Quality control method of biological medicine

Abstract

The invention belongs to the field of biological detection, and relates to a quality control method of a biological medicine. The establishment of the invention obviously expands the research and development possibility of a botulinum toxin detection method, has important application value in the aspects of food and environment safety screening, laboratory diagnosis of clinical suspected cases and the like, and also can provide sequence reference for the development of recombinant antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, and in particular relates to a quality control method for biological pharmaceuticals. Background Technology

[0002] Quality control of biopharmaceuticals is of paramount importance. Due to the complex structure and sensitive processing of biopharmaceutical active ingredients, even minor changes from production to purification can lead to product heterogeneity or potential risks, directly impacting patient safety and efficacy. A rigorous quality control system, covering the entire process from raw materials to finished product, is the core means of ensuring the purity, potency, and consistency of each batch. It not only meets the mandatory requirements of drug regulatory regulations but is also the cornerstone for preventing risks, protecting public health, and maintaining the product's lifeline. Taking botulinum toxin type A as an example, it is a protein complex containing botulinum neurotoxin type A, non-toxic non-hemagglutinating components (NTNH), hemagglutinin 70 (HA70), and hemagglutinin 33 (HA33). New botulinum toxin type A products are obtained through chromatographic purification of botulinum neurotoxin type A. Their composition should be botulinum neurotoxin type A and should not contain non-toxic non-hemagglutinating components (NTNH), hemagglutinin 70 (HA70), or hemagglutinin 33 (HA33). Therefore, testing for these components is mandatory during the production of botulinum neurotoxin type A products.

[0003] Currently, the main problems in the detection and application of botulinum toxin and its antibodies are as follows: First, in the research and development and production of botulinum toxin-related products (such as therapeutic agents and diagnostic reagents), antibodies targeting toxin complex proteins or neurotoxin proteins are inevitably used. Whether from commercial sources or self-developed antibodies, there is a lack of standards and effective quality control methods. This makes it difficult to accurately verify the true specificity and binding activity of antibodies against toxin complexes or specific protein components.

[0004] Second, there is a lack of commercially available antibodies targeting the complete set of botulinum toxin components. When it is necessary to develop antibodies in-house, there is a lack of commercially available specific component proteins as immunogens, and there is no efficient and specific purification method to obtain specific component proteins (such as hemagglutinin) in their natural state.

[0005] Third, the lack of the aforementioned key reagents (antibodies and specific component proteins) also hinders the establishment and application of many important detection methods. For example, it is difficult to use immunological methods to monitor the integrity and purity of toxin complex proteins, identify different components of botulinum toxin products, or verify the correct expression of botulinum toxin components.

[0006] In summary, the existing technologies mainly have the following problems: (1) lack of antibody resources: lack of high-quality antibodies against key components of toxin complexes (such as hemagglutinin); (2) lack of quality control and purification tools: lack of standardized quality control methods and efficient purification tools; (3) limited detection methods: due to insufficient core reagents, it is difficult to establish reliable detection schemes that are suitable for multiple scenarios. Summary of the Invention

[0007] To address at least some of the technical problems in the prior art, the present invention uses a natural botulinum toxin complex as an antigen to prepare an antibody against hemagglutinin 70. This antibody or its antigen-binding fragment exhibits good sensitivity, specificity, affinity, and stability. Specifically, the present invention includes the following:

[0008] In one aspect, the present invention provides a quality control method for a biopharmaceutical, comprising the steps of contacting a monoclonal antibody or a functional fragment thereof with the biopharmaceutical to be tested and performing an immunological detection, wherein the immunological detection is selected from at least one of enzyme-linked immunosorbent assay (ELISA), capillary immunoelectrophoresis, and molecular interaction assays, and the monoclonal antibody comprises a heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3 and / or a light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0009] In some embodiments, according to the quality control method for biopharmaceuticals of the present invention, the biopharmaceutical to be tested contains botulinum toxin type A.

