurate correlates well with the loss of polysorbate 20 during oxidative degradation, both when oxidation is artificially induced and when the oxidation of polysorbate 20 occurs naturally. Thus, according to the present invention, it is possible to distinguish between non-oxidative degradation and oxidative degradation in the degradation of polysorbate 20 in aqueous formulations. When observing the degradation of polysorbate 20 that correlates with a simultaneous increase in the relative level of polyethylene glycol-6-laurate, oxidative degradation of polysorbate 20 is clearly present. When the degradation of polysorbate 20 is observed but no simultaneous increase in the relative level of polyethylene glycol-6-laurate occurs, non-oxidative degradation of polysorbate 20 is clearly present. So far, analytical tools that enable the distinction between hydrolytic and oxidative degradation of polysorbate 20 by direct analytical means have not been described in the prior art. Therefore, the relative level of polyethylene glycol-6-laurate functions as a marker for the presence of oxidative degradation pathways in aqueous formulations. The oxidative marker can also indicate oxidative degradation in aqueous biopharmaceutical formulations, both in the presence and absence of proteins, such as pharmaceutical active ingredients (APIs) like monoclonal antibodies. The additional quantification of the oxidative marker polyethylene glycol-6-laurate also makes it possible to estimate the degree of oxidative degradation and thus draw conclusions about the polysorbate 20 remaining in the aqueous formulation.

[0132] Based on the method of the present invention, it is further possible to predict the expected oxidative degradation of polysorbate 20 in the same or very similar aqueous formulations based on the estimation of the oxidative degradation of polysorbate 20 in an aqueous formulation at a specific time. This can be particularly important for the development of aqueous formulations, especially aqueous biopharmaceutical formulations. In the present invention, it is also possible to determine the degree of oxidative degradation of polysorbate 20 in several aqueous formulations, especially each aqueous formulation contains the same amount of polysorbate 20. This enables continuous tests to be carried out in many aqueous formulations, for example, enabling quality control or stability control. In this way, optimized formulation compositions can also be created. Accordingly, the present invention provides a first time method using an analytical method for monitoring, detecting, and optionally quantifying an analyte not previously described, namely polyethylene glycol-6-laurate, in an aqueous formulation, such as an aqueous biopharmaceutical formulation, to observe and estimate the oxidative degradation of polysorbate 20.

[0133] List of abbreviations

Table 1

[0134] Experiment 1. Materials and methods 1.1 Materials All experiments were carried out with polysorbate 20 high purity (PS20 HP) (Croda, Edison, NJ, USA). As polyethylene glycol-6-laurate (PEG-6 laurate), synthetic polyethylene glycol-6-laurate with a purity of 95% and the formula C 24 H 48 O8, molecular weight 464.65 g / mol, synthesized by ChiroBlock GmbH, Bitterfeld-Wolfen, Germany, was used. All other chemicals used were of analytical grade and obtained from commercial sources.

[0135] a) Monoclonal antibody Four monoclonal antibodies (mAbs) in a specific formulation were used: The mAb-1 formulation contains mAb-1, which is an IgG1-type antibody. The mAb-1 used in the mAb-1 formulation was prepared by different variants, namely Variant A and Variant B, thereby optimizing the downstream process of Variant B for the removal of host cell impurities. Variant A of the mAb-1 formulation is concentrated at 65 mg / mL and formulated at pH 6.0 with 0.200 g / mL of polysorbate 20, 11.4 mM L-histidine, 12.8 mM L-histidine HCl + 2H2O, 213.0 mM mannitol, 28.3 mM sucrose. Variant B of the mAb-1 formulation is concentrated at 65 mg / mL and formulated at pH 6.0 with 0.200 g / mL of polysorbate 20, 11.4 mM L-histidine, 12.8 mM L-histidine HCl + 2H2O, 43.9 mM mannitol, 184 mM sucrose.

