EP3639030A1 - Procédé de détection d'agrégats de substances biothérapeutiques dans un échantillon - Google Patents

Procédé de détection d'agrégats de substances biothérapeutiques dans un échantillon

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Publication number
EP3639030A1
EP3639030A1 EP18731354.9A EP18731354A EP3639030A1 EP 3639030 A1 EP3639030 A1 EP 3639030A1 EP 18731354 A EP18731354 A EP 18731354A EP 3639030 A1 EP3639030 A1 EP 3639030A1
Authority
EP
European Patent Office
Prior art keywords
dad
aggregates
rch
biotherapeutic
substrate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18731354.9A
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German (de)
English (en)
Inventor
Christian ZAFIU
Andreas Kulawik
Olivier BANNACH
Dieter Willbold
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Original Assignee
Forschungszentrum Juelich GmbH
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Filing date
Publication date
Application filed by Forschungszentrum Juelich GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP3639030A1 publication Critical patent/EP3639030A1/fr
Pending legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence

Definitions

  • the invention relates to a method for detecting aggregates of biotherapeutic substances in a sample.
  • Biopharmaceuticals are used in biologics and their counterfeit biosimilars and are the category of therapeutics that are produced in living organisms. These products include, among others, recombinant proteins and antibodies. These products play a key role in the treatment of various diseases such as diabetes, various cancers and inflammatory diseases.
  • biopharmaceuticals are highly attractive therapeutics because proteins and antibodies have outstanding activities and specificities in terms of their effects.
  • the greatest challenges are physical and chemical stability. Misfolding of such proteins, and subsequently, aggregation, may occur during each and every phase of the product cycle of such a therapeutic agent. These stages include expression, folding, purification, sterilization, shipping, storage and delivery of the product.
  • Homogeneous protein aggregates are defined as any self-associated protein species, where the monomer is the smallest naturally occurring unit or non-self-assembled protein species. represents unit. Aggregates are classified according to the following five characteristics: size, reversibility (dissociation), conformation, chemical modification, and morphology [1].
  • the smallest aggregate unit corresponds to two monomers (dimer), whereby the number of monomers is not limited to the upper limit [1]. Disadvantageously, immune responses have been found even for the smallest aggregate units of biopharmaceutical protein products [2].
  • heterogeneous aggregates in which the protein can associate monomer with one or more other protein species or even organic or inorganic contaminants.
  • the underlying mechanisms that can cause or enhance an immune response by aggregates are different. These include a) increased cross-linking of B-cell receptors, which may cause their activation [3], b) increased antigen uptake, processing and presentation with subsequent triggering of immunostimulatory signals [4]. Such mechanisms can stimulate T cells to produce antibodies.
  • the greatest clinical risk of aggregate-induced immune response depends on the maintenance or degeneration of monomer epitopes in the aggregate. In the case of preservation of the epitopes, antibodies which were originally directed only against the aggregate can also bind monomers and reduce or neutralize their action. In the case of degeneration, the antibodies will target exclusively aggregates while not interfering with the drug activity of the native protein.
  • LO light attenuation
  • DIPA Dynamic Imaging Particle Analysis
  • MFI micro-flow imaging
  • CC electrochemical method of the Coulter counter
  • Aggregates between 0.1 pm and 1 ⁇ In this size range complex systems for the detection of small aggregates are used. These include size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and asymmetric field-flow fractionation (AF4). To increase the sensitivity of such devices, they are often coupled to a mass spectrometer. Although these techniques allow the quantification and distribution of aggregates. It is disadvantageous to determine the composition and these processes are only of limited suitability for high-throughput applications.
  • the object of the invention is to provide a highly sensitive method for detecting aggregates of biotherapeutic substances in a sample.
  • Another object of the invention is to provide a kit for carrying out the method.
  • the object is achieved by a method for detecting aggregates of biotherapeutic substances in a sample, comprising the following steps: a. Applying the sample to be examined to a substrate, b. Add probes suitable for detection by
  • step b) Detection of the labeled aggregates of biotherapeutic substances wherein step b) can be carried out before step a).
  • the method for the detection and in particular for the quantitative and / or qualitative determination of homogeneous and heterogeneous aggregates is characterized in that aggregates of and in biopharmaceutical products contain at least one binding site for a probe.
  • the aggregate also comprises at least one binding site for a capture molecule.
  • the method then comprises the steps of: a) immobilizing catcher molecules on a substrate, b) contacting the sample with a biopharmaceutical product
  • Solution with the catcher molecules c) immobilizing monomers and / or aggregates of one or more of themselves d) contacting a probe with the monomers and / or aggregates; e) attaching the probe to the monomers and / or aggregates, wherein the probe is capable of binding to the capture molecules is to generate a defined signal and the steps b) and d) can take place simultaneously or the step d) before the step b) can take place.
