Polypeptide molecule as well as preparation method and application thereof

Abstract

The present invention provides a polypeptide molecule comprising n copies of peptide monomers, where n is any integer from 2 to 14. According to the present invention, a certain number of multi-copy target sequences are repeatedly inserted in a single expression region to obtain the complex formed by connecting a plurality of monomer proteins (polypeptides); the stability of the obtained multi-copy protein (polypeptide) is obviously superior to that of a single-copy protein (polypeptide). The stability is improved, and meanwhile, no allogenic material is introduced. And the antigen reactivity of the protein (polypeptide) is not influenced. The method can be used for storing polypeptide or protein in a non-freeze-drying mode and preparing a liquid polypeptide quality control substance with good stability.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a polypeptide molecule, a preparation method thereof, and an application thereof. Background Art

[0002] Proteins or polypeptides must maintain a certain three-dimensional conformation to exhibit antigenicity and bind to corresponding antibodies. Since proteins or polypeptides themselves have active groups, they are prone to react with other substances in the environment, resulting in degradation or conformational changes of the proteins or polypeptides, loss of antigenicity, short half-life, and poor stability in vivo or in vitro.

[0003] Currently, the commonly adopted method is to freeze-dry proteins or polypeptides to improve stability. When conducting detections, commercially available polypeptide quality control substances are all stored in the form of lyophilized powder. For example, C-peptide is an important indicator for diabetes detection. However, C-peptide itself is very unstable, with a half-life in blood of only dozens of minutes. Commercially available C-peptide quality control substances are all stored in the form of lyophilized powder.

[0004] In addition, PCT reflects the activity degree of the systemic inflammatory response. The content of PCT in normal human serum is extremely low (<0.05 mg / L), and its concentration does not increase or slightly increases in diseases such as local infection, viral infection, chronic non-specific inflammation, cancer fever, graft-host rejection reaction, or autoimmune diseases. However, when there are severe infections of bacteria, fungi, parasites, as well as sepsis and multiple organ failure, the level of PCT in plasma significantly increases. Therefore, the measurement result of PCT can be used as an acute parameter to differentially diagnose bacterial and non-bacterial infections and inflammations. For example, it is used for the diagnosis, stratification, treatment monitoring, and prognosis evaluation of bacterial infectious sepsis. However, PCT has a short existence time in plasma, with a half-life of 22 - 29 hours. Its stability in ordinary buffer is also not good, and quality control substances are all stored in the form of lyophilized powder. However, lyophilization will affect the antigen activity of proteins or polypeptides. At the same time, the uniformity of lyophilized powder is relatively poor, affecting the detection effect.

[0005] Moreover, there may be situations where refrigeration is unavailable in some regions. In terms of transportation, cold chain transportation has high costs and high operation requirements, all of which pose higher requirements for the stability of proteins.

[0006] On the other hand, as an important part of the detection system, the quality control substance of polypeptide monomer, if its stability cannot meet the corresponding requirements, will affect the production, transportation, and storage of the reagent kit. Currently, in order to improve the stability of the quality control substance, generally, a lyophilized preparation is prepared by a freeze-drying method. However, the polypeptide monomer is unstable during the freeze-drying process, resulting in a low recovery rate. Therefore, finding a highly efficient and stable calibration preparation method is of great significance for promoting the detection application of small-molecule polypeptides. Summary of the Invention

[0007] To solve at least one of the above problems, the present disclosure provides a polypeptide molecule comprising n (2 ≤ n ≤ 14) copies of a target peptide monomer. The present disclosure also provides a reagent containing the polypeptide molecule and its applications.

[0008] According to one aspect of the present disclosure, there is provided a polypeptide molecule having n copies of a peptide monomer, where n is any integer from 2 to 14. In some embodiments, n can be selected from any integer in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

[0009] In some embodiments, n can be any integer from 2 to 10.

[0010] In some embodiments, the peptide monomer has less than 4000 amino acid residues.

[0011] In some embodiments, the peptide monomer can have less than 200, less than 300, less than 400, less than 500, less than 600, less than 700, less than 800, less than 900, less than 1000, less than 1100, less than 1200, less than 1300, less than 1400, less than 1500, less than 1600, less than 1700, less than 1800, less than 1900, less than 2000, less than 2100, less than 2200, less than 2300, less than 2400, less than 2500, less than 2600, less than 2700, less than 2800, less than 2900, less than 3000, less than 3100, less than 3200, less than 3300, less than 3400, less than 3500, less than 3600, less than 3700, less than 3800, less than 3900, or less than 4000 amino acid residues.