[0136] The mAb-2 formulation consisting of mAb-2 is an IgG1-type antibody but is concentrated at 50 mg / mL and formulated at pH 5.5 with 0.400 mg / mL of polysorbate 20, 4.23 mM acetic acid, 15.8 mM sodium acetate trihydrate, 220 mM glycine, 20 mM trehalose dihydrate. The mAb-3 formulation consisting of mAb-3 is an IgG1-type antibody but is concentrated at 60 mg / mL and formulated at pH 5.5 with 0.400 mg / mL of polysorbate 20, 45 mM acetate, 150 mM sucrose, 25 mM L-arginine HCL. The placebo for the mAb-1 formulation is a formulation containing 0.200 g / mL of polysorbate 20, 11.4 mM L-histidine, 12.8 mM L-histidine HCl + 2H2O, 43.9 mM mannitol, 184 mM sucrose, pH 6.0 and does not contain mAb-1. The mAb-4 formulation consisting of mAb-4 is an IgG4-type antibody, but it is concentrated at 80 mg / mL and formulated at 0.400 mg / mL of polysorbate 20, 10 mM acetate, 240 mM trehalose, pH 5.2. The placebo for the mAb-4 formulation is a formulation containing 0.400 g / mL of polysorbate 20, 10 mM acetate, 240 mM trehalose, pH 5.2 and does not contain mAb-4.

[0137] b) mAb stability study The stability study of mAb-1 and its corresponding placebo was carried out in 20R glass vials (content: 20 mL) under storage conditions of 25 °C (room temperature) for 3 months. The stability study of mAb-2 was carried out in 20R glass vials under storage conditions of 25 °C (room temperature) / 60% RH (relative humidity) for 6 months. The stability study of mAb-4 and its corresponding placebo was carried out in 6R glass vials (content: 3 mL) under storage conditions of 25 °C (room temperature) / 60% RH (relative humidity) for 6 months.

[0138] c) Preparation of samples of artificially stressed polysorbate 20 Experiment for the identification of PEG-6-laurate: 1.5 mM 2,2’-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) was added to an aqueous polysorbate 20 solution at 0.040 mg / mL. The polysorbate 20 solution was stressed at 50 °C for 40 hours. For correlation with the degradation of total polysorbate 20: 1.5 mM 2,2’-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) was added to an aqueous polysorbate 20 solution at 0.040 mg / mL. The polysorbate 20 solution was stressed at 50 °C for 35 hours. Samples were taken from the solution at 0, 1, 3, 8, 12, 24, and 35 hours and immediately frozen at -20 °C to stop the degradation at each time point.

[0139] 1.2 Method a) Screening of species of untreated polysorbate 20 (LC-MS POE) via liquid chromatography-mass spectrometry Liquid chromatography: The method for characterizing polysorbate used reversed-phase chromatography (Kinetex XB-C C18, 2.6 μm, 2.1×150 mm, Phenomenex) operating at 50 °C with a flow rate of 0.250 mL / min for the separation of polysorbate species in the order of these high hydrophobicities (1200 series, Agilent) using water as mobile phase (MP) A and 1% formic acid in acetonitrile (ACN) as mobile phase (MP) B. The gradient of the analysis started with 5% of MP B and went to 100% of MP B in 20 minutes with a linear gradient until 25 minutes. MP B was decreased to 5% in 0.1 minute at 25.1 minutes, and column washing and re-equilibration steps with 5% of MP B continued until 30 minutes. The column load was usually a 1-2 μg polysorbate sample in ACN.

[0140] Mass spectrometry: The separated species were then ionized with a heated electrospray ionization (HESI) source (Ion Max ion source with HESI probe, Thermo Fisher Scientific). The instrument was operated in positive ionization mode at a source voltage of 4 kV and 310 °C. Fatty acid-specific fragments generated by subsequent in-source fragmentation were detected with a mass spectrometer (LTQ, Thermo Fisher Scientific). The capillary temperature was 275 °C and it was operated at 43 kV. Full scan spectra were collected over the m / z range of 100 - 2000 amu (atomic mass unit). The fatty acid esters of polysorbate 20 were evaluated by generating extracted ion chromatograms (EIC or XIC) from the full scan data, and the peak areas of the selected compounds were obtained using LCquan™ (software for quantitative analysis of liquid chromatography). Fragments of species containing the same fatty acid differed by their characteristic retention times. b) High-resolution mass spectrometry (HR-MS) Liquid chromatography: Refer to the above chapter a) (LC-MS POE) for chromatographic conditions.