  • steps b) and d) take place simultaneously, then steps c) and e) are also carried out simultaneously.
  • step d) is carried out before step b
  • immobilization of monomers and aggregates labeled with probes on the substrate thus takes place in step c). Consequently, step e) also takes place before steps b) and c).
  • the "quantitative determination” initially means the determination of the concentration of the aggregates, and therefore also the determination of their presence and / or absence.
  • the quantitative determination also means the selective quantification of aggregate compositions. Such quantification can be done via the corresponding probes.
  • the "qualitative determination” means the characterization of the aggregate composition.
  • the aggregates are labeled with one or more probes useful for detection.
  • the probes contain an affinity molecule which recognizes and binds to a binding site of the aggregate or its monomer.
  • the probes contain at least one detection molecule or moiety which is bound to the affine molecule or moiety and is detectable or measurable by chemical or physical methods.
  • the probes may in one alternative have identical affine molecules or moieties with different detection molecules (or parts).
  • different affine molecules or parts of molecules may be combined with different detection molecules or parts, or alternatively different affine molecules or parts with identical detection molecules or parts. It is also possible to use mixtures of different probes.
  • a spatially resolved determination of the probe signal ie a spatially resolved detection of the signal emitted by the probe. Accordingly, in this embodiment of the invention, methods based on a non-spatially resolved signal, such as ELISA or sandwich ELISA, are excluded.
  • the spatially resolved determination of the probe signal is based on the examination of a small volume element in the region of Femtolitern below a Femtoliters, or a volume range above the contact surface of the capture molecules with a height of 500 nm, preferably 300 nm, more preferably 250 nm, in particular 200 nm, but also 150 nm and 90 nm.
  • aggregates are either homogeneous aggregates consisting of at least two identical monomer units, or heterogeneous aggregates consisting of at least two different monomer units. In the case of heterogeneous aggregates, both monomers can be identical in their primary sequence, but differ in their conformation.
  • the material of the substrate is selected from the group consisting of or consisting of plastic, silicon and silicon dioxide.
  • glass is used as the substrate.
  • the capture molecules are covalently bound to the substrate.
  • a substrate which has a hydrophilic surface.
  • this is achieved by applying a hydrophilic layer, before step a), to the substrate.
  • the capture molecules covalently bind to the substrate or to the hydrophilic layer with which the substrate is loaded.
  • the hydrophilic layer is a biomolecule-repellent layer so as to minimize nonspecific binding of biomolecules to the substrate.
  • the capture molecules preferably covalently, are optionally immobilized on this layer. These are affinitive to a feature of the monomers or their aggregates.
  • the scavenger molecules may all be identical, or mixtures of different scavenger molecules.
  • the same molecules are used as catcher molecules and probes, preferably the capture molecules do not contain a detection molecule or parts of the molecule.
  • the hydrophilic layer is selected from the group consisting of or consisting of PEG, poly-lysine, preferably poly-D-lysine, and dextran or derivatives thereof, preferably carboxymethyl-dextran (CMD).
  • CMD carboxymethyl-dextran
  • Derivatives in the context of the invention are compounds which differ in some substituents from the parent compounds, the substituents being inert to the process according to the invention.
  • the surface of the substrate is first hydroxylated before application of the hydrophilic layer and then functionalized with suitable chemical groups, preferably amino groups. This functionalization with amino groups takes place in an alternative by contacting the substrate with amino silanes, preferably APTES (3-aminopropyltrietoxysilane), or with ethanolamine.
  • the substrate In an alternative, bringing the substrate into contact with aminosilanes, preferably APTES, in the gas phase; the optionally pretreated substrate is thus vaporized with the aminosilanes.
  • aminosilanes preferably APTES
  • the substrate is treated with an aqueous solution of CMD (in a concentration of 10 mg / ml or 20 mg / ml) and with a catalyst for the covalent coupling, optionally N- Ethyl N- (3-dimethylaminopropyl) carbodiimide (EDC), (200mM) and N-hydroxysuccinimide (NHS), (50mM) were incubated, followed by washing.
  • EDC Ethyl N- (3-dimethylaminopropyl) carbodiimide
  • NHS N-hydroxysuccinimide
  • the substrate used may be microtiter plates, preferably with a glass bottom. Because when using polystyrene frames the use of concentrated
  • Covalent, preferably covalent, catcher molecules are immobilized on this hydrophilic layer which are affine towards a characteristic (eg proteins) of the aggregate.
  • the capture molecules may all be identical or mixtures of different capture molecules.
  • the capture molecules preferably antibodies to monomers of the aggregate, optionally after activation of the CMD-coated support by a mixture of EDC / NHS, preferably 200 or 50 mM, are immobilized on the substrate.
  • the substrates or carriers are optionally rinsed with buffer.
  • the substrate is blocked with protein or peptide containing solutions prior to application of the sample and washed with buffer.
  • the sample to be measured is brought into contact with the substrate thus prepared and optionally incubated.
