Method for determining the charge state of proteins

Abstract

The invention relates to a method for analyzing charge variants of proteins using microtiter plates, at least some of the wells of which have different pH values and / or salt concentrations, and further using functionalized surfaces.

Description

Method for determining the charge state of proteins

[0001] The application relates to methods for analyzing charge variants of proteins using microtiter plates whose wells have at least partially different pH values ​​and / or salt concentrations and using functionalized surfaces.

[0002] The production of proteins, and in particular the production and development of biopharmaceuticals, including virus particle-based therapeutics, is of great economic importance. However, the production of proteins, and especially biopharmaceuticals, is very complex and time-consuming. Biopharmaceuticals are more heterogeneous in terms of their physical properties than chemically produced drugs, so-called small molecule drugs.

[0003] Biopharmaceuticals, including virus particle-based therapeutics, are generally pharmaceutically useful and effective proteins, such as antibodies and virus particles.

[0004] During the production and handling of proteins, and especially biopharmaceuticals, undesirable by-products may arise due to mis-synthesis, degradation or other reactions, which must be identified and separated, as these undesirable products often affect the biological activity of the protein.

[0005] The by-products are usually incorrectly assembled, e.g. mispaired proteins or fragments of these proteins or different charge variants of the target protein.

[0006] The different charge states of the charge variants of the biopharmaceutical, in particular antibodies, Fc fusion proteins, antibody fragments, bi- and multispecific antibodies as well as virus particle-based therapeutics, can lead to significant differences in the physico-chemical behavior, such as structure, stability, binding affinity and thus influence the therapeutic efficacy.

[0007] When producing proteins, especially biopharmaceuticals, it is therefore important to find cell lines and cultivation conditions that minimize assembly errors and charge variations. This so-called upstream development often generates hundreds of samples that must be tested for suitability in a short period of time.

[0008] The same applies to so-called downstream development, in which the purification of proteins, especially biopharmaceuticals, is optimized.

[0009] Mispairing occurs, for example, when the different amino acid chains produced after the expression of bi- or multispecific antibodies are not assembled in the desired manner. Despite various approaches to optimize assembly and shift it toward the desired target protein, this mispairing remains a major problem.

[0010] The primary goal in developing processes for the production and purification of proteins, especially biopharmaceuticals, should therefore be to find conditions under which as few byproducts as possible are formed. This must be determined experimentally and requires close monitoring of the experimental setup (e.g., cell culture) to enable timely changes to the experimental conditions.

[0011] Viral particles used in gene therapy, for example, can also be charged differently. Loading the viral particles with nucleic acids (DNA or RNA), for example, results in the viral particle surface being more negatively charged than uncharged or only partially charged viral particles. Since the loading of viral particles with nucleic acids is essential for the therapeutic effect, it is very important to ensure that the viral particles are loaded during viral particle production processes. Virus particles not loaded with nucleic acids are non-functional byproducts and must be removed.

[0012] Various methods are available for detecting the presence or absence of nucleic acids in virus particles. These include PCR methods in combination with ELISA methods, cryo-electron microscopy, dynamic Light scattering (DLS), analytical ultracentrifugation, or multi-step methods with various biosensors using biolayer interferometry. Furthermore, nucleic acids influence the charge of the viral capsid, making separation based on their charge possible in ion exchange chromatography.

[0013] New processes, tools and devices are constantly being developed to simplify the production methods and handling of proteins, especially biopharmaceuticals, and thus make them more cost-effective.

[0014] The present invention aims to develop methods for rapidly detecting charge variants of a target protein, namely antibodies, Fc fusion proteins, antibody fragments, bi- and multispecific antibodies, and virus particle-based therapeutics, as well as incorrectly assembled (e.g., mismatched or fragmented) proteins. The latter is based on the fact that the target proteins and the proteins incorrectly assembled during their production very often have different charges and thus different isoelectric points.

[0015] Methods and apparatus for detecting and quantifying proteins based on their physical parameters are known.

[0016] A method frequently used to characterize and purify biological molecules is column chromatography. It can be performed under both atmospheric and elevated pressure. The latter often requires expensive equipment such as HPLC or FPLC.

[0017] Another method is based on the use of biosensors, as described, for example, in US 5,804,453 B.

[0018] A biosensor is a device that combines a biological component with a physical or chemical transducer to generate a measurable signal in response to a target analyte. Biosensors can utilize various physical measurement principles to detect the presence of target analytes. Examples include piezoelectric biosensors, optical Detection methods based on interferometry or surface plasmon resonance as well as calorimetric measurement methods.

[0019] Biosensors that use interferometric methods are described, for example, in US Pat. No. 5,804,453 B. Biosensors that use piezoelectric methods are also known and are described, for example, in EP2017613A1.

[0020] Biosensors that use interferometric methods are particularly frequently used in combination with microtiter plates because the biosensors are easy to manufacture and, due to their shape, can be easily immersed into the wells of the microtiter plates.

[0021] The biosensors used in so-called biolayer interferometry have two reflective surfaces on which a phase shift is measured when biomolecules attach to the second surface, further away from the light source.

[0022] Biosensor surfaces must be functionalized for target analyte binding. The surfaces are often functionalized with amines to enable further coupling of proteins or peptides via an amide bond, as described, for example, in WO 2008 / 033535.

[0023] Functionalization is achieved through bifunctional linkers, which, on the one hand, react with the surface of the biosensor and, on the other hand, provide a functionality that enables the binding of the actual binding molecule (e.g., an antibody). For glass surfaces, these can be siloxanes, a classic method for functionalizing glass surfaces. For polymer surfaces, copolymers of, for example, polystyrene and divinylbenzene can be used, which provide reactive groups for further functionalization of the polymer surfaces. These coupling processes are also suitable for introducing groups for cation or anion exchange.

[0024] While column chromatographic methods are used to detect and, if necessary, identify charge variants and incorrectly assembled proteins, the use of biosensors is not yet known.

[0025] The disadvantage of column chromatography methods is always that pre-cleaning of the samples is necessary before analysis. This is important because otherwise the column material is irreversibly inactivated very quickly and because foreign proteins cannot be distinguished from the target protein by the commonly used UV absorption measurement at, for example, 280 nm. Regeneration of the column material between runs is also necessary and time-consuming.

[0026] First, the sample is adsorbed onto a so-called stationary phase (the column material, which can consist of various materials). By adding eluent (mobile phase(s)), i.e., solutions with different properties (salt content / pH), the ionic interactions of the analytes with the stationary phase are weakened, and the adsorbed molecules are desorbed and eluted.