[0012] In some embodiments, the peptide monomer has less than 1000 amino acid residues.

[0013] In some embodiments, the peptide monomer has less than 200 amino acid residues.

[0014] In some embodiments, two adjacent peptide monomers can be directly linked or linked through a linking peptide.

[0015] In some embodiments, the linking peptide is a flexible linking peptide.

[0016] In some specific embodiments, the peptide monomer has less than 50 amino acid residues, and two adjacent peptide monomers are directly linked; or,

[0017] The peptide monomer has ≥50 amino acid residues, and two adjacent peptide monomers are linked by the linking peptide.

[0018] In some specific embodiments, the linking peptide has 0 to 100 amino acid residues;

[0019] In some specific embodiments, the amino acid sequence of the linking peptide is selected from one or more of the amino acid sequences shown by (G n S) m , (G) n or (EA3K) n , where n and m are each independently selected from integers of 0 to 5.

[0020] In some specific embodiments, the amino acid sequence of the linking peptide is selected from the amino acid sequences shown by any one of SEQ ID NO:7 - 11, or an amino acid sequence having at least 85% sequence identity therewith.

[0021] In some embodiments, the peptide monomer is selected from one or more of hormones, growth factors / cytokines, proteases, receptor molecules, monoclonal antibodies or fragments thereof.

[0022] In some embodiments, the peptide monomer is insulin C - peptide or procalcitonin.

[0023] In some embodiments, the polypeptide molecule is 2 to 14 copies of insulin C - peptide or procalcitonin;

[0024] In some embodiments, the insulin C - peptide may have the amino acid sequence shown by SEQ ID NO:1, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity therewith.

[0025] In some embodiments, the polypeptide molecule has the amino acid sequence shown by SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity therewith.

[0026] In some embodiments, the procalcitonin may have an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0027] In some embodiments, the polypeptide molecule has an amino acid sequence as shown in SEQ ID NO:14, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0028] In some embodiments, the peptide monomer or the polypeptide molecule may be modified by one or more of polyethylene glycol modification, glycosylation modification, hydroxyethylation modification, or removal of O-glycosylation, etc.

[0029] According to another aspect of the present disclosure, there is provided a nucleic acid molecule encoding the polypeptide molecule of the present disclosure.

[0030] In some embodiments, the nucleic acid molecule encodes 2 to 14 copies of insulin C peptide or procalcitonin.

[0031] In some embodiments, the insulin C peptide has a nucleotide sequence as shown in SEQ ID NO:2, or a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0032] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:4, or a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0033] In some embodiments, the procalcitonin has a nucleotide sequence as shown in SEQ ID NO: 13, or a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0034] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 15, or a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0035] According to another aspect of the present disclosure, there is provided a liquid quality control substance containing the polypeptide molecule of the present disclosure. The polypeptide molecule of the present disclosure can have excellent stability under liquid conditions and retains immunogenicity.

[0036] In some embodiments, the liquid quality control substance further comprises: PBS buffer and serum albumin.

[0037] In some embodiments, the liquid quality control substance comprises 0.5% - 5% (w / v) serum albumin. In a specific embodiment, the liquid quality control substance comprises 1% (w / v) serum albumin.

[0038] In some embodiments, the serum albumin is selected from one or more of bovine serum albumin, horse serum albumin, porcine serum albumin, rabbit serum albumin, goat serum albumin or chicken serum.

[0039] In some embodiments, the bovine serum albumin is selected from fetal bovine serum albumin, newborn bovine serum albumin, calf serum albumin or adult bovine serum albumin.

[0040] In some embodiments, the liquid quality control substance includes, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9% or 5% (w / v) of serum albumin.

[0041] In some embodiments, the pH value of the PBS buffer is selected from any value in the range of 7.0 to 8.5.

[0042] In some embodiments, the pH value of the PBS buffer is selected from any value of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4 or 8.5.

[0043] In some embodiments, the quality control substance includes reference materials (RM), calibrators, quality control samples, reference products or control products, etc.

[0044] According to another aspect of the present disclosure, there is provided an application of the above-mentioned polypeptide molecule or the above-mentioned liquid quality control substance of the present disclosure in detection.

[0045] In some embodiments, during the use of the quality control substance, it can be serially diluted to obtain a set of standard samples, which can be used for quantitative detection.

[0046] In some specific embodiments, the detection includes but is not limited to immunoassay.

[0047] In some specific embodiments, the immunoassay includes but is not limited to affinity chromatography, liquid chromatography, colorimetry, immunochemistry.

[0048] In some specific embodiments, the immunoassay includes but is not limited to colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA) or chemiluminescence immunoassay (CLIA).

[0049] According to another aspect of the present disclosure, there is provided a method for preparing the polypeptide molecule of the present disclosure, the method comprising: repeatedly inserting multiple copies of the target sequence into a single expression region of the genome of a host cell for expression to obtain a complex composed of multiple monomeric proteins (polypeptides) linked together.

[0050] In some embodiments, the host cell may include, but is not limited to, any animal cell, fungal cell, or prokaryotic cell. In some embodiments, the animal cell may be selected from the mouse myeloma (NSO) cell line, Chinese hamster ovary (CHO) cell line, and kidney epithelial cells of African green monkey (Vero).

[0051] In some embodiments, the fungal cell may be selected from yeast cells, such as Pichia pastoris, Hansenula anomala, Saccharomyces cerevisiae, etc.

[0052] In some embodiments, the prokaryotic cell may be selected from Escherichia coli, Bacillus subtilis, etc.

[0053] The present disclosure improves the stability of a protein or polypeptide by preparing multiple copies of the polypeptide molecule. In a single expression region, a certain number of multiple copies of the target sequence are repeatedly inserted to obtain a complex composed of multiple monomeric proteins (polypeptides) linked together. The stability of the obtained multiple-copy protein (polypeptide) is significantly better than that of the single-copy protein (polypeptide). The polypeptide molecule can be stored in a liquid environment in a non-lyophilized manner and has long-term stability. Moreover, when improving the stability, no foreign substances are introduced, and the antigen reactivity of the protein (polypeptide) is not affected.

[0054] The copy number of the polypeptide molecule of the present invention is within a certain range, with the optimal stability and the smallest decrease in antigen reactivity. Beyond a certain range, the stability will instead decrease.

[0055] The present invention uses the multiple-copy stabilization technology to successfully achieve the stability of liquid-preserved C-peptide at the level of freeze-dried powder, and a liquid polypeptide quality control substance with good stability can be prepared. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Shows the long-term stability curve of the multiple-copy C-peptide (liquid) of the present disclosure.

[0057] Figure 2 Shows the long-term stability curve of the 15-copy C-peptide (liquid) of the present disclosure.

[0058] Figure 3 Shows the long-term stability curve of the 5-copy PCT (liquid) of the present disclosure.

[0059] Figure 4 Shows the long-term stability curve of the 4-copy C-peptide calibrator 2 with a final concentration of 0.5 ng / mL of the present disclosure.

[0060] Figure 5 Shows the long-term stability curve of the 4-copy C-peptide calibrator 3 with a final concentration of 1 ng / mL of the present disclosure.

[0061] Figure 6 Shows the long-term stability curve of the 4-copy C-peptide calibrator 4 with a final concentration of 5 ng / mL of the present disclosure.

[0062] Figure 7 Shows the long-term stability curve of the 4-copy C-peptide calibrator 5 with a final concentration of 12 ng / mL of the present disclosure.

[0063] Figure 8 Shows the long-term stability curve of the 4-copy C-peptide calibrator 6 with a final concentration of 30 ng / mL of the present disclosure. Detailed implementation manners

[0064] For the purpose of making the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies have also been described in many publications.

[0065] Definition

[0066] Unless otherwise defined, all technical terms and scientific and technical terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of explaining this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0067] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents.

[0068] In this article, the terms "polypeptide molecule" and "polypeptide", "peptide" can be used interchangeably and generally refer to polymers of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two parts covalently linked together.

[0069] As used herein, the term "peptide monomer" refers to a polypeptide molecule composed of at least two amino acids, wherein the amino acids are linked by a peptide bond between the carboxyl group of one amino acid and the amino group of the next amino acid. A "peptide monomer" includes only one polypeptide molecule. For example, it is in no way a multimer (such as a dimer, trimer, tetramer, pentamer, etc.). A "peptide monomer" may also contain non-protein ligands, such as inorganic iron (Fe), nickel (Ni), iron-sulfur centers (FeS), or other organic ligands, such as carbon monoxide (CO), cyanide (CN), or flavin. The definition of a "peptide monomer" does not consider the length of the polypeptide or the manner in which the polypeptide is produced.

[0070] As used herein, the term "multi-copy protein" means a protein containing at least 2 copies of a peptide monomer. In some specific embodiments, the protein contains 2 to 100 copies of the peptide monomer. For example, the protein contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 copies of the peptide monomer.

[0071] As used herein, the term "C-peptide" includes all forms of insulin C-peptide, including natural or synthetic peptides. Such insulin C-peptides can be of human origin or can be from other animal species and genera, preferably mammals. Thus, variants and modified forms of natural insulin C-peptide are included as long as the insulin C-peptide activity is retained. In the art, it is known to modify protein or peptide sequences while retaining their useful activities, and this can be achieved by using standard techniques in the art that are well-described in the literature, such as random or site-directed mutagenesis, nucleic acid cleavage and ligation, etc. Thus, functional equivalent variants or derivatives of the natural insulin C-peptide sequence can be readily prepared according to techniques well-known in the art, and they contain peptide sequences having the functional (e.g., biological) activity of natural insulin C-peptide. All such analogs, variants, derivatives, or fragments of insulin C-peptide are specifically included within the scope of the present invention and are encompassed by the term "insulin C-peptide".

[0072] As used herein, the term "immunogenicity" refers to the ability of a substance, whether alone or when linked to a carrier, in the presence or absence of an adjuvant, to elicit, initiate, stimulate, or induce an immune response against a specific antigen, or to modify, enhance, increase, or prolong a pre-existing immune response against a specific antigen.

[0073] As used herein, the term "procalcitonin" or "PCT" is a protein composed of 116 amino acids with a molecular weight of approximately 13 kDa. It is the propeptide of calcitonin (CT), has no calcitonin-like hormonal activity, and its molecule consists of calcitonin, katacalcin, and an N-terminal fragment containing 57 amino acids.

[0074] As used herein, the term "linker peptide" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. The domains in the bispecific fusion polypeptides of the present invention can be linked by linker peptides, where each linker peptide is fused to and / or otherwise linked (e.g., via peptide bonds) to at least two polypeptides or domains. Examples of linker peptide classifications include flexible linker peptides, rigid linker peptides, and in vivo cleavable linker peptides, etc. Rigid linker peptides are composed of amino acid residues that easily form stable secondary structures. In many cases, due to their ability to form relatively stable secondary structures, they can more effectively separate functional domains and maintain their independent functions compared to flexible linker peptides. When the spatial separation of functional domains is crucial for the stability and biological activity of the fusion protein, rigid linker peptides can be selected. Many natural rigid linker peptides form α-helical structures. The commonly used rigid α-helical linker peptide is (EA3K)n (n ≤ 6). Due to the presence of hydrogen bonds inside and its tight backbone, the α-helical structure is rigid and stable. In vivo cleavable linker peptides are mostly used in biopharmaceuticals and are generally not used for the construction of fusion proteins. For example, in vivo cleavable disulfide linker peptides. Flexible linker peptides and rigid linker peptides are generally not preferentially degraded in vivo. These stable covalent linker peptides link functional domains together as a whole molecule and exhibit many advantages in biopharmaceuticals, such as prolonging the plasma half-life, etc.

[0075] As used herein, the term "flexible linker peptide" is composed of small non-polar amino acids (such as glycine) or polar amino acids (such as serine or threonine). Small amino acids can provide more flexibility, enabling the linked functional domains not to interfere with each other and thus better play their roles. Polar amino acids such as serine and threonine can form hydrogen bonds with water molecules, thus ensuring the stability of the linker peptide in aqueous solution while reducing the adverse reactions between the linker peptide and the protein region.

[0076] In some embodiments, the amino acid sequences of all the linker peptides present in the bispecific fusion polypeptides of the present invention are the same. In other embodiments, the amino acid sequences of at least two linker peptides present in the bispecific fusion polypeptides of the present invention are different. The linker peptide should have a length suitable for linking two or more monomer domains in this way, and the linker peptide can ensure that the different domains it links are correctly folded and properly presented, so as to exert their biological activity functions. In different embodiments, the linker peptide has a flexible conformation. Suitable flexible linker peptides include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker peptide may be selected from (G n S) m , (G) n , (EA3K) n or (XP) n , where X is any amino acid, preferably alanine, lysine or glutamic acid, and n and m each independently selected from integers from 0 to 5. For example, n is selected from 0, 1, 2, 3, 4 or 5, and m is selected from 1, 2, 3, 4 or 5. In some embodiments, the linker peptide may also include a linker peptide containing an amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, etc.

[0077] The "percent sequence identity" or "identity percent" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for the optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid exists in both the target sequence and the reference sequence. Since a gap is not a nucleotide or an amino acid, a gap present in the target sequence is not counted. Similarly, a gap present in the reference sequence is not counted because nucleotides or amino acids from the reference sequence are not counted while nucleotides or amino acids from the target sequence are counted. At least 85% sequence identity includes continuous segments having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the full length of the sequence.

[0078] Methods for aligning comparison sequences are well known in the art. Various programs and alignment algorithms are described in "Smith and Waterman, Adv. Appl. Math. 2" 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48" 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85" 2444, 1988; Higgins and Sharp, Gene, 73" 237-44, 1988; Higgins and Sharp, CABIOS 5" 151-3, 1989; Corpet et al., Nuc. Acids Res. 16" 10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24" 307-31, 1994; Altschul et al., J. Mol. Biol. 215" 403-10, 1990, presenting detailed considerations of sequence alignment methods and homology calculations.

[0079] In the present disclosure, the term "nucleotide sequence" encompasses an oligonucleotide chain or an oligonucleotide chain complementary thereto.

[0080] In the present disclosure, the term "complementary" refers to the concept of sequence complementarity between regions of two polynucleotide chains or between two regions of the same polynucleotide chain. It is known that an adenine base in a first region of a polynucleotide can form specific hydrogen bonds ("base pairs") with a base (if the base is thymine or uracil) in a second region of a polynucleotide that is antiparallel to the first region. Similarly, it is known that a cytosine base in a first polynucleotide chain can base pair with a base (if the base is guanine) in a second polynucleotide chain that is antiparallel to the first region. Two regions are complementary if at least one nucleotide in the first region can base pair with a base in the second region when the first region of the polynucleotide is arranged antiparallel to the second region of the same or another different polynucleotide. Thus, two complementary polynucleotides do not need to base pair at every nucleotide position. "Complementary" refers to 100% or "perfect" complementarity of a first polynucleotide with a second polynucleotide and thus forms base pairs at every nucleotide site. "Complementary" also refers to a first polynucleotide that is not 100% complementary (e.g., 90%, or 80% or 70%, or 60%, or 50% complementary) and contains mismatched nucleotides at one or more nucleotide positions.

[0081] In this text, the terms "lyophilization" and "freeze-drying" are used as synonyms in the context of the present invention. It refers to the process of first freezing the substance to be dried, usually in liquid form, and then sublimating it into a solid state in a vacuum environment to remove ice or the frozen solvent.

[0082] As used herein, the term "serial dilution" includes a series of specific samples (e.g., antibodies) with decreasing concentrations. Generally, a serial dilution is produced by the following process: mixing a measured amount of the starting concentration sample or reagent with a diluent (e.g., dilution buffer) to produce a sample or reagent with a lower concentration, and repeating this process a sufficient number of times to obtain the desired number of serial dilutions. The sample or reagent can be serially diluted at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, or 1000-fold to produce a serial dilution containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 samples or reagents with decreasing concentrations.

[0083] In this text, the term "quality control substance" includes reference materials (RM), calibrators, control materials, reference products or control products, etc. Quality control substances play an important role in the research and development, production, traceability of measurement values, and use process of detection products.

[0084] The so-called "reference material" is also known as a reference standard, reference substance or reference sample. It refers to a homogeneous and stable substance with one or more specified characteristic quantity values suitable for the measurement of specific purposes or the inspection of nominal characteristics. For example, a series of known biomarker solutions with known concentrations or contents, such as proteins or polypeptides.

[0085] The so-called "calibrator" is a measurement reference substance used to calibrate a measurement system to establish the traceability of the measurement results of the metrological system. The matrix of the calibrator should be as consistent as possible with the actual test sample. It has homogeneity, stability and interchangeability.

[0086] The so-called "control material", its purpose is to evaluate or verify measurement precision and measurement accuracy, or performance characteristics such as analysis deviation caused by changes in reagents or analytical instruments. The control material should be homogeneous and stable and can be stored for a certain period of time if conditions permit. It can be a liquid or lyophilized material containing one or more components (analytes) with known concentrations.

[0087] The so-called "reference product" or "reference reagent" refers to a quality control substance used to quantitatively detect the biological potency and biological activity of certain products, and its potency is expressed in specific activity units.

[0088] The "reference substance" refers to a quality control substance containing a single component, a combined component or a mixed component, which is used for identification, inspection and content determination in the inspection of biological products.

[0089] The present disclosure provides a polