METHOD FOR THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF PROTEIN AGREGATES

Description

[0001] The application relates to a method for the qualitative and quantitative determination of protein aggregates using a specially equipped microtiter plate and aggregate-specific fluorescent dyes.

[0002] Aggregates of biological systems, also called high molecular weight species (HMWS), are complexes of similar or different molecules. They are formed by the aggregation of several molecules, resulting in di-, tri-, and oligomers.

[0003] Protein aggregation usually occurs when two or more proteins bind together via hydrophobic sites. During (partial) unfolding of proteins, the hydrophobic regions, mostly located within the protein structure, become accessible for interactions. If binding with another protein occurs, aggregation takes place.

[0004] Proteins exhibit varying degrees of aggregation due to their stability. Less stable proteins aggregate more readily than stable proteins. Protein stability is influenced by the matrix in which the protein is embedded. Destabilization leads to at least a partial disruption of protein folding, which can be triggered by physical or chemical factors. These triggers include temperature changes, such as those occurring during freezing and melting cycles, the application of physical forces (e.g., shear forces), and the composition of the matrix in which the proteins are embedded. In particular, there is a correlation between the buffer substances and their concentrations, the pH value, the protein concentration, the presence of denaturing agents, and the formation of aggregates.

[0005] Aggregation frequently occurs during the production of pharmaceutically active biological agents (so-called biologics), because biologics are often sensitive, complex proteins that are also predominantly produced temporarily in cell culture. At the same time, cell culture production involves conditions and process steps, each of which can individually trigger aggregation.

[0006] Protein aggregates are usually undesirable byproducts in the production of biologics because they can exhibit immunogenic activity profiles. Therefore, the presence of aggregates is rigorously tested during pharmaceutical drug approval. Although the process of protein aggregation is dynamic and partially reversible under certain conditions, it is often a progressive process.

[0007] Avoiding aggregation is a key objective in the production of and handling of proteins. Therefore, biological systems and reaction conditions that minimize aggregate formation must be identified during the development of biological processes.

[0008] It is therefore desirable to quickly identify conditions that promote aggregation in order to optimize them in such a way as to avoid aggregation.

[0009] The need to determine the occurrence of protein aggregates arises as early as the initial stages of process development, namely cell line development and the so-called upstream development. Here, for example, many monoclonal cell lines are produced and then examined for productivity and product quality, including their tendency to aggregate. It is often crucial to identify cell lines or cell culture conditions that tend to produce aggregated proteins and to eliminate these from the development process. Frequently, hundreds of samples are generated at this stage of process development, all of which must be examined for aggregation within a short timeframe.

[0010] Furthermore, many samples are also generated in downstream development, where the various steps of product purification are optimized.

[0011] However, effective control in the production and purification of proteins, as well as in the selection of cell lines, yeast or bacterial strains, and cell culture conditions for protein production, can only be achieved if the result of the control is available quickly and reliably in order to make short-term changes to the process parameters.

[0012] Methods for protein aggregate measurements are well-established. The most frequently used method is size exclusion chromatography (SEC). In SEC, a purified protein solution is applied to a stationary phase, where the proteins are separated based on their different sizes and therefore elute at different times. The eluates are typically detected by measuring their absorbance at a wavelength of 280 nm. This method allows for the identification and quantification of molecules of different sizes by determining the areas of the eluate peaks / fractions in the chromatogram. SEC usually requires purified samples, which makes the measurement technically and time-consuming, and it cannot detect aggregates larger than approximately 0.1 µm (100 nm).

[0013] Other methods for determining protein aggregates include gel electrophoresis, which also separates molecules based on their size, and DLS, or dynamic light scattering. DLS is a method that detects the presence of aggregates based on the light scattering of larger molecules. Here, too, the samples must be purified before measurement, and DLS is not suitable for protein aggregates larger than 1 µm.

[0014] Fluorescent dyes are now also used for the detection of aggregates. These dyes are designed to interact with the hydrophobic regions of the aggregated proteins, triggering fluorescence emission upon excitation. These protein-dye interactions range from physical / chemical bonding to charge-transfer mechanisms and are described, for example, in EP 2 507 319 A, WO2011 / 065980A3 and US2021 / 0009809 A1.