[0141] Mass spectrometry: The separated species were ionized with a heated electrospray ionization (HESI) source (Ion Max ion source with HESI probe, Thermo Fisher Scientific). The instrument was operated in positive ion mode at a source voltage of 4 kV and 310 °C. Fatty acid-specific fragments generated by subsequent in-source fragmentation were detected with a mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific). The capillary temperature was 275 °C and it was operated at 9 kV. Full scan spectra with a resolution of 30000 were collected over the m / z range of 100.0 - 2000.0 amu. The structures were identified via exact determination of the monoisotopic peak.

[0142] c) Quantification of the oxidation marker PEG-6 laurate (P6L) (LCMS P6L) via liquid chromatography mass spectrometry Liquid chromatography: Refer to the above chapter a) (LC-MS POE) for chromatographic conditions of the LC-MS P6L method. The column load was a 1 μg polysorbate sample in ACN.

[0143] Mass spectrometry: Next, the separated seeds were ionized with a heated electrospray ionization (HESI) source (Ion Max ion source with HESI probe, Thermo Fisher Scientific). The apparatus was operated in positive ionization mode at a source voltage of 4 kV and 310 °C. The generated ions were detected with a mass spectrometer (LTQ, Thermo Fisher Scientific). The capillary temperature was 275 °C and it was operated at 43 kV. Full scan spectra were collected over the m / z range of 300.0 - 550.0 amu. PEG-6-laurate was evaluated by generating an extracted ion chromatogram (EIC) from the full scan data and the peak area of the selected compound was obtained using LCquan™ (software for quantitative analysis of liquid chromatography).

[0144] d) Quantification of polysorbate 20 via high performance liquid chromatography charged aerosol detection (HPLC-CAD) Samples of mAb and placebo were diluted 1:2 with water at polysorbate 20 concentrations within the calibration range of the method. An external calibration curve was prepared using an aqueous polysorbate 20 solution. The injection volume was 10 μL. HPLC-CAD was measured with a CAD Veo RS (Thermo Fisher Scientific) coupled to an Agilent 1290 LC system (Agilent). Chromatographic separation was performed with the aid of a mixed-mode column Oasis MAX online (20 × 2.1 mm, 30 μm, water) at a column temperature of 1 mL / mL and 30 °C. MP A consisted of 10 mM ammonium formate, 20% 2-propanol, pH 3; MP B consisted of acetonitrile / 2-propanol 50 / 50% (v / v). The gradient for the analysis started isocratic at 0% MP B and was followed by a gradient of 0.1 min to 10% MP B at 14.0 - 14.1 min. At 20 min, MP B was increased to 0% and re-equilibrated until 25 min. CAD was operated at an evaporation temperature of 70 °C, a detector power function of 1.0, a filter constant of 5 s and a data collection rate of 10 Hz. The recorded chromatograms were integrated and the polysorbate 20 concentration was calculated using a second order regression (Empower).

[0145] e) Determination of free fatty acids (FFA) via liquid chromatography mass spectrometry (LC-MS FFA) The method for characterizing polysorbate determined free fatty acids, namely lauric acid, myristic acid, palmitic acid, stearic acid and oleic acid, via LC-MS. Samples of mAb and polysorbate 20 were diluted with water to a final concentration of 0.040 mg / mL of polysorbate 20. The injection volume was 20 μL. For chromatographic separation, an RP column (Jupiter C18, 3 μm, 2 × 150 mm, Phenomenex) at 50 °C and an HPLC system (LC-30AD, SIL-30 AC, Shimadzu) with a flow rate of 0.300 mL / min were used. 10 mM ammonium acetate was used as MP A and 2-propanol was used as MP B. The gradient of the analysis started at 50% of MP B until 2.5 minutes and became 100% of MP B with a linear gradient in 1 minute. At 3.5 minutes, MP B increased to 100% with a gradient of 0.1 minute. MP B was decreased to 50% with a gradient of 0.1 minute at 5 minutes, and the column washing and re-equilibration steps continued with 50% of MP B until 11 minutes.