  • Differently formulated solutions of the biopharmaceutical product, of the product in cell supernatants and culture media or endogenous fluids can be used as the sample to be investigated.
  • the sample is selected from CSF, blood, plasma and urine.
  • the samples may undergo different processing steps known to those skilled in the art.
  • the sample is applied directly to the substrate (uncoated substrate), optionally by covalent bonding on the optionally activated surface of the substrate.
  • the sample is pretreated by one or more of the following methods:
  • enzymes for example nuclease, lipases
  • the sample is brought into contact with the substrate immediately and / or without pretreatment.
  • Unspecific bound substances can be removed by washing steps.
  • immobilized aggregates are labeled with one or more probes useful for further detection.
  • the individual steps can also be carried out in a different order according to the invention.
  • Suitable washing steps remove excess probes that are not bound to the aggregates.
  • these excess probes are not removed. This eliminates the last washing steps and there is no equilibrium shift in the direction of the dissociation of the aggregate-probe complexes or Connections take place. Due to the spatially resolved detection, the excess probes are not detected during the evaluation and do not affect the measurement.
  • sample-catcher molecule complexes are chemically fixed.
  • detection probe-probe-capture molecule complexes are chemically fixed and thus also the sample-capture molecule complexes.
  • the binding sites of the aggregate are epitopes and the capture molecules and probes are antibodies.
  • capture molecules and probes may be identical.
  • capture molecules and probes differ. So z. B. different antibodies are used as catcher molecules and as probes.
  • capture molecules and probes are used, which are identical to each other with the exception of the eventual (dye) label.
  • various probes are used which are identical to each other except for the eventual (dye) label.
  • At least two or more different capture molecules and / or probes are used which contain different antibodies and optionally also have different dye labeling.
  • two or more probes are labeled with appropriate dyes such that a FRET, a so-called resonant energy transfer promoter, takes place, one dye being excited and another being emitted nearby, both dyes being different molecules is.
  • the probes are characterized in that they emit an optically detectable signal selected from the group consisting of fluorescence, Phosphorescence, bioluminescence, chemiluminescence and electrochemiluminescence emission, and absorption.
  • the probes are thus labeled with fluorescent dyes.
  • fluorescent dye the dyes known to those skilled in the art can be used.
  • fluorescent biomolecules preferably GFP (green fluorescence protein), conjugates and / or fusion proteins thereof, and also fluorescent nanoparticles, preferably quantum dots.
  • catcher molecules for later quality control of the surface, for example uniformity of the coating with catcher molecules, catcher molecules, labeled with fluorescent dyes, can be used.
  • a dye is preferably used which does not interfere with the detection.
  • the immobilized and labeled aggregates are detected by imaging the surface (eg laser microscopy).
  • the highest possible spatial resolution determines a high number of pixels, whereby the sensitivity and the selectivity of the assay can be increased because structural features can be mapped and analyzed.
  • the specific signal increases in front of the background signal (eg nonspecifically bound probes).
  • the detection is preferably carried out with confocal fluorescence microscopy, fluorescence correlation spectroscopy (FCS), in particular in combination with cross-correlation and laser scanning microscope (LSM).
  • FCS fluorescence correlation spectroscopy
  • LSM laser scanning microscope
  • the detection or detection is carried out in an alternative of the present invention with a confocal laser scanning microscope.
  • a laser focus as z.
  • FCS Fluorescence Correlation Spectroscopy System
  • the detection or the detection by means of spatially resolving fluorescence microscopy, preferably by a TIRF microscope done so like the corresponding super-resolution variants of it, such as STORM, dSTORM.
  • the probes can be selected such that in the case of heterogeneous agregates, the presence of individual components or conformations does not affect the measurement result.
  • the probes can be selected such that homogeneous and heterogeneous aggregates and different heterogeneous aggregates can be determined in one measurement.
  • the spatially resolved information (eg, the fluorescence intensity) of all inserted and detected probes is used to determine e.g. For example, determine the number of aggregates, their size and their characteristics.
  • Other image analysis options include z. For example, the search for local intensity maxima in order to obtain the number of detected aggregates from the image information.
  • standards such as: B. internal and / or external standards are used. These standards may also be used to calibrate the measurement to determine the size distribution, quantity and / or composition of aggregates of biopharmaceutical products.
  • Another object of the present invention is to provide standards which have a narrow size distribution and consist of two or more identical or different polypeptide sequences.
  • the polypeptide sequences may also be the native monomeric form of the biopharmaceutical agent.
  • the standard consists of a mixture of different size distributions.
  • the standard is marked.
  • the standard consists of two or more monomers covalently linked together.