[0010] In some embodiments, the quality control method for biopharmaceuticals according to the present invention includes: a) The monoclonal antibody against hemagglutinin 70 is coated onto the surface of a solid-phase support; b) Add a sample containing the test biopharmaceutical, allowing the test biopharmaceutical to bind with the coated monoclonal antibody to form a first complex; c) Add an enzyme-labeled detection antibody, wherein the detection antibody forms a second complex with the first complex; d) Add the substrate corresponding to the enzyme to initiate a colorimetric reaction; e) Detect the colorimetric signal and determine the presence of the hemagglutinin 70 or quantify the hemagglutinin 70 based on the signal.

[0011] In some embodiments, the quality control method for biopharmaceuticals according to the present invention includes: forming a sandwich complex in a capillary with the monoclonal antibody, the biopharmaceutical to be tested, and the detection antibody, and catalyzing a detection solution to generate a signal that can be captured by a detector, thereby determining the presence of hemagglutinin 70 or quantifying the hemagglutinin 70.

[0012] In one aspect, the present invention provides a detection kit for detecting botulinum toxin type A complex or the hemagglutinin 70 component therein, comprising an antibody against hemagglutinin 70 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof being specifically capable of binding to hemagglutinin 70 in botulinum toxin type A complex, comprising heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3 and / or light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0013] In one aspect of the present invention, an antibody against hemagglutinin 70 or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof is capable of specifically binding to hemagglutinin 70 in a botulinum toxin type A complex, comprising a heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3 and / or a light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III): (I) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7 and / or the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof according to the present invention comprises Fab, Fab', F(ab)2, F(ab')2, scFv or scFv Fc fragments.

[0016] In one aspect, the present invention provides a nucleic acid molecule that encodes the aforementioned antibody or its antigen-binding fragment.

[0017] In one aspect, the present invention provides a carrier molecule comprising the aforementioned nucleic acid molecule.

[0018] In one aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned carrier molecule.

[0019] In one aspect, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0020] In one aspect, the present invention provides the use of the method, or the kit, or the antibody or its antigen-binding fragment, wherein the use includes quality control and monitoring of botulinum toxin type A production or botulinum toxin type A standards.

[0021] This invention purifies hemagglutinin 70 using natural strains and prepares an antibody that retains the natural high affinity and native variable-constant region pairing, successfully filling a gap in the domestic and international markets for this specific natural antibody. This not only effectively solves the problem of purifying natural toxin component proteins, providing a key tool for related purification work, but also further enriches the antibody library of component proteins of botulinum toxin complexes. Based on this antibody, combined with known detection methods in the art (including but not limited to capillary immunoelectrophoresis), standard patterns can be established for detection, providing a means for antibody quality control. The establishment of this invention significantly expands the development possibilities of botulinum toxin detection methods, and has important application value in food and environmental safety screening, laboratory diagnosis of suspected clinical cases, etc., while also providing sequence references for the development of recombinant antibodies. Attached Figure Description

[0022] Figure 1 The results of SDS-PAGE of hemagglutinin 70 antigen are shown.

[0023] Figure 2 The results of the identification of the antihemagglutinin 70 monoclonal antibody subclass are shown.

[0024] Figure 3 The results of capillary immunoelectrophoresis of the complex of antihemagglutinin 70 and natural botulinum toxin type A are shown.

[0025] Figure 4 The results of the affinity assay for the antihemagglutinin 70 monoclonal antibody are shown. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0029] This specification provides the specific sequences of the corresponding antibodies. In accordance with relevant regulations, this application also provides a computer-readable sequence listing. It should be noted that the sequences in the computer-readable sequence listing are for reference only. In the event of any discrepancy between the sequences in this specification and the sequences in the computer-readable sequence listing, the sequences in this specification shall prevail.

[0030] Ⅰ Definition The term "botulinum toxin type A" specifically refers to a 150 kDa active protein with neurotoxicity, i.e., a neurotoxin. It consists of a heavy chain (approximately 100 kDa, responsible for specifically binding to receptors on the surface of neurons) and a light chain (approximately 50 kDa, possessing zinc endopeptidase activity) covalently linked by disulfide bonds. The light chain is the direct functional unit responsible for producing the neurotoxic effect.