[0027] Cation exchange chromatography (CEX) uses negatively charged column material combined with a pH and / or salt gradient. When a pH gradient is used, the sample starts with a low pH value. At this value, the biological molecules (e.g., proteins) in the sample are relatively highly profaned and thus positively charged. This results in very good binding of the molecules to the column material. As the pH value increases, the biological molecules are deprotonated, lose their positive charge, no longer bind to the stationary phase, and elute successively and depending on their charge. Anion exchange chromatography (AEX) works in a similar way, but with positively charged column material.

[0028] Together with or instead of the pH gradient, a salt gradient can also be used to separate different molecules. This involves starting with low salt concentrations and increasing the salt concentration during the chromatographic separation. As a result, the salt (e.g., NaCl), especially the cations (e.g., Na+), increasingly bind to the negatively charged Column material and displaces the positively charged biological molecules bound there, which then elute from the column material.

[0029] A pH gradient or a salt gradient refers to a change in pH or hydrogen ion concentration and / or salt concentration over a certain spatial or temporal range.

[0030] Mixed-mode ion chromatography is also used for certain complex protein separations. Mixed-mode ion chromatography is a further development of ion exchange chromatography. It uses surfaces (e.g., in the form of particles) that contain both positively and negatively charged groups. Another option is multi-mode ion exchange chromatography, which uses surfaces (e.g., in the form of particles) that can form hydrophobic interactions and hydrogen bonds in addition to ionic interactions.

[0031] The object of the present invention is to provide devices and methods with which proteins, such as biopharmaceuticals, in particular antibodies, Fc fusion proteins, antibody fragments, bi- and multispecific antibodies, and virus particle-based therapeutics, can be efficiently and rapidly determined qualitatively and quantitatively directly and without prior sample purification. Furthermore, the measurement should be simple and preferably feasible using means routinely available in the laboratory.

[0032] The inventors have now discovered the methods and uses, as well as devices protected by the patent claims, with which proteins can be characterized and separated based on their charge differences. This allows users to quickly and easily detect byproducts based on their charge state. The disadvantages of the aforementioned methods (lengthy purification processes prior to the actual analysis, etc.) are thus avoided.

[0033] The invention particularly relates to a method for analyzing charge variants of proteins using microtiter plates whose wells have at least partially different pH values ​​and / or salt concentrations and using functionalized surfaces.

[0034] The invention is preferably used for the analysis of charge variants of biopharmaceuticals, in particular therapeutically active proteins, such as antibodies and protein formats derived therefrom, such as Fc fusion proteins, antibody fragments, bi- and multispecific antibodies, and virus particle-based therapeutics.

[0035] The functionalized surfaces are preferably ion exchanger particles, in particular anion or cation exchangers, mixed-mode and multi-mode ion exchangers, or sensors, such as biosensors, whose measuring surface is designed in such a way that charged molecules are bound and whose shape is such that they can be immersed in the wells of microtiter plates.

[0036] If marker molecules, such as mono- or polyclonal antibodies or fragments derived therefrom, such as nanobodies or single-domain antibodies, as well as peptides and RNA- or DNA-based aptamers, are used in the method according to the invention, the biopharmaceutical, the target protein, has binding sites for the marker molecule(s). The marker molecules can further comprise dyes, such as fluorescent dyes. Such markers are referred to below as fluorescent markers.

[0037] The use of markers, especially fluorescent markers in combination with the PAIA plate, has the advantage that other proteins that can bind to the functionalized surfaces due to their charge but are not bound by the marker molecule(s) are not detected. Such proteins include, for example, host cell proteins, as they are typically present in cell culture supernatants, or misassemblies during the production of bi- or multispecific antibodies that no longer have a binding site for the marker molecule(s).

[0038] Marker molecules are molecules that can specifically bind to the target molecules. Examples of marker molecules are mono- or polyclonal antibodies, or derived fragments, such as the so-called nanobodies or single-domain antibodies as well as peptides and RNA- or DNA-based aptamers.

[0039] Fluorescent markers are marker molecules to which a fluorescent dye is bound. The dyes can be covalently bound to the fluorescent markers or, for example, via the well-known streptavidin-biotin system, in which a fluorescently labeled streptavidin or other fluorescently labeled biotin-binding proteins such as avidin or neutravidin is coupled to a biotinylated marker molecule.

[0040] A person skilled in the art will readily determine which fluorescent markers are particularly suitable for the method and use according to the invention. Examples of fluorescent markers that can be used according to the invention are Affibody conjugated with Alexa 647 from Affibody AB or Nano-Secondary® alpaca anti-human IgG / anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 (Art. No. CTK0101 - ChromoTek GmbH). Other fluorescent markers are commercially available, e.g. from Jackson Immunoresearch or Abeam. These are already coupled with a fluorescent dye and can be used directly. Alternatively, monoclonal or polyclonal antibodies can be purchased from these companies and coupled with various commercially available dyes, e.g. fluorescein, as well as dyes from Biotium, Atto-Tec, and ThermoFisher.

[0041] When selecting fluorescent markers, it is important to ensure that the fluorescent marker does not bind directly to the functionalized surfaces under any of the assay conditions used (pH and salt). This is easily determined by the expert, for example, by examining mixtures of the surfaces with the fluorescent marker under fluorescence microscopy.

[0042] The application of the method mainly relates to the analysis, i.e. the identification and, if necessary, quantification of differently charged proteins (here also called “charge variants of proteins”). Proteins are, for example, biopharmaceuticals (i.e. therapeutically active proteins), such as antibodies, Fc fusion proteins, antibody fragments or bi- and multi-specific antibodies as well as virus particle-based therapeutics.

[0043] Charge variants can be the same protein (i.e., a protein with the same amino acid sequence) that has multiple charge states. For example, antibodies that have different charges due to different glycosylation, amidation, or oxidation of amino acids.

[0044] Charge variants also include incorrectly assembled proteins. These have different amino acid sequences, or different types of protein domains, and possibly also a different number of protein domains, such as the light and heavy chains in antibodies, and are therefore often differently charged and have different isoelectric points. Likewise, differently charged virus particles fall under the term "charge variants" if they are differently charged due to their loading with nucleic acids (DNA or RNA), their glycosylation, or an amidation or oxidation of the amino acids of their capsid proteins.