  • the standard consists of a nanoparticle on the surface of which two or more monomers or a polypeptide sequence are covalently bound and the polypeptide sequence is identical in a partial region with the sequence of the monomer of the biotherapeutic substances.
  • the present invention also provides a kit containing one or more of the following components:
  • Substrate if appropriate with hydrophilic surface, catcher molecule, probe, standard, substrate with catcher molecule, solutions, buffer.
  • the compounds and / or components of the kit of the present invention may be packaged in containers, optionally with / in buffers and / or solution. Alternatively, some components may be packaged in the same container. In addition or alternatively, one or more of the components could be attached to a solid support, such as a solid support. As a glass plate, a chip or a nylon membrane or to the well of a microtiter plate, be adsorbed. Further, the kit may include instructions for using the kit for any of the embodiments.
  • the above-described catcher molecules are immobilized on the substrate.
  • the kit may contain solutions and / or buffers.
  • the biomolecule-repellent surface eg dextran surface
  • the catcher molecules immobilized thereon, they can be covered with a solution or a buffer.
  • the solution contains one or more biocides which increase the durability of the surface.
  • Another object of the present invention is the use of the method according to the invention for the detection of homogeneous and heterogeneous aggregates. and of biopharmaceutical products in any sample, for the quantification (titer determination) of homogeneous and heterogeneous aggregates of and in biopharmaceutical products, direct and / or absolute quantification of the number of particles.
  • Another object of the present invention is the use of the method according to the invention for the optimization and monitoring of process steps during the production of biopharmaceutical active ingredients and / or quality determination of end products.
  • Another object of the present invention is the use of the inventive method for the detection of homogeneous and heterogeneous aggregates of biopharmaceutical products in clinical tests, studies and in therapy monitoring.
  • samples are measured according to the method according to the invention and the results compared.
  • Fig. 1 aggregates (hatched bars) and monomers (white bars) of the human IgG antibody as a sample.
  • FIG. 1 shows aggregates (shaded bars) and monomers (white bars) of the human IgG antibody (isotype control, ThermoFisher Scietific, RF237824) in a decadal dilution series.
  • the antibody as it was used was used and diluted in saline phosphate buffer (PBS) at pH 7.4.
  • PBS saline phosphate buffer
  • the sample (5 mg / ml) was heated for 10 min at 70 ° C and diluted after reaching 25 ° C decadent.
  • the surface of the microtiter plate was constructed or functionalized.
  • the plate was placed in a desiccator, in which a bowl with
  • reaction chamber was washed three times with water followed by 20 ⁇ each of an aqueous 200 mM EDC solution (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, Sigma) and 50 mM NHS (N-hydroxysuccinimide, Sigma) for 30 minutes. This gives the hydrophilic coating with PEG functionality for coupling biomolecules.
  • the plate was again washed three times with deionized water. Thereafter, the reaction chamber was coated with clone 8A4 (Thermo-Fischer), a monoclonal antibody as a capture molecule that specifically binds the CH2 domain in the FC portion of human antibodies (20 ⁇ , 10 pg / ml in PBS, 1 hour). Subsequently, the reaction chamber was treated with the wash program consisting of three washes each time and empty eyes with TBS with 0.1% Tween-20 and TBS.
  • clone 8A4 Thermo-Fischer
  • reaction chamber was coated overnight with 50 ⁇ M Smartblock (Candor Bioscience GmbH) at room temperature (RT) and, after the lapse of time, again three times with saline tris (hydroxymethyl) aminomethane (TBS;
  • the samples were sequentially diluted in Tris (hydroxymethyl) aminomethane (TRIS) buffer with Hoechst Stain dye (1 pg ml-1) and incubated for one hour. Thereafter, in a threefold version, 20 ⁇ l of sample were applied to the reaction chamber and incubated at room temperature for 1 hour. After incubation, the reaction chamber was washed three times with TBS and 20 ⁇ detection antibody was added.
  • the detection antibodies were each labeled with a type of fluorescent dye: 8A4 (ThermoFisher Scientific, MA1-81864,) was separately labeled with fluorescent dyes CF488 and with CF633. These probe antibodies or detection antibodies were diluted together in TBS to a final concentration of 1.25 ng / ml for each antibody. They specifically bind epitopes of the monomers and aggregates of the biotherapeutic substance.
  • the maximum laser power (100%), an exposure time of 500 ms and a gain value of 800 were selected.
  • the image data was evaluated afterwards.
  • Intensity thresholds were set for each channel at 0.0001% gray levels of the averaged negative control in the corresponding channel.
  • the intensity threshold value was first applied for each image in each channel, and images of the same position in both values were compared with one another. Only those pixels per image were counted where in both channels the pixel is at the exact same position above the intensity threshold of the channel. Finally, the number of
  • This series of calibrations can then serve as an introduction to more complex detection methods of biotherapeutic substances, in the specific case of IgG, which can be present as a biotherapeutic substance in solution of a pharmaceutical preparation as a sample and should be examined for aggregates.
  • the fluorescent probe antibody can not bind to a capture molecule bound monomer because its binding site is occupied by the capture molecule. Instead, it only binds to the monomer epitopes of the aggregates. This is thus quantifiable in the manner shown.