[0015] The use of aggregate-specific fluorescent markers also only yields reliable results if the samples have been pre-purified. Attempts on unpurified samples led to unsatisfactory results, as described by Oshinbolu et al. in "Evaluation of fluorescent dyes to measure protein aggregation within mammalian cell culture supernatants." Journal of Chemical Technology & Biotechnology 93.3 (2018): 909-917) and Paul, Albert Jesuran, et al. in "Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants." Analytical and Bioanalytical Chemistry 407.16 (2015): 4849-4856.

[0016] Oshinbolu et al. describe that it is not possible to measure aggregation in cell culture supernatants using Sypro-Orange. Paul et al. used the dyes Bis-ANS and Thioflavin T and arrived at similar results, namely that the proteins must be purified before dye treatment. The substances present in the culture medium and cell supernatant pose a particular problem here.

[0017] Paul et al., in "High-throughput analysis for sub-visible mAb aggregate particles using automated fluorescence microscopy imaging", Anal. Bioanal. Chem (2017), 409, 4149-4156, also describe the use of fluorescence spectroscopy with aggregate-specific fluorescent markers to determine aggregates, all larger than 1 µm, with regard to their occurrence and size distribution in purified samples. Paul et al. do not address the quantitative determination of aggregates in culture medium or cell supernatant.

[0018] So far, no method is known that allows protein aggregates to be determined quickly and reliably without having to purify the protein-containing sample.

[0019] The object of the present invention is therefore to provide a method for the efficient and rapid qualitative and quantitative determination of protein aggregates directly and without prior sample purification. The measurement should also be simple and quick to perform using equipment routinely available in the laboratory. The invention also relates to the use of the measuring chambers described in WO 2015 / 135840 A1 in the method according to the invention.

[0020] US 2010 / 0028935 describes microtiter plates featuring multiple wells with convex bottoms. It is suggested that these plates be used for microbiological and enzymatic analyses of blood, plasma, serum, or cell culture samples. This publication does not address aggregate detection.

[0021] The inventors have now found the method, as defined in the claims and described in the description, which avoids the aforementioned disadvantages.

[0022] In particular, the method can be used advantageously in the development of cell culture processes for the recombinant (technical) production of proteins, especially antibodies, and in the development of downstream processes.

[0023] The procedure is easy to perform and is based on measuring the difference in fluorescence emission of aggregate-specific fluorescent markers.

[0024] The sample containing the target protein can be measured directly; purification is not necessary. The inventive method allows even those aggregate-specific fluorescent markers for the detection of protein aggregates to be used that otherwise only yield reliable results with purified samples (see Oshinbolu (2018) and Paul (2015)).

[0025] For the process according to the invention, the microtiter plates described in WO 2015 / 135840 A1 with specially equipped measuring chambers are used. In this respect, full reference is made to that publication and its disclosure. In contrast to the disclosure in WO 2015 / 135840 A1, in which fluorescent dyes are used that fluoresce in both the unbound and bound states after excitation, the present invention uses aggregate-specific fluorescent dyes which, as described below, fluoresce only when bound to aggregates. These microtiter plates, preferably in SBS format, have specially designed wells as measuring chambers.They include a projection that tapers upwards from the base, at least slightly, the base / cross-section of which has any desired shape, and the end of the projection facing away from the base of the device is pointed, flat, or convex, so that no test components, especially no particles, can accumulate there and interfere with the beam path for subsequent fluorescence measurement. Advantageously, the upper end of the projection has a diameter of less than 50 µm.

[0026] The sample containing the target protein is, after extraction and without further treatment, added in portions to the wells of the aforementioned specially equipped microtiter plate, in which the other experimental components are already present or are added. These experimental components include at least one aggregate-specific fluorescent dye, optionally buffer, and optionally functionalized particles.

[0027] For the method according to the invention, equally sized portions are taken from the target protein-containing sample. The minimum number of such portions is two. The number of portions depends on the number of measurement points per sample that are desired, for example, to determine the measurement deviation.

[0028] If two portions of the protein-containing sample are taken, an aggregate-specific fluorescent dye is added to each portion. More than one aggregate-specific fluorescent dye can also be added. One of these portions contains functionalized particles as additional experimental components.

