EP1454144A1

EP1454144A1 - Method for conducting the quantitatively analytical determination of aggregates

Info

Publication number: EP1454144A1
Application number: EP02795236A
Authority: EP
Inventors: Birgit Hecks; Martin Pitschke; Claudia Clemens; Jessica Langer
Original assignee: Evotec Technologies GmbH
Current assignee: PerkinElmer Cellular Technologies Germany GmbH
Priority date: 2001-12-21
Filing date: 2002-12-19
Publication date: 2004-09-08
Also published as: WO2003054548A1; EP1321771A1

Abstract

The invention relates to a method for conducting the quantitatively analytical determination of the content of aggregates of substances in mixtures involving the following steps: adding at least one probe provided in the form of a protein, protein fragment, protein analog, peptide, peptide fragment and/or peptide analog, which is capable of binding to the aggregate, to the mixture to be examined, whereby the at least one probe and/or the at least one aggregate have/has at least one detectable property; associating the at least one probe on at least one aggregate before a self-aggregation of the at least on probe prevails, whereby the at least one detectable property undergoes a change during the association of the at least one probe and the at least one aggregate, and; determining the at least one detectable property and/or the modification thereof by using physical methods, whereby, during this determination, the measurement signal obtained is immediately reduced to the level of the background signal, which is caused by non-associated probes and/or aggregates both of which having a detectable property.

Description

Method for the quantitative analytical determination of aggregates

The present invention relates to a method for the quantitative analytical determination of the content of aggregates of substances in mixtures.

There are many substances that are converted into aggregates depending on their concentration and the environment. This occurs particularly frequently with proteins, protein fragments, protein analogs, peptides, peptide fragments and / or peptide analogs.

For example, peptide production, especially the successful synthesis of peptides that tend to aggregate, is often associated with great difficulties (Nilsson MR et al., (2001) Synthesis and purification of amyfoidogenic peptides, Anal Biochem 288 (1): 76-82) they, such as peptide hormones (insulin, glucagon, ANF), are converted into aggregates, in particular depending on the concentration and the environment.

Comparable problems also arise in the genetic engineering of proteins, for example when expressed in prokaryotes or eukaryotes. It is therefore often difficult to obtain soluble and active proteins. In many cases, overexpression leads to protein deposition, e.g. B. in prokaryotes for the production of "inclusion bodies". These are insoluble aggregates of incorrectly folded protein (Par ar, AA, (2000) Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli, Protein Expr. Purif. 20 (2): 152-161), which must be solubilized or removed in the course of protein purification.

Since the activity of these substances is reduced or even completely lost through aggregation, this can lead to a variety of problems. For example, in the manufacture of drugs / therapeutics containing peptides or proteins, aggregate formation is a problem because it is important that the specified amount of substance is also active. In addition, aggregates that are administered intravenously can lead to side effects. As an example here are peptide hormones as therapeutic agents and antibodies as therapeutic agents and diagnostic agents, e.g. As for non-Hodgkin lymphoma or Crohn's disease ^Λ to call (Borrebaeck CA et ai, (2001) Human therapeutic antibodies, Curr Opin Pharmacol 1 (4): 404-8). In addition, peptides or proteins are often used in these therapeutic agents in very high concentrations, which further increases the likelihood of the formation of aggregates. In addition, it is assumed that aggregates, for example when injecting the above-mentioned therapeutic agents, in particular insulin, cause swelling at the injection site, for example, and thus have a negative medical effect. A control of the aggregate content is therefore essential to monitor the quality of a drug / therapeutic.

But also in research, it is necessary for the informative value of the investigations that the quantitative determination of the content of aggregate in mixtures, in particular solutions, makes it possible to precisely determine the proportion of active substance. Undetermined quantities of aggregate could lead to incorrect test results.

For example, it was shown that contamination with peptide aggregates was responsible for the different results of experiments with neuronal cell cultures in the case of incubations with amyloid-ß-peptides. Depending on the batch used and the manufacturer, different results were obtained (May P.C., et al. (1992) beta-amyloid peptide in vitro toxicity: lot-to-lot variability. Neurobiol Aging 3: 605-607). The peptide aggregates had a neurotoxic and thus negative influence on the survival rate of the cells.

It is therefore important for these isolated proteins or petids, in particular synthetically produced peptides and genetically engineered proteins, to determine their actual content of aggregates. The quality of synthesized peptides is currently being assessed, ie the proportion of aggregates is determined, for example by HPLC, light scattering and mass spectrometry (MALDI). FTIR (Fourier transform infrared spectroscopy), CD (circular dicroism) and EM (electron microscopy) are used to detect amyloid fibrils (Bόuchard M., et al (2000), Formation of Insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy. Protein Be 9 (10): 1960-1967). Although the methods for determining the proportion of aggregates listed here allow a quantitative determination of a content of up to 1 ng aggregate, they are often very time-consuming and costly and often require complex sample preparation, which can influence the aggregate proportion of the sample. Therefore, if at all, they can only be used satisfactorily on a laboratory scale.