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Abstract

L'invention concerne un procédé de détection d'agrégats de substances biothérapeutiques, comprenant les étapes consistant à : a. appliquer l'échantillon à analyser sur un substrat, b. ajouter des sondes qui sont qualifiées pour la détection et qui marquent les agrégats de substances biothérapeutiques par liaison spécifiques à des derniers et c. détecter les agrégats, marqués, des substances biothérapeutiques, l'étape a) étant exécutée avant l'étape b). L'invention concerne également un kit permettant la mise en oeuvre du procédé.
EP18731354.9A 2017-06-13 2018-05-15 Procédé de détection d'agrégats de substances biothérapeutiques dans un échantillon Pending EP3639030A1 (fr)

Applications Claiming Priority (3)

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DE102017005544 2017-06-13
DE102017010455.7A DE102017010455A1 (de) 2017-06-13 2017-11-13 Verfahren zum Nachweis von Aggregaten biotherapeutischer Substanzen in einer Probe
PCT/DE2018/000139 WO2018228622A1 (fr) 2017-06-13 2018-05-15 Procédé de détection d'agrégats de substances biothérapeutiques dans un échantillon

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EP3639030A1 true EP3639030A1 (fr) 2020-04-22

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US (1) US20200200748A1 (fr)
EP (1) EP3639030A1 (fr)
JP (1) JP2020523554A (fr)
CN (1) CN110832323A (fr)
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WO (1) WO2018228622A1 (fr)

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EP3438649B1 (fr) * 2017-07-31 2020-03-11 Vestel Elektronik Sanayi ve Ticaret A.S. Étiquette d'identfication et procédé d'identification d'un objet
DE102020003794A1 (de) * 2020-06-25 2021-12-30 Forschungszentrum Jülich GmbH Verfahren, Verwendung des Verfahrens sowle Kit zum Nachweis von Bioindikatoren in einer Probe

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US20050208050A1 (en) * 2001-11-09 2005-09-22 Gerd Multhaup Compounds for the diagnosis/prevention/treatment of alzheimer's disease
US8658374B2 (en) * 2002-02-28 2014-02-25 Microsens Biphage Limited Binding of aggregated forms of proteins
US8190374B2 (en) * 2003-04-25 2012-05-29 Stephen Eliot Zweig Method and device to detect therapeutic protein immunogenicity
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DE102011057021A1 (de) * 2011-12-23 2013-06-27 Forschungszentrum Jülich GmbH Verfahren zur selektiven Quantifizierung von A-Beta-Aggregaten
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EP3071597B1 (fr) * 2013-11-21 2020-07-29 F.Hoffmann-La Roche Ag Anticorps anti-alpha-synucleine et procédés d'utilisation
DE102015003404B4 (de) * 2015-03-18 2021-10-07 Forschungszentrum Jülich GmbH Verfahren zur Herstellung eines Standards für den Nachweis von Proteinaggregaten einer Proteinfehlfaltungserkrankung sowie Standard und dessen Verwendung

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DE102017010455A1 (de) 2018-12-13
JP2020523554A (ja) 2020-08-06
CN110832323A (zh) 2020-02-21
WO2018228622A1 (fr) 2018-12-20
US20200200748A1 (en) 2020-06-25
WO2018228622A8 (fr) 2019-12-26

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