[0031] The term "type A botulinum toxin complex" refers to the complete, naturally occurring form secreted by Clostridium botulinum. It is a large protein complex formed by non-covalent binding of botulinum toxin type A (a neurotoxin) to a variety of accessory proteins. These accessory proteins mainly include non-toxic non-hemagglutinin proteins and hemagglutinin protein complexes, which together constitute a stable "protective shell" that protects and delivers the neurotoxin.

[0032] Hemagglutinin 70 is one of the key subunits (approximately 70 kDa) of the hemagglutinin protein complex that constitutes the botulinum toxin type A complex. As a structural component of the complex's "protective shell," it plays a crucial role in the toxin's resistance to degradation in the upper gastrointestinal tract and in mediating intestinal absorption, but it itself does not possess neurotoxicity.

[0033] In this article, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a particular antigen. Antibodies typically contain variable and constant regions in each of their heavy and light chains. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody moiety that binds to the antigen.

[0034] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of this invention consists of "framework" regions interspersed among three "complementarity-determining regions (CDRs)". The framework regions are used to modulate the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0035] In this document, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the sequences of this invention. To determine sequence identity, sequence alignment can be performed, which can be done in various ways known to those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms required to achieve optimal alignment across the full-length sequences being compared.

[0036] In this invention, variant antibody sequences obtained by modification, substitution, deletion, or addition of one or more amino acids are also within the scope of protection of this invention. The term "modification" refers to any chemical modification of the amino acid sequence. The term "substitution" refers to replacing one or more amino acids with different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to a change in the amino acid sequence resulting in an increase of one or more amino acids compared to the naturally occurring molecule. It should be noted that in the variant antibodies provided by this invention, the modification, substitution, deletion, or addition preferably occurs in regions other than the variable region, such as the frame region or constant region of the antibody, and the modified, substituted, deleted, or added antibodies or their antigen-binding fragments still retain the desired functional properties of the antibodies or their antigen-binding fragments of this invention, or have improved antigen-binding properties.

[0037] In this invention, conserved amino acid substitutions are preferred, and these conserved antibody variants are preferably generated by amino acid substitutions according to the following table: In this article, the term "monoclonal antibody," sometimes also called "mAb," refers to an immunoglobulin derived from a pure cell line, possessing the same structure and chemical properties, and specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (which typically contain different antibodies targeting different determinants); each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are obtained through hybridoma or recombinant engineered cell culture, free from contamination by other immunoglobulins. This characteristic contrasts with polyclonal antibody products, which generally comprise antibodies targeting different antigenic determinants. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a homogeneous group of antibodies, but this should not be interpreted as requiring any special methods to produce the antibody.

[0038] In this article, the term "antigen-binding fragment" generally refers to one or more fragments in an antibody that perform the function of specifically binding antigens. It can be understood that the antigen-binding function of an antibody can be achieved by the full-length fragment of the antibody, or by the following: a heavy chain containing fragments including Fab, Fab', F(ab)2, F(ab')2, or scFv, or a light chain containing fragments including Fab, Fab', F(ab)2, F(ab')2, or scFv.

[0039] In this document, the terms “binding,” “specific binding,” “targeting,” and “containing” are used interchangeably and generally refer to non-covalent interactions occurring between an immunoglobulin molecule and an antigen specific to said immunoglobulin. The strength or affinity of an immunobinding interaction can be expressed as a dissociation constant (Kd), where a smaller Kd represents a higher affinity. “Affinity” refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, when used herein, “binding affinity” refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment with an antigen). The affinity of molecule X for its partner Y is generally expressed as a binding dissociation equilibrium constant. Affinity can be measured using methods commonly known in the art, including those known in the prior art and those described herein.

[0040] In this document, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0041] Once the coding sequence of the antibody or its antigen-binding fragment described in this invention is obtained, recombinant technology can be used to obtain the antibody or its antigen-binding fragment in large quantities. An exemplary method is to clone its coding gene into a vector, transform it into cells, and then isolate it from the proliferated host cells using conventional methods.