[0146] FFA was ionized with HESI (Ion Max ion source with HESI probe, Thermo Fisher Scientific) and analyzed with a mass spectrometer (LTQ, Thermo Fisher Scientific) in negative mode. The instrument was operated at a source voltage of 3.5 kV and 310 °C in positive ionization mode. Fatty acids were detected with a mass spectrometer (LTQ, Thermo Fisher Scientific). The capillary temperature was 275 °C and it was operated at -49 kV. Full scan spectra were collected over the m / z range of 150.0 - 300.0 amu. Finally, the extracted ion chromatogram (EIC) was generated from the full scan data using LCquan™ (software for quantitative analysis of liquid chromatography). Quantification determination was performed by using an FFA calibration standard (10 - 1500 ng / mL).

[0147] 2. Characterization of the implementation of the LC-MS P6L method To verify whether a method based on LC-MS is a reliable and suitable test method, the method was investigated for linearity, specificity, accuracy, and precision by procedures well-known in the art. Linearity: First, the linearity of the method was examined by evaluating the coefficient of determination (R 2 ) of the standard curve. The R 2 evaluation of the standard curve of PEG-6-laurate at 1 - 25 ng / mL reveals good linearity of the method (R 2 = 0.9979). The standard curve of the LC-MS P6L method is shown in Figure 8. Figure 8 shows the standard curve of PEG-6-laurate by plotting the detection area (LC-MS P6L) (x-axis) of the LC-MS analysis against the concentration of the standard solution (1 - 25 ng / mL) (y-axis). The linear fit of the standard curve shows a coefficient of determination (R 2 ) of 0.9979. Therefore, the method has good linearity.

[0148] Specificity: Specificity analysis using mAb-3 spiked with synthetic PEG-6-laurate and placebo against mAb-3 did not reveal interference of API, polysorbate 20, or other formulation compounds with the signal of the spiked PEG-6-laurate. That is, PEG-6-laurate was detected in the spiked samples, but no interfering signals were detected in the control solutions. Since only PEG-6-laurate was detected in the spiked samples, the specificity of the LC-MS P6L method is obtained. The results are summarized in Table 1.

[0149] Accuracy and Precision: The evaluation of precision and accuracy was performed via the analysis of NBE solutions spiked with synthetic PEG-6-laurate at 2.5 ng / nL and 20 ng / nL, in triplicate (n = 3) prepared independently for each concentration (for each PEG-6-laurate concentration). The results of the precision evaluation show low relative standard deviations (%RSD) for both levels of PEG-6-laurate samples (%RSD at 2.5 ng / mL of PEG-6-laurate: 2%; %RSD at 20 ng / mL: 3%). Furthermore, the relative mean recovery rates for both PEG-6-laurate samples with respect to the target level (spike concentration) vary between 90 - 104%, showing good results and indicating good precision and accuracy of the LC-MS P6L method. The results are also summarized in Table 1. Table 1 below shows the results of the evaluation of the specificity, precision, and accuracy parameters of the method for detecting and quantifying PEG-6-laurate (P6L).

[0150]

Table 2

[0151] In summary, the characterization of the LC-MS-based method of the present invention for the quantification of PEG-6-laurate shows good performance, is specific, and can determine the level of PEG-6-laurate in a stringent and accurate NBE solution. Subsequently, the method LC-MS P6L can be used to identify critical PEG-6-laurate thresholds in NBE, thereby providing an analytical tool during formulation and process development for aqueous formulations such as biopharmaceuticals.

[0152] Cited References TIFF0007699302000005.tif95170 TIFF0007699302000006.tif120166 The present invention includes aspects disclosed in the following sentences.

[0153] Sentence 1. The following steps: Step 1 of preparing a first sample of the aqueous formulation to be tested, which functions as a reference sample; Step 2 of preparing another sample and any additional samples of the aqueous formulation to be tested; Separating polyethylene glycol-6-laurate and using a method based on liquid chromatography-mass spectrometry to determine the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, from the reference sample of Step 1, the other sample of Step 2, and any additional samples, respectively; Step 3 of determining the relative peak area of polyethylene glycol-6-laurate based on the peak area of polyethylene glycol-6-laurate in the other sample and each of any additional samples with respect to the peak area of polyethylene glycol-6-laurate in the reference sample, wherein the relative peak area is proportional to the degree of oxidative degradation of polysorbate 20; and Step 4 of optionally quantifying the compound polyethylene glycol-6-laurate based on the relative peak area of the other sample and each of any additional samples. A method for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20.