When cleaning proteins from "inclusion bodies", the detection of protein aggregation is often carried out via a scattering measurement (dynamic light scattering) of the solution and, by comparison, the determination of the absorption at 400 nm (US Dept Commerce / NOAA / NMFS / NWFSC / Molecular Biology Protocols: Preparation of active proteins from inclusion bodies, Laurant Vuillard & Alasdalr Freeman, University of St Andrews UK).

In general, methods such as scatter measurements and absorption determinations can be carried out comparatively quickly, but they are relatively unspecific and insensitive, ie the aggregate content must be at least 1% of the protein content in order to be detectable. In addition, the inherent turbidity of the medium or the presence of other non-aggregate components in the sample influences the measurement result and leads to inaccurate or even incorrect results. Thus, this method is only conditional for a qualitative aggregate detection, but not for a quantitative detection, e.g. B. can be used in the control of therapeutic agents (medications). The same applies to gel electrophoretic detection methods, which likewise have only a low sensitivity and are therefore only suitable for the qualitative detection of higher aggregate concentrations. In addition, the aggregates must be subjected to gel electrophoretic analysis. be partially solubilized or denatured. The aggregate verification is therefore only indirectly via the unspecific staining of the individual aggregate components. In addition, this time-consuming sample preparation, in particular re-buffering, changing the salt conditions, pH or concentration, disturbs the equilibrium existing in the sample to be tested and can significantly influence the degree of aggregation. The maximum sensitivity that can be achieved with gel electrophoretic methods is therefore, depending on the staining technique, only a maximum of 1 ng, for example for silver staining.

Also, a method for the detection of diseases associated with protein deposits described in the international patent application WO 99/15903 only discloses qualitative diagnostic detection of the aggregates. In the method disclosed there, for example, the association of substructures of pathological protein deposits and / or the association of pathological protein deposits with substructures of pathological protein deposits and / or pathological protein deposits is determined. If the measured value reaches or exceeds a certain threshold value, an illness can be assumed.

All of the methods listed here are due to the z. T. high effort either only satisfactorily applicable on a laboratory scale or not suitable for a quantitative analytical determination due to a lack of sensitivity and in particular specificity. The methods are therefore also, for example, for monitoring an industrial synthesis of molecules that tend to aggregate, e.g. B. proteins, protein fragments, protein analogs, in particular protein and / or protein fragments corresponding structures and / or peptides, peptide fragments and / or peptide analogs, in particular peptides and / or Structures corresponding to peptide fragments or also for quality control of therapeutic agents, for example, are not suitable.

The object of the present invention is therefore to provide a highly specific and sensitive method for the quantitative analytical determination of the content of aggregates of substances in mixtures, which the above-mentioned. Does not have disadvantages. This procedure should also be particularly suitable for routine use in quality control.

In addition, it is also an object of the present invention to provide a method for identifying and optimizing substances which can influence the formation of aggregates.

These objects were achieved by a method for the quantitative analytical determination of the content of aggregates of substances with the features of claim 1 and a method for the identification and optimization of substances that can influence aggregate formation with the features of claim 16. Preferred embodiments are in described the subclaims.

Figure 1 shows the principle of germ-induced multimerization.

Figure 2 shows the FIDA signal as a function of the concentration

Protein aggregates and in pictures A and B the background signal as well as the specific measurement signal. Figure 3 shows the results of an experiment to investigate different batches of a peptide. Figure 4 shows the results of an investigation of different batches of an antibody. Figure 5 shows the results of homologous and heterologous

Antibody detection. The method according to the invention for the quantitative analytical determination of the content of aggregates of substances in mixtures is characterized by the following steps:

Adding at least one probe in the form of a protein, protein fragment, protein analogs, peptide, peptide fragment and / or peptide analogs, which is able to bind to the aggregate, to the mixture to be examined; wherein the at least one probe and / or the at least one aggregate have at least one detectable property,

Association of the at least one probe with at least one aggregate before self-aggregation of the at least one probe predominates, the at least one detectable property undergoing a change when the at least one probe and the at least one aggregate are associated,

Determination of the at least one detectable property and / or its change by means of physical methods, with this determination the measurement signal obtained being immediately reduced by the background signal caused by non-associated probes and / or aggregates having a detectable property.

The background signal can be determined by various methods. In general, the quantitative determination of the background signal is an essential aspect of the method according to the invention. For example, to carry out the method it may be necessary to add an excess of probe, which may be, for example, 10 ⁵ -10 ⁷ compared to the aggregate. It is therefore particularly important, if the probe has a property to be detected, to determine the signal of the unbound probe quantitatively. One method to quantitatively determine the background signal is the FIDA (Fluorescence Intensity Distribution Analysis) one-component analysis. An example of this method is shown in Figure 2. With the help of this one-component analysis (Fida_Eval), the dominant background of the probe or the aggregate provided with a detectable property (secondary image A and B, channels with a black background) is determined. The cut-off limit is the value at which the fit curve intersects the value 1 of the Y axis for the one-component analysis. The measurement channels on the right in the diagram (secondary picture A and B, channels with a gray background) then give the specific measurement signal of the probe-aggregate complexes formed by the association.

Another possibility of determining the background signal is that the background signal is given as a Poisson distribution of the measured values of the probe or an aggregate that has only one property to be detected. This method is particularly suitable for higher aggregate concentrations, especially at concentrations> 50 fM.