[0042] In this document, the term "vector" refers to an artificial construct capable of delivering and preferably expressing one or more target genes or sequences in a host cell. The vectors used in this invention are not limited and may be expression vectors, viral vectors, etc. In some embodiments, the vector contains a target gene encoding an antibody of the present invention or an antigen-binding fragment thereof, a promoter, a terminator, or optionally, a marker gene. Known vectors or self-constructed vectors may be used. Known vectors include plasmid vectors, lentiviral vectors, adenovirus vectors, AAV viral vectors, etc.

[0043] In this document, the term "host cell" refers to any cell type suitable for transformation, transfection, transduction, etc., using nucleic acid constructs or expression vectors containing the nucleic acid molecules of the present invention. Host cells include any offspring of the parent cell that differ from the parent cell due to mutations occurring during replication.

[0044] In this document, the term "kit" refers to a combination of reagents and other materials. A kit is intended to contain reagents such as buffers, protein stabilizing agents, signal generation systems (e.g., fluorescence signal generation systems), antibodies or antigen-binding fragments thereof, control proteins, and test containers (e.g., microtiter plates, etc.). The term "kit" is not limited to a specific combination of reagents and / or other materials; for example, a kit may also include instructions for using the reagents. Kits can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers, along with instructions for performing the assay. Kits can be prepared using a variety of methods known in the art.

[0045] II. Detailed Description of Implementation Methods In a first aspect, the present invention provides a method for detecting botulinum toxin type A complex, the method comprising the step of contacting a sample to be tested with an antibody or antigen-binding fragment thereof against hemagglutinin 70; wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to hemagglutinin 70 in the botulinum toxin type A complex, and comprises a heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3 and / or a light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0046] In some embodiments, the method of the present invention is for non-diagnostic purposes, such as its use in the research and development and production process of botulinum toxin, especially type A related products (such as therapeutic preparations, diagnostic reagents). Therefore, the present invention also provides the application of the above-mentioned antibodies, diagnostic reagents, detection products or detection methods in the quality control and monitoring of type A botulinum neurotoxin production or type A botulinum neurotoxin standards.

[0047] In some implementations, the method includes the step (1) of contacting the sample to be tested with the aforementioned antibody or its antigen-binding fragment.

[0048] In some implementations, the sample to be tested can be a tissue sample or a fluid sample.

[0049] In some embodiments, the method further includes a step (2) of detecting whether the antibody or its antigen-binding fragment binds to the botulinum toxin type A complex in the test sample to form an immune complex.

[0050] In some implementations, step (2) is performed by enzyme-linked immunosorbent assay (ELISA), immunochromatography, chemiluminescent immunoassay, or immunofluorescence assay.

[0051] Secondly, the present invention provides a detection kit for detecting botulinum toxin type A complex or the hemagglutinin 70 component therein, comprising an antibody against hemagglutinin 70 or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to hemagglutinin 70 in the botulinum toxin type A complex, comprising heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3 and / or light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0052] In some implementations, the detection may take the form of enzyme-linked immunosorbent assay (ELISA), immunochromatographic test strips, immunofluorescence assay, or chemiluminescent immunoassay.

[0053] In some embodiments, the kit further comprises at least one component selected from the following: a solid-phase carrier, a conjugate, a chromogenic substrate, a buffer, a negative control, a positive control, or instructions for use, wherein the instructions describe a method or procedure for detecting hemagglutinin 70 or botulinum toxin type A in a sample to be tested using the antibody of the present invention.

[0054] Thirdly, the present invention provides an antibody against hemagglutinin 70 or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof being capable of specifically binding to hemagglutinin 70 in botulinum toxin type A complex, comprising heavy chain CDR1-3 as shown in SEQ ID NO: 1-3 and / or light chain CDR1-3 as shown in SEQ ID NO: 4-6.

[0055] In some embodiments, the antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III): (I) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7 and / or the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

[0056] In some embodiments, the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, or a murine antibody, and the antigen-binding fragment comprises Fab, Fab', F(ab)2, F(ab')2, scFv, or scFv Fc fragments.