[0154] 2. The method according to sentence 1, wherein the prepared reference sample is an aqueous solution containing polysorbate 20 and the content of polysorbate 20 is the same as that of the aqueous formulation to be tested. The method according to sentence 1. 3. The method according to sentence 1, wherein in Step 2, two, three or more additional samples of the aqueous formulation containing polysorbate 20 are taken at successive time intervals T1, T2, T3.... which can be of the same length or different lengths. The method according to sentence 1.

[0155] 4. The relative peak area of polyethylene glycol-6-laurate for the other sample and each of any additional samples in Step 3 is - forming a difference in the peak areas obtained by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak areas of polyethylene glycol-6-laurate of the other sample and any additional sample, respectively; or - normalizing the peak areas obtained by setting the peak area of polyethylene glycol-6-laurate of the reference sample to 100% and calculating the peak areas of polyethylene glycol-6-laurate of the other sample and any additional sample based thereon, respectively, for normalization determined by the method according to sentence 1.

[0156] 5. The following steps: Step 1a of preparing a sample of each aqueous formulation to be inspected; Separating polyethylene glycol-6-laurate and using a method based on liquid chromatography and mass spectrometry to determine the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, from each sample of step 1a; Step 2a of comparing the peak areas of the samples with each other, wherein the change in the peak area of the sample is proportional to the degree of oxidative degradation of polysorbate 20; and Step 3a of optionally quantifying the compound polyethylene glycol-6-laurate based on the peak areas obtained for each sample A method for determining the degree of oxidative degradation of polysorbate 20 in several aqueous formulations each containing polysorbate 20, particularly when each aqueous formulation contains the same amount of polysorbate 20.

[0157] 6. The relative peak areas of polyethylene glycol-6-laurate of each sample are - Forming a difference in the obtained peak areas by subtracting the peak area of polyethylene glycol-6-laurate of another sample obtained from a reference sample from the peak area of polyethylene glycol-6-laurate of the sample; Or - Normalizing the obtained peak areas by setting the peak area of polyethylene glycol-6-laurate of the sample obtained from the reference sample to 100% and calculating the peak area of polyethylene glycol-6-laurate of another sample based on this for normalization Determined for the reference sample by The method according to sentence 5.

[0158] 7. The following conditions: - A polar solvent is added to the polysorbate-containing sample to be investigated before performing liquid chromatography, especially the solvent used in liquid chromatography; - Liquid chromatography is used as separation chromatography, especially in the form of column chromatography, more specifically as reverse-phase chromatography; - Liquid chromatography is used as high-performance liquid chromatography and gradient elution can be used; - Liquid chromatography is used as ultra-high-performance liquid chromatography and gradient elution can be used; - Mass spectrometry is selected to be high-resolution mass spectrometry especially for the purpose of structure elucidation; - Polyethylene glycol-6-laurate is separated by liquid chromatography using hydrophobicity; and / or - The quantification in step 4 or step 3a is performed by external calibration using a standard solution of polyethylene glycol-6-laurate Satisfying one, two or more of The method according to any one of the previous sentences 1 to 6.

[0159] 8. For the data evaluation of a method based on liquid chromatography mass spectrometry, an extracted ion chromatogram is created from a mass-to-charge ratio that includes the range of about 487 amu, particularly includes or consists of the range of 486.5 - 487.5 amu. The method according to any one of the preceding sentences 1 - 7. 9. Step 5, step 5' or step 4a includes estimating to what extent polysorbate 20 has already been oxidatively decomposed in the sample based on the inverse proportional relationship between the polyethylene glycol-6-laurate level and the polysorbate 20 level. The method according to any one of the preceding sentences 1 - 8. 10. An aqueous biopharmaceutical formulation is used as an aqueous formulation. The method according to any one of the preceding sentences 1 - 9. 11. The polysorbate 20 present is - intended for pharmaceutical use, particularly for use in drugs; and / or - suitable for parenteral administration. The method according to any one of the preceding sentences 1 - 10.

[0160] 12. An aqueous formulation contains one or more proteins, and the protein can be selected from monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, single-chain antibodies or antibody fragments thereof, such as Fv, scFv, Fab, Fab’, scFab, F(ab’)2, Fab2, Fc and Fc’ fragments, heavy and light immunoglobulin chains and their constant, variable or hypervariable regions and Fv and Fd fragments, bispecific antibodies, trispecific antibodies, scFv-Fc, minibodies, or single-domain antibodies, or mixtures thereof. The protein can be selected from therapeutic proteins used for the prevention or treatment of diseases or disorders. The method according to any one of the preceding sentences 1 - 11.