In this method, the property to be detected is first determined using the single-labeled probe or the single-labeled aggregate without the addition of aggregates or probes. The width of a Poisson distribution, which optimally describes this calibration measurement, is determined on the basis of the measurement curve determined therefrom. The background signal is determined in the method according to the invention with these values for describing the Poisson distribution from the calibration measurement. All signals outside the Poisson distribution result in the specific measurement signal.

In addition, it may also be desirable for the background signal to be determined by a combination of the FIDA (Fluorescence Intensity Distribution Analysis) one-component analysis and a Poisson distribution of the measured values of the probe or the aggregate that has a property to be detected. The method according to the invention generally represents a method which makes it possible, in particular, to structure substances, in particular comprising proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments, in particular to examine synthetically produced peptides and genetically engineered proteins for contamination with aggregates.

The aggregates preferably belong to the same type of protein, protein fragments, protein analogs, in particular structures and / or protein fragments corresponding structures, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments, such as the substance.

The invention. The method is suitable for a quantitative analytical determination of the concentration of the aggregates and thus allows highly sensitive quality control for aggregate contamination, which has not been possible until now.

The method according to the invention is therefore also particularly suitable for quality control of therapeutic agents, process validation of biotechnologically and / or chemically synthesized and / or isolated native or synthetic proteins, protein fragments, protein analogs, in particular structures and peptides, peptide fragments and / or peptide analogs corresponding to proteins, particularly suitable structures corresponding to peptides and / or peptide fragments.

With the aid of the method according to the invention, it is even possible to detect "inclusion bodies" which arise when proteins are expressed in prokaryotes. Furthermore, it enables the monitoring of long-term effects during the storage of substances which contain, for example, structures corresponding to proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments, structures corresponding to peptides, peptide fragments and / or peptide analogs, in particular peptides and / or peptide fragments and an optimization of the product stability.

The principle of the method according to the invention is shown in Figure 1. The method according to the invention takes advantage of the sequence of the aggregate formation process. The process initially begins with a slow so-called “nucleating” reaction. Individual monomers, in particular proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments, have corresponding structures, peptides, peptide fragments and / or peptide analogs, in particular peptides and / or peptide fragments corresponding structures, to form small aggregates containing a few monomers, so-called nuclei (self-aggregation / "de novo" aggregation). The equilibrium is clearly on the side of the monomers. In the second step, on the other hand, in which further monomers are attached to the germ in a rapid reaction, the equilibrium lies on the side of the product. This process is called "germ-induced multimerization". Large aggregates are formed.

The speed of the second reaction is used in the determination of aggregates by means of the method according to the invention.

According to the method according to the invention, aggregates, which consist in particular of proteins, protein fragments, protein analogs, in particular structures and structures corresponding to proteins and / or protein fragments, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments, are carried out with the aid of probes that can support an aggregation process, in particular at least one probe that recognizes and binds to the aggregates. According to the invention, it can be preferred that the aggregates are detected in the liquid phase. It may therefore be desirable for a substance to be examined for aggregates to be in a liquid or to be introduced into a liquid before determining the association of the at least one probe with at least one aggregate.

The at least one probe is preferably a monomeric or oligomeric unit of proteins, protein fragments, protein analogs, in particular structures corresponding to proteins and / or protein fragments, in particular antibodies, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments.

According to the invention, the probe can have structures corresponding to the same (homologous detection) or a different type (heterologous detection) of the monomeric or oligomeric units, in particular proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments, peptides, peptide fragments and / or peptide analogs, in particular Structures of the at least one aggregate belonging to peptides and / or peptide fragments belong. So z. B. the probe can be a specific antibody, the aggregates consisting of the same (homologous detection) or another antibody (heterologous detection).

It is therefore also preferred if the probe corresponds to the same (homologous detection) or a different type (heterologous detection) of the monomeric or oligomeric units, in particular proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments, structures, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments, of the at least one substance to be examined belongs. In particular by using proteins, protein fragments, proteins and / or protein fragments that are homologous to the aggregates, in particular antibodies, peptides, peptide fragments and / or peptides and / or peptide fragments, structures as probes or the combined use with other probes, such as specific antibodies , false positive detections of aggregates and thus, for example, an inaccurate quantification, especially in biological media, can be suppressed.

However, it is desirable according to the invention that the at least one probe is heterologous to monomeric or oligomeric units of the at least one aggregate and / or the substance. An example of this is the determination of aggregates with antibodies using the antigen-antibody interactions.

A comparison of heterologous with homologous detection is shown in Figure 5 using the example of antibodies. Here the detection of the aggregates via homologous or heterologous binding shows comparable results. In general, however, the antibody batches examined have very different aggregate contents.

This ensures that the probes are present as monomers or also for the preparation of an aggregate-free solution, treatment of the at least one probe and / or the aggregates, in particular the proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments, can be carried out according to the invention, in particular antibodies, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments with physical methods such as ultrasound, exposure to temperature changes, centrifugation, chemical methods, such as treatment with solutions of different ionic strength, treatment with solutions of chaotropic ions, treatment with detergents and / or enzymes, in particular proteases. According to the invention, it is desirable if the at least one detectable property of the at least one probe and / or the at least one aggregate, either already in the at least one probe and / or the at least one aggregate, in particular in the proteins, protein fragments, protein analogs, in particular proteins and / or structures corresponding to protein fragments, in particular antibodies, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments, is contained or subsequently introduced.

Detectable properties include, for example, their size and extent. Properties such as molecular weight, structure measurable by circular dichroism, optical properties such as luminescence, in particular fluorescence or absorption, but also radioactivity can also be used to determine the association of the at least one probe and / or the at least one aggregate.

According to the invention, the detectable properties are also produced, for example, by fluorescent labels, which can be low-molecular but also high-molecular groups. For example, labeled antibodies that are bound to the aggregates can label them. The association of the probes can then be measured accordingly. Different colored markings of different probes can be made and a coincidence analysis can be carried out on them, for example by cross correlation. It is also possible to provide the probes with appropriate labels, which are either low-molecular fluorescent ligands and / or corresponding label conjugates.

It is preferred according to the invention that the at least one detectable property is specific for the at least one probe and / or the at least one aggregate. According to the invention, at least one monomeric or oligomeric probe, in a preferred form of the method according to the invention, a labeled monomeric probe which has a detectable property is therefore added to a substance to be investigated. If the substance to be examined contains aggregates (germs), the probes attach to them and large, in particular labeled, aggregates are formed.

It is desirable that at least one detectable property undergoes a change in the association of the at least one probe and the at least one aggregate.

It is also possible that the aggregates (germs) already have a label, in particular due to an intrinsically detectable property, in such a case it is desirable to use probes without detectable properties or with another detectable property.

According to the invention, the at least one detectable property and / or its change is preferably determined by means of physical methods, in particular spectroscopic methods.

Fluorimetric methods, such as confocal fluorescence spectroscopy, are preferred as possible methods for determining the at least one detectable property of the probes or aggregates.

Fluorescence Correlation Spectroscopy (FCS), FCS in combination with cross correlation, FIDA (Fluorescence intensity distribution analysis), FIMDA, FILDA, cFLA and 2D-FIDA with the associated evaluation methods.

The combination with extremely sensitive detection methods using confocal optics, for example fluorimetric methods such as FIDA (Fluorescence intensity distribution analysis), FIMDA or FILDA for example, leads to a corresponding one Amplification of the measurement signal. For example, when using the FIDA detection method, the molecules are differentiated in terms of their specific brightness. This method is also independent of molecular weight (PCT / EP97 / 05619; Kask et al. 1999 Procl. Natl. Sei. USA. 96.13756). According to the invention, for example, the differences in brightness between the small, low-luminance monomers and the large, intensely fluorescent aggregates are determined. The measurement takes place here immediately after adding the probe to the sample. This ensures that the measured aggregates were already present as germs in the test solution and were not created by the probe self-aggregating.

The detection parameters, which are determined from the detectable properties of the probes or aggregates and / or a change in the detectable properties, include translation diffusion speeds, lifetime of excited states, polarization of radiation, energy transfer, quantum efficiency, molecular brightness, number of particles or concentration, and intensity differences.

For example, become molecular. If brightnesses are determined, it is desirable in the case of slowly diffusing complexes of probes and aggregate to detect them by means of a scanning process.

However, if the association of the probe with aggregates (germs) takes a back seat to the self-aggregation of the probe, a reliable measurement is no longer guaranteed. Relatively short measuring times in the range of seconds to hours are therefore necessary. The times in which the measurements are taken naturally depend on the respective measurement conditions, but are easy to determine in preliminary tests. In particular, the concentration of the aggregate (germ) or the probe should be mentioned as parameters which can influence the measuring time. For example, if the probe concentrations are comparatively high compared to the aggregate concentration, the self-aggregation of the probes will start sooner than would be the case if the probes were at a low concentration are present or even the probe concentration is of a similar order of magnitude as the aggregate concentration or below.

In general, measuring times of well under an hour, in particular less than 30 minutes, are possible with the method according to the invention. As a rule, the measurements are even carried out within a few seconds, in particular 1 to 60 seconds, preferably 20 to 40 seconds, particularly preferably 30 seconds. This is particularly advantageous for the use of the method in routine operation, especially in quality control.

In order to increase the sensitivity of the method and to increase the analysis speed, it may generally be desirable to "scan" the sample by means of a confocal volume element moved through the measurement solution.

In addition, concentrations of aggregates (germs) of up to <0.3fM can be qualitatively detected and quantified using the method according to the invention. The method according to the invention thus has a sensitivity which is increased by up to a factor of 1000 compared to the known methods.

In addition, only small measurement volumes of less than <100 μl are required, preferably volumes of <20 μl or even <10 μl.

The aggregates can also be detected directly as intact aggregates and do not have to be broken up and split into their individual components. Complex preparations and sample preparation methods are not necessary or are avoided in order to be able to examine the solution to be analyzed in its original state (native) if possible (avoiding artifacts).

Figure 2 shows an example of the method according to the invention, the use of FIDA detection depending on the aggregate concentration. It is clearly too recognize that clear and specific signals can be obtained and quantified even in concentration ranges of less than 1 fM, even less than 0.3 fM. The smaller secondary figures A and B illustrate the quantitative determination of the background signal using FIDA (Fluorescence Intensity Distribution Analysis) one-component analysis.