[0057] Fourthly, the present invention provides a nucleic acid molecule that encodes the aforementioned antibody or its antigen-binding fragment.

[0058] Fifthly, the present invention provides a carrier molecule comprising the aforementioned nucleic acid molecule.

[0059] In a sixth aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or the aforementioned carrier molecule.

[0060] In a seventh aspect, the present invention provides a method for preparing the aforementioned antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is prepared by artificial synthesis or genetic engineering.

[0061] In some embodiments, the method of the present invention further includes the step of isolating and purifying the monoclonal antibody using an immunogenic fragment, immunizing animals, and hybridoma cell cultures, wherein the immunogen used is derived from a natural botulinum toxin type A complex. Although hybridoma technology is used in specific embodiments, this should not be construed as requiring any special method to produce the antibody. Those skilled in the art will understand that the antibody of the present invention can be obtained, for example, but not limited to, direct amino acid synthesis, or by constructing a recombinant vector expressing the antibody of the present invention, transferring the vector into a genetic engineering expression system for genetic engineering expression, and then culturing and purifying it to obtain the antibody of the present invention.

[0062] In a preferred embodiment, the antibody of the present invention can be prepared by the following steps: (1) Construct a recombinant vector expressing the antibody heavy chain with the amino acid sequence shown in SEQ ID NO: 9 and / or the antibody light chain with the amino acid sequence shown in SEQ ID NO: 10; (2) The vector is transformed into host cells and cultured under conditions suitable for antibody expression; (3) Collect antibodies and purify them.

[0063] In some embodiments, the natural botulinum toxin type A complex is obtained by culturing Clostridium botulinum type A Hall strain.

[0064] In some preferred embodiments, the preparation method includes the following steps: (1) Antigen preparation: Clostridium botulinum type A was cultured, natural botulinum toxin type A complex was purified, and hemagglutinin 70 protein was isolated from the complex; (2) Animal immunization: Immunize mammals with the hemagglutinin 70 protein obtained in step (1); (3) Cell fusion and screening: Spleen cells from immunized animals were fused with myeloma cells to obtain hybridoma cells, and hybridoma cell lines that could secrete monoclonal antibodies that specifically bind to hemagglutinin 70 were screened. (4) Antibody production: Culture the hybridoma cell line, collect and purify monoclonal antibodies.

[0065] Eighthly, the present invention provides the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a product for detecting botulinum toxin type A complex or the hemagglutinin 70 component therein.

[0066] Example 1 I. Preparation and Purification of Antigens The natural botulinum toxin type A complex was obtained by culturing Clostridium botulinum Hall strain in trypsin-yeast dialysis medium at 35°C for 5 days, followed by purification through sterile filtration, acid precipitation, extraction, concentration and anion exchange chromatography. The complex was subjected to electrophoresis, and the band corresponding to the subunit hemagglutinin 70 was recovered, extracted and preserved by gel extraction technology.

[0067] II. Preparation of anti-hemagglutinin 70 monoclonal antibodies using hybridoma technology 1. Animal immunization Emulsified hemagglutinin 70 with an equal amount of Freund's adjuvant, the mixture was used to immunize 6-8 week old Balb / c mice. The mice were injected subcutaneously into the groin at 0, 3, 5, 7 and 9 weeks of age, with doses of 40 μg / mouse and 140 μL / mouse, respectively. After the fifth immunization, mice with higher titers (whose antiserum had an OD450 value of 0.983 after 10,000-fold dilution) were selected by indirect ELISA and then given a pulse immunization two weeks later at 100 μg / mouse.