[0045] In the method according to the invention, specially prepared test vessels, in particular microtiter plates, are used.

[0046] If fluorescent markers are used in the method according to the invention, the PAIA plate as defined here is used.

[0047] When biosensors are used as functionalized surfaces in the method according to the invention, PAIA plates are not used.

[0048] The advantage of the invention is that a large number of samples can be tested for the presence of charge variants in a relatively short time. Due to the simple and, if desired, automated procedure, a measurement result for more than 100 samples is generally available in less than 60 minutes. The automation available for microtiter plates (e.g., pipetting robots or automatic readout processes) can be utilized.

[0049] Microtiter plates are well-known and usually rectangular, with a varying number of wells, also called cavities, or recesses. These wells are usually arranged in rows parallel to the long side of the microtiter plate and in columns parallel to the short side.

[0050] Letters are usually used to designate rows and numbers to designate columns. For example, the wells of a 384-well plate are arranged in 16 rows (from A to P) and 24 columns (from 1 to 24). A schematic drawing of such a 384-well plate is shown in Fig. 1.

[0051] The wells of a 96-well plate are arranged in 8 rows (A to H) and 12 columns (1 to 12). This allows each well to be uniquely identified: The first row of a 96-well plate contains 12 wells, namely wells A1 to A12. The second row contains wells B1 to B12, and so on.

[0052] The special preparation of the microtiter plate concerns one or more wells or, in the case of the PAIA plate, as described in more detail below, one or more wells that form a measuring chamber.

[0053] The wells of the microtiter plate are prepared as follows:

[0054] In a first step, the desired number of wells are filled with aqueous buffer solutions with different pH values ​​and / or different salt concentrations. These solutions contain different salt concentrations and pH values.

[0055] The pH differences between the wells can be varied depending on the requirements of the experiment (assay). This also applies to the number of wells across which the assay extends. Figures 1b and 1c show a possible configuration with a 12-point or 12-step gradient.

[0056] The intervals between the pH values ​​are chosen as required by the accuracy of the experiment. Intervals between the pH values ​​in the individual wells in the range of 0.02 to 1, 0.05 to 1.5, or 0.03 to 2 are advantageous. Larger intervals are also possible.

[0057] Alternatively or additionally, the buffer solution can have different salt concentrations. The intervals between the salt concentrations are selected as required for the accuracy of the experiment. Intervals between the concentrations in the individual wells in the range of 0.5 up to 500 mmol / L or 0.5 to 100 mmol / L or 1 to 90 mmol / L or 0.8 to 20 mmol / L or 1.5 to 50 mmol / L or 10 to 75 mmol / L. Salts suitable for preparing such salt gradients in buffers are typically alkali and alkaline earth metal salts such as NaCl, KCl, MgCl, CaCl2, MgSO4, and mixtures or combinations thereof. Any suitable salts may be used.

[0058] If the buffer solutions contain different salt concentrations and pH values, this is a combination of pH and salt gradients. This combination is referred to in the literature as a "salt-mediated pH gradient" and is used to optimize the separation performance of chromatography.

[0059] Goyon et al. (2020) describe different mixing regimes consisting of three buffers, starting with a 20 mM MES buffer (2-(N-morpholino)ethanesulfonic acid) at pH 5.6 (buffer A), a buffer consisting of 20 mM MES and 20 mM potassium HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid) at pH 6.8 (buffer B), and a 20 mM HEPES buffer at pH 8.2 and 100 mM potassium chloride as the salt component.

[0060] For example, if charge variants with very different charges are to be differentiated in a mixture, it is important to cover a wide pH range with the gradient in order to detect all charge variants. In this case, it is advantageous if the distance between the pH values ​​(pH steps) is approximately 0.2 to 1.5 pH units. These steps can, but do not have to, be the same from step to step. For such tests, at least eight different values ​​are usually required (a so-called 8-step gradient).

[0061] If the task requires the differentiation of different charge variants with only small charge differences in a mixture, the use of smaller pH differences between the steps of the pH gradient, e.g., 0.02 to 0.1 or 0.1-0.4 pH units, is advantageous.

[0062] It is also advantageous to increase the number of gradient wells to cover a sufficiently wide pH range simultaneously. The same applies to concentration differences when forming a salt gradient.

[0063] The above statements regarding the differentiation of charge variants with large or small charge differences apply accordingly to the concentration differences in the formation of a salt gradient, ie in order to improve the resolution in a salt gradient, the concentration differences between the individual wells in the salt gradient must be small.

[0064] The above applies analogously if salt and pH gradients are used in the process according to the invention.

[0065] The water can now be removed from the wells by drying, preferably at elevated temperature, i.e. temperature greater than 18 °C, at normal pressure or under reduced pressure

[0066] This has the advantage that the prepared microtiter plate can be stored. Of course, the contents of the wells with the various pH values ​​and / or salt concentrations must be recorded. This can be done separately or on the plate.

[0067] It has proven advantageous for experimental performance if buffers with different pH values ​​and / or salt concentrations are placed in wells arranged next to or below each other, creating rows or columns with increasing or decreasing pH and / or salt concentrations. This simplifies the evaluation of the results.

[0068] Buffers with different pH values ​​and / or salt concentrations are most easily obtained by mixing two buffers with different pH values ​​and / or salt concentrations. If buffer A with a low pH value and / or salt concentration and buffer B with a high pH value and / or salt concentration are available, the desired pH value or salt concentration can be adjusted by selecting an appropriate mixing ratio.

[0069] Buffers with different pH values ​​and salt concentrations can be prepared by mixing two buffers, a buffer A and a buffer B. Both Buffers have different pH values ​​and one of the buffers also contains a certain salt concentration.

[0070] All common and well-known chemicals can be used as buffer substances, and the choice depends on the application of the assay. Common buffer systems for handling proteins include PBS (phosphate-based saline buffer), ammonium acetate, and citrate-based buffers.

[0071] A suitable buffer system is the CX-1 buffer system from Thermo Fisher Scientific, which provides a highly linear buffer gradient. It consists of two buffers with pH values ​​of 5.6 and 10.2, each containing four zwitterionic buffer substances. The desired pH values ​​are adjusted by mixing these two buffers in different volume ratios.