[0161] 13. A process for producing a recombinant protein, comprising the following steps: Step a) of culturing eukaryotic cells that express the recombinant protein in cell culture; Step b) of harvesting the recombinant protein; Step c) of purifying the recombinant protein using an aqueous formulation; and Step d) of formulating the recombinant protein into a pharmaceutically acceptable aqueous formulation suitable for administration using polysorbate 20; Step e) of obtaining at least one sample containing the recombinant protein and polysorbate 20 from step d); comprising, further comprising performing a method for detecting oxidative degradation of polysorbate 20 as described in any one of the preceding sentences 1 to 9 in the sample obtained in step e), wherein the sample from step d) can be a drug substance sample or a pharmaceutical sample; a process.

[0162] 14. A method for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation or several aqueous formulations each containing polysorbate 20, in particular the use of polyethylene glycol-6-laurate as an oxidation marker in the method as described in any one of the preceding sentences 1 to 12. 15. Use of the method as described in any one of the preceding sentences 1 to 12 as a stability test during the development, manufacture, shelf life, or storage period of an aqueous formulation or several aqueous formulations each containing polysorbate 20 for determining the degree of oxidative degradation of polysorbate 20 in the aqueous formulation.

Claims

1. A method for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20, comprising the following steps: Step 1 of preparing a first sample of the aqueous formulation to be tested that functions as a reference sample; Step 2 of preparing a second sample of the aqueous formulation to be tested and any additional samples; and From the reference sample of Step 1, and the second sample and each additional sample of Step 2, respectively, polyethylene glycol-6-laurate is separated, and a method based on liquid chromatography-mass spectrometry for determining the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, is used. Step 3 of determining the relative peak area of polyethylene glycol-6-laurate based on the peak area of polyethylene glycol-6-laurate of the second sample and each additional sample with respect to the peak area of polyethylene glycol-6-laurate of the reference sample, wherein the relative peak area is proportional to the degree of oxidative degradation of polysorbate 20. A method comprising the above.

2. The method according to claim 1, further comprising Step 4 of quantifying the amount of polyethylene glycol-6-laurate in the second sample and each additional sample based on the relative peak area of each sample.

3. The method according to claim 1, characterized in that the prepared reference sample is an aqueous solution containing polysorbate 20, and the content of polysorbate 20 is the same as that of the aqueous formulation to be tested. The method according to claim 1.

4. In Step 2, two, three or more additional samples of the aqueous formulation containing polysorbate 20 are taken at consecutive time intervals T1, T2, T3 that can be of the same length or different lengths. Characterized by the above. The method according to claim 1.

5. The relative peak area of polyethylene glycol-6-laurate of the second sample and each additional sample in Step 3 is - calculating the difference in the obtained peak areas by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak area of polyethylene glycol-6-laurate of the second sample and from the peak area of polyethylene glycol-6-laurate of each of any further samples, respectively; or - normalizing the obtained peak areas by setting the peak area of polyethylene glycol-6-laurate of the reference sample to 100% and calculating and normalizing the peak area of polyethylene glycol-6-laurate of each of the second sample and any further samples based thereon characterized in being determined by the method according to claim 1.

6. A method for determining the degree of oxidative degradation of polysorbate 20 in several aqueous formulations each containing polysorbate 20, in particular in several aqueous formulations in which each aqueous formulation contains the same amount of polysorbate 20, the following steps: Step 1a of preparing a sample of each aqueous formulation to be examined; and separating polyethylene glycol-6-laurate and using a method based on liquid chromatography and mass spectrometry to determine the peak area of polyethylene glycol-6-laurate, which is an oxidation marker for the oxidative degradation of polysorbate 20, from each sample of step 1a Step 2a of comparing the peak areas of the samples with each other, wherein the difference in the peak areas of the samples is proportional to the degree of oxidative degradation of polysorbate 20 in each sample; A method comprising.