[0068] 2. Preparation and screening of hybridoma cells SP2 / 0 myeloma cells were cultured statically in a 37°C, 5.0% CO2 incubator, with regular medium changes and timely passage. Under aseptic conditions, feeder cells were prepared by repeatedly flushing the peritoneal cavity of normal Balb / c mice with complete 1640 medium. Spleen cells from immunized mice were isolated under aseptic conditions and mixed with SP2 / 0 myeloma cells at a 5:1 ratio. Cell fusion was performed using the PEG method, and the fused cell suspension was dropped into 96-well plates pre-coated with feeder cells. After 2 weeks of culture, positive clones were screened using microscopic observation combined with ELISA (using 2% BSA as blocking medium, HRP-labeled goat anti-mouse secondary antibody as detection antibody, and TMB as the chromogenic substrate). Positive wells were then subjected to tertiary cloning using limiting dilution at a density of 7-10 cells per well to obtain hybridoma cell lines stably secreting specific antibodies.

[0069] The heavy chain amino acid sequence of the obtained antihemagglutinin 70 antibody is shown in SEQ ID NO: 9, and its heavy chain CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, 2 and 3, respectively.

[0070] EVQLVESGGGLVQPKGSLKLSCAASGFIFNTYAVHWVRQAPGQGLEWVARIRSKSGNYGTNYADSVKDRFTISRDDSQNMLYLQMNNLKTEDTATYYCVGGNPFAYWGQGTL VTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0071] The light chain amino acid sequence of the obtained anti-hemagglutinin 70 antibody is shown in SEQ ID NO: 10, and its light chains CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 4, 5 and 6, respectively.

[0072] DVQITQSPSYLAASPGDTISINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLHSGIPSRFSGSGSGTDFTLTISGLEPEDFALYYCQQHYEYPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0073] III. Preparation and Purification of Monoclonal Antibodies Antibodies were prepared using an in vivo induction method. Balb / c mice were sensitized with liquid paraffin (0.5 mL / mouse), and 1×10⁻⁶ antibodies were collected after one week. 6 Hybridoma cells were injected intraperitoneally, and ascites was collected 10 days later and purified using a Protein-G affinity chromatography column.

[0074] The purified monoclonal antibodies were subclassed using a subclass identification kit. The immunoreactivity of the monoclonal antibody-natural botulinum toxin type A complex was identified by capillary immunoelectrophoresis. The affinity of the monoclonal antibody was analyzed using biomembrane interference technology. Results are as follows: Figure 2-4 As shown in Table 1.

[0075] Table 1 Example 2 1. Testing Item 1: Quality Control of the Fermentation and Cultivation Process of Botulinum Toxin Products The fermentation process development focuses on cell growth curves and toxicity testing, while the production process aims to improve purity and protect structure. Immunological testing of botulinum toxin is an auxiliary method for toxicity testing and a primary method for analyzing the structure and composition of toxins at various fermentation time points, including the immunological testing of HA70.

[0076] The test subject was botulinum toxin fermentation broth. Qualitative and quantitative detection were performed using the HA70 monoclonal antibody and conventional ELISA method. Conventional capillary immunoelectrophoresis was used to detect the proportion of HA70 components in the fermentation broth at various time points. As reference data for structural analysis, molecular interaction analysis was used to analyze the differences in HA70 among different batches, which could also be used for quantitative detection.

[0077] 2. Testing Item 2: Quality Control of the Botulinum Toxin Product Purification Process Type A botulinum toxin complex can be purified by chromatography to obtain type A botulinum neurotoxin, while NTNH, HA70, and HA33 are removed. The purification process must consider the removal rate of product-related impurities, and process control strategies should be developed based on the results. Therefore, HA70 detection is a mandatory component of the botulinum neurotoxin product purification process. Since product-related impurities are usually present at low concentrations, methods with good specificity, exclusivity, and sensitivity must be employed. This invention has found that using the HA70 monoclonal antibody from Example 1, the purified neurotoxin can be detected at the picogram level by capillary immunoelectrophoresis.

[0078] 3. Test Item 3: Impurity Analysis of Botulinum Toxin Products Potential impurities generated during the production process, storage, and / or identified in stability study batches, including product-related impurities, must have their sources clearly identified to ensure safe levels of removal and residue. The monoclonal antibody in Example 1 can be used to conduct impurity isolation and identification studies. HA70 monoclonal antibody can serve as a primary tool for identifying HA70 in product stability studies.