[0072] A 12-stage buffer system with narrow pH intervals between buffers P1 to P12, consisting of the buffer substances Na2HPO4 (buffer A) and NaH2PO4 (buffer B) in a concentration of 0.02 M each, can be obtained by mixing buffers with pH 5.7 and pH 8.1 as follows. For example: P1 is the buffer with pH 5.8, P2 is buffer 2 with pH 6.0, etc. Table 1: Example of a 12-stage buffer system composed of two PBS buffers

[0073] The preparation of such multi-step (quasilinear) buffer gradients is known and there are now configurators on the Internet (see e.g. https: / / www.aatbio.com / resources / buffer-preparations-and-recipes / phosphate-buffer-ph- 5-8-to-7-4).

[0074] To produce salt gradients, i.e., buffers with different salt concentrations, buffer systems with dissolved salts in different concentrations are also used and mixed. The pH of these solutions remains the same.

[0075] In a particular embodiment, the invention relates to a method for analyzing charge variants of proteins using the specially prepared PAIA plates (i.e., in whose wells there are different pH values ​​and / or salt concentrations, as described above) using anion or cation exchangers, mixed-mode or multi-mode ion exchangers as functionalized surfaces and fluorescent markers as markers.

[0076] The PAIA plate is a microtiter plate in SBS format with a specific number of wells designed as measuring chambers. The raised portion (referred to as a structural element in WO 2015 / 135840 A1) is shaped so that its base area occupies at least 50% of the floor area of ​​a measuring chamber or well. These plates are known and described in WO 2015 / 135840 A1. They are manufactured and marketed under the trade name PAIAplate 384 by PAIA Biotech GmbH. These plates have 384 wells.

[0077] It goes without saying that the PAIA plate to be used can have more or fewer than 384 wells.

[0078] Preferably, the elevation has a square base that tapers (at least slightly) towards the top and has the shape of a four-sided pyramid. The upper end of the elevation is pointed, flat, or convex, preferably tapered. Advantageously, the upper end of the elevation has a diameter of less than 50 μm, so that no test components are deposited there. The height of the elevation is at least 10% and at most 50% of the height of the Measuring chamber or well, preferably at least 15% and at most 30%. The bottom of the measuring chamber or well is opaque except for the bottom of the protrusion. The protrusion, however, is translucent and forms a measurement window used to measure fluorescence emission, as described in detail in WO 2015 / 135840 A1.

[0079] The PAIA plate is in particular equipped as follows: The desired number of wells (some or all) of the microtiter plate are designed as measuring chambers, in which there is a raised area which is shaped such that its base area takes up at least 50% of the bottom area of ​​a measuring chamber or well and preferably has a square base which tapers at least slightly towards the top and has the shape of a four-sided pyramid, wherein the upper end of the raised area is pointed, flat or convex and advantageously the upper end of the raised area has a diameter of less than 50 pm so that no test components are deposited there, wherein the height of the raised area is at least 10% and at most 50% of the height of the measuring chamber or well and the bottom of the measuring chamber orof the well is opaque down to the bottom of the bump and the bump is translucent and forms a measurement window used to measure fluorescence emission.

[0080] Regarding the measurement windows, reference is made to the description in WO 2015 / 132840 A1, which is incorporated by reference in its entirety. For fluorescence measurement, the measuring chamber or microtiter plate according to the invention is illuminated from below with excitation light, which is then directed into the sample via the raised area. Commercially available fluorescence readers can be used to read the fluorescence signal.

[0081] The following devices can be used as fluorescence readers according to the invention: The devices of the Spectramax series from Molecular Devices, the systems from Tecan (Satire, the Infinite series, or SPARK), readers from BMG Labtech (Omega, Clariostar, Pherastar) and fluorescence microscopes, e.g. from SynenTec (Cellavista and NyONE).

[0082] The invention using the PAIA plate can be advantageously used, in particular, in the development of cell culture methods for producing proteins, in particular therapeutically active proteins, such as antibodies, Fc fusion proteins, antibody fragments, bi- and multispecific antibodies, and virus particle-based therapeutics. The invention therefore also relates to the use of PAIA plates for the analysis, i.e., detection and identification of charge variants of proteins, in particular therapeutically active proteins, such as antibodies, Fc fusion proteins, antibody fragments, bi- and multispecific antibodies, and virus particle-based therapeutics.

[0083] In one embodiment of the invention, the method according to the invention uses a microtiter plate in whose wells buffer substances and / or salts are present, which, in dissolved form, result in different pH values ​​and / or different salt contents. Such plates are produced by introducing buffer solutions and / or salt solutions with different pH values ​​and / or salt contents into the wells of the plates and drying them at elevated temperature, advantageously at temperatures above 18°C, preferably above 25°C, in particular at 30°C, at atmospheric pressure or reduced pressure.

[0084] The invention also relates to a PAIA plate in which buffer solutions with different pH values ​​and / or salt concentrations are present in one or more of the wells and in which anion or cation exchangers, mixed-mode or multi-mode ion exchangers and optionally fluorescent markers are introduced into the optionally functionalized surfaces.

[0085] The method according to the invention using the PAIA plate can be carried out as follows: (a) Addition of one aliquot of the protein sample to each of the wells pretreated with buffers with different pH values ​​and / or salt concentrations; (b) addition of anion or cation exchangers, mixed-mode or multi-mode ion exchangers that bind charge-specifically to the protein in the form of particles; (c) adding one or more fluorescent labels selected to specifically bind to the protein to be determined, the charge variants of which are to be determined; (d) mixing; (e) measuring the intensity of the unbound fluorescent markers after settling of the anion or cation exchangers, mixed-mode or multi-mode ion exchangers; (f) Correlating the measurement with the pH and / or salinity present in the wells to determine the different charge variants of the protein.

[0086] In the above process, steps (a), (b), and (c) can be performed in any order. If steps (b) and / or (c) are performed before step (a), in any order, a drying step can be provided after step (b) and / or step (c). In the drying step, the liquid introduced with the test components is removed, preferably by drying at elevated temperature, as described here.

[0087] If a drying step is inserted, the process can be interrupted for any length of time after the drying step and continued with step (a).

[0088] The invention relates to the PAIA plate in which, after the introduction of the buffer solutions, possibly the functionalized surfaces and / or fluorescent markers, the liquid components (water from the buffer solution, etc.) are removed by drying.

[0089] The invention therefore also relates to a PAIA plate with dried substances, namely buffer substances, optionally functionalized surfaces and / or optionally fluorescent markers, wherein in some of the wells arranged in columns or rows there is a decreasing or increasing pH value and / or salt concentration upon addition of sample solution.

