Eco-friendly liquid chromatography organic solvent

Abstract

Herein is reported a method for the separation or / and analysis of polypeptides on a reversed-phase chromatography material, wherein the separation is achieved by applying a gradient to the chromatography material, wherein the gradient is from an aqueous Eluent A to an organic Eluent B, wherein the aqueous Eluent A is water, and wherein the organic Eluent B is a mixture of ethanol and ethyl acetate at a volume ratio in the range of from 60:40 to 99:1.

Description

[0001] Eco-friendly liquid chromatography organic solvent

[0002] The current invention is in the field of analytical methods. More specifically, herein is reported an eco-friendly liquid chromatography (LC) solvent, wherein the use of acetonitrile has been avoided, i.e., acetonitrile has been replaced by a mixture of ethanol and ethyl acetate.

[0003] Background of the Invention

[0004] High performance liquid chromatography (HPLC) is an indispensable technique applied in the development and quality control of virtually all bio-therapeutic drugs on the market today [1-3], While HPLC offers an array of powerful separation modes, the reversed-phase chromatography (RP) approach is among the most widely used. RP’s comprehensive field of application encompasses almost all drug formats of the biopharmaceutical industry, including, therapeutic proteins [4-9], antibodydrug conjugates [7-8, 10] and, more recently, oligonucleotide drugs [11-14] and recombinant adeno-associated viral particles (rAAVps) for gene therapy [15-17],

[0005] One major discipline of biopharmaceutical analysis which is gaining immense momentum in recent years is intact protein analysis by liquid chromatographymass spectrometry (LC-MS) [18-21], For advancing this powerful technique, scientists are faced with the challenge to carefully fine tune their methods to optimize both separation and mass spectrometric (MS) detection performance for a wide range of analytes. To address this challenge, much effort has been directed toward finding alternative ion-pairing agents for higher analyte recovery and separation power, while mitigating ion suppression inherent to the potent ion-pairing agent trifluoroacetic acid (TFA) [22-25],

[0006] Recently, LC-MS analysis of therapeutic proteins using difluoroacetic acid (DFA) as the single acidifier or in combination with formic acid (FA) for improved mass spectrometric sensitivity while maintaining optimal chromatographic resolution was reported [17, 26-28],

[0007] Additionally, recently developed polyphenol-functionalized, wide-pore superficially porous particles stationary phase [8] now allows protein separation under milder conditions, i.e., lower temperature and lower to no TFA content [29-31],

[0008] While sample preparation protocols, column dimensions, solid phase chemistries, particle size, HPLC-parameters and eluent modifiers vastly differ across the known LC-MS methods, almost all RP applications that are suitable for bio-therapeutics utilize the same mobile phases, namely water and acetonitrile (ACN). Variants thereof encompass the addition of small percentages of methanol [7], n-butanol

[0032] or isopropanol

[0027] to acetonitrile for use as Eluent B.

[0009] When it comes to RP bio-therapeutics separation, the primary focus has been on performance, as reflected in the methods reported in the art

[0033] , while the eco- friendliness of the method, especially of the employed solvents, was not considered.

[0010] With increasing environmental awareness and climate concerns, scientist have embraced their responsibility, creating the discipline of “Green Chemistry” [34-35], Although, this concept historically focuses primarily on chemical synthesis, waste disposal, recycling- and preparative applications, the field of “Green Analytical Chemistry” (GAC) is receiving ever-growing attention

[0036] , Specific to liquid chromatography (LC), the reduced solvent consumption requirements were enabled predominantly by technological advancement, such as, smaller column inner diameters and lengths that deliver the required performance, at lower flow rates, as well as smaller particle sizes that enable shorter run times at identical or increased resolving power

[0036] ,

[0011] Ambrogelly et al. (mAbs 4 (2012) 701-709) reported the characterization of antibody variants during process development with a RP-HPLC using a mobile phase B which consisted of 0.2% trifluoroacetic acid in 90% acetonitrile (v:v) and a mobile phase A which consisted of 0.2% trifluoroacetic acid in water (v:v).

[0012] Shen et al.

[0051] reported that alternative solvents can make preparative liquid chromatography greener by replacing the best traditional eluent, i.e. methanol-THF- water (2: 1 : 7), for the analysis of five ginkgo terpene trilactones by HPLC by two ternary mixtures of acetone-ethyl acetate-water (20 .25: 3.75: 76) and ethanol-ethyl acetate- water (9.5: 7.5: 83).

[0013] Kobayashi et al. (Chrom. 37 (2016) 133-139) reported about the effect of acidic additives on peak capacity and detectivity in peptide analysis using nano-flow LC / MS with low-density ODS modified monolithic silica capillary columns.

[0014] Therefore, in order to achieve a more holistic approach, the complete replacement of hazardous chemicals is desirable

[0037] , Summary of the Invention

[0015] The current invention is based, at least in part, on the finding that the chromatographic separation, such as, e.g., the reversed phase chromatographic separation, of compounds of interest, such as, e.g., (therapeutic) proteins, (therapeutic) monoclonal antibodies (mAbs) and / or subunits or conjugates, such as PEGylated antibodies or antibody-drug conjugates or derivatives thereof, can be achieved using either ethanol or ethyl acetate or a combination thereof as mobile phase. It has to be pointed out that the proteinaceous part of a conjugate or derivatives determines the separation behavior of the compound.

[0016] The invention is based, at least in part, on the finding that acetonitrile as Eluent B in a method for the separation or / and analysis of a compound of interest on a reversed- phase chromatography material using a gradient from an aqueous Eluent A to Eluent B can be replaced by ethanol or ethanol-ethyl acetate-mixtures without negative effect on the chromatographic resolution or separation (run) time but with even improved chromatographic resolution and shortened separation (run) times. This is especially true for Eluent B comprising 70 % (v / v) or more ethanol and correspondingly 30 % (v / v) or less ethyl acetate. Thus, the Eluent B according to the current invention comprises ethanol and ethyl acetate at a ratio of and including 99: 1 to 60:40, preferably 85: 15 to 70:30, more preferably about 70:30 or about 85: 15.

[0017] The current invention comprises at least the following embodiments:

[0018] 1. A method for the separation or / and analysis of a compound of interest on a reversed-phase chromatography material, wherein the separation is achieved by applying a gradient, preferably a solvent gradient, to the chromatography material, wherein the gradient is from an aqueous Eluent A to an organic Eluent B, wherein the aqueous Eluent A comprises or is water, and wherein the organic Eluent B comprises or is either i) a mixture of ethanol and ethyl acetate at a volume ratio in the range of from 60:40 to 100:0, or ii) a mixture of methanol and methyl acetate at a volume ratio in the range of from 60:40 to 100:0.

[0019] 2. Use of an organic solvent comprising ethanol and ethyl acetate as eluent in a reversed-phase liquid chromatography-UV or in a reversed-phase liquid chromatography-mass spectrometry method for improving UV-detection or mass spectrometric detection of a compound of interest or fragment or derivative thereof compared to an eluent comprising acetonitrile.

[0020] 3. Use of an organic solvent comprising ethanol and ethyl acetate as eluent in a reversed-phase liquid chromatography mass spectrometry method for increasing ionization yield of a compound of interest or fragment thereof compared to an eluent comprising acetonitrile.

[0021] 4. Use of an organic solvent comprising ethanol and ethyl acetate as eluent in a reversed-phase liquid chromatography mass spectrometry method for reducing organic solvent consumption compared to acetonitrile as organic solvent under otherwise identical conditions.

[0022] 5. Use of an organic solvent comprising ethanol and ethyl acetate as eluent in a reversed-phase liquid chromatography mass spectrometry method for reducing wear of chromatography systems compared to acetonitrile as organic solvent under otherwise identical conditions.

[0023] 6. The use according to any one of embodiments 2 to 5, wherein the organic solvent comprises about 75 % (v / v) water, about 17 % (v / v) ethanol and about 8 % (v / v) ethyl acetate.

[0024] 7. The method according to embodiment 1 or the use according to any one of embodiments 2 to 6, wherein the organic Eluent B or the organic solvent has an ethanol to ethyl acetate ratio of about 10 to 3.

[0025] 8. The method according to any one of embodiments 1 and 7 or the use according to any one of embodiments 2 to 7, wherein the organic Eluent B or the organic solvent further comprises about 0.1 % (v / v) trifluoroacetic acid or difluoroacetic acid or formic acid or about 0.1 % (v / v) of a mixture of 0.05 % (v / v) each of two of them, or up to 1 % (v / v) acetic acid or about 1-50 mM cyanoacetic acid. a. The method according to any one of embodiments 1 and 7 to 8 or the use according to any one of embodiments 2 to 8, wherein the organic Eluent B or the organic solvent further comprises about 3-20 mM cyanoacetic acid. . The method according to any one of embodiments 1 and 7 to 8, wherein the aqueous Eluent A comprises deionized, ultra-pure water (PWA) or water for analysis. 0. The method according to any one of embodiments 1 and 7 to 9, wherein the aqueous Eluent A further comprises about 0.05 % (v / v) trifluoroacetic acid and / or difluoroacetic acid and / or formic acid or a mixture of 0.05 % (v / v) of two of them, or up to 1 % (v / v) acetic acid or about 1-50 mM cyanoacetic acid. 0a. The method according to any one of embodiments 1 and 7 to 10, wherein the aqueous Eluent A further comprises about 3-20 mM cyanoacetic acid. 1. The method according to any one of embodiments 1 and 7 to 10 or the use according to any one of embodiments 2 to 8, wherein the method or the use is at a temperature in the range and including 40 °C to 90 °C, preferably in the range and including 65 °C to 80 °C, more preferably at about 75 °C. 2. The method according to any one of embodiments 1 and 7 to 11 or the use according to any one of embodiments 2 to 8 and 11, wherein the reversed- phase chromatography material is selected from a C4, C8, Cl 8, diphenyl, polyphenyl and monolithic material, in one preferred embodiment the reversed-phase chromatography material is a polyphenyl-derivatized material. 3. The method according to any one of embodiments 1 and 7 to 12 or the use according to any one of embodiments 2 to 8 and 11 to 12, wherein the compound of interest is an antibody or an antibody -fragment. 4. The method according to any one of embodiments 1 and 7 to 13 or the use according to any one of embodiments 2 to 8 and 11 to 13, wherein the compound of interest is a polypeptide and the method or the use is for intact polypeptide mass analysis or polypeptide subunit mass analysis or for reduced polypeptide mass analysis, in one preferred embodiment the compound of interest is an antibody, antibody fragment or antibody derivative. 5. The method according to any one of embodiments 1 and 7 to 14 or the use according to any one of embodiments 2 to 8 and 11 to 14, wherein the organic Eluent B or the organic solvent has an ethanol to ethyl acetate ratio in the range of and including 60:40 to 99: 1, preferably in the range of and including 65:35 to 95:5, more preferably in the range of 67:33 to 90: 10, even more preferably in the range and including 70:30 to 85: 15, most preferably of about 70:30 or about 85 : 15 or about 99: 1.

[0026] 16. The method according to any one of embodiments 1 and 7 to 15 or the use according to any one of embodiments 2 to 8 and 11 to 14, wherein the method or the use comprises a gradient from 15 to 50 % (v / v) organic Eluent B at an ethanol to ethyl acetate ratio of about 85 : 15 or a gradient from 20 to 45 % (v / v) Eluent B at an ethanol to ethyl acetate ratio of about 70:30.

[0027] 16a. The method according to any one of embodiments 1 and 7 to 16 or the use according to any one of embodiments 2 to 8 and 11 to 14, wherein the method or the use comprises a gradient from 30 to 42 % (v / v) organic Eluent B at an ethanol to ethyl acetate ratio of about 85 : 15 or a gradient from 22 to 34 % (v / v) Eluent B at an ethanol to ethyl acetate ratio of about 70:30.

[0028] 17. The method or use according to any one of embodiments 1 to 16, wherein the compound of interest is selected form the group consisting of glycosylated or non-glycosylated proteins, glycated or non-glycated, naturally or artificially modified (oxidation, glycol-engineering, reduction, chemical labelling, alkylation, de-glycosylation, chemical or physical stress treatment) compounds, which have been chemically and / or enzymatically treated for the purpose of sample preparation prior to analysis or be analyzed in their native state.