[0079] 4. Testing Item 4: Environmental Control of Botulinum Toxin Product Manufacturing Site The removal of NTNH, HA70, and HA33 during the production process poses a potential risk of contaminating the plant environment and may thus become process-related impurities. Therefore, environmental disinfection of the production site is crucial, and strict environmental monitoring is also required. The HA70 monoclonal antibody in Example 1 is the main tool for monitoring potential HA70 in the environment. Since the content of residual HA70 in the environment is low, high-sensitivity capillary immunoelectrophoresis or other high-sensitivity immunoassay methods can be used.

[0080] The following is merely an illustrative description of the quality control method. Those skilled in the art will understand that, given that the present invention has shown that HA70 monoclonal antibody can be used as a detection antibody, the specific detection parameters or other detection reagents involved in various immunoassay processes can be adjusted and selected as needed, and are not particularly limited in this regard.

[0081] I. Capillary Immunoelectrophoresis (1) Add the monoclonal antibody, the sample to be tested, the detection antibody and the detection solution into the detection plate of the capillary immunoelectrophoresis apparatus respectively; (2) The sample to be tested is bound to the monoclonal antibody in the capillary assembly and then further bound to the detection antibody to form a complex, and passes through the capillary in different zones; (3) The detection antibody catalyzes the detection solution, and the presence of hemagglutinin 70 is determined or the hemagglutinin 70 is quantified based on the signal captured by the detector.

[0082] In the above steps, known commercially available enzyme-labeled antibodies can be used for detection. The enzymes used for labeling include, but are not limited to, horseradish peroxidase, alkaline phosphatase, or β-carotene. Galactosidase.

[0083] In the above steps, the detection solution includes a chemiluminescent agent or a colorimetric agent, and the detection solution corresponding to horseradish peroxidase includes luminol, o-phenylenediamine, tetramethylbenzidine, and 5-ethylhexylene. Aminosalicylic acid and 2,2 Hydrazine 2(3) Ethyl benzothiazolium 6 At least one of the diammonium salts of sulfonic acid (SAF), and the detection solution corresponding to the alkaline phosphatase includes AMPPD, acridinium ester, and 4... At least one of methyl umbelliferone phosphates, the β The detection solution corresponding to galactosidase includes 5 bromine 4 chlorine 3 Indole β D galactoside, Nitrophenyl phosphate and 4 Methyl umbelliferone R At least one of D-galactoside.

[0084] Capillary immunoelectrophoresis can be performed using equipment known in the art, such as the Protein Simple Jess multifunctional fully automated protein blot quantitative analysis system from Simple™ Western Technology, which includes the detection plate, capillary assembly, and detector used in this embodiment. Those skilled in the art will understand that other types of capillary immunoelectrophoresis instruments, or even microfluidic devices containing capillaries, can be used as long as it is possible to form a sandwich complex of monoclonal antibody, test sample, and detection antibody in the capillary and catalyze the detection solution to generate a signal that can be captured by the detector. In addition to the monoclonal antibody and its corresponding detection antibody, other related reagents can be those disclosed in CN119224316A, such as working solutions and buffer solutions.

[0085] II. ELISA Testing Exemplary ELISA detection methods include: a) Coating a monoclonal antibody against hemagglutinin 70 onto the surface of a solid-phase carrier; b) Add a sample containing the test biopharmaceutical, allowing the test biopharmaceutical to bind with the coated monoclonal antibody to form a first complex; c) Add an enzyme-labeled detection antibody, wherein the detection antibody forms a second complex with the first complex; d) Add the substrate corresponding to the enzyme to initiate a colorimetric reaction; e) Detect the colorimetric signal and determine the presence of the hemagglutinin 70 or quantify the hemagglutinin 70 based on the signal.

[0086] In the above steps, known commercially available enzyme-labeled antibodies can be used for detection. The enzymes used for labeling include, but are not limited to, horseradish peroxidase, alkaline phosphatase, or β-carotene. Galactosidase.