[0090] The invention further relates to a so-called kit of parts comprising a PAIA plate with dried substances, namely buffer substances and functionalized surfaces and a fluorescent marker, wherein in some of the wells arranged in columns or rows there is a decreasing or increasing pH value and / or salt concentration upon addition of sample solution.

[0091] The invention can also be implemented using biosensors as functionalized surfaces in microtiter plates. In this case, the use of a PAIA plate is not intended.

[0092] Therefore, the invention also relates to a method for analyzing charge variants of proteins using microtiter plates whose wells have at least partially different pH values ​​and / or salt concentrations, and using biosensors as functionalized surfaces. The biosensors are equipped to act as anion and cation exchangers or as mixed-mode or multi-mode ion exchangers. The method can also include the addition of marker molecules.

[0093] The procedure then includes the following steps (a) Use of the specially pretreated microtiter plate in which wells have different pH values ​​and / or salt concentrations, whereby not every well of the plate needs to be occupied, and (b) Addition of one aliquot of the protein sample to each of the wells pretreated with buffers of different pH values ​​and / or salt concentrations; (c) Introduction of the biosensor into the treated and sampled wells of the microtiter plate; (d) mixing; (e) measuring the triggered signal of the biosensor; (f) Correlating the measurement with the pH and / or salinity present in the wells to determine the different charge variants of the protein.

[0094] The method according to the invention for analyzing charge variants of proteins using biosensors can also be carried out as follows: (1 ) Using the specially prepared microtiter plate in whose wells a suitable pH and / or salt gradient is present, wherein the pH or salt gradient is present in dried form or is freshly introduced; (2) Addition of one aliquot of the protein sample to each of the wells pretreated with buffers of different pH values ​​and / or salt concentrations; (3) introducing the biosensors with ion exchange surfaces into the wells with the pH and / or salt gradient; (4) Shaking the microtiter plate, which accelerates the reaction, and simultaneous (real-time) measurement of binding to the ion exchange surfaces on the biosensors; (5) When using non-purified samples containing foreign proteins or when the measurement signal of the target molecule is too low, shaking is stopped after reaching the maximum measurement signal (i.e., biosensors are saturated); (6) The biosensors are removed from the microtiter plate and placed in a second microtiter plate containing the same gradient buffer and additionally a marker molecule that specifically binds to the target molecule; (7) Shaking the microtiter plate again and simultaneously measuring the binding of the marker molecule to the target proteins already bound to the ion exchange surfaces of the biosensors; (8) Determination of the binding of the target protein to the ion exchange surfaces in all wells of the gradient either via the signal measured in step (4) or the signal measured in step (7) and determination of the charge profile of the sample.

[0095] The detection of charge variants using biosensors can be achieved by applying ion exchange surfaces to biosensors, which are placed in the wells of microtiter plates (not PAIA plates) with different pH values ​​or salt concentrations and to which the binding of the target proteins is measured.

[0096] Optical methods such as biolayer interferometry are primarily used for this purpose. This involves a phase shift in the light when molecules bind to the surface of the biosensor. This phase shift is measurable and depends on the number and mass of the molecules bound to the surface.

[0097] Measurement with biosensors can be performed simultaneously, i.e., with multiple biosensors. Alternatively, it can be performed sequentially, i.e., a biosensor is introduced successively into the various wells of the microtiter plate containing a gradient level and is regenerated and washed before each measurement of a new well.

[0098] Biosensor regeneration is a well-known process. Modern devices are capable of measuring with multiple biosensors simultaneously in different wells, thus achieving significantly higher sample throughput.

[0099] If only small amounts of a protein are bound during the measurement, or if very small proteins are measured, and therefore only a very weak measurement signal is generated, it is common practice for biosensors to bind a marker that specifically binds to the target molecule, e.g., an antibody with a sufficiently large molecular mass, to the surface after the target molecule has bound to the surface to amplify the signal. If this is not sufficient to amplify the signal, another marker (referred to here as a binder, e.g., a secondary antibody) that specifically binds to the primary antibody can also bind to the biosensor.

[0100] The same approach is used when protein binding results in the binding of other unwanted molecules and proteins ("non-target molecules") to the surface. This can be the case, for example, if the sample has not been purified and contains foreign proteins that also bind to the surface.

[0101] In this case, the use of a specific marker molecule, such as an antibody that specifically binds to the target protein, allows the measurement of a phase shift due to the binding of the marker molecule. This binding depends only on the amount of target molecules bound to the surface. Thus, the measurement signal is specific to the target molecule.

[0102] The sample containing the protein with the charge variants is usually added in portions to the wells of the microtiter plate, which can also be a PAIA plate, without further treatment (e.g., purification). However, dilution of the sample with water is advantageous to reduce the concentration of buffer substances that could interfere with the experiment.

[0103] Portioning is done manually or automatically with an automated pipetting robot (liquid handler).

[0104] When using the PAIA plate, it can be advantageous for efficiency if the fluorescent marker(s) and / or the functionalized surfaces are already dried in the PAIA plate.

[0105] The special feature of the invention is that when carrying out the method, different pH values ​​and / or salt concentrations are present in the wells of the microtiter plate arranged in rows or columns.

[0106] A particular advantage of the invention is that the samples can be measured directly (if necessary after dilution with water). Purification prior to performing the procedures (the assays) is not required.

[0107] The pH and / or salt concentration present in the wells are crucial for the binding properties of the respective charge variant of the protein to the functionalized surface(s). Measurement with the biosensor or—when using the PAIA plate—the fluorescence intensity of the unbound fluorescent markers provides a value from which the charge state of the measured protein can be deduced.

[0108] In the method according to the invention, the functionalized surfaces are selected so that they can bind the protein with the charge variants. Anion or cation exchangers, mixed-mode or multi-mode ion exchangers, and biosensors whose measuring surfaces have the same or similar functionalities as the aforementioned anion or cation exchangers, mixed-mode or multi-mode ion exchangers can be used according to the invention. - TI -

[0109] Anion exchangers are well-known and are classified according to their ionic strength into strong and weak anion exchangers. Strong anion exchangers are those with positively charged functional groups, e.g., quaternary aminoethyl groups, quaternary aminomethyl groups, or triethylaminomethyl groups. Weak anion exchangers often contain diethylaminoethyl groups.

[0110] Strong anion exchangers include Macro-Prep High Q Support and UNOsphere Q Media, both available from Bio-Rad, or QAE Sephadex A-25 (Cytiva), Capto Q (Cytiva), POROS XQ Strong Anion Exchange Resin (ThermoFisher).