[0029] 18. The method or use according to any one of embodiments 1 to 17, wherein the compound of interest is a therapeutic protein or a therapeutic antibody or a fragment, conjugate or derivative thereof.

[0030] 19. The method or use according to any one of embodiments 1 to 18, wherein an analytical or semi-preparative or preparative liquid chromatography column is used.

[0031] 20. The method or use according to any one of embodiments 1 to 19, wherein a chromatography column that has an inner diameter from about 20 pm to 100 mm, a column length of from 5 mm to 750 mm and a flow rate ranging from 0.05 pL / min to 10 L / min is applied. 21. The method or use according to any one of embodiments 1 to 20, wherein the reversed-phase chromatography material has a particle size of less than 2 pm to 100 pm with a pores size suitable for the separation of the compound of interest.

[0032] 22. The method or use according to any one of embodiments 1 to 21, wherein the reversed-phase chromatography material is a polyphenyl chromatography material with a particle size of 2.7 pm and is inside a chromatography column that has an inner diameter of 2.1 mm, a length of 150 mm and is operated with a flow of 300 pL / min.

[0033] 23. The method according to any one of embodiments 1 and 7 to 22, wherein the method is a liquid chromatography method.

[0034] 24. The method or use according to any one of embodiments 2 to 23, wherein the liquid chromatography is a high performance liquid chromatography or an ultra-high performance liquid chromatography.

[0035] 25. The method or use according to any one of embodiments 2 to 24, wherein the liquid chromatography is performed at a pressure of 1 to 1000 bar.

[0036] In addition to the various embodiments depicted and embodimented, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and embodimented herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0037] Descrintion of the Figures

[0038] Figure 1 LC-MS Total Ion Chromatograms (TIC) of IdeS (FabRICATOR™) digested trastuzumab using different Eluent B at at otherwise identical chromatographic and mass spectrometric settings; 100 % (v / v) ethanol (i, yellow), 15 % (v / v) ethyl acetate in ethanol (ii, orange), 30 % (v / v) ethyl acetate in ethanol (iii, blue) and acetonitrile (iv, black), each containing 0.05 % (v / v) DFA and 0.05 % (v / v) FA respectively. Gradient separation with 20 % (v / v) Eluent B to 50 % (v / v) Eluent B in 30 minutes at 300 pL / minute flow rate; for all runs, the elution order was found to be glycosylated Fc / 2 followed by F(ab’)2; shown is the chromatographic range from minute 2 to minute 30; MS detection using a time of flight (TOF) analyzer and electrospray ionization (ESI) at 5.5 kV and 325 °C source temperature.

[0039] Figure 2 LC-MS Total Ion Chromatograms of deglycosylated antibodies (bivalent, monospecific, full-length IgG antibody trastuzumab, bivalent, bispecific, full-length CrossMab and NISTmAb); 6-4 runs using 30 % (v / v) EtOAc in EtOH as Eluent B (including 0.05 % (v / v) FA and 0.05 % (v / v) DFA) and 1-3 using acetonitrile as Eluent B (including 0.05 % (v / v) FA and 0.05 % (v / v) DFA) under identical LC conditions; time of flight, mass spectrometric detection was performed via ESI ionization at 325 °C source temperature for 30 % (v / v) EtOAc in EtOH and at 400 °C for ACN; 15 % intensity offset between runs in the graph was introduced to allow presentation in one graph.

[0040] Figure 3 LC-MS Total Ion Chromatograms of deglycosylated and reduced trastuzumab antibody, using (i, yellow) ethanol, (ii, orange) 15 % (v / v) EtOAc in EtOH, (iii, blue) 30 % (v / v) EtOAc in EtOH as Eluent B (always including 0.05 % (v / v) FA and 0.05 % (v / v) DFA) and (iv, black) using ACN as Eluent B (including 0.05 % (v / v) FA and 0.05 % (v / v) DFA) under identical LC conditions; time of flight, mass spectrometric detection was performed via ESI ionization under identical conditions for all runs.

[0041] Figure 4 LC-MS Total Ion Chromatograms of deglycosylated-reduced bispecific antibody (bsAb), using (i, green, top trace, 0.05 % (v / v) DFA and FA each in 30 % (v / v) EtOAc in EtOH, (ii, orange, upper middle trace) 15.89 mmol / L cyanoacetic acid (CNA) in 30 % (v / v) EtOAc in EtOH, 15 % (v / v) EtOAc in EtOH, (iii, blue, middle lower trace) 10 mmol / L cyanoacetic acid (CNA) in 30 % (v / v) EtOAc in EtOH and (iv, black, bottom trace) using 5 mmol / L cyanoacetic acid (CNA) in 30 % (v / v) EtOAc in EtOH as Eluent B with the respective aqueous solution as Eluent A under identical LC conditions; time of flight, mass spectrometric detection was performed via ESI ionization under identical conditions for all runs.

[0042] Figure 5: LC-MS Total Ion Chromatograms of an antibody mixture comprising an aqueous buffered solution of three different antibodies, including a 146.4 kDa monoclonal antibody (mAb), a 147.2 kDa mAb and a 192.89 kDa bispecific antibody (bsAb), using (i, blue, upper trace) 15.89 mmol / L cyanoacetic acid in 30 % (v / v) EtOAc in EtOH as Eluent B, (ii, black, lower trace) 15.89 mmol / L cyanoacetic acid in acetonitrile as Eluent B with the respective aqueous solution as Eluent A under identical LC conditions; time of flight, mass spectrometric detection was performed via ESI ionization under identical conditions for all runs. Shown with approximately 4E+07 counts offset between the two baselines for illustration purpose.

[0043] Figure 6: LC-UV chromatograms of an antibody mixture comprising an aqueous buffered solution of three different antibodies, including a 146.4 kDa mAb, a 147.2 kDa mAb and a 192.89 kDabsAb, using (i, blue, upper trace) 15.89 mmol / L CNA in 30 % (v / v) EtOAc in EtOH as Eluent B, (ii, black, lower trace) 15.89 mmol / L CNA in acetonitrile as Eluent B with the respective aqueous solution as Eluent A under identical LC conditions; UV detection was performed at 280 nm under identical conditions for all runs. Shown with approximately 50 mAu offset between the two baselines for illustration purpose.

[0044] Figure 7: LC-MS Total Ion Chromatograms of deglycosylated-intact bsAb, using (i, blue, upper trace) 10 mmol / L CNA in 30 % (v / v) EtOAc in EtOH as Eluent B, (ii, black, lower trace) 0.05 % (v / v) DFA and 0.05 % (v / v) FA in 30 % (v / v) EtOAc in EtOH as Eluent B with the respective aqueous solution as Eluent A under identical LC conditions; time of flight, mass spectrometric detection was performed via ESI ionization under identical conditions for all runs.

[0045] Figure 8: Averaged ESI-MS spectra of deglycosylated-intact bsAb LC-MS main peak, using (i, blue, upper trace) 10 mmol / L cyanoacetic acid in 30 % (v / v) EtOAc in EtOH as Eluent B, (ii, black, lower trace) 0.05 % (v / v) DFA and 0.05 % (v / v) FA in 30 % (v / v) EtOAc in EtOH as Eluent B with the respective aqueous solution as Eluent A under identical LC conditions; time of flight, mass spectrometric detection was performed via ESI ionization under identical conditions for all runs. Averaging time ranges are: (i, blue, upper trace) 7.66 min to 8.19 min and (ii, black, lower trace) 8.63 min to 9.48 min.

[0046] Figure 9: Boxplot distribution comparison of mAb-A derived peptide intensities for each run across the experimental sets and for different eluent types. Median values are displayed in the boxes in white, p-values are displayed on top.

[0047] Figure 10: Boxplot distribution comparison of mAb-A derived total sums of peptide intensities for each run across the experimental sets and for different eluent types. Median values are displayed in the boxes in white, p-values are displayed on top.

[0048] Figure 11: Comparison of mAb-A heavy chain sequence coverage in percent across the experimental sets and for different eluent types. Mean values are displayed in the boxes, maximum values are displayed on top of the boxes and p-values are displayed in the upper section of the plot.

[0049] Figure 12: Comparison of mAb-B (0.5 mass percent spike protein) sequence coverage in percent across the experimental sets and for different eluent types. Mean values are displayed in the boxes, maximum values are displayed on top of the boxes and p-values are displayed in the upper section of the plot.

[0050] Detailed Descrintion of the Invention

[0051] With the increasing awareness for and of environmental impact, scientists have begun to actively challenge their routines and behavior toward what is called “Green Chemistry”.

[0052] Analytical chemistry may often not be perceived as a major source of pollution. However, with chromatography being the working horse in terms of analytical methods in bio-therapeutics development and research, solvents and eluents used for performing such experiments are the major waste generating consumables. Often, hazardous and environmentally xenobiotic solvents and / or modifiers need to be used to achieve the required performance, especially for RP separations.

[0053] The current invention is based, at least in part, on the finding that the separation of bio-therapeutics, such as, e.g., therapeutic proteins or antibodies, can be achieved using mixtures of ethanol and ethyl acetate as organic eluent.

[0054] It has been found by the current inventors that at least mixtures of ethyl acetate and ethanol can be used as eco-friendly and improved alternatives compared to the most commonly used organic solvent for RP, namely, ACN. Amongst other things, the mixture according to the current invention is improved compared to the prior art standard ACN eluent in terms of separation efficiency, i.e., resolution or / and separation time, as well as mass spectrometry detection was improved and less organic solvent was consumed. Additionally, the properties of the solvent according to the current invention allows UV detection at a wavelength of 280 nm, which is the standard detection wavelength for proteinaceous analytes, that is not possible using, e.g., acetone or other ketones.

[0055] DEFINITIONS

[0056] Human immunoglobulins are mainly glycosylated at the asparagine residue at about position 297 (Asn297) of the heavy chain CH2 domain or in the FAB region with a more or less fucosylated biantennary complex oligosaccharide (immunoglobulin amino acid residue numbering according to Kabat, see below). The biantennary glycostructure can be terminated by up to two consecutive galactose (Gal) residues in each arm. The arms are denoted (1,6) and (1,3) according to the glycoside bond to the central mannose residue. The glycostructure denoted as GO comprises no galactose residue. The glycostructure denoted as G1 contains one or more galactose residues in one arm. The glycostructure denoted as G2 contains one or more galactose residues in each arm (Raju, T.S., Bioprocess Int. 1 (2003) 44-53). Human constant heavy chain regions are reported in detail by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and by Brueggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T.W., et al., Methods Enzymol. 178 (1989) 515- 527. CHO type glycosylation of immunoglobulin Fc parts is e.g. described by Routier, F.H., Glycoconj. J. 14 (1997) 201-207. The term “immunoglobulin” denotes and encompasses the various forms of immunoglobulins such as human immunoglobulins, humanized immunoglobulins, chimeric immunoglobulins, or T-cell antigen depleted immunoglobulins (see e.g. WO 98 / 33523, WO 98 / 52976, and WO 00 / 34317). In one embodiment the antibody in the methods as reported herein is a human or humanized antibody. Genetic engineering of immunoglobulins is e.g. described in Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244; Riechmann, L., et al., Nature 332 (1988) 323-327; Neuberger, M.S., et al., Nature 314 (1985) 268-270; Lonberg, N., Nat. Biotechnol. 23 (2005) 1117-1125.

[0057] An immunoglobulin in general comprises two so called full length light chain polypeptides (light chain) and two so called full length heavy chain polypeptides (heavy chain). Each of the full length heavy and light chain polypeptides contains a variable domain (variable region) (generally the amino terminal portion of the full length polypeptide chain) comprising binding regions which interact with an antigen. Each of the full length heavy and light chain polypeptides comprises a constant region (generally the carboxyl terminal portion). The constant region of the full length heavy chain mediates the binding of the immunoglobulin i) to cells bearing a Fc gamma receptor (FcyR), such as phagocytic cells, or ii) to cells bearing the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also mediates the binding to some factors including factors of the classical complement system such as component (Clq). The variable domain of a full length immunoglobulin’s light or heavy chain in turn comprises different segments, i.e. four framework regions (FR) and three hypervariable regions (CDR). A “full length immunoglobulin heavy chain” is a polypeptide consisting in N-terminal to C-terminal direction of an immunoglobulin heavy chain variable domain (VH), an immunoglobulin constant domain 1 (CHI), an immunoglobulin hinge region, an immunoglobulin constant domain 2 (CH2), an immunoglobulin constant domain 3 (CH3), and optionally an immunoglobulin constant domain 4 (CH4) in case of an immunoglobulin of the subclass IgE. A “full length immunoglobulin light chain” is a polypeptide consisting in N-terminal to C-terminal direction of an immunoglobulin light chain variable domain (VL), and an immunoglobulin light chain constant domain (CL). The full length immunoglobulin chains a linked together via inter-polypeptide disulfide bonds between the CL-domain and the CHI domain and between the hinge regions of the full length immunoglobulin heavy chains.

[0058] The term “immunoglobulin fragment” denotes within this application a polypeptide comprising at least the CH2 domain and the CH3 domain of a full length immunoglobulin heavy chain. An immunoglobulin fragment may also comprise additional non-immunoglobulin derived amino acid sequences.

[0059] It has been reported in recent years that the glycosylation pattern of immunoglobulins, i.e., the saccharide composition and multitude of attached glycostructures, has a strong influence on the biological properties (see, e.g., Jefferis, R., Biotechnol. Prog. 21 (2005) 11-16). Immunoglobulins produced by mammalian cells contain 2-3 % by mass oligosaccharides (Taniguchi, T., et al., Biochem. 24 (1985) 5551-5557). This is equivalent e.g. in an immunoglobulin of class G (IgG) to 2.3 oligosaccharide residues in an IgG of mouse origin (Mizuochi, T., et al., Arch. Biochem. Biophys. 257 (1987) 387-394) and to 2.8 oligosaccharide residues in an IgG of human origin (Parekh, R.B., et al., Nature 316 (1985) 452-457), whereof generally two are located in the Fc-region at Asn297 and the remaining in the variable region (Saba, J. A., et al., Anal. Biochem. 305 (2002) 16-31).

[0060] The term “glycostructure” as used within this application denotes a single, defined N- or O-linked oligosaccharide at a specified amino acid residue. Thus, the term “immunoglobulin with a G1 glycostructure” denotes an immunoglobulin comprising at the asparagine amino acid residue at about amino acid position 297 according to the Kabat numbering scheme or in the FAB region a biantennary oligosaccharide comprising only one terminal galactose residue at the non-reducing ends of the oligosaccharide. The term “oligosaccharide” as used within this application denotes a polymeric saccharide comprising two or more covalently linked monosaccharide units.

[0061] For the notation of the different N- or O-linked oligosaccharides in the current invention the individual sugar residues are listed from the non-reducing end to the reducing end of the oligosaccharide molecule. The longest sugar chain was chosen as basic chain for the notation. The reducing end of an N- or O-linked oligosaccharide is the monosaccharide residue, which is directly bound to the amino acid of the amino acid backbone of the immunoglobulin, whereas the end of an N- or O-linked oligosaccharide, which is located at the opposite terminus as the reducing end of the basic chain, is termed non-reducing end.

[0062] As used herein, the term "protein" includes any amino acid polymer having covalently linked amide bonds. Proteins comprise 100 or more amino acid residues. Smaller amino acid polymers are denoted as polypeptides and peptides, respectively. Proteins may comprise two or more amino acid polymer chains. A protein may contain one or multiple polypeptides to form a single functioning biomolecule. In one embodiment, the protein is an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a host-cell protein or any combinations thereof.

[0063] As used herein, a “bio-therapeutic drug” or “therapeutic molecule” includes at least one active ingredient which can be fully or partially biological in nature and which has pharmaceutical activity. In one embodiment, the pharmaceutical product can comprise a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a Fc-region of an antibody, an enzyme product, a cytokine, a growth factor, a protein pharmaceutical product, a toxin, a nucleic acid, DNA, RNA, a chemical compound, a cell, a tissue, or any other pharmaceutically active compound.

[0064] As used herein, a "denaturation solution" includes alkaline solution, acid solution, or solution containing urea, guanidinium chloride, oxidizing agents, reducing agents, organic solvents or other chaotropic substances.

[0065] As used herein, "peptide mapping" refers to a technology for confirming a protein's primary structure, e.g., amino acid sequence. Peptide mapping can be used for identification, primary structural characterization, and quality assurance / quality control (QA / QC). The unknown protein of interest can be first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as coupling a liquid chromatography with tandem mass spectrometry (LC-MS / MS)-based peptide mapping platform for confirmation of protein primary structure.

[0066] In some aspects, the mass spectrometer in the method or use of the present invention is a high resolution mass spectrometer (HRMS) or low resolution mass spectrometer (LRMS) using different physical principles to determine a molecules mass to charge ration. This includes, but is not limited to time of flight mass spectrometers (TOF- MS), multi reflection time of flight mass spectrometers (MR-TOF-MS), single- or triple quadrupole mass spectrometers (QDa-MS, QQQ-MS), ion trap mass spectrometers (IT -MS), Orbitrap™ mass spectrometers, ion mobility mass spectrometers (IMS-MS), Fourier-transform ion cyclotron mass spectrometers (FT- ICR-MS) or magnetic resonance mass spectrometers (MRMS), magnetic sector field mass spectrometer or hybrid instruments, such as, e.g., Q-TOF, QQQ-TOF, lonTrap- Orbitrap, Q-FT-ICR, etc. The sample can be introduced through different ionization techniques, which include, but are not limited to: electrospray ionization (ESI), nanoelectrospray ionization (nESI), atmospheric pressure chemical ionization (APCI), desorption electro spray ionization (DESI), electrosonic spray ionization (ESSI), wherein the mass spectrometer is coupled to a liquid chromatography system, wherein the mass spectrometer is capable of performing LC-MS (liquid chromatography-mass spectrometry) or a MS / MS-(meaning: liquid chromatography-tandem -mass spectrometry) analyses (the latter is often described as tandem MS) or LC-MS / MS / MS or LC-MS3. For this purpose, the instrument may be operated in various scan modes including but not limited to: full ion scan (FullMS), all ion fragmentation (AIF), data dependent acquisition (DDA), precursor ion scan (PI), neutral loss scan (NL), product ion scan (EPI), data independent aqueous (DIA), single ion monitoring or selective ion monitoring (SIM), selective reaction monitoring (SRM) multiple reaction monitoring (MRM), parallel reaction monitoring (PRM). The molecular ion fragmentation in these various scan modes may be provoke by the means of different fragmentation techniques, such as: collision induced dissociation (CID), higher energy collisional dissociation (HCD), collision activated dissociation (CAD) in-source fragmentation or in-source decay (ISD), electron transfer dissociation (ETD), electron detachment dissociation (EDD), electron capture dissociation (ECD) electron activated dissociation (EAD), ultraviolet photo dissociation (UVPD), infrared multiphoton dissociation (IRMPD), negative electron transfer dissociation (NETD), laser induced dissociation (LID) and / or surface induced dissociation (SIM). A combination or series of the mentioned techniques are possible in multi-stage MS / MS or MSnexperiments.

[0067] As used herein, a "mass spectrometer" includes a device capable of identifying specific molecular species and determining their accurate masses through measuring the analyte’s mass to charge ratio. The term is meant to include any molecular detector into which a compound of interest, such as, e.g., a polypeptide or peptide, may be eluted or injected for detection and / or characterization. A mass spectrometer includes three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into the gas phase and ionized concurrently (as in electrospray ionization). The choice of ion source depends on the intended application.

[0068] As used herein, the term "liquid chromatography" refers to a process in which a chemical mixture carried by a liquid mobile phase can be separated into components as a result of differential adsorption of the chemical entities comprised in the mixture as they flow around or over a stationary liquid or solid phase. Non-limiting examples of chromatography include traditional reversed-phased (RP), ion exchange (IEX), mixed mode chromatography and normal phase chromatography (NP).

[0069] As used herein, the term "electrospray ionization" or "ESI" refers to the process of spray ionization in which either cations or anions in solution are transferred to the gas phase via formation and desolvation at atmospheric pressure of a stream of highly charged droplets that result from applying a potential difference between the tip of the electrospray needle containing the solution and a counter electrode. There are generally three major steps in the production of gas-phase ions from electrolyte ions in solution. These are: (a) production of charged droplets at the ES emitter needle; (b) shrinkage of charged droplets by solvent evaporation and repeated droplet disintegrations leading to small highly charged droplets capable of producing gasphase ions; and (c) the mechanism by which gas-phase ions are produced from very small and highly charged droplets. Stages (a)-(c) generally occur in the atmospheric pressure region of the apparatus. In some exemplary embodiments, the electrospray ionization mass spectrometer can be a nano-electrospray ionization mass spectrometer.

[0070] As used herein, the term "triple quadruple mass spectrometer" refers to a tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass-resolving) radio frequency (RF), only quadrupole between them to act as a cell for collision-induced dissociation. In a triple quadrupole mass spectrometer, a peptide sample is injected onto an LC device coupled with a MS instrument. The first quadrupole can be used as a mass filter to isolate peptides with a targeted m / z. The second quadrupole serves as a collision cell to break the peptide into fragments. The third quadrupole serves as a second mass filter for specified m / z fragments from the initial parent peptide.

[0071] As used herein, the term "tandem mass spectrometry" includes a technique where structural information on sample molecules can be obtained by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules can be transferred into gas phase and ionized in their intact state and that they can be induced to disintegrate in some predictable and controllable fashion after the first mass selection step. Multistage MS / MS, or MSn, can be performed by first selecting and isolating a precursor ion (MS2), fragmenting it, isolating a primary fragment ion (MS3), fragmenting it, isolating a secondary fragment, and so on as long as one can obtain meaningful information or the fragment ion signal can be detectable. Tandem MS have been successfully performed with a wide variety of analyzer combinations. What analyzers to combine for a certain application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. The two major categories of tandem MS methods are tandem- in-space and tandem-in-time, but there are also hybrids where tandem-in-time analyzers are coupled in space or with tandem-in-space analyzers. A tandem-in- space mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. Specific m / z separation functions can be designed so that in one section of the instrument ions are selected, dissociated in an intermediate region, and the product ions are then transmitted to another analyzer for m / z separation and data acquisition. In tandem-in-time mass spectrometer ions produced in the ion source can be trapped, isolated, fragmented, and m / z separated in the same physical device.

[0072] In some embodiments, the method or use according to the invention comprises a tandem mass spectrometry (MS / MS) technique or direct MS detection. In some embodiments, the method or use according to the current invention comprises an LC- MS / MS technique. For example, the mass spectrometry technique can be: single quadrupole SM (LC-Q),LC- Triple quadrupole MS (LC-TQ or LC-QQQ), or LC- ToF, or LC-QToF, or LC-ToF / ToF, or LC-TQToF or LC-Orbitrap, LC ion trap (LC- IT), LC-MRMS or hybrid LC-IT-Orbitrap, or LC-FT-ICR or hybrid instruments which combine two or more mass detection or mass filtering devices. In some embodiments, the mass spectrometry technique comprises selective reaction monitoring (SRM) analysis. In some embodiments, the SRM analysis comprises monitoring the IgG constant region.

[0073] LC-MS methodology

[0074] For example, after sample preparation, a monoclonal antibody sample, such as a trypsin digested immunoglobulin sample, can be subjected to a mass spectrometry (MS) technique, either directly or after separation on a high-performance liquid chromatography column (HPLC). HPLC-MS / MS is an analytical technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry and is suitable for detection and potential identification of chemicals in a complex mixture. Any LC-MS instrument can be used. In some embodiments, a reversed-phase liquid chromatography HPLC column can be utilized. Any suitable reversed-phase liquid chromatography HPLC column can be used. The ion mass spectrum can be analyzed for one or more peaks corresponding to one or more heavy or light chain peptides in the sample. In some embodiments, the ratio is determined by the peak area of the selected ion peak(s).