[0029] If more than two portions are taken from the protein-containing sample, it is advantageous to take an even number of portions, mix them with the experimental components, and additionally treat half of these mixtures with functionalized particles. This results in one mixture with and one mixture without functionalized particles. For determining the difference in fluorescence emission, it is important that at least one of the portions has not been treated with functionalized particles. Instead of pairwise comparison, it is also conceivable to compare and evaluate each of the particle-treated samples with only one sample that has not been treated with particles.

[0030] After portioning the protein-containing sample solution and mixing it with the experimental components, the sample portions are usually mixed at room temperature using a so-called shaker.

[0031] The mixture containing a portion of the sample, aggregate-specific fluorescent dye and functionalized particles is hereinafter referred to as PP-A and the other mixture containing a portion of the sample and aggregate-specific fluorescent dye is hereinafter referred to as PP-B.

[0032] PP-A and PP-B contain the same amount of sample, the same amount and type of aggregate-specific fluorescent dye, and, if applicable, the same amount and type of other experimental components, e.g., buffer, except for the presence or absence of the functionalized particles. PP-A and PP-B are always treated identically, and it is advantageous if the PP-A and PP-B mixtures are prepared in the wells of the same microtiter plate.

[0033] The mixing sequence is irrelevant for the success of the process. However, it can be advantageous for the efficiency of the process if wells already loaded with functionalized particles are used for the production of the PP-A mixture.

[0034] The use of the specially equipped microtiter plate has the advantage that, after mixing the sample portions with the experimental components and settling the functionalized particles, the mixtures can be used directly for fluorescence measurement without further treatment (e.g., aliquoting measuring solutions and transferring them into measuring vessels (cuvettes or microtiter plates suitable for fluorescence measurement)).

[0035] Preferred microtiter plates according to the invention are those in SBS format with 384 wells, configured as measuring chambers according to the invention, in which the raised area (referred to as a structural element in WO 2015 / 135840 A1) is shaped such that its base area occupies at least 50% of the base area of ​​a measuring chamber or well. Preferably, the raised area has a square base, tapers (at least slightly) upwards, and is shaped like a four-sided pyramid. The upper end preferably tapers to a point. The height of the raised area 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 base of the measuring chamber or well is opaque except for the base area of ​​the raised area. The raised area, however, is transparent and forms a measuring window which, as described in detail in WO 2015 / 135840 A1, is used to measure fluorescence emission.

[0036] The production of such microtiter plates is achieved in particular by using commercially available microtiter plates whose base is replaced by a translucent (transparent) and non-fluorescent film (e.g., made of polypropylene) into which the corresponding raised areas are embossed in the arrangement corresponding to the SBS format. The required opacity of the base, with the exception of the base of the raised area, is achieved by applying a dark, preferably black, color.

[0037] Regarding Measurement windowReference is made to the description in WO 2015 / 135840 A1, and full reference is made to it in this regard. For fluorescence measurement, the measuring chamber or the microtiter plate according to the invention is irradiated from below with excitation light, which is then directed into the sample via the raised area. Ordinary fluorescence readers can be used to read out the fluorescence signal.

[0038] In one embodiment of the invention, the inventive method uses a microtiter plate equipped with the inventive measuring chambers, which already contains at least partially dried functionalized particles. Such plates are produced by introducing a suspension with a defined quantity of functionalized particles into the wells of the plates and drying them at 35 °C. In one embodiment, a maximum of 50% of the wells are coated with particles, while the remaining wells contain no particles.

[0039] If plates containing dried-out functionalized particles are used, these particles must be reconstituted before adding the sample solution. The sample portion is sufficient for this purpose.

[0040] Microtiter plates that can be used preferably according to the invention are available from PAIA Biotech GmbH under the trade name PAIAplate 384.

[0041] Target proteins, i.e., those proteins whose aggregation state is to be determined, can be any isolated protein. Economically important proteins are primarily biologics, especially antibodies and molecules derived from them, such as Fc fusion proteins and bi-, tri-, or tetraspecific antibody formats.

[0042] The speed and ease of execution of the procedure are also advantageous in media development for cell line development, where identifying the right cell line and determining the appropriate culture conditions are of great importance. Only the right cell lines, maintained under optimal culture conditions, produce large quantities of high-quality proteins with little or no protein aggregation.