With the help of this one-component analysis (Fida_Eval), the dominant background of the probe or the aggregate provided with a detectable property (secondary image A and B, channels with a black background) is determined. The cut-off limit is the value at which the fit curve for the one-component analysis intersects the value 1 of the Y axis. The measurement channels to the right of this limit in the diagrams (secondary picture A and B, channels with a gray background) then give the specific measurement signal of the probe-aggregate complexes formed by the association. It is preferred to quantify the specific measurement signal by summing the channels or by integrating them.

Figures 3 and 4 show the use of the method according to the invention for examining the quality of different batches of peptides or antibodies. It can be clearly seen that different batches from the same manufacturer or from different manufacturers have large differences in the aggregate content and thus in the actual concentration of active peptide or antibody monomer.

The method according to the invention can also be used to determine substances which prevent aggregate formation or can even dissolve aggregates which have already formed. Such substances, so-called stabilizers, can, in particular, increase the shelf life of, in particular, proteins, protein fragments, protein analogs, in particular structures and structures corresponding to proteins and / or protein fragments, in particular antibodies, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments for the stabilization of medication / therapeutics in particular important. In general, substances such as detergents, solvents, proteins, peptides, serum albumin are suitable. However, with regard to use in medication / therapeutic agents, there is the problem that these are often not usable in the human or animal organism, such as. B. DMSO or SDS or they include, such as serum albumin, the possibility of disease transmission.

It may also be desirable to find substances that accelerate the process of aggregate formation and increase the aggregate content, for example for the purification, concentration and / or separation of molecules that tend to aggregate, in particular proteins, protein fragments, protein analogs, in particular structures corresponding to proteins and / or protein fragments , in particular antibodies, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments.

If, for example, molecules which are only present in a low concentration in a solution are to be concentrated, at least one such inductor, for example a metal ion, can be added according to the invention which brings about an aggregation of these molecules. The aggregates can now be separated from the solution by conventional physical and / or chemical separation methods, for example centrifugation and / or HPLC, and can then be dissolved again in a smaller volume. To completely solve these aggregates, it may be desirable to use physical methods such as ultrasound, exposure to temperature changes and / or chemical methods.

The identification and optimization of substances which can influence aggregate formation, in particular inductors and / or stabilizers, can be carried out according to the invention as follows:

Providing at least one aggregate-free solution and / or a solution with a known aggregate content, in particular containing proteins, Protein fragments, protein analogs, in particular proteins and / or

Structures corresponding to protein fragments, peptides, peptide fragments and / or peptide analogs, in particular structures corresponding to peptides and / or peptide fragments,

Introducing at least one that potentially influences aggregate formation

Substance,

Mixing the at least one solution,

Determination of the aggregate content with the help of the invention

Method for the quantitative analytical determination of the content of

Aggregates of substances in mixtures,

Determination of the properties of the at least one substance that influence aggregate formation by comparing the aggregate content thereof

Solution with the at least one reference sample.

It may be preferred according to the invention to carry out an incubation of the solution after the solution has been mixed. The incubation conditions, in particular incubation time and temperature, can be adapted to the test conditions.

To produce an aggregate-free solution, it may be desirable according to the invention to expose the solution to physical methods such as ultrasound, exposure to temperature changes, centrifugation and / or chemical methods, or to substances which dissolve aggregates, in particular detergents, enzymes, in particular proteases and / or solutions of different ionic strength and / or chaotropic ions.

The reference sample is preferably an aggregate-free solution and / or solution with a known aggregate content of the same protein, protein fragments, protein analogs, in particular protein and / or protein fragments with a corresponding structure, peptides, peptide fragments and / or peptide analogs, in particular peptide and / or peptide fragments Structure, before adding the at least one aggregate-influencing substance.

It is also desirable that the reference sample be incubated. Preferably, the incubation ^conditions' (time, temperature) are identical with those of the reference sample of the solution.

According to the invention, when a substance which reduces the formation of aggregates, so-called stabilizers, is added, the aggregate content of the solution is significantly lower than that of the reference sample.

According to the invention, the term “aggregate-reducing substance” also includes substances that completely inhibit aggregate formation.

After addition, substances that can dissolve aggregates cause a significant decrease in the aggregate content of the solution compared to the reference sample.

These substances that dissolve aggregates are also distinguished by the fact that, after addition, they bring about a significant reduction in the aggregate content of the solution compared to a reference sample that contains a substance that reduces the formation of aggregates.

The addition of a substance that accelerates the formation of aggregates, so-called inductors, causes a significant increase in the aggregate content of the solution compared to the reference sample.

In general, it may be desirable to test different concentrations of the potential substance that influences the formation of aggregates in a series of experiments. According to the invention, it can be at least. a substance which influences the formation of aggregates, in particular a detergent, a chaotropic substance, salt, lipid, lipid derivative, nucleic acid, nucleic acid derivative, carbohydrate or its derivative, solvent, protein, protein fragment, protein analog, in particular structure and peptide corresponding to protein and / or protein fragment, Act peptide fragment and / or peptide analog, in particular peptide and / or peptide fragment corresponding structure.