[0075] SPECIFIC EMBODIMENTS OF THE CURRENT INVENTION

[0076] The current invention is based, at least in part, on the finding that the separation of compounds of interest, e.g., bio-therapeutics, such as, e.g., proteins, can be achieved using mixtures of ethanol and ethyl acetate or organic eluent.

[0077] The method according to the current invention is applicable to peptides, polypeptides and proteins as their building blocks are the same, i.e. all of them are biopolymers consisting of amino acid residues linked by amide bonds. Thus, the difference is the number of residues. This has an influence on the total number of interactions with the stationary phase but not regarding the solvent employed for the HPLC separation. This has been shown, e.g., by Fekete and Guillarme (LCGC Europe, 25 (2012) 540- 550) which used RP-HPLC with a gradient of the mobile phase A which consisted of 0.1% TFA in water and the mobile phase B which consisted of 0.1% TFA in acetonitrile for the analysis of the therapeutic protein filgrastim as well as the recombinant monoclonal antibodies rituximab and bevacizumab, each of them as full-length antibody, reduced into individual chains and as enzymatic digest.

[0078] It has been found that mixtures of ethyl acetate and ethanol can be used as eco- friendly alternatives to the most commonly used organic solvent for RP-LC, namely, acetonitrile (ACN). Especially as these compounds can be converted without substantive changes in the sewage treatment plant into methane (biogas), which in turn can be used for the generation of electricity and heat. Thus, the ethanol and ethyl acetate mixture used as mobile phase according to the current invention would ultimately be processed into biogas in the anaerobic microbial process during wastewater treatment. Thereby profoundly changing the processing of analytical chromatography waste making energy consuming and environmentally concerning incineration obsolete.

[0079] It has further been found that a mobile phase comprising or consisting of the mixture according to the current invention is unexpectedly improved compared to the prior art standard ACN eluent at least in terms of separation efficiency and mass spectrometry detection as these were unexpectedly improved and unexpectedly less organic solvent was consumed (e.g., faster separation). A person skilled in the art did not expect that an Eluent with improved performance properties compared to ACN could be found. Additionally, the properties of the solvent according to the current invention allows UV detection at a wavelength of 280 nm, which is not possible using, e.g., ketones, such as acetone.

[0080] The current invention is further based on the finding that the environmentally critical solvent modifiers, i.e., ion paring reagents such as di- and trifluoroacetic acid can be replaced by the modifier cyanoacetic acid (malonic mononitrile, CNA) which resulted in an unexpected improvement of the solvent according to the current invention.

[0081] RP necessitates the use of non-polar solvents, i.e., typically organic compounds. Much research embarked on the search for alternative eluents to ACN for RP application

[0038] ,

[0082] Acetone and methanol were deemed to be suitable for peptide-RP-separation

[0025] , Although acetone showed higher elution strength for peptides than acetonitrile and even greater than methanol, the high UV-cut off of acetone at 300 nm posed a major drawback for UV detection. In HPLC analysis that is often used in combination with LC-MS. Proteins are typically detected at 280 nm UV wavelength. Mass spectrometry for protein analysis is typical employed in combination with upstream UV detection, i.e., (U)HPLC-UV-LC. However, both ethanol and ethyl acetate with their respective UV cut-offs at 205 nm and below 260 nm (e.g., 256 nm), respectively, enable UV detection and, thus, can be employed in a UHPLC-UV-MS setup.

[0083] A combination of 50 % (v / v) ethanol and acetone was also tested for peptide separation

[0039] , Like for acetone and methanol, the limitations with respect to the applicability of UV detection was a major drawback and prevented their used.

[0084] Although glycerol, has been successfully applied to RP separation of antiviral drugs

[0040] , due to its non-volatility, it is not suitable for use in LC-MS applications which necessitates the effective evaporation and full desolvation of the analyte that has to be eventually is transferred in the gas phase for it to be analyzed.

[0085] Ethanol / water combinations have been applied for the separation of small molecules [41-42], However, ethanol’s higher viscosity resulted in significantly higher backpressures in the chromatography system and thereby created new challenges although the UV cut-off is at an acceptable 210 nm [43-45], With respect to small molecule RP-HPLC separation only, ethyl lactate [46-47], propylene carbonate [48-49], dimethyl carbonate

[0050] and ethyl acetate [51-53] have been tested in combination with methanol

[0054] or ethanol

[0053] but only found suitable for isocratic elution

[0055] ,

[0086] “Wassergefahrdungsklasse (WGK)” or ‘water hazard class” (WHC) is a classification system issued by the German Umweltbundesamt (UBA) that is accessible to the public via: https: / / webrigoletto.uba.de / Rigoletto /

[0087] Since 2017 the UBA categorizes substances into 5 WHC groups based on a rating system that accounts for a substances' chemo-physical properties, biological and toxicological profile: non-hazardous to waters (nwg, former WHC-class 0). This class is exclusively used for food grade material meant for humans and / or animal consumption; hazardous to water in general (awg), e.g., slurry (mixture of water and manure), yelp, silage; slightly hazardous to waters (WHC class 1), e.g., acetic acid, sodium hydroxide solution, alcohol, hydrogen peroxide; obviously hazardous to water (WHC class 2), e.g., iodine, sodium hypochlorite, heating oil; and highly hazardous to water (WHC class 3), e.g., benzene, used motor oil, chlorinated hydrocarbons).

[0088] These classifications have major implications regarding the handling, transport and storage as well as documentation practices for a given compound.

[0089] For example, acetonitrile has a WGK of 2; Identification-number 8, (current status 2017); (https: / / webrigoletto.uba.de / Rigoletto / Home / SearchDetail / 8); cyanoacetic acid has a WGK of 1; Identification-number 2521, (current status 2017); https: / / webrigoletto.uba.de / Rigoletto / Home / SearchDetail / 2521); fluoroacetic acid has a WGK of 3 ; Identification-number 156, (current status 2017); (https: / / webrigoletto.uba.de / Rigoletto / Home / SearchDetail / 156); difluoroacetic acid is not listed, therefore it should be put into class 3; trifluoroacetic acid has a WGK of 2; Identification-number 729, (current status 2017); (https: / / webrigoleto.uba.de / Rigoletto / Honie / SearchDetail / 729); formic acid has a WGK of 1; Identification-number 210, (current status 2017); (https: / / webrigoleto.uba.de / Rigoletto / Home / SearchDetail / 210); ethanol has a WGK of 1; Identification-number 96, (current status 2017); (https: / / webrigoleto.uba.de / Rigoletto / Home / SearchDetail / 96); ethyl acetate has a WGK of 1; Identification-number 95, (current status 2017); (https: / / webrigoleto.uba.de / Rigoletto / Home / SearchDetail / 95).

[0090] Thus, the current invention is based, at least in part, on the finding that the separation of polypeptides, e.g., of bio-therapeutics, such as, e.g., proteins, can be achieved using either ethanol or ethyl acetate or a combination thereof as organic eluent. Thereby the use of acetonitrile can be abolished. The favorable environmental and health profile, its availability and chemical properties makes the mixture according to the current invention an improved solvent for protein analytical chemistry.

[0091] The HPLC eluent according to the current invention, composed of a mixture of ethanol and ethyl acetate in combination with the acidic modifier cyanoacetic acid (malonic acid mononitrile) are compounds that are rated at WHC class 1. This exemplifies the environmental and sustainability advantage over the art of the current invention. The most widely used solvent (gold standard) for RP-HPLC protein separation, i.e. mixtures of trifluoroacetic acid with acetonitrile, are classified as obviously hazardous to water with both compounds at WHC 2. DFA was not rated by the Umweltbundesamt, however - following their guideline of self-assessment based on similarity - is either WHC 2 (as trifluoroacetic acid) of WHC 3 (strongly hazardous to water) for monofluoroacetic acid.

[0092] Both ethyl acetate and ethanol can be derived from renewable feedstock and are natural compounds present in the biosphere, while acetonitrile is xenobiotic, i.e., foreign to nature and thus has to be chemically synthesized

[0056] , M. Tobiszewski, et al., elaborated on the original CHEMS 1 scoring algorithm

[0057] for a comprehensive scoring system that takes both environmental and health effects, e.g., acute and chronic toxicity, carcinogenicity, biodegradability, etc., into account to give solvent total hazard values (tHV) and total analytical hazard values (taHV). Here, ethanol scores 4.1 in tHV and 7.2 taHV which is comparable to ethyl acetate’s 5.0 tHV and 7.3 taHV, while acetonitrile is rated at a concerning 18.8 tHV and 26.8 taHV respectively

[0058] . Ethanol and ethyl acetate can be mixed in any ratio, i.e., these are fully miscible with each other. Thus, in the Eluent B ethanol and ethyl acetate can have any ratio, such as, e.g., from 50:50 to 99: 1, in one embodiment from 60:40 to 99: 1, in one embodiment from 65:35 to 99: 1, in one preferred embodiment from about 70:30 to 99: 1, in one more preferred embodiment from 70:30 to 85: 15, in one most preferred embodiment about 70:30 or about 85: 15.

[0093] However, it has to be taken into account that mixing Eluent B of the current invention with water as Eluent A independent of the ratio is possible. At room temperature mixing of an Eluent B comprising ethanol and ethyl acetate with an Eluent A comprising only water is possible at any ratio, i.e., Eluent A: Eluent B from 99: 1 to 1 :99, only in case Eluent B has at most 30 % (v / v) ethyl acetate.

[0094] The current invention is exemplified in the following with three applications, i.e., intact mass, subunit and reduced monoclonal antibody (mAb) analysis. This is presented solely to exemplify the invention and shall not be construed as a limitation. The true scope of the invention is set forth in the appended claims.

[0095] The separation of peptides, polypeptide or proteins using either ethanol or ethyl acetate or a combination thereof has not been reported prior to the current invention.

[0096] As can be seen from the boxplots in Figures 9 to 12 that with an eluent according to the current invention compared to acetonitrile an improved analytical behavior of the substances to be analyzed can be seen, such as higher peptide intensities (about 2-fold) or / and sequence coverage.

[0097] In principle, highly organic eluent combinations are required to effectively elute the analyte, i.e., a bio-therapeutic or fragment thereof, from an RP-LC column and to enable high ionization yield, ensuring high quality mass spectrometric analysis results in an RP-LC-MS gradient elution method.

[0098] Despite its favorable ecological and health-hazard profile, the suitability of ethyl acetate (EtOAc) for gradient elution, however, is limited by its non-miscibility with water. In contrast, the most commonly used organic phases for RP-LC, namely methanol and acetonitrile, are fully miscible with water at any ratio. Naturally, any in situ or in system de-mixing of the mobile phase with the inherent formation of two phases would be disastrous for the LC system and any chromatographic separation. A ternary mixing diagram of water, ethanol and ethyl acetate was reported by Tang et al.,

[0059] showing that these three compounds are not freely miscible at any ratio. Especially, mixtures with high % (v / v) of water are prone to phase separation at 40 °C and de-mix readily resulting in the formation of distinct and separated phases (2-phases system). However, mixtures with a high % (v / v) water are required for ensuring adsorption of, e.g., a protein analyte to a RP column material and, thus, enabling chromatographic separation.

[0099] The current invention is based, at least in part, on the finding that ethyl acetate (EtOAc) is fully miscible with ethanol (EtOH) and that EtOH can act as a mediator between the non-miscible pair of EtOAc and water allowing the use of this ternary system as LC-MS solvents.

[0100] The current invention is based, at least in part, on the finding that in the range from 20 °C to 80 °C a composition comprising about 75 % (v / v) water, about 17 % (v / v) ethanol and about 8 % (v / v) ethyl acetate is especially suitable as LC-MS elution solvent. This mixture has an EtOAc to EtOH ratio of about 3 to 10.

[0101] The invention is based, at least in part, on the finding that the mixture of about 75 % (v / v) water, about 17 % (v / v) ethanol and about 8 % (v / v) ethyl acetate provides for maximum elution strength and full miscibility.