[0043] The target organism in which the protein is to be produced must be genetically modified so that it can produce the foreign protein. This involves, among other things, introducing the gene sequence that codes for the target protein and other gene sequences important for its production into the target organism (transfection or transformation). This process often leads to random integration of the target DNA and to many genetically diverse cell lines and bacterial or yeast strains that are suited to producing the target protein to varying degrees, for example, because growth is inhibited or because the target protein cannot be produced efficiently. Therefore, the selection of the most suitable cell lines or bacterial or yeast strains is an important part of process development.

[0044] The same applies to monitoring the temporal development of product quality during a reactor run, for example, when it comes to optimizing the feeding strategy for the cell culture or optimizing the culture conditions (composition of the nutrient medium, temperature, pH value, supply of oxygen and other additives such as vitamins, amino acids and sugars). Here, too, the speed and ease of execution of the method for determining protein aggregation are a great advantage.

[0045] The present method is particularly useful in the production of pharmaceutically active biologics manufactured in various expression systems. Examples of such expression systems include cell culture systems in which animal, human, or plant cells are propagated and used to produce proteins outside the organism. Cell lines used in such cell culture systems include, for example, CHO cell lines (Chinese Ovarian Hamster), human HEK cell lines (Human Embryonic Kidney), cell lines from mice, insects (e.g., Sf-9 or Sf-21), and plants, e.g., from maize or tobacco. Alternative expression systems include bacterial systems, such as the E. coli system ( Escherichia coli ), systems based on yeast cells, such as pichia pastoris or saccharomyces cerevisiae , as well as systems based on fungi (e.g. aspergillus niger ) .

[0046] As part of biological process development, the number of cell lines is successively reduced, inefficient cell lines are eliminated, and the culture volume is continuously increased (so-called upscaling). Simultaneously, the processes in the bioreactors are monitored more and more closely (e.g., oxygen content, pH value, metabolic products, and cell growth) to determine the optimal conditions for the final reactor volume. To optimize these conditions, various culture media with different compositions are used, and a wide range of additives, such as vitamins, amino acids, salts, trace elements, proteins and peptides, fatty acids, lipids, and sugars, are tested to determine whether they improve cell health and growth, as well as product yield and quality. This process can generate hundreds of samples, in which the product must be characterized and its aggregation state analyzed.

[0047] The method according to the invention is particularly suitable for use in the development of expression systems, in particular cell culture systems for the production of biologics, especially therapeutically effective antibodies.

[0048] The method according to the invention is equally suitable for use in cell line development, in particular for finding and optimizing culture conditions for the production of biologics, such as therapeutic antibodies, etc., in large quantities and of good quality.

[0049] The method according to the invention is also particularly suitable for use in process control during the manufacture of biologics, especially therapeutically effective antibodies, particularly for monitoring product quality during production by an expression system (also called reactor run).

[0050] The process uses special fluorescent dyes capable of detecting aggregates, such as aggregate-specific fluorescent dyes (also called fluorescent markers) described, for example, in WO2011 / 065980A3 and US2021 / 0009809 A1, EP2507319 A2, and WO 96 / 36882 A. These fluorescent dyes can be used alone, in combination, or as a composition containing several dyes. A person skilled in the art can determine, without significant technical effort, which aggregate-specific fluorescent dyes are particularly suitable for the process according to the invention. In their unbound state, these fluorescent dyes exhibit only negligible fluorescence emission upon excitation. They can only be excited to fluorescence upon binding to aggregates.