The process according to the invention is explained in more detail by the following examples:

example 1

Determination of the specific measurement signal depending on the aggregate concentration using the example of an Aß aggregate concentration series.

material

DMSO (Sigma, order no.D-8779, lot no.38H3447) Na phosphate buffer (0.02M; pH7.0) => (sol. Ϊ)

Aß1-42 peptide (delivery 13.09.1999 with Fmoc protective group, # 2301, synthesized by Margit Ellis, AG Biopolymers, Center for Molecular Biology, University of Heidelberg) fluorescence-labeled with EVOblue ³⁰ (KS-01-06, 09.05.2000, Evotec OAI) in 0.02M Na phosphate buffer pH 7.0 with 0.2% SDS => (solution 2) final concentration peptide 50 nM synthetic Aß aggregates

Implementation / measurement approach

The synthetic Aß aggregates are diluted in solution 1 so that eight solutions with aggregate concentrations between 3.2 fM and 400 fM are formed. For the individual measurement batches, 18 μl solution 1 are mixed with 1 μl one of the aggregate concentrations and 1 μl solution 2. The measurement batch is mixed well, applied to a chamber of the sample holder and measured directly using FIDA. 10 measurements of 30 seconds each are carried out.

Measurement conditions:

Measuring instrument: The solutions were measured using an INSIGHT fluorescence correlation spectrometer from Evotec Technologies GmbH, equipped with a detection facility for FIDA. The excitation depends on the dye used and was carried out depending on the dye used. For EVOblue ³⁰ , the excitation took place at a wavelength of 633 nm.

The fluorescent light was collected through a microscope objective, each solution to be measured was scanned with a frequency of 20-25 Hz and a scanning distance of 150 μm and was recorded convocally on a photon counter, which delivers a signal pulse for each photon detected. For each solution to be measured, 10 measurements of 30 s duration were carried out and averaged. The signal pulses were recorded with a channel width of 40 μs. This resulted in 750,000 measurement channels per measurement,

Data analysis:

Determination of the background signal

Depending on the aggregate concentration, the background signal was determined using one of the methods described below:

At low aggregate concentrations, the background signal was quantified using a FIDA (Fluorescence Intensity Distribution Analysis) one-component analysis. An example of this method is shown in Figure 2. With the help of this one-component analysis (Fida_Eval), the dominant background of the probe or the aggregate provided with a detectable property (secondary image A and B, channels with a black background) is determined. The cut-off limit is the value at which the fit curve intersects the value 1 of the Y axis for the one-component analysis. The measurement channels on the right in the diagram (secondary picture A and B, channels with a gray background) then give the specific measurement signal of the probe-aggregate complexes formed by the association.

At high aggregate concentrations, another method for determining the background signal is more suitable. The background signal is here as Poisson distribution of the measured values of the probe or an aggregate having only one property to be detected is given.

The specific measurement signal can be quantified by summing the channels or integrating them.

Result:

Figure 2 shows the specific measurement signal as a function of the aggregate concentration. It can be clearly seen that even in concentration ranges of less than 1 fM, even less than 0.3 fM, clear and specific signals are obtained and closed. are quantify.

Example 2:

Quality control of various batches of Aß peptides

Different batches of peptides are tested for the presence of aggregates.

Material:

Different batches of Aß peptides from different manufacturers:

Aß1-42 from Bachern, Lot No. 516817

Aß1-42 from Calbiochem, Lot No. D04765-4

Aß1-42 Delivery from 13.09.1999 with Fmoc protective group, # 2301, synthesized by Margit Ellis, AG Biopolymers, Center for Molecular Biology,

University of Heidelberg,

Aliquot-wise removal of the protective group, investigation of three different aliquots Aß1-42 delivery from August 18, 1999 with Fmoc-Hec1 protective group, synthesized by Dr. Peter Henklein, Faculty of Medicine (Charite), Institute of Biochemistry d. Humboldt University Berlin

Aß18-42 + 0.5 nmol nicotine, SD-06.08 from 6.7.1999 synthesized by Evotec OAi, Labeling Chemistry

DMSO, Sigma, Order No. D-8779, Lot No. 38H3447

Na phosphate buffer (0.02M; pH 7.0) => (solution 1)

Aß1-42 peptide (delivery 13.09.1999 with Fmoc protective group, # 2301, synthesized by Margit Ellis, AG Biopolymers, Center for Molecular Biology, University of Heidelberg) fluorescence-labeled with EVOblue ³⁰ (KS-01-06, 09.05.2000, Evotec OAI) in 0.02M Na phosphate buffer pH 7.0 with 0.2% SDS => (solution 2) final concentration peptide 50 nM

Execution:

The peptides tested here are highly hydrophobic and therefore poorly soluble in buffer. For this reason, they are first solved in DMSO. The peptides to be tested are each dissolved in 1mM in DMSO. Based on this, the following dilution series results:

^■ »1: 1 in 50% DMSO (=> 500μM)

^■ »1: 50 in solution 1 (=> 10μM)

the mixture for measurement:

19μl peptide solution (10μM) + 1 μl solution 2

The measurement batch is mixed well, applied to a chamber of the sample holder and measured using FIDA, taking into account the background signal, as described in Example 1. 10 measurements of 30 seconds each are carried out.