[0102] Optimal and stable mixing properties were found for the “30 % (v / v) mixture” comprising 30 % (v / v) EtOAc and 70 % (v / v) EtOH for Eluent B. The mixture can be acidified with 0.05 % (v / v) of each DFA and FA, respectively, or 3-20 mM CNA. Thereby an Eluent B was obtained that comprises EtOAc as a strong eluent and allows access to low % (v / v) Eluent B chromatographic ranges that are indispensably vital for RP-LC bio-therapeutic separation. Eluent A was deionized, ultra-pure water (PWA) comprising 0.05 % (v / v) of each DFA and FA, respectively, or CNA.

[0103] In a first set of experiments the analysis of the glycosylation patterns of a therapeutic antibody by RP -LC-MS analysis of enzymatically cleaved protein chains was performed.

[0104] First, the analysis of such sub units of the monoclonal antibody trastuzumab was performed. The RP-LC-column (polyphenyl-ligand, 2.7 pm particle size, 2.1x150 mm) was run at 75 °C and 300 pL / min flow rate. About 2 pg of the digested antibody was injected and separated by a gradient starting at 20 % (v / v) Eluent B, increasing to 50 % (v / v) Eluent B in 40.5 minutes using different compositions for Eluent B as depicted in the following Table 1.

[0105] Table 1: Eluent compositions

[0106] The total intensities of antibody-subunits and glycan structures detected with eluent composition 1 to 4 are shown in the following Table 2.

[0107] An antibody subunit and glycosylation analysis post IdeS mediated enzymatic cleavage at the antibody hinge region was performed (see Example 1). The results are presented in the following.

[0108] Table 2: Absolute intensities of antibody-subunits and glycan structures detected using different eluents for chromatographic separation.

[0109] From deconvoluted mass spectra at identical computing conditions using the PMI Intact™ software suite. The respective stacked chromatograms are shown in Figure 1 for the Eluent B range from 21.5 % (v / v) to 42 % (v / v) of each chromatogram.

[0110] As can be seen in Figure 1 and from the corresponding data presented in Table 2, all eluent composition containing ethanol in Eluent B yielded significantly higher signal intensities. That is, a surprisingly significantly higher mass spectrometric intensity under otherwise identical chromatographic and mass spectrometric conditions, i.e., temperature, flow rate, source voltage etc., was achieved using an Eluent B comprising ethanol alone or in a mixture with ethyl acetate. This is independent of the respective subunits and glycan-species, all showing an about 2-fold increase in intensity compared to the separation with acetonitrile as Eluent B.

[0111] Without being bound by this theory, it is assumed that this is at least in part due to the slightly lower boiling point of both ethanol and ethyl acetate at roughly 78.4 °C and 77.1 °C respectively, compared to approx. 82 °C for acetonitrile at atmospheric pressure, likely resulting in a faster desolvation and, thus, enhanced ion yield in electrospray ionization (ESI).

[0112] It is important to highlight that the relative glycostructure distribution found using any of the solvents according to the current invention was comparable, hence the analysis results are consistent with the use of ACN as Eluent B.

[0113] Increasing the ion source temperature from 325 °C to 400 °C for acetonitrile as Eluent B did not yield any higher intensities.

[0114] This clearly shows the improved mass spectrometric properties of the solvent mixture according to the current invention.

[0115] It has to be pointed out that when attempting to mix 50:50 (v / v) combinations of EtOAc and EtOH with large amounts of water, de-mixing, i.e., phase separation, occurred at room temperature (RT), as evident by the formerly clear solutions turning cloudy and the occurrence of opalescent droplets that clearly indicate the presence of two phases.

[0116] It has further been found that with 30 % (v / v) EtOAc in Eluent B a significantly reduces back pressure in the chromatography system in comparison with using pure ethanol as Eluent B could be achieved. A significantly higher backpressure is one of the major drawback when using pure ethanol as the organic phase in RP-HPLC. Said backpressure is notably higher for ethanol than for acetonitrile, given ethanol’s higher viscosity. That is, using an Eluent B comprising 30 % (v / v) EtOAc resulted in a pressure peak reduction by approximately 50 bar of total system backpressure while with 15 % (v / v) EtOAc a roughly 25 bar reduction of backpressure was achieved in comparison to using ethanol only.

[0117] Typical columns and UHPLC on the market today that are used in the art are capable of withstanding system pressure of around 1000 bar. For prolonged use, this pressure limit should not be exceeded. Eluent viscosity and backpressure decreases with column temperature and rises with flowrate. The typical LC-MS peptide analysis experiment in the art using a 2.1 mm inner diameter sub 2 pm particle size column is performed at 0.3 mL / min flow rate. Backpressure is the highest at 65 % Eluent B in the gradient profile, hence it was monitored at this isocratic mixing ratio with PWA as Eluent A. Peptide separation and analysis is typically performed at moderate, but not to high column temperatures. As high temperate are linked to on- column degradation of the peptides and less retention as they reduce analyte affinity to the solid phase. To avoid this, the columns are typically operated at around 50 °C in such an experiment.

[0118] As evident from the data in Table 9, ethanol as Eluent B results in system backpressures that exceed 1000 bar at the desired settings (0.3 mL / min and 50 °C). Some UHPLC systems therefore will not be able to perform the intended experiment, as sequenced runs are typically automatically halted when safe pressure limits are exceeded. The addition of 10 to 30 % (v / v) ethyl or methyl acetate according to the current invention, however, drastically reduces system back pressures. Therefore, it enables safe operation of the system well below 1000 bar and with the desired analysis settings. In some examples, the pressure difference between identical settings to pure ethanol are well over 100 bar.

[0119] The invention is further based, at least in part, on the unexpected finding that high- EtOAc fraction Eluent B resulted in a much higher elution strength, which can be seen by the reduced analyte retention time for the identical gradient and temperature compared to acetonitrile. Unexpectedly and surprisingly, although the overall retention time decreases for higher EtOAc fractions in Eluent B, the chromatographic resolution is fully maintained. This can be seen by improved chromatographic resolution values for the Fc / 2 and F(ab’)2 peaks in the chromatographic data presented in Table 3. Additionally, these values are higher for all ethanol containing Eluent B compositions when compared to acetonitrile as Eluent B. This shows an improved chromatographic performance of the mixture according to the current invention. Furthermore, this improvement is achieved with neglectable peak widening. Chromatographic performance descriptors for Fc / 2 and F(ab’)2 subunits separation using different mobile phase are shown in the following Table 3.

[0120] Table 3: Chromatographic performance descriptors for Fc and F(ab’)2 subunits separation using different Eluent B. Corresponds to chromatograms shown in Figure 1.

[0121] VF0 5 / l, FWHM, full width at half maximum and Rsresolution as per US-Pharmacopeia definition

[0060] ,

[0122] Thus, the faster elution at significantly lower % (v / v) Eluent B values results in a reduction of Eluent B consumption, i.e., of the environmentally concerning and costly organic mobile phase.

[0123] The invention is based, at least in part, on the finding that the improved gradientrange determined for different antibody separations was found to be 36 - 48 % (v / v) Eluent B using ethanol, 30 - 42 % (v / v) Eluent B using 15 % (v / v) EtOAc / EtOH (equally to ACN) and 22 - 34 % (v / v) Eluent B using the 30 % (v / v) EtOAcZEtOH mixture.

[0124] The reduction of Eluent B consumption has been calculated using Thermo Chromeleon™ 6 software package, taking into account a real-world separation scenario reflected as a 30 min. gradient separation at the values mentioned above, followed by a 1 min. ramp to 99 % (v / v) Eluent B for a 2 min. wash and 5 min. slope and 3 min. equilibration at the starting conditions, at a 300 pL / min. flow rate. The solvent consumption estimates are 5.78 mL for ethanol only, 5.2 mL for 15 % (v / v) EtOAc / EtOH or ACN and 4.43 mL for 30 % (v / v) EtOAc / EtOH. Based on 30 % (v / v) EtOAc / EtOH set to 100 % that corresponds to 117.4 % when using 15 % (v / v) EtOAc / EtOH or ACN and 130.5% when using pure ethanol.

[0125] The data presented demonstrates the applicability of ethanol and the combination of ethyl acetate and ethanol for the analyses of molecules of interest, especially of therapeutic proteins (bio-therapeutics) and subunits thereof. No distinct drawback was observed in comparison with the traditionally solvent comprising acetonitrile, which presents high environmental and health-concerns, also at a higher financial cost.

[0126] Given the less aggressive nature of ethanol and ethyl acetate against polymers, it can also be expected that less wear or damage on chromatography systems and, thus, less downtime and need for maintenance will occur. Which also represents another advantage in sustainability.

[0127] It has been frequently acknowledged in the art that acetonitrile mobile phases cause ruby-sapphire inlet check valves to stick shut requiring frequent checks and replacements, described as “sticky check valves”. This causes excessive instrument down times and service costs. It is generally assumed that ACN’s ability to polymerize under prolonged exposure to heat and / or light, or its origin as a byproduct from the polymer industry and thus potential polymeric impurities are the source of this frequent problem. EtOH and EtOAc at the other hand have no ability to from solid by-products and, thus, it can be expected to see enhanced check-valve and pump head life times that translate to less instrument failure and lower maintenance cost, enabling more sustainable operation of the instrument when using a method according to the current invention.

[0128] An analysis of deglycosylated antibodies at the intact level has been performed (Example 2). The results are presented in the following.

[0129] Despite the higher source temperature applied for the ACN runs (to account for acetonitrile’ s higher boiling point in comparison to EtOAc and EtOH), the respective intensities of all analyzed antibodies was found to be substantially higher for the EtOAc / EtOH mixture according to the current invention. Additionally the chromatographic performance was improved with the Eluent B according to the current invention, as evident by the significantly shorter elution time, at lower % B and similar-width or even narrower FWHM peak widths for the analyzed biomolecules. This further confirms that the 30 % (v / v) EtOAc in EtOH mixture according to the current invention is an improved eluent for the LC-MS analysis of intact antibodies in comparison to the standard acetonitrile based solvent.

[0130] Table 4: Chromatographic and mass spectrometric performance data corresponding to then intact deglycosylated analysis of different antibodies as depicted in Figure 2.

[0131] An analysis of deglycosylated trastuzumab at the reduced antibody level has been performed (Example 3). The results are shown in the following and Figure 3.

[0132] Table 5: Chromatographic and mass spectrometric performance data corresponding to the deglycosylated, reduced analysis of trastuzumab as depicted in Figure 3. HC meaning heavy chain, LC, meaning light chain.

[0133] The findings as outlined above for the analysis of intact antibodies have been confirmed for the analysis of the respective reduced forms. As can be seen from the data in the Table 5 above, for all runs with EtOH containing Eluent B according to the current invention, the MS signal intensities were significantly enhanced (approx. 2-fold) in comparison to the runs with acetonitrile as Eluent B. At the same time, the chromatographic separation efficiency was maintained of even improved.

[0134] The run with 30 % (v / v) EtOAc content in Eluent B according to the current invention showed the fastest elution at the lowest % B range, i.e., a much faster gradient at significantly lower Eluent B consumption which results in vastly reduced chemical waste production can be achieved using these mixtures according to the current invention. This translates to an improved analysis with less valuable consumables used and reduced waste output at lower health risks for the operators.

[0135] The run with 15 % (v / v) EtOAc content in Eluent B according to the current invention showed the best chromatographic resolution. Moreover, all tested Eluent B variants, i.e., 100 % (v / v) ethanol, or mixtures of ethanol and ethyl acetate in the range from 99: 1 to 70:30 showed an improved chromatographic resolution.

[0136] In a further experiment (Example 4) the acidic modifier has been changed from DFA / FA to CNA. In more detail different acidic modifier variants of Eluent B (30 % (v / v) EtOAc) were tested:

[0137] - 5 mM cyanoacetic acid,

[0138] - 10 mM cyanoacetic acid, - 15.89 mM cyanoacetic acid, and

[0139] - 0.05 % (v / v) FA + 0.05 % (v / v) DFA.

[0140] For each of the above, aqueous counterparts - used as eluent A - using Millipore® ultra-pure water were produced at identical acidic modifier concentrations.

[0141] The results are shown in Tables 6 and 7. Table 6: Chromatographic and mass spectrometric data corresponding to the analysis of deglycosylated, reduced bsAb as depicted in Figure 4. HC meaning heavy chain, LC, meaning light chain. FWHM meaning Full width at half height.