[0051] Aggregate-specific fluorescent markers are commercially available, such as aggregate-specific fluorescent dyes from the following classes: Molecular rotors from the class of substituted julolidines, e.g., 9-(2,2-dicyanovinyl)julolidines (DCVJ) and 9-(2-carboxy-2-cyanovinyl)julolidines (CCVJ); substituted naphthalenes or bis-naphthalenes, e.g., 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (Bis-ANS); merocyanines, such as N-(3-sulfopropyl)-4-(p-dihexylaminostyryl)pyridinium and as described in WO 96 / 36882 A, in particular those with the following formulas where in the aforementioned formulas n represents 3 or 4 and m represents an integer from 2 to 10. Some of the aforementioned fluorescent dyes are available under the following trade names: Sypro® - Orange, Sypro® - Red, or ProLite Orange; these dyes are preferably used. Aggregate-specific dyes as described in EP 2 507 319 A, in particular those with the following formulas (I) - (VII), in which X may represent the following ions: perchlorate (ClO₄⁻), sulfate (SO₄²⁻), alkanesulfonates, arylsulfonates, phosphates, tosylates, mesylates, tetrafluoroborates, and halogen ions such as chloride, iodide, bromide, or fluoride, as well as any mixtures of these anions. Benzothiazoles, such as those available under the trade name Thioflavin T®; phenoxazines, such as those available under the trade name Nile Red or Nile Blue Oxazone; sulfonated azo fluorescent dyes, such as those available under the trade name CongoRed or Chrysamine-G, a derivative of CongoRed; or 2-[4-(Dimethylamino)styryl]-1-methylpyridinium iodide (DASPMI or 2-Di-1-ASP), N-(3-Sulfopropyl)-4-(p-dihexylaminostyryl)pyridinium (e.g., available from Interchim).

[0052] In the process according to the invention, the functionalized particles are selected such that they can bind the target protein(s), regardless of whether the proteins are present as monomeric or oligomeric aggregates. The use of the functionalized particles in the process according to the invention causes the target protein to be immobilized and settle at the bottom of the measuring chamber.

[0053] Usable functionalized particles are, in particular, those described in WO2015 / 135840 A1. For example, particles functionalized with one or more of the following proteins or chemicals, namely protein A, protein L or protein G, strepTactin, streptavidin, avidin or neutravidin, which may be complexed with biotinylated affinity ligands.

[0054] The aforementioned functionalized particles are common reagents in biochemical practice and are often used for the purification of proteins, e.g. as column material for analytical HPLC columns or as column material on an industrial scale.

[0055] Examples of functionalized particles that can be used according to the invention are Protein A particles from Repligen, which are marketed under the trade name "CaptivA™<" (e.g., CaptivA PriMAB™<). These are cross-linked agarose particles with recombinant native Staphylococcus Protein A (rSPA) from E. coli immobilized on them. Other examples of Protein A particles include Affi-Gel® Protein A resin or Affi-Prep® Protein A resin from Bio-Rad, Poros Mab capture A select affinity chromatography resin from Thermo Fisher, Ultra-linked rProtein A resin from Sino Biological Inc., Immobilized Recombinant Protein A Resin from Generon, Protein A Agarose from Sigma Aldrich, Mabselect protein A resins from GE Healthcare, ProSep® Ultra Plus Chromatography Resin from Merck-Millipore, Praesto AP, APc or Praesto Jetted A50 from Purolite, and TOYOPEARL® AF-rProtein A HC-650F from Tosoh Biosciences.

[0056] Preferably, the particles usable according to the invention have mean diameters in the range of about 20 to 200 µm. Particles preferred according to the invention have mean diameters in the range of 20 to 80 µm, 50 to 100 µm, 40 to 200 µm, 50 to 150 µm and 50 to 200 µm.

[0057] Portioning is done manually or automatically with an automated pipetting robot (liquid handler).

[0058] The advantage of the method according to the invention lies particularly in the fact that many samples can be examined for the presence of aggregates in a relatively short time. Due to the simple and, if desired, automated procedure, measurement results for more than 100 samples simultaneously are usually obtained in less than 100 minutes.

[0059] The following devices, for example, can be used as fluorescence readers according to the invention: The devices of the Spectramax series from Molecular Devices, the systems from Tecan (Safire, the Infinite series, or SPARK), readers from BMG Labtech (Omega, Clariostar, Pherastar) and fluorescence microscopes, e.g. from SynenTec (Cellavista and NyONE).

[0060] The invention is explained with reference to the examples shown in the figures below and described further below, without limiting the invention thereto. Fig. 1 - Graphical representation of the measured values ​​determined in Example 1, with indication of the standard deviation based on 6 measurements per aggregate content. The y-axis shows the difference in the measured fluorescence intensities (measured emission from PP-B minus measured emission from PP-A) and the x-axis shows the aggregate content of the sample solution used. Fig. 2- Graphical representation of the measured values ​​obtained in Example 2, with indication of the standard deviation based on 6 measurements per aggregate content. The y-axis shows the difference in the measured fluorescence intensities (measured emission from PP-B minus measured emission from PP-A) and the x-axis shows the aggregate content of the sample solution used. Fig. 3 - Graphical comparison of the values ​​obtained in examples 1 and 2. Example 1

[0061] Establishing a calibration curve with aggregated antibodies in an aqueous buffer solution: For aggregation measurement, trastuzumab (an IgG1 antibody) from Roche is used and aggregated. Antibody aggregation is achieved by subjecting the trastuzumab in buffer to a 15-minute temperature treatment at 75 °C. The solution is shaken during the temperature treatment. The degree of aggregation of the resulting trastuzumab sample is determined by SEC.