As a control, 1 μl solution 2 is measured in 19 μl solution 1.

Result:

Figure 3 shows the results of the test series graphically. There is a clear distinction between (a) batches free of aggregates (good quality) and (b) batches containing aggregates (poor quality). While in good batches only the low fluorescent digits of the monomers can be seen, the _^ bad batches are characterized by the presence of large fluorescent aggregates. The latter is evident in the significantly increased FIDA signal intensity.

Example 3:

Quality control of different antibody production batches

Antibodies of various types and batches are tested for the presence of aggregates.

Materials:

Na phosphate buffer (0.02M; pH 7.0) => (solution 1)

Antibody batches from the following manufacturers:

Anti-Aß 6E10 monoclonal antibody, lgG1, product number: 320-02, lot

No .: 306, Signet Pathology

Anti-alpha-synuclein (human, monoclonal) 15G7, Council IgG2a, product

No .: 804-258-L001, batch no. L05992, Alexis Biochemicals

Anti-alpha-synuclein (rabbit, polyclonal), catalog no .: 44-444, lot no .:

0101, Biosource International

Anti-CystatinC antibody (rabbit anti-human), code no .: A 0451, lot

No .: 028, DAKO

Dilution of the individual antibodies in solution 1, so that an antibody concentration of 500 μg / ml results.

with EVOblue ³⁰ (KS-01-06, dated 09.05.2000, Evotec OAI) fluorescence-labeled antibodies of the batch to be tested in 0.02M Na phosphate buffer pH 7.0 with 0.2% SDS => (solution 3) final concentration antibody 50 nM (A part of the batch of antibodies to be tested is fluorescence-labeled and then serves as a detection probe.)

Execution:

Measurement batch: 18 μl solution 1 + 1 μl antibody + 1 μl solution 3 The measurement batch is mixed well, applied to a chamber of the sample holder and measured using FIDA, taking into account the background signal, as described in Example 1. 10 measurements of 30 seconds each are carried out.

As a control, 1 μl solution 3 is measured in 19 μl solution 1.

Result:

Figure 4 shows the results of the test series. Two different groups of antibody batches can be seen. One batch with good quality (no aggregates) is characterized by low FIDA signal intensities. In poor quality batches, aggregate contamination leads to a significant increase in FIDA signal intensity.

Example 4:

Heterologous and homologous detection of antibody aggregates

With the help of a fluorescence-labeled antibody probe, both aggregates of the same batch (homologous binding) and aggregates of different antibody batches (heterologous binding) can be detected.

Three different batches of antibodies are measured with two different fluorescence-labeled antibody probes.

Materials:

Na phosphate buffer (0.02M; pH 7.0) => (solution 1)

Antibody batches from the manufacturers:

Anti-alpha-synuclein, rabbit PAb, polyclonal, cat. No. 44-444, lot no. 0101, from Biosource International, 0.1mg / 0.2ml Anti-ß-amyloid42, rabbit PAb, cat.no.44-344, lot no.44344132, Biosource International (0.391 mg / ml)

Anti-rabbit IgG (whole rholecule), develöped in goat, cat.no.R-2004, lot no.79H9240, Sigma

with EVOblue ³⁰ (KS-01-06, dated 09.05.2000, Evotec OAI) fluorescence-labeled antibodies (anti-alpha-synuclein, rabbit PAb, polyclonal, cat. no. 44-444, lot no. 0101, Biosource International, 0.1mg / 0.2ml) 1: 5 in 0.02M Na phosphate buffer pH 7.0 diluted with 0.2% SDS => (solution 4).

with EVOblue ³⁰ (KS-01-06, dated 09.05.2000, Evotec OAI) fluorescence-labeled antibodies (Anti-rabbit IgG (whole molecule), develöped in goat, cat.no.R- 2004, lot no.79H9240, Sigma ) 1: 100 diluted in 0.02M Na phosphate buffer pH 7.0 ^J with 0.2% SDS => (solution 5).

Execution:

the mixture for measurement:

13 μl solution 1 + 2 μl 8% SDS + 4 μl antibody + 1 μ | Sol. 4 or Sol. 5

The following is measured as a control:

17μl solution 1 + 2μl 8% SDS + 1 μl solution 4 or solution 5 Result:

Figure 5 shows the results of the measurements. The three antibody batches examined are characterized by very different aggregate contents. The detection of the aggregates via homologous or heterologous binding shows comparable results.

Homologous binding means that part of an antibody batch is fluorescence-labeled and serves as a probe for the detection of the aggregates in the remaining batch. Batches A and C (gray bars) were examined in this way. Batch A has many aggregates, while Batch C is almost aggregate-free.

In heterologous binding, an antibody is fluorescently labeled and serves as a probe for a wide variety of antibody batches. Batches A and B were detected with fluorescence-labeled antibody C and batches B and C with fluorescence-labeled antibody A.

The numerous aggregates in batch A can also be demonstrated by heterologous binding. Batch C has virtually no aggregates even with heterologous detection. Batch B, which was examined with two different heterologous binding probes, shows a low aggregate content in both cases.