[0142] Table 7: Chromatographic performance descriptors for individual peak pairs using different eluent compositions, calculated from the data in Table 6. Chromatographic resolution, (Rs) as per US-Pharmacopeia definition

[0060] , The data presented in Tables 6 and 7 above shows that cyanoacetic acid (CNA) dissolved in the eluent according to the current invention, composed of ethanol and ethyl acetate, allowed for the efficient separation of complex biological molecules, i.e., proteins in this instance. The chromatographic separation performance was improved, as evident by higher resolution values between each pair of peaks in Table 7 in comparison to a comparative eluent using the environmentally concerning fluorinated compound difluoroacetic acid. This is especially evident when using a concentration of 10 mM cyanoacetic acid or higher, which is one preferred embodiment of the current invention. Additionally, a more rapid elution at comparable or even reduced FWHM peak widths was achieved when using CNA as acidic modified in the eluent according to the current invention. This allows for quicker separation and therefore for a further reduction of organic solvent used and waste generated. The current invention is thus further based, at least in part, on the finding that that cyanoacetic acid (malonic acid mononitrile, CNA) in combination with a mixture of ethanol and ethyl acetate provides for potent ion pairing that allows for the RP- (U)HPLC gradient separation with UV and / or mass spectrometric detection of large biomolecules. In contrast in the prior art this could only be achieved by using environmentally problematic perfluorinated and fluorinated ion pairing agents, i.e., mainly DFA and TFA.

[0143] In a comparative example (Example 5) acetonitrile with cyanoacetic acid and the eluent according to the current invention with cyanoacetic acid has been compared. The results are shown in Figures 5 and 6 as well as Table 8.

[0144] Table 8: Chromatographic and mass spectrometric data corresponding to the analysis of the antibody standard mix as depicted in Figure 5.

[0145] Chromatographic performance descriptors for individual peak pairs using different Eluent compositions. Chromatographic resolution, (Rs) as per US-Pharmacopeia definition

[0060] ,

[0146] As shown in Figure 5 and Figure 6 and the data presented in Table 8, ethanol and ethyl acetate based eluents according to the current invention comprising cyanoacetic acid as acidic modifier outperformed acetonitrile as solvent with CNA as acidic modifier at the identical concentration.

[0147] This can be seen from the increased resolution between the peaks and their increased peak height in the UC mass spec trace at smaller peak FWHM. The acetonitrile based solvent resulted in later elution of the species, with less selectivity. The solvent according to the current invention with CNA as acidic modifier showed improved selectivity, evident by resolving additional peaks between the main peaks at identical gradient slope compared to acetonitrile. Comparably high gas flows were chosen for ESI-MS detection, in order to account for the inferior ionization properties of acetonitrile vs. the ethyl acetate / ethanol based eluent according to the current invention. However, even under these conditions, the eluent according to the current invention proved to generate substantially higher intensity mass spectra. It can also be seen that cyanoacetic acid in combination with the eluent according to the current invention is more efficient at eluting large biomolecules from the RP-LC column in comparison to ACN as can be seen by the increased intensity of the UV-LC chromatogram in Figure 6.

[0148] Thus, the current invention is based, at least in part, on the finding that cyanoacetic acid in combination with the eluent according to the current invention provides for an advantage for both UV and MS detection in protein separation and separation of large molecule mixtures.

[0149] In a further example (Example 6) the eluent according to the current invention with cyanoacetic acid and with FA / DFA has been compared.

[0150] The results are shown in Figures 7 and 8.

[0151] As can be seen from the chromatogram and the absolute intensity mass spectrum shown in Figure 7 and Figure 8, respectively, cyanoacetic acid as the acidic modifier in combination with an ethanol and ethyl acetate based eluent according to the current invention provides for improved methods, such as, e.g., with respect to signal intensity, compared to the acidic modifier FA+DFA at comparable concentration. From the data it can be seen that cyanoacetic acid in combination with the eluent according to the current invention provides for improved ionizing of compound in ESLMS detection, yielding substantially higher intensity mass spectra. It is noteworthy that cyanoacetic acid generated higher charge states than DFA for the large protein molecular-ions (see higher averaged charge states mass spectra across charge envelope in the respective sum spectra in Figure 8), which improves mass accuracy and enables measurements at lower tier instruments.

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[0211] ***

[0212] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended embodiments. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0213] Examnles

[0214] Materials

[0215] Acetonitrile (ACN) LiChrosolv® for LC-MS, formic acid (FA) 98-100 %, LiChropur® for LC-MS and ethyl acetate (EtOAc) LiChrosolv® hypergrade for LC- MS, guanidinium hydrochloride (Gua-HCl) BioUltra™ for molecular biology, > 99.5 % and Trizma® (TRIS-HC1), tris-(hydroxymethyl)- aminomethane hydrochloride, BioUltra™ for molecular biology, > 99.0 % (Tris- HC1) and cyanoacetic acid [malonic mononitrile, CNA] 99% were from Supelco® and Sigma Aldrich® (Merck, Darmstadt, Germany), difluoroacetic acid (DFA) (available from Waters), ethanol (EtOH) Rotisolv®, > 99.95 %, Ultra LC-MS was purchased from Carl Roth (Karlsruhe, Germany), 1,4-Dithiotreitol (DTT) crystalline powder > 97 %, PNGase F of Flavobacterium Meningosepticum, recombinant from E. Coli (Cat. No. 113651770012), bivalent, monospecific full-length IgGl antibody trastuzumab (Herceptin™) and bivalent, bispecific full-length IgGl CrossMab reference standard were supplied by Roche Diagnostics GmbH (Mannheim, Germany), FabRICATOR™ lyophilized powder (A0-FR1-020) was from Genovis (Lund, Sweden). NISTmAb #8671 monoclonal antibody reference material was from NIST (Gaithersburg, MD, United States). The APCI positive TOF calibration solution was supplied by AB Sciex (Toronto, Canada). Ultra-pure deionized water (PWA) was produced in-house using a Sartorius Arium™ Pro system (Sartorius AG, Gottingen, Germany).

[0216] Instrumentation (Example 1 to 3)

[0217] For biomolecule analysis the following equipment was used: The RP-HPLC separation was performed using a BioResolve™ Polyphenyl column with the dimensions: 450 A, 2.7 pm, 2.1x150 mm, purchased from Waters Corporation (Milford, MA, United States). The chromatography was performed on a Vanquish™ Horizon UHPLC system which was equipped with a Vanquish™ Diode Array Detectorand and a Vanquish™ LightPipe™ Flow Cells for UV monitoring at 280 nm (Thermo Fisher Scientific Inc., Waltham, MA, United States). Mass spectrometric analysis was carried out on an AB Sciex TripleToF™ 6600 device, which was equipped with a DuoSource™ APCI&TSI ion source (AB Sciex Pte. Ltd., Toronto, Canada). The LC-column was coupled to an ASI 600 series adjustable flow splitter (ASI Inc., Richmond, CA, United States) which directed one third of the LC flow to the mass spectrometer’s TSI source and the remaining flow to the UV detector for parallel detection with minimal post column dispersion effect. The TOF mass analyzer was calibrated at the start of each sequence and automatically after every three samples utilizing the Sciex APCI positive calibration solution, which was introduced by the Sciex Calibrant Delivery System (CDS) at 200 pL / min via the APCI probe (AB Sciex, Toronto, Canada). The calibration was performed under identical mass spectrometer settings as the subsequent analysis runs. Solvent composition (experiment 1 to 3)

[0218] The solvents used in all experiments were produced by mixing: for solvent 1 : 1000 mL ethanol with 500 pL DFA and FA respectively; for solvent 2: 700 mL ethanol plus 300 mL ethyl acetate with 500 pL DFA and FA respectively; for solvent 3: 850 mL ethanol plus 150 mL ethyl acetate with 500 pL DFA and FA respectively; for solvent 4: 1000 mL acetonitrile with 500 pL DFA and FA respectively.

[0219] Example 1

[0220] Antibody subunit and glycosylation analysis post-IdeS enzymatic cleavage at hinge region

[0221] Sample preparation

[0222] For subunit analysis, trastuzumab reference standard solution was diluted to 1.25 pg / pL, of which 100 pL was combined with 25 pL FabRICATOR™ solution (lO units / pL in PWA) and subsequently incubated at 37 °C for 60 minutes. The reaction was stopped by acidification through addition of Eluent A at ratio of 1 :5 (v / v), thereby yielding a sample concentration of 0.2 pg / pL. All samples were analyzed immediately, with cooling at 4 °C in the LC auto sampler, or stored at -80 °C until further analysis.

[0223] LC-MS settings

[0224] For sub unit analysis, the following mass spectrometric settings were applied: Ion source gas 1 (GS1) and source gas 2 (GS2) were set to 45 and 35 auxiliary units, respectively. Curtain gas (CUR) was set to 45 units, while the probe temperature was 325 °C and the TSI spray voltage 5.5 kV, the time bins to sum parameter was at 100. A declustering potential (DP) of 80 arbitrary units and 14.5 units collision energy (CE) at a mass range from 600 to 3000 m / z were applied.

[0225] The RP-column (Waters BioResolve™ Polyphenyl, 2.1x150 mm) was run at 75 °C and 300 pL / min flow rate. 2 pg of the FabRICATOR™ digested antibody (10 pL injection volume) was injected using the Vanquish™ Horizon UHPLC system and separated by a scouting gradient starting at 20% B, increasing to 50% B in 40.5 minutes using each of the eluents.

[0226] Examnle 2

[0227] Analysis of deglycosylated antibodies at the intact level

[0228] Sample preparation

[0229] For intact-deglycosylated monoclonal antibody (mAb) analysis, the antibody samples were deglycosylated as follows: the respective antibody was diluted to a concentration of 1 pg / pL in digestion buffer (0.1 M TRIS-HC1, pH 7.8) in a volume of 125 pL. Subsequently, 2 pL PNGaseF solution (2.5 units / pL in purified water (PWA)) was added and incubated overnight (18 hours) at 37 °C. 50 pL of the resulting digested solution was then acidified and adjusted to a final concentration of 0.2 pg / pL using Eluent A (0.05 % (v / v) FA, and 0.05 % (v / v) DFA in PWA).

[0230] LC-MS Analysis

[0231] For intact deglycosylated mAb analysis, the following mass spectrometric settings were applied: Ion source gas 1 (GS1) and source gas 2 (GS2) were set to 45 and 35 auxiliary units, respectively. Curtain gas (CUR) was set to 45 units, while the probe temperature was 325 °C and the TSI spray voltage 5.5 kV, the time bins to sum parameter was at 100. A declustering potential (DP) of 250 arbitrary units and 35 collision energy (CE) with a mass range from 600 to 5000 m / z were applied. ACN runs were performed at both 325 °C and 400 °C probe temperature respectively.

[0232] For all runs, the RP-column (Waters BioResolve™ Polyphenyl, 2.1x150 mm) was run at 80 °C and 300 pL / min flow rate. 2 pg of the deglycosylated antibody was injected (10 pL injection volume) using the Vanquish™ Horizon UHPLC system and separated by a scouting gradient starting at 20% B, increasing to 50% B in 15 minutes using each of the eluents.

[0233] Examnle 3

[0234] Analysis of deglycosylated trastuzumab at the reduced antibody level

[0235] Sample preparation

[0236] For the reduced-deglycosylated mAb preparation, 40 pL of the deglycosylated sample solution from example 2 was mixed with 250 pL denaturing buffer (8.0 M Gua-HCl, 0.4 M TRIS-HC1, pH 8.0) and 10 pL DTT solution (0.1 mg / pL in denaturing buffer) and incubated at 50 °C for 60 minutes. The reaction was stopped by adding 100 pL of Eluent A to the solution, resulting in a final concentration of approx. 0.1 pg / pL.

[0237] LC-MS Analysis

[0238] For deglycosylated-reduced mAb analysis, the following mass spectrometric settings were applied: Ion source gas 1 (GS1) and source gas 2 (GS2) were set to 45 and 35 auxiliary units, respectively. Curtain gas (CUR) was set to 45 units, while the probe temperature was 325 °C and the TSI spray voltage 5.5 kV, the time bins to sum parameter was at 100. A declustering potential (DP) of 80 arbitrary units and 14.5 collision energy (CE) with a mass range from 600 to 3000 m / z were applied.