[0062] A diluted solution of SYPRO-Orange is used as a fluorescent marker. For this purpose, the 5000x SYPRO-Orange concentrate in DMSO from Thermo Fisher (Art. No. S6650) was diluted with 1x PBS buffer to a concentration of 55x.

[0063] 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.

[0064] The wells (cups) of a PAIAplate 384, available from PAIA Biotech GmbH and manufactured as described in WO 2015 / 135840 A1, are used as reaction and measuring vessels.

[0065] The mixtures are prepared in the wells of the 384-well PAIAplate. The working volume of a well is 15 µL–110 µL.

[0066] CaptivA beads from Repligen, with an average diameter of approximately 90 µm, are used as functionalized particles. These particles are functionalized with Protein A and are commercially available.

[0067] To prepare the wells and the plate, CaptivA Protein A particles are pipetted in buffer into the appropriate number of wells, and buffer is added to the appropriate number of other wells. The plate is then stored at 35 °C for 48 hours to allow the water to evaporate from the buffer and the particles to dry.

[0068] For the measurement series, the partially aggregated trastuzumab samples prepared as described above are mixed with non-aggregated trastuzumab in predefined ratios, resulting in six different sample solutions with aggregate concentrations of 27%, 22%, 16%, 11%, 5%, and 1%. The total trastuzumab concentration is 0.5 mg / mL in each case.

[0069] 40 µl of each of these 6 sample solutions are pipetted into six wells A, containing the PP-A mixture (which contains particles), and into six wells B, containing the PP-B mixture (which does not contain particles). 20 µl of the diluted 55x-SYPRO-Orange solution is then added to each well. This yields six values ​​for each sample solution, each with a specific aggregate content.

[0070] The microtiter plate is shaken on a shaker (Bioshake from QInstruments) for 90 minutes at room temperature and 1400 rpm. Afterwards, the microtiter plate is briefly centrifuged to allow the particles to settle in wells A. The plate is then used for fluorescence measurement.

[0071] The fluorescence intensities in the wells of type A and in the wells of type B are determined from below, i.e. by bottom reading, in a Tecan Safire fluorescence plate reader at an excitation wavelength of 480 nm and an emission wavelength of 580 nm.

[0072] The difference in fluorescence intensities from Wells B and Wells A is determined as a measure of the aggregation state. The higher the difference, the more aggregates are present in the sample. The values ​​of a PP-A mixture and a PP-B mixture are always compared.

[0073] To calibrate the measurement, for example, to quantitatively determine the aggregates, the obtained fluorescence intensity values ​​can be plotted against the previously set aggregate concentrations. The values ​​can then be fitted with a linear function to obtain a calibration function. The corresponding calibration curve is shown in Fig. 1 The R2 value is 0.9979. The values ​​are based on 6 measurements per aggregate content. Example 2 Creating a calibration curve using aggregated antibodies in cell culture supernatant

[0074] The procedure is the same as described in Example 1, with the difference that CHO cell culture supernatant, also referred to here as CCS (cell culture supernatant), is used instead of PBS buffer. The CHO cell culture supernatant originates from an expression system for antibody production. Its composition is unknown, but it can be assumed to contain buffer substances, biological substances derived from the culture, nutrients, and salts. The corresponding calibration curve is shown in Fig. 2 The R2 value is 0.9972. The values ​​are based on 6 measurements per aggregate content.

[0075] The values ​​obtained in Examples 1 and 2 were compared. The data are shown in Table 1.