7. The method according to claim 6, further comprising step 3a of quantifying the amount of polyethylene glycol-6-laurate in each sample based on the peak area obtained for each sample.

8. The relative peak area of polyethylene glycol-6-laurate of each sample is - calculating the difference in the obtained peak areas by subtracting the peak area of polyethylene glycol-6-laurate of the reference sample from the peak area of polyethylene glycol-6-laurate of each sample of step 2a; or - normalizing the obtained peak areas by setting the peak area of polyethylene glycol-6-laurate in the reference sample to 100% and calculating and normalizing the peak areas of polyethylene glycol-6-laurate in each sample of step 2a based thereon as determined for the reference sample The method according to claim 6 **Claim 9** The following conditions - a polar solvent is added to the polysorbate-containing sample to be examined before performing liquid chromatography, in particular the solvent used in liquid chromatography - liquid chromatography is used as separation chromatography, in particular in the form of column chromatography, more specifically as reverse-phase chromatography - liquid chromatography is used as high-performance liquid chromatography and gradient elution can be used - liquid chromatography is used as ultra-high-performance liquid chromatography and gradient elution can be used - the mass spectrometry method is selected to be high-resolution mass spectrometry, in particular for the purpose of structure elucidation - polyethylene glycol-6-laurate is separated by liquid chromatography using hydrophobicity; and / or - the quantification in step 4 or step 3a is carried out by external calibration using a standard solution of polyethylene glycol-6-laurate characterized by satisfying one, two or more of the above The method according to claim 1 or 6 **Claim 10** For the data evaluation of a method based on liquid chromatography-mass spectrometry, the extracted ion chromatogram is created from a mass-to-charge ratio that includes the range of about 487 amu, in particular includes or consists of the range of 486.5 - 487.5 amu The method according to claim 1 or 6 **Claim 11** Performing an additional step 5 following step 4 or step 3, or an additional step 4a following step 3a or step 2a, each of which includes estimating to what extent polysorbate 20 has already been oxidized and decomposed in the sample based on the inverse proportional relationship between the polyethylene glycol-6-laurate level and the polysorbate 20 level The method according to claim 1, 2, 6 or 7 **Claim 12** An aqueous biopharmaceutical formulation, characterized in that it is used as an aqueous formulation, The method according to claim 1 or 6.

13. The polysorbate 20 present in the sample, - Intended for pharmaceutical use, particularly for use in drugs; and / or - Suitable for parenteral administration Characterized by The method according to claim 1 or 6.

14. The aqueous formulation contains one or more proteins, and the proteins can be selected from monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, single-chain antibodies or antibody fragments thereof, such as Fv, scFv, Fab, Fab’, scFab, F(ab’)2, Fab2, Fc and Fc’ fragments, immunoglobulin heavy and light chains and their constant, variable or hypervariable regions and Fv and Fd fragments, bispecific antibodies, trispecific antibodies, scFv-Fc, minibodies, or single-domain antibodies, or mixtures thereof, Characterized in that the protein can be selected from therapeutic proteins used for the prevention or treatment of diseases or disorders, The method according to claim 1 or 6.

15. As a stability test for determining the degree of oxidative degradation of polysorbate 20 in the aqueous formulation during the development, manufacture, shelf life, or storage period of an aqueous formulation containing polysorbate 20 or several aqueous formulations, characterized by The method according to claim 1 or 6.

16. A process for producing a recombinant protein, comprising the following steps: Step a) culturing eukaryotic cells expressing the recombinant protein in cell culture; Step b) harvesting the recombinant protein; Step c) purifying the recombinant protein using an aqueous formulation; Step d) formulating the recombinant protein into a pharmaceutically acceptable aqueous formulation suitable for administration using polysorbate 20; and Step e) obtaining at least one sample of the pharmaceutically acceptable aqueous formulation of step d); Including Further comprising performing a method for detecting the degree of oxidative degradation of polysorbate 20 according to claim 1 or 6 on the sample obtained in step e), wherein the sample from step d) can be a drug substance sample or a pharmaceutical sample, Process.

17. Use of polyethylene glycol-6-laurate as an oxidation marker in a method for determining the degree of oxidative degradation of polysorbate 20 in an aqueous formulation containing polysorbate 20 or in several aqueous formulations, said method being the method according to claim 1 or 6, said use.

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