[0111] Weak anion exchangers include DEAE Sepharose Fast Flow (Cytiva), Macro-Prep DEAE Resin (Bio-Rad), TOYOPEARL DEAE-650 (Tosoh), DEAE Ceramic HyperD (Sartorius), Fractogel® EMD DEAE (Merck).

[0112] Cation exchangers are also well known and are also divided into strong and weak cation exchangers. The former contain, for example, sulfonate groups (S), sulfoethyl groups (SE), or sulfopropyl groups (SP) on their surface. Weak cation exchangers contain negatively charged groups, such as carboxymethyl groups (CM).

[0113] Strong cation exchangers include SP Sepharose Fast Flow (Cytiva), SP Sephadex C-25 (Cytiva), TOYOPEARL SP-650 (Tosoh), Macro-Prep High S (Bio-Rad).

[0114] Weak cation exchangers are well known and commercially available, such as Macro-Prep CM (Bio-Rad), CM Sepharose High Performance FF (Cytiva), G-Sep™ CM Agarose Fast Flow (G-Bioscience).

[0115] Mixed-mode and multi-mode ion exchangers are well known and commercially available.

[0116] Mixed-mode ion exchangers include, for example, the CHT (Ceramic Hydroxyapatite) column material Type I and Type II from Bio-Rad, on whose surface negatively charged phosphate groups and positively charged calcium ions are available for interaction.

[0117] Another mixed-mode or multi-mode (cation) exchanger is the material TOYOPEARL® MX-Trp-650M from Tosoh Bioscience, which features negatively charged carboxyl groups as well as hydrophobic tryptophan groups on its surface for hydrophobic interactions. Another mixed-mode ion exchanger is a weak cation exchanger with hydrophobic groups, available as Capto MMC column material from Cytiva. Another example is Capto Adhere (also from Cytiva), a strong anion exchanger with additional hydrophobic aromatic groups.

[0118] Anion or cation exchangers, mixed-mode or multi-mode ion exchangers usable according to the invention preferably have average diameters in the range of approximately 5 to 200 pm. The particles preferably have average diameters in the range of 5 to 150 pm or 10 to 100 pm.

[0119] Description of the characters: Fig. 1 a shows a schematic labeling of the wells in a 384-well microtiter plate. Fig. 1 b shows a schematic representation of a 384-well plate with a 12-step pH gradient. The 12-step gradient is present in each column. Untreated wells are located in columns 5, 10, 14, 18, and 22-24. Fig. 1 c shows a schematic representation of a 384-well plate with a 12-step pH gradient. The 12-step gradient is present twice per row (columns 1-12 and columns 13-24). There are no untreated wells. Fig. 2 shows the document scheme from Example 1 . Fig. 3 refers to Example 1 and shows the measured fluorescence intensities of the unbound fluorescent markers of V1-A trastumab (HER) - unstressed and V1 -B trastumab (HER) - heat-treated as a function of pH. Fig. 4 Fig. refers to Example 1 and shows the fluorescence intensity of V1 -C Trastumab (HER) - unstressed and V1 -D Trastumab (HER) - heat-treated measured in diluted cell culture supernatant. Fig. 5a refers to Example 1 and represents a comparative example. It shows the result of the chromatographic evaluation, namely a CEX chromatogram of the unstressed trastuzumab sample 2 mg / mL in H2O. Fig. 5b refers to Example 1 and represents a comparative example. It shows the result of the chromatographic evaluation, namely a CEX chromatogram of the trastuzumab sample 2 mg / mL in H2O stressed at 35 degrees. Fig. 6 refers to Example 2 and shows the course of the fluorescence intensities as a function of the pH values ​​shown on the x-axis. Fig. 7 refers to Example 2 and shows a plot of the point-to-point slope as a function of pH

[0120] Labeling of the figures Fig. 1 a: Schematic labeling of the wells in a 384-well plate. Fig. 1 b: Schematic representation of a 384-well plate with a 12-step pH gradient. Fig. 1 c: Schematic representation of a 384-well plate with a 12-step pH gradient. Fig. 2: Receipt scheme from example 1. Fig. 3: Example 1 : Fluorescence intensities of the samples - samples measured in H2O Fig. 4: Example 1 : Fluorescence intensities of the samples - samples measured in diluted cell culture supernatant Fig. 5a: Example 1 - Comparative example: Chromatographic separation of unstressed samples in H2O Fig. 5b: Example 1 - Comparative example: Chromatographic separation of stressed samples in H2O Fig. 6: Example 2 - Course of fluorescence intensities depending on the pH value Fig. 7: Example 2 - Representation of the point-to-point slope as a function of pH

[0121] The invention is explained with reference to the examples shown in the figures and described below, without limiting the invention thereto. Test examples:

[0122] Unless otherwise stated, in the following examples 1 and 2 The following aids and reagents are used:

[0123] The wells of a PAIAplate 384 are used as reaction and measurement chambers. It is available from PAIA Biotech GmbH and is manufactured as described in WO 2015 / 135840 A1. The working volume of one well is 15 pL–110 pL.

[0124] Unless otherwise stated, a phosphate-buffered saline solution, a so-called PBS buffer, is used, adjusted to a pH of 7.4. The PBS buffer contains 137 mM NaCl, 2.7 mM KCl, and 12 mM phosphate.

[0125] A strong cation exchanger in the form of particles, namely CaptoSP ImpRes particles (Cytiva, Art. No. 17546802) with an average diameter of approximately 10 pm, is used as the functionalized surface. The particles carry ligands with sulfonic acid groups.

[0126] Unless otherwise stated, the measuring chambers are treated as follows before measurement: Preparation of the measuring chambers / microtiter plate

[0127] A 9% particle working solution with a concentration of 10% [v / v] of LPS is prepared from the cation exchange particle stock solution (slurry) by adding water and LPS (Liquid Plater Sealer, Candor).

[0128] 30 pL of this particle working solution is pipetted into the corresponding wells of the PAIA plate. 7.5 pL of a buffer solution with different pH values ​​is added to each of these wells (more details on the pH values ​​can be found in the tables in Examples 1 and 2). The buffer addition is carried out according to a specific schedule so that it is possible to track which buffer pH was added to which well.