[0076] The compared values ​​are shown in the following table and in Fig. 3 shown: Trastuzumab aggregate content at a trastuzumab concentration of 0.5 mg / mL Relative fluorescence units Example 1 Example 2 1% 61 1 5% 5426 6467 11% 12304 12167 16% 18276 18703 22% 24122 23871 27% 28841 29146

[0077] It was found that the measurements are very comparable despite the significantly different medium (buffer in example 1 and cell culture supernatant in example 2), so that the procedure can be carried out flexibly and independently of the purity of the sample.

Claims

1. A method for the qualitative and quantitative determination of protein aggregates using aggregate-specific fluorescent dyes, comprising the following steps: a. Portioning the sample into at least two equally sized portions, PP-A and PP-B, and transferring the portions into specially designed wells, containing a measurement chamber, including an upwardly tapering elevation whose base area covers at least 50% of the bottom area of the well, wherein the height of the elevation is at least 10% and at most 50% of the height of the measurement chamber, wherein the bottom of the well is opaque except for the base area of the elevation, while the elevation is transparent and forms the measurement window for the fluorescence measurement, b. Preparing a mixture comprising the PP-A portion, at least one aggregate-specific fluorescent marker, and functionalized particles capable of binding the target protein(s), regardless of whether the proteins are present as monomers, oligomers or aggregates, in a well A, and preparing the same mixture in a further mixing vessel using the PP-B portion, but without functionalized particles; c. mixing both mixtures and allowing the functionalized particles to settle; d. Measuring the fluorescence emission of mixtures PP-A and PP-B; e. Calculating the difference between the measured emission of mixture PP-B and that of mixture PP-A to determine the protein aggregation.

2. Method according to the preceding claim, characterized in that the functionalized particles are functionalized with one or more of the proteins or chemicals listed below , namely Protein A, Protein L, or Protein G, StrepTactin, Streptavidin, Avidin, or Neutravidin, which are optionally complexed with biotinylated affinity ligands.

3. Method according to any one of the preceding claims, characterized in that the functionalized particles are present in the well designated for receiving the PP-A portion even before step a.

4. Method according to any one of the preceding claims, characterized in that the protein aggregates are quantified using calibration curves.

5. A method according to any one of the preceding claims, characterized in that the aggregate-specific fluorescent markers are selected from the following classes of fluorescent dyes: - molecular rotors from the class of substituted julolidines, 9-(2,2-dicyanovinyl)julolidines (DCJV), and 9-(2-carboxy-2-cyanovinyl)julolidines (CCVJ); - substituted naphthalenes or bis-naphthalenes, 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (Bis-ANS); - merocyanines, such as N-(3-sulfopropyl)-4-(p-dihexylaminostyryl)pyridinium, as well as molecules having the following formulas wherein n is 3 or 4 and m is an integer from 2 to 10; - molecules having one of the formulae (I), (II), (III), (IV), (V), (VI), or (VII) wherein X- may represent the following ions: perchlorate (ClO4- ), sulfate (SO42- ), alkanesulfonates, arylsulfonates, phosphates, tosylates, mesylates, tetrafluoroborates, as well as halide ions such as chloride, iodide, bromide, or fluoride, and any mixtures of these anions - benzothiazoles, thioflavin T; - phenoxazines or -oxazones, Nile red or Nile blue; - Sulfonated azo fluorescent dyes or derivatives thereof, Congo Red or Chrysamine-G; and - 2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DASPMI or 2-Di-1-ASP), N-(3-sulfopropyl)-4-(p-dihexylaminostyryl)pyridinium.

6. Method according to any one of the preceding claims, characterized in that the functionalized particles have an average diameter in the range of about 20 to 200 µm and are functionalized with one or more of the following proteins or chemicals, namely Protein A, Protein L, or Protein G, StrepTactin, streptavidin, avidin, or neutravidin, which are optionally complexed with biotinylated affinity ligands.

7. Method according to any one of the preceding claims for reaction control in the production of pharmaceutically active proteins.

8. Method according to any one of the preceding claims 1 to 7 for use in the development of expression systems, in particular cell culture systems for the production of biologics, in particular therapeutically active antibodies.

9. Method according to any one of the preceding claims 1 to 7, for use in cell line development, in particular the identification and optimization of culture conditions for the production of biologics, in particular therapeutically active antibodies.

10. Method according to any one of the preceding claims 1 to 7, for process control in the production of biologics, in particular therapeutically active antibodies, suitable in particular for monitoring product quality during production by an expression system.