[0239] For all runs, the RP-column (Waters BioResolve™ Polyphenyl, 2.1x150 mm) was run at 65 °C and 300 pL / min flow rate. 1 pg of the deglycosylated-reduced digested antibody (10 pL injection volume) was injected using the Vanquish™ Horizon UHPLC system and separated by a scouting gradient starting at 20% B, increasing to 50% B in 40.5 minutes using each of the eluents.

[0240] Example 4

[0241] Characterization of cyanoacetic acid (malonic mononitrile) as acidic modifier

[0242] Eluent composition

[0243] The solvents used as Eluent B in the experiments were produced by mixing as follows: for solvent 1 (5 mM cyanoacetic acid): 700 mL ethanol and 300 ml ethyl acetate with 425.3 mg cyanoacetic acid; for solvent 2 (10 mM cyanoacetic acid): 700 mL ethanol plus 300 mL ethyl acetate with 850.6 mg cyanoacetic acid; for solvent 3 (15.89 mM cyanoacetic acid): 700 mL ethanol plus 300 mL ethyl acetate with 1325 mg cyanoacetic acid with; for solvent 4 (0.05 % (v / v) FA + 0.05 % (v / v) DFA) 700 mL ethanol plus 300 mL ethyl acetate with 500 pL formic acid (FA) and 500 pL difluoroacetic acid (DFA). For each of the above, aqueous counterparts - used as Eluent A - using Millipore® ultra-pure water were produced at identical acidic modifier concentrations. Aqueous 15.89 mmol / L cyanoacetic acid results in a solution with pH value of 2.25 (24°C), for 10 mmol / L with a pH value of 2.32 and for 5 mmol / L with a pH value of 2.55.

[0244] Sample preparation

[0245] Intact-deglycosylated bispecific antibody (bsAb) analysis

[0246] The antibody has a molecular weight of approx. 193 kDa. The sample was deglycosylated as follows: 1 pL of the respective 100 pg / pL antibody reference standard (100 pg bsAb) was diluted in 247 pL phosphate buffer (10 mmol / L, pH 7.2). Subsequently, 2 pL PNGaseF solution (2.5 units / pL in purified water (PWA)) was added and incubated overnight (24 hours) at 37 °C.

[0247] Reduced bsAb analysis

[0248] Forty pL of the deglycosylated bsAb solution were incubated with 40 pL freshly prepared denaturation / reduction solution consisting of 8.0 M Guanidinium-HCl, 0.4 M TRIS-HC1, 0.04 M DTT, pH 8.0. The sample was incubated at 37 °C for 60 min. The reduction was stopped by adding 80 pL Eluent A, resulting in a 0.1 pg / pL reduced-deglycosylated bsAb solution of which 10 pL (1 pg) were injected on the HPLC-MS system for analysis.

[0249] LC-MS Analysis

[0250] Instrumentation

[0251] For bsAb analysis the following equipment was used: The RP-HPLC separation was performed using a BioResolve™ Polyphenyl column with the dimensions: 450 A, 2.7 pm, 2.1 x 150 mm, purchased from Waters Corporation (Milford, MA, United States). The chromatography was performed on a Vanquish™ Horizon UHPLC system which was equipped with a Vanquish™ diode Array Detector and a Vanquish™ LightPipe™ Flow Cells for UV monitoring at 280 nm (Thermo Fisher Scientific Inc., Waltham, MA, United States). Mass spectrometric analysis was carried out on an AB Sciex TripleToF™ 6600 device, which was equipped with a duoSource™ PCI&TSI ion source (AB Sciex Pte. Ltd., Toronto, Canada). The LC- column was coupled to an ASI 600 series adjustable flow splitter (ASI Inc., Richmond, CA, United States) which directed 1 / 6, i.e., 50 pL / min of the LC flow to the mass spectrometer’s TSI source and the remaining flow to the UV detector (at 280 nm) for parallel detection with minimal post column dispersion effect. The TOF mass analyzer was calibrated at the start of each sequence and automatically after every three samples utilizing a ESI calibration solution (G1969-85000, Agilent Technologies, Santa Clara, CA, US) which was introduced by the Sciex Calibrant Delivery System (CDS) at 200 pL / min via the APCI probe (AB Sciex, Toronto, Canada). The calibration was performed under identical mass spectrometer settings as the subsequent analysis runs.

[0252] LC-MS settings

[0253] For deglycosylated-reduced bsAb analysis, the following mass spectrometric settings were applied: Ion source gas 1 (GS1) and source gas 2 (GS2) were set to 25 and 25 auxiliary units. Curtain gas (CUR) was set to 10 units, while the probe temperature was 325 °C and the TSI spray voltage 5.5 kV, the ‘time bins to sum’ parameter was at 100. A declustering potential (DP) of 80 arbitrary units and 14.5 collision energy (CE) with a mass range from 600 to 3600 m / z were applied.

[0254] For all runs, the RP-column (Waters BioResolve™ Polyphenyl, 2.1x150 mm) was run at 75 °C and 300 pL / min flow rate. 1 pg of the deglycosylated-reduced antibody (10 pL injection volume) was injected using the Vanquish™ Horizon UHPLC system and separated by a separation gradient starting at 20 % Eluent B, increasing to 40 % Eluent B in 17.5 minutes using ethanol and ethyl acetate based eluents as outlined above.

[0255] The LC-column was coupled to an ASI 600 series adjustable flow splitter (ASI Inc., Richmond, A, United States) which directed 1 / 6, i.e., 50 pL / min of the LC flow to the mass spectrometer’s TSI source and the remaining flow to the UV detector for parallel detection at 280 nm optimized for minimal post column dispersion effect.

[0256] The results are shown in Tables 5 and 6.

[0257] Example 5

[0258] Comparison ACN with cyanoacetic acid and EtOH+EtOAc + cyanoacetic acid for intact Ab analysis

[0259] Solvent composition

[0260] The solvents used as Eluent B were produced by mixing: for solvent 1 (15.89 mM cyanoacetic acid): mixing 700 mL ethanol plus 300 mL ethyl acetate with 1325 mg cyanoacetic acid; for solvent 2 (15.89 mM cyanoacetic acid in ACN): 1000 mL acetonitrile with 1325 mg cyanoacetic acid.

[0261] For the solvents above an aqueous counterpart - used as Eluent A - using Millipore® ultra-pure water was produced at identical acidic modifier concentration. Aqueous 15.89 mmol / L cyanoacetic acid results in a solution with pH value of 2.25 (24 °C).

[0262] Sample preparation

[0263] For separation of intact-deglycosylated bispecific antibodies (Ab), an in-house produced antibody mix comprised of three different antibody-type molecules was used. The mix comprised of an aqueous buffered solution of three different antibodies and antibody derivatives, including a 146.4 kDa mAb, a 147.2 kDa mAb and a 192.89 kDa bsAb, each concentrated a 0.43 pg / pL. The sample was diluted 1 :2 vol / vol using Eluent A, for pre-conditioning the sample.

[0264] LC-MS Analysis

[0265] Instrumentation

[0266] For antibody mix LC-MS analysis the following equipment was used: The RP-HPLC separation was performed using a BioResolve™ Polyphenyl column with the dimensions: 450 A, 2.7 pm, 2.1 x 150 mm, purchased from Waters Corporation (Milford, MA, United States). The chromatography was performed on a Vanquish™ Horizon UHPLC system which was equipped with a Vanquish™ diode Array Detector and a Vanquish™ LightPipe™ Flow Cells for UV monitoring at 280 nm (Thermo Fisher Scientific Inc., Waltham, MA, United States). Mass spectrometric analysis was carried out on an AB Sciex TripleToF™ 6600 device, which was equipped with a duoSource™ PCI&TSI ion source (AB Sciex Pte. Ltd., Toronto, Canada). The LC-column was coupled to an ASI 600 series adjustable flow splitter (ASI Inc., Richmond, CA, United States) which directed 1 / 3, i.e., 100 pL / min of the LC flow to the mass spectrometer’s TSI source and the remaining flow to the UV detector (at 280 nm) for parallel detection with minimal post column dispersion effect. The TOF mass analyzer was calibrated at the start of each sequence and automatically after every three samples utilizing a ESI calibration solution (G1969- 85000, Agilent Technology, Santa Clara, CA, US) which was introduced by the Sciex Calibrant Delivery System (CDS) at 200 pL / min via the APCI probe (AB Sciex, Toronto, Canada). The calibration was performed under identical mass spectrometer settings as the subsequent analysis runs.

[0267] LC-MS settings

[0268] For deglycosylated-intact bsAb analysis, the following mass spectrometric settings were applied: Ion source gas 1 (GS1) and source gas 2 (GS2) were set to 45 and 35 auxiliary units. Curtain gas (CUR) was set to 35 units, while the probe temperature was 325 °C and the TSI spray voltage 5.5 kV, the ‘time bins to sum’ parameter was at 100. A declustering potential (DP) of 250 arbitrary units and 50 collision energy (CE) with a mass range from 600 to 5000 m / z were applied.

[0269] For all runs, the RP-column (Waters BioResolve™ Polyphenyl, 2.1x150 mm) was run at 75 °C and 300 pL / min flow rate. 10 pL of the deglycosylated-intact Ab-mix was injected using the Vanquish™ Horizon UHPLC system and separated by a separation gradient starting at 20 % Eluent B, increasing to 40 % Eluent B in 17.5 minutes using the ethanol and ethyl acetate-based eluents as outlined above. For acetonitrile-based Eluent B, a gradient from 25 % to 45 % Eluent B in 17.5 min was used.

[0270] In both cases the gradient slope is identical.

[0271] The results are shown in Figures 5 and 6 and Table 8.

[0272] Example 6

[0273] Comparison of EtOH+EtOAc + FA / DFA and EtOH+EtOAc + CNA as eluent

[0274] Solvent composition

[0275] The solvents used as Eluent B were produced by mixing: for solvent 1 (10 mM cyanoacetic acid): 700 mL ethanol plus 300 mL ethyl acetate with 850.6 mg cyanoacetic acid: for solvent 2 (0.05 % (v / v) FA + 0.05 % (v / v) DFA) 700 mL ethanol plus 300 mL ethyl acetate with 500 pL formic acid (FA) and 500 pL difluoroacetic acid (DFA).

[0276] For each of the above, aqueous counterparts - used as Eluent A - using Millipore® ultra-pure water were produced at identical acidic modifier concentrations. Aqueous 15.89 mmol / L cyanoacetic acid results in a solution with pH value of 2.25 (24°C) and (0.05 % (v / v) FA + 0.05 % (v / v) DFA) results in a solution with pH 2.18 (24°C).

[0277] Sample preparation

[0278] For intact-deglycosylated bispecific antibody (bsAb) analysis. The antibody used has a molecular weight of approx. 193 kDa. The sample was deglycosylated as follows: One pL of the respective 100 pg / pL antibody reference standard (100 pg bsAb) was diluted in 247 pL phosphate buffer (10 mmol / 1, pH 7.2). Subsequently, 2 pL PNGaseF solution (2.5 units / pL in purified water (PWA)) was added and incubated overnight (24 hours) at 37°C.

[0279] For intact-deglycosylated bsAb analysis, the resulting 0.4 pg / pL deglycosylated bsAb solution was diluted to 0.1 pg / pL using Eluent A of which 10 pL (1 pg) were injected on the HPLC-MS system for analysis.