Claims

claims

1. Method for the quantitative analytical determination of the content of aggregates of substances in mixtures with the following steps:

2. The method according to claim 1, characterized in that the background signal is determined with the aid of a FIDA (Fluorescence Intensity Distribution Analysis) one-component analysis of the probe or an aggregate having only one property to be detected.

3. The method according to claim 1, characterized in that the background signal is given as a Poisson distribution of the measured values of the probe or an aggregate having only one property to be detected.

4. The method according to claim 1, characterized in that the background signal is determined by a combination of the FIDA (Fluorescence Intensity Distribution Analysis) one-component analysis and a Poisson distribution of the measured values of the probe or a unit having a property to be detected.

5. The method according to at least one of claims 1 to 4, characterized in that the substances are proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments corresponding structures, peptides, peptide fragments and / or peptide analogs, in particular peptides and / or Structures corresponding to peptide fragments.

6. The method according to at least one of claims 1 to 5, characterized in that the at least one aggregate of proteins, protein fragments, protein analogs, in particular proteins and / or protein fragments corresponding structures, peptides, peptide fragments and / or peptide analogs, in particular peptides and / or peptide fragments corresponding structures.

7. The method according to at least one of claims 1 to 6, characterized in that the aggregates are detected in the liquid phase.

8. The method according to at least one of claims 1 to 7, characterized in that the at least one probe is homologous to monomeric or oligomeric units of the at least one aggregate.

9. The method according to at least one of claims 1 to 8, characterized in that the at least one probe is heterologous to monomeric or oligomeric units of the at least one aggregate.

10. The method according to at least one of claims 1 to 9, characterized in that the at least one detectable property is either already contained in the at least one probe and / or the at least one unit or is introduced subsequently.

11. The method according to at least one of claims 1 to 10, characterized in that the at least one detectable property is specific for the at least one probe and / or the at least one unit.

12. The method according to at least one of claims 1 to 11, characterized in that the at least one detectable property is size, extension, molecular weight, structure, circular dichroism, optical properties such as luminescence, in particular fluorescence, absorption, radioactivity.

13. The method according to at least one of claims 1 to 12, characterized in that the physical methods are spectroscopic methods.

14. The method according to at least one of claims 1 to 13, characterized in that the determination of the at least one detectable property and / or its change by means of fluorometric methods, such as confocal fluorescence spectroscopy, fluorescence correlation spectroscopy (FCS), FCS in combination with cross-correlation , Fluorescence Intensity Distribution Analysis (FIDA), FIMDA, FILDA, cFLA, 2D-FIDA.

^"15. A method according to any one of claims 1 to 14 characterized in that be determined from the detectable characteristics parameters such as translational diffusion rates, rotational diffusion rates, lifetimes of excited states, polarization of radiation, energy transfer, quantum yield, molecular brightnesses, particle number or concentration, intensity differences.

16. A method for identifying and optimizing substances that can influence aggregate formation, with the following steps:

Provision of at least one aggregate-free solution and / or a solution with a known aggregate content,

Introduction of at least one substance which potentially influences aggregate formation,

Mixing the at least one solution,

Determination of the aggregate content using the method according to at least one of claims 1 to 15,

Determination of the properties of the at least one substance that influence aggregate formation by comparing the aggregate content of this solution with the at least one reference sample.

17. The method according to claim 16, characterized in that it is appropriate at ^'the aggregate proteins, protein fragments, protein analogues, in particular proteins and / or protein fragments corresponding structures, peptides, peptide fragments and / or peptide analogues, in particular peptides and / or peptide fragments structures, These

18. The method according to claim 16 and / or 17, characterized in that the method is characterized in that an incubation of the solution is carried out after the mixing.

19. The method according to at least one of claims 16 to 18, characterized in that the reference sample is preferably an aggregate-free solution and / or solution with a known aggregate content of the same protein, protein fragments, protein analogs, in particular protein and / or protein fragment corresponding structure, Peptide, peptide fragments and / or peptide analogs, in particular structure corresponding to peptide and / or peptide fragment, acts before adding the substance influencing at least one aggregate formation.

20. The method according to claim 19, characterized in that the reference sample is also incubated.

21. The method according to at least one of claims 16 to 20, characterized in that the method is characterized in that a substance which reduces aggregate formation causes a significantly lower aggregate content of the solution compared to the reference sample.

22. The method according to at least one of claims 16 to 21, characterized in that the method is characterized in that an aggregate accelerating substance causes a significant increase in the aggregate content compared to the reference sample.

23. The method according to at least one of claims 16 to 22, characterized in that the method is characterized in that an aggregate-dissolving substance has a significant decrease in the aggregate content of the solution compared to the reference sample and / or a reference sample which contains an aggregate-reducing substance , causes.

24. The method according to at least one of claims 16 to 23, characterized in that the substance influencing at least one aggregate formation is a detergent, a chaotropic substance, salt, Lipid, lipid derivative, nucleic acid, nucleic acid derivative, carbohydrate or its derivative, solvent, protein, protein fragment, protein analog, in particular protein and / or protein fragment corresponding structure, peptide, peptide fragment and / or peptide analog, in particular peptide and / or peptide fragment corresponding structure.