[0129] After adding the buffer, mix briefly. The liquid is then removed from the plate. To do this, the plate is dried at 35 °C for 48 hours in a drying cabinet or similar. After drying, the titer plate can be stored or used directly for measurement. Example 1 : Measurement of charge variants (CVAs) of thermally stressed antibody samples Preparation of the buffer gradient:

[0130] A pH gradient system CX-1 from Thermo Fisher Scientific (item no. 302779) is used. This system consists of four different buffer substances and is characterized by the fact that defined pH values ​​can be set by mixing two buffers (Buffer A and Buffer B), resulting in a pH gradient with intervals between the pH values ​​of 0.2–0.3.

[0131] Buffer A has a pH of 5.6 and buffer B has a pH of 10.2.

[0132] The following mixtures are used to produce a 12-point gradient in the range of 7.3 to 10.2. Table 2: 12-point gradient, prepared with CX-1 buffer system from Thermo Fisher Scientific

[0133] The measurement chambers in the PAIA plate are prepared as described above, with 7.5 μL of buffer solutions P1 to P12 added to each well containing particles, as shown in Table 2. The buffer addition is carried out according to a specific schedule so that it is possible to track which buffer P1 to P12 is present in each well. Alternatively, the well containing each buffer P1 to P12 can be noted.

[0134] The following samples were tested; the target protein is the antibody trastuzumab: V1 -A: Trastuzumab (HER, Herceptin) stored at 4 °C; sample diluted with water before measurement. V1 -B: Trastuzumab stored for 14 days at 35 °C; sample diluted with water before measurement. V1 -C: Trastuzumab (HER, Herceptin) stored at 4 °C; sample diluted with cell culture supernatant (CSC) before measurement. V1 -D: Trastuzumab (HER, Herceptin) stored for 14 days at 35 °C; sample diluted with cell culture supernatant (CSC) before measurement.

[0135] Heat treatment produces acidic charge variants of trastuzumab.

[0136] The trastuzumab samples are diluted with water or in cell culture supernatant diluted with water (dilution 1:5) to a concentration of 150 pg / mL.

[0137] 1 OpL of this sample solution was added to 3 of the wells treated with P1 to P12. This means that 36 wells are occupied and one receives three wells per buffer P1 to P12. Data values. 50 pL of the fluorescent marker (Affibody conjugated with Alexa 647 (Affibody AB, Sweden)) is added to these wells and mixed.

[0138] 50pL of the above-mentioned fluorescent marker, which is present at a concentration of 12 nM in a PBS buffer with 50 ppm BSA, is added.

[0139] The layout of the plate is shown as an example in Fig. 2.

[0140] The entire microtiter plate was shaken at room temperature at 1400 rpm on an Eppendorf Thermomixer Comfort for 30 minutes and then centrifuged at 500 xg for one minute.

[0141] The fluorescence intensity is then measured from below (bottom reading) in each measuring chamber in a Tecan Satire fluorescence plate reader at an excitation wavelength of 630 nm and an emission wavelength of 665 nm.

[0142] Figures 3 and 4 illustrate the differences with regard to the existing loading variants, ie the CVA profiles of the temperature-treated samples V1-B and V1-D compared to the non-temperature-treated samples V1-A and V1-C.

[0143] The curves of the temperature-treated and thus stressed samples show a shift of the fluorescence increase to lower (acidic) pH values, i.e. there is a proportion of trastuzumab antibodies in these samples that are detached from the particles (or bind more weakly to the particles) even in slightly acidic buffers, which suggests that these antibodies are acidic charge variants of trastuzumab.

[0144] To verify the results, the same samples are analyzed using conventional cation exchange chromatography. The system uses a 250 x 4 mm Thermo ProPac WCX-10 cation exchange column and the ThermoFisher CX-1 buffer system with a flow rate of 1 mL / min. The starting buffer consists of 100% Buffer A, to which Buffer B is successively added over the first 15 minutes until a concentration of 50% B is reached. Over the next 2 minutes, the proportion of Buffer B is increased to 100%. This results in a pH gradient buffer from 5.6 to 10.2. The detection of the eluted molecules is performed by Absorption measurement at 280 nm. The results are shown in Figures 5a and 5b.

[0145] The results of the experiments using the present invention and the conventional method (CEX chromatography) are in good agreement. The measurements of the samples in water and the diluted cell culture supernatant are also consistent.

[0146] Looking at the results of conventional cation exchange chromatography, it is striking that the main peak, i.e., the most abundant charge variant in the sample, elutes from the column after a retention time of 16.54 minutes in the unstressed trastuzumab sample. The peak area is 870,278 AU.

[0147] Before this main peak, a rise in the baselines can be seen, indicating the presence of some acidic charge variants, as well as an acidic peak at a retention time of 14.92 minutes. The acidic variants have a total peak area of ​​526,724 AU.

[0148] In this sample, a basic charge variant is also registered at a retention time of 17.23 minutes with a peak area of ​​278,435 AU.

[0149] In the sample stressed at 35 °C, the main peak (retention time 16.55 minutes) is significantly smaller than in the unstressed sample and has a significantly smaller peak area (227,437 AU).

[0150] The acidic charge variants are significantly more present in the stressed sample and show a cumulative peak area of ​​1,157,381 AU.

[0151] The basic peak detected in both samples has a virtually identical peak area in both chromatograms.

[0152] This means that acidic charge variants are generated by the temperature treatment. This has also been described in the literature (Spanov et al., "Change of charge variant composition of trastuzumab upon stressing at physiological conditions," Journal of Chromatography A 1655 (2021) 462506). Example 2: Determination of two antibodies with different isoelectric points

[0153] The buffer solutions with different pH values ​​are prepared as described in Example 1, using the 8-stage buffer system shown in Table 3 - as shown below. Table 3: 8-point gradient, prepared with CX-1 buffer system from Thermo Fisher Scientific

[0154] The fluorescent marker used is Nano-Secondary® alpaca anti-human IgG / anti-rabbit IgG, recombinant VHH, Alexa Fluor® 647 (Art. No. CTK0101 - ChromoTek GmbH). This is used at a concentration of 5 nM in a PBS buffer containing 50 ppm BSA.

[0155] The two monoclonal antibodies bevacizumab (Bev) and rituximab (Rit), as well as three mixtures of these two molecules in the ratios 75:25, 50:50, and 25:75, were used as biological analytes. The total antibody concentration in all samples was 50 pg / mL.