[0280] LC-MS Analysis

[0281] Instrumentation

[0282] For bsAb analysis the following equipment was used: The RP-HPLC separation was performed sing a BioResolve™ Polyphenyl column with the dimensions: 450 A, 2.7 pm, 2.1 x 150 mm, purchased from Waters Corporation (Milford, MA, United States). The chromatography was performed on a Vanquish™ Horizon UHPLC system which was equipped with a Vanquish™ diode Array Detector and a Vanquish™ LightPipe™ Flow Cells for UV monitoring at 280 nm (Thermo Fisher Scientific Inc., Waltham, MA, United States). Mass spectrometric analysis was carried out on an AB Sciex TripleToF™ 6600 device, which was equipped with a duoSource™ PCI&TSI ion source (AB Sciex Pte. Ltd., Toronto, Canada). The LC- column was coupled to an ASI 600 series adjustable flow splitter (ASI Inc., Richmond, CA, United States) which directed 1 / 6, i.e., 50 pL / min of the LC flow to the mass spectrometer’s TSI source and the remaining flow to the UV detector (at 280 nm) for parallel detection with minimal post column dispersion effect. The TOF mass analyzer was calibrated at the start of each sequence and automatically after every three samples utilizing a ESI calibration solution (G1969-85000, Agilent Technology, Santa Clara, CA, US) which was introduced by the Sciex Calibrant Delivery System (CDS) at 200 pL / min via the APCI probe (AB Sciex, Toronto, Canada). The calibration was performed under identical mass spectrometer settings as the subsequent analysis runs. LC-MS settings

[0283] For deglycosylated-intact bsAb analysis, the following mass spectrometric settings were applied: Ion source gas 1 (GS1) and source gas 2 (GS2) were set to 25 and 25 auxiliary units. Curtain gas (CUR) was set to 10 units, while the probe temperature was 325 °C and the TSI spray voltage 5.5 kV, the ‘time bins to sum’ parameter was at 100. A declustering potential (DP) of 250 arbitrary units and 50 collision energy (CE) with a mass range from 600 to 4000 m / z were applied.

[0284] For all runs, the RP-column (Waters BioResolve™ Polyphenyl, 2.1x150 mm) was run at 75 °C and 300 pL / min flow rate. One pg of the deglycosylated-intact bsAb (10 pL injection volume) was injected using the Vanquish™ Horizon UHPLC system and separated by a separation gradient starting at 20 % Eluent B, increasing to 40 % Eluent B in 17.5 minutes.

[0285] Example 7

[0286] Back pressure Testing

[0287] Chemicals

[0288] Illustra™ NAP-PD10 columns (Cytiva Germany, Dreieich, Germany), iodoacetic acid (IAA); L-methionine >99% was from Agros Organics™ (Thermo Fisher Scientific); trypsin modified sequencing grade, product ID: 11418025001 was supplied by Roche (Roche Diagnostics GmbH, Mannheim, Germany); DBA >99.5% (dibutylamine) and HFIP > 99% (l,l,l,3,3,3-hexafluoro-2-propanol) were from Sigma Aldrich.

[0289] Instrumentation:

[0290] Agilent series bio 1290 UHPLC system (Agilent Technologies, Santa Clara, CA, United States) equipped with a binary pump and a variable wavelength UV detector was directly hyphenated to an Orbitrap Fusion™ Tribrid mass spectrometer from Thermo Scientific (Waltham, MA, United States) downstream of the UV detection cell. The mass spectrometer was operating with the standard electron spray ion source. Back pressure Testing

[0291] The back pressure behavior of different solvent compositions were investigated in the typical range for the intended LC-MS experiment for peptide separation and analysis.

[0292] Table 9: System back pressure on the Agilent UHPLC system with a 1.7 pm,

[0293] 2.1 x 150 mm UHPLC column at different flow rates and Eluent B compositions in relation to different temperatures. Isocratic flow at 65 % Eluent B for all runs.

[0294] Typical columns and UHPLC on the market today that are used in the art are capable of withstanding system pressure of around 1000 bar. For prolonged use, this pressure limit should not be exceeded. Eluent viscosity and backpressure decreases with column temperature and rises with flowrate. The typical LC-MS peptide analysis experiment in the art using a 2.1 mm inner diameter sub 2 pm particle size column is performed at 0.3 mL / min flow rate. Backpressure is the highest at 65% Eluent B in the gradient profile, hence it was monitored at this isocratic mixing ratio with PWA as Eluent A. Peptide separation and analysis is typically performed at moderate, but not to high column temperatures. As high temperate are linked to on- column degradation of the peptides and less retention as they reduce analyte affinity to the solid phase. To avoid this, the columns are typically operated at around 50 °C in such an experiment.

[0295] As evident from the data in Table 9, ethanol as Eluent B results in system backpressures that exceed 1000 bar at the desired settings (0.3 mL / min and 50 °C). Some UHPLC systems therefore will not be able to perform the intended experiment, as sequenced runs are typically automatically halted when safe pressure limits are exceeded. The addition of 10 to 30 % (v / v) ethyl or methyl acetate according to the current invention, however, drastically reduces system back pressures. Therefore, it enables safe operation of the system well below 1000 bar and with the desired analysis settings. In some examples, the pressure difference between identical settings to pure ethanol are well over 100 bar.

[0296] Example 8

[0297] LC-MS analysis of antibody derived peptides

[0298] Sample preparation of mAb mixtures tryptic digest

[0299] A 143.1 kDa humanized antibody solution (mAb-A) was spiked with 0.5 w / w-% of a second 146.4 kDa humanized antibody (mAb-B) to with a total protein concentration of 10 mg / mL. The mixture was further denatured is a Guanidine buffer, chemically reduced using DTT (reaction time 1 hour at 37 °C) and alkylated using iodoacetic acid (IAA) (reaction time 15 minutes at room temperature in the dark). The IAA surplus was finally quenched using additional DTT. Prior to tryptic digestion, the mixture was buffer exchanged using Illustra™ NAP-PD10 columns (Cytiva Germany, Dreieich, Germany), using 0.1 M Tris-HCl buffer pH 7. Modified Trypsin sequencing grade was reconstituted in 10 mM hydrochloric acid and added to the mAb solution. Enzyme was added at an enzyme / substrate ratio of roughly 15: 1 and incubated at 37 °C for 1 hour. The reaction was quenched by adding roughly 7 % (v / v) of 54 mM L-methionine dissolved in aqueous 80 % (v / v) formic acid. The tryptic digest was transferred as aliquots into in LC vials and stored at -80 °C until use.

[0300] Peptides derived from the tryptic digestion were injected on an Acquity BEH C18 reversed phase 130 A, 1.7 pm, 2.1 x 150 mm UHPLC column from Waters Corporation (Milford, MA, United States). The column was operated at 0.3 mL / min flow rate and 55 °C column temperature. The separation gradient for solvent 1 was 1 to 35 % Eluent B in 45 min. with a linear slope to 65 % Eluent B at minute 50. For solvent 2 and 3 the gradient was from 1 to 28 % Eluent B in 45 min. with a linear slope to 65 % Eluent B at minute 50. UV detection was performed at 280 nm detection wavelength. Mass spectrometric detection was performed at varying source parameters as stated in the following.

[0301] To investigate the behavior of different solvent system an experimental design with varying parameters was used, with changes along instrument inlet capillary temperature and sprayer (emitter) temperature. Both are relevant parameters that have an impact with respect to efficiency in analyte ionization and ion transportation into the mass spectrometer.

[0302] For comparison the following eluents were analyzed Solvent 1 : acetonitrile with 0.5. % (v / v) FA

[0303] Solvent 2: 10 % (v / v) MetOAc in EtOH with 0.5. % (v / v) FA

[0304] Solvent 3: 10 % (v / v) EtOAc in EtOH with 0.5. % (v / v) FA

[0305] For each of the solvents a set of 16 experiments were performed with a design of experiment series of settings to assess the performance under a range of conditions as outlined in Table 10. For each run, the MS tune settings were as follows, spray voltage was 3.500 V in positive mode, resolution was set to 60.000, scan range was m / z 200 to m / z 2.000, maximum injection time was 100 milliseconds, RF lens setting was at 60 % and AGC target at 50 %, HCD collision energy was at 32 %. MSI was collected in the Orbitrap mass analyzer, while MS2 was detected in the ion trap. MS2 threshold was 5.000.

[0306] Table 10: Mass spectrometric ion source settings for each eluent type. The collected raw data was analyzed using the Byos Protein metric software suite Version: v5.8.24 (Dotmatics Ltd, Boston MA, United States). A “Byonic” MS / MS analysis was performed setting the tolerance to 7.0 ppm precursor and fragment mass tolerance to 20 ppm. The false detection rate was limited to 1 %, fully specific cleavage at R and K residues C-terminally, for trypsin with a maximum of one miscleavage. The m / z integration window for the MS extract option was 18 ppm.

[0307] The processed and annotated data was filtered for wild type peptides only, meaning only unmodified peptide were included in the post-processing data evaluation. Total intensities were exported for each peptide hit and plotted in the graphs shown in Figures 9 to 12.

[0308] From the boxplots it can be seen that with Eluent B according to the current invention compared to acetonitrile as Eluent B an improved analytical behavior of the substances to be analyzed can be seen, such as higher peptide intensities (about 2-fold) or / and sequence coverage.

Claims

Patent Claims1. A method for the separation of polypeptides on a reversed-phase chromatography material, wherein the separation is achieved by applying a gradient to the chromatography material, wherein the gradient is from an aqueous Eluent A to an organic Eluent B, wherein the aqueous Eluent A comprises water, and wherein the organic Eluent B comprises a mixture of ethanol and ethyl acetate at a volume ratio in the range of from 60:40 to 99: 1.

2. Use of an organic solvent comprising ethanol and ethyl acetate as eluent in a reversed-phase liquid chromatography method for improving detection of polypeptides or fragments thereof compared to an eluent comprising acetonitrile.

3. Use of an organic solvent comprising ethanol and ethyl acetate as eluent in a reversed-phase liquid chromatography-mass spectrometry method for increasing ionization yield of polypeptides or fragments or derivatives thereof compared to an eluent comprising acetonitrile.

4. The use according to any one of claims 2 to 3, wherein the organic solvent comprises about 75 % (v / v) water, about 17 % (v / v) ethanol and about 8 % (v / v) ethyl acetate5. The method according to claim 1 or the use according to any one of claims 2 to 4, wherein the organic Eluent B or the organic solvent has an ethanol to ethyl acetate ratio of about 10 to 3.

6. The method according to any one of claims 1 and 5 or the use according to any one of claims 2 to 5, wherein the organic Eluent B or the organic solvent further comprises about 0.05 % (v / v) trifluoroacetic acid and / or difluoroacetic acid and / or formic acid or about 1-50 mM cyanoacetic acid.

7. The method according to any one of claims 1 and 5 to 6, wherein the aqueous Eluent A comprises deionized, ultra-pure water.

8. The method according to any one of claims 1 and 5 to 7, wherein the aqueous Eluent A further comprises about 0.05 % (v / v) trifluoroacetic acid and / or difluoroacetic acid and / or formic acid of each in a mixture thereof or about 0.1 % (v / v) trifluoroacetic acid or difluoroacetic acid or formic acid about 1- 50 mM cyanoacetic acid when used as individual acidifier.9 The method according to any one of claims 1 and 5 to 8 or the use according to any one of claims 2 to 6, wherein the method or the use is at about 75 °C.

10. The method according to any one of claims 1 and 5 to 9 or the use according to any one of claims 2 to 6 and 9, wherein the reversed-phase chromatography material comprises a polyphenyl-ligand.

11. The method according to any one of claims 1 and 5 to 10 or the use according to any one of claims 2 to 6 and 9 to 10, wherein the polypeptide is an antibody or an antibody-fragment.

12. The method according to any one of claims 1 and 5 to 11 or the use according to any one of claims 2 to 6 and 9 to 11, wherein the method or use is for intact polypeptide mass analysis or polypeptide subunit mass analysis or for reduced polypeptide mass analysis.

13. The method according to any one of claims 1 and 5 to 12 or the use according to any one of claims 2 to 6 and 9 to 12, wherein the organic Eluent B or the organic solvent has an ethanol to ethyl acetate ratio in the range and including 69:31 to 99: 1.

14. The method according to any one of claims 1 and 5 to 13 or the use according to any one of claims 2 to 6 and 9 to 12, wherein the method or use comprises a gradient from 30 to 42 % (v / v) organic Eluent B or a gradient from 22 to 34 % (v / v) Eluent B.

15. The method according to any one of claims 1 and 5 to 14 or the use according to any one of claims 2 to 6 and 9 to 12 or 14, wherein the organic Eluent B or the organic solvent has an ethanol to ethyl acetate ratio in the range of and including 70:30 to 85: 15.

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