[0087] In the above steps, the detection solution includes a chemiluminescent agent or a colorimetric agent, and the detection solution corresponding to horseradish peroxidase includes luminol, o-phenylenediamine, tetramethylbenzidine, and 5-ethylhexylene. Aminosalicylic acid and 2,2 Hydrazine 2(3) Ethyl benzothiazolium 6 At least one of the diammonium salts of sulfonic acid (SAF), and the detection solution corresponding to the alkaline phosphatase includes AMPPD, acridinium ester, and 4... At least one of methyl umbelliferone phosphates, the β The detection solution corresponding to galactosidase includes 5 bromine 4 chlorine 3 Indole β D galactoside, Nitrophenyl phosphate and 4 Methyl umbelliferone R At least one of D-galactoside.

[0088] In addition to monoclonal antibodies and their corresponding detection antibodies, other related reagents can be those publicly available in commercially available ELISA kits, such as working solutions and buffer solutions.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A quality control method for biological pharmaceuticals, characterized in that, The method includes the steps of contacting a monoclonal antibody or a functional fragment thereof with a biopharmaceutical to be tested and performing an immunological detection, wherein the immunological detection is selected from at least one of enzyme-linked immunosorbent assay (ELISA), capillary immunoelectrophoresis, and molecular interaction assays, and the monoclonal antibody comprises a heavy chain CDR1-3 with an amino acid sequence as shown in SEQ ID NO: 1-3 and / or a light chain CDR1-3 with an amino acid sequence as shown in SEQ ID NO: 4-6.

2. The quality control method for biological pharmaceuticals according to claim 1, characterized in that, The tested biological drug contains botulinum toxin type A.

3. The quality control method for biological pharmaceuticals according to claim 2, characterized in that, The method includes: a) The monoclonal antibody against hemagglutinin 70 is coated onto the surface of a solid-phase carrier; b) Add a sample containing the test biopharmaceutical, allowing the test biopharmaceutical to bind with the coated monoclonal antibody to form a first complex; c) Add an enzyme-labeled detection antibody, wherein the detection antibody forms a second complex with the first complex; d) Add the substrate corresponding to the enzyme to initiate a colorimetric reaction; e) Detect the colorimetric signal and determine the presence of the hemagglutinin 70 or quantify the hemagglutinin 70 based on the signal.

4. The quality control method for biological pharmaceuticals according to claim 2, characterized in that, The method includes: forming a sandwich complex in a capillary with the monoclonal antibody, the biopharmaceutical to be tested, and the detection antibody, and catalyzing the detection solution to generate a signal that can be captured by a detector, thereby determining the presence of hemagglutinin 70 or quantifying the hemagglutinin 70.

5. A detection kit for detecting botulinum toxin type A complex or its hemagglutinin 70 component, characterized in that, It contains an antibody against hemagglutinin 70 or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof being capable of specifically binding to hemagglutinin 70 in botulinum toxin type A complex, which contains heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3 and / or light chain CDR1-3 with amino acid sequences as shown in SEQ ID NO: 4-6.

6. An antibody against hemagglutinin 70 or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment can specifically bind to hemagglutinin 70 in botulinum toxin type A complex, which comprises heavy chain CDR1-3 as shown in SEQ ID NO: 1-3 and / or light chain CDR1-3 as shown in SEQ ID NO: 4-6.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I)-(III): (I) The amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 7 and / or the amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8; (II) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in (I) and has the same function; (III) An amino acid sequence that has the same function as the amino acid sequence shown in (I) or (II) obtained by modifying, substituting, deleting or adding one or more amino acids.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein, The antigen-binding fragment includes Fab, Fab', F(ab)2, F(ab')2, scFv, or scFv Fc fragments.

9. The method for preparing the antibody or its antigen-binding fragment according to claim 6 or 7, characterized in that, The antibody or its antigen-binding fragment is prepared by artificial synthesis or genetic engineering.

10. According to claim 1 4. The use of the method according to any one of claims, or the kit according to claim 5, or the antibody or its antigen-binding fragment according to claim 6 or 7, characterized in that, The applications include quality control and monitoring of botulinum toxin type A production or botulinum toxin type A standard.

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