[0156] 10 μl of each of these sample solutions are added to three wells with different pH values ​​(a total of 24 wells per sample) and mixed with 50 μl of the fluorescent marker solution. The entire microtiter plate is incubated at Shaken at room temperature for 30 minutes at 1400 rpm in an Eppendorf Thermomixer Comfort and then centrifuged for one minute at 500 xg.

[0157] The fluorescence intensity is then measured from below (bottom reading) in each measuring chamber in a Tecan Satire fluorescence plate reader at an excitation wavelength of 630 nm and an emission wavelength of 665 nm.

[0158] The diagrams in Fig. 6 show the fluorescence intensity curves of the different samples as a function of the pH value in the wells.

[0159] The sample with 100% Bev shows the largest difference in fluorescence intensity between pH values ​​6.9 and 7.6. As the Bev content in the samples decreases, this difference decreases, and at the same time, the increasing Rit content in these samples causes the fluorescence intensity to increase sharply between pH values ​​8.9 and 9.5.

[0160] This means that Bev is detached from the particles at pH values ​​of approximately 7, while Rit only at pH values ​​of approximately 9. This means that Bev is a significantly more acidic antibody than Rit. This is consistent with the isoelectric points of 8.5 for Bev and 9.5 for Rit reported in the literature. (See Goyon et al. 2017: Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies, Journal of Chromatography B Volumes 1065-1066, 15 October 2017, pp. 119-128).

[0161] If the point-to-point gradient of the samples is plotted instead of the fluorescence intensity, a chromatogram-like representation is obtained in which peaks are clearly visible (Fig. 7). The height of the peaks also corresponds to the mixing ratios set in the samples, allowing quantitative statements to be made.

Claims

Claims: 1 . Method for the analysis of charge variants of proteins using microtiter plates whose wells have at least partially different pH values ​​and / or salt concentrations and using functionalized surfaces.

2. The method according to claim 1, wherein the proteins are therapeutically active proteins, such as antibodies, Fc fusion proteins, antibody fragments, bi- and multispecific antibodies and virus particle-based therapeutics.

3. The method of claim 1, wherein the charge variants comprise mispaired and misassembled proteins and their fragments that occur during production or virus particle-based therapeutics that have a faulty nucleic acid load.

4. The method according to claim 1 or 2, wherein the proteins are virus particles which may be loaded with nucleic acids.

5. Method according to one of the preceding claims, wherein the wells of the microtiter plate are prepared as follows: Filling the wells in the microtiter plate with aqueous buffer solutions having different pH values, with the pH values ​​in the individual wells being in the range of 0.02 to 1.5, whereby not all wells need to be filled; and / or Filling the wells in the microtiter plate with aqueous buffer solutions having different salt concentrations, whereby the distances between the concentrations in the individual wells are in the range of 0.5 to 500 mmol / L, whereby not all wells have to be filled; or filling the wells in the microtiter plate with aqueous buffer solutions having different pH values ​​and salt concentrations, whereby the The pH values ​​in the individual wells are in the range of 0.02 to 1.5 and the salt concentration intervals are in the range of 0.5 to 500 mmol / L, whereby not all wells need to be filled; and optionally removal of water from the wells.

6. The method according to claim 5, wherein the buffers with different pH values ​​and / or salt concentrations are introduced into wells located next to or below one another, so that rows or columns with increasing or decreasing pH value and / or salt content are formed.

7. Method according to one of the preceding claims, using marker molecules that bind specifically to the protein whose charge variants are analyzed.

8. The method according to claim 7, wherein the marker molecules are mono- or polyclonal antibodies or nanobodies or single-domain antibodies derived therefrom, as well as peptides and RNA- or DNA-based aptamers, and these, optionally coupled to fluorescent dyes, form fluorescent markers.

9. Method according to one of the preceding claims, wherein the functionalized surfaces are anion or cation exchangers, mixed-mode or multi-mode ion exchangers, the marker molecules are fluorescent markers, and the microtiter plate is equipped as follows: Some or all of the wells of the microtiter plate are designed as measuring chambers; in which there is a raised portion which is shaped such that its base area occupies at least 50% of the base area of ​​a measuring chamber or well, and preferably a square base area which tapers at least slightly towards the top and has the shape of a four-sided pyramid, wherein the upper end of the raised portion is pointed, flat or convex, and advantageously the upper end of the raised portion has a diameter of less than 50 pm so that no test components are deposited there, whereby the height of the elevation is at least 10% and at most 50% of the height of the measuring chamber or well and the bottom of the measuring chamber or well is opaque up to the bottom of the elevation and the elevation is translucent and forms a measuring window which is used to measure the fluorescence emission.

10. The method according to claim 9, comprising the following steps: a. Using the microtiter plate whose wells have been at least partially pretreated with the method according to claim 5 or 6; b. Adding an aliquot of the protein sample to each of the pretreated wells; c. Adding anion or cation exchangers, mixed-mode or multi-mode ion exchangers, which bind charge-specifically to the protein in the form of particles; d. Adding one or more fluorescent markers selected to bind specifically to the protein whose charge variants are to be determined; e. Mixing; f. After settling the anion or cation exchangers, mixed-mode or multi-mode ion exchangers, measuring the intensity of the unbound fluorescent markers; g. Correlating the measurement with the pH values ​​and / or salt contents present in the wells in order to determine the different charge variants of the protein.

11. A method according to any one of claims 1 to 8, wherein the functionalized surfaces are biosensors capable of acting as anion and cation exchangers or as mixed-mode or multi-mode ion exchangers; and optionally, marker molecules are present, in particular mono- or polyclonal antibodies or nanobodies or single-domain antibodies derived therefrom, as well as peptides and RNA- or DNA-based aptamers.

12. The method according to claim 11, comprising the following steps: a. Using the microtiter plate whose wells have been at least partially pretreated with the method according to claim 5 or 6; b. Adding an aliquot of the protein sample to each of the wells pretreated with buffers having different pH values ​​and / or salt concentrations; c. Introducing the biosensor into the treated and sample-containing wells of the microtiter plate; d. Mixing; e. Measuring the elicited signal from the biosensor; f. Correlating the measurement with the pH value and / or salt content present in the wells to determine the different charge variants of the protein.

13. Microtiter plate produced by one of the processes according to claim 5 or 6 in the dried state.

14. Microtiter plate according to claim 13, wherein the following process step is carried out before drying, namely addition of anion or cation exchanger, mixed-mode or multi-mode ion exchanger in the form of particles.

15. Kit containing the microtiter plates according to claim 13 or 14 and a marker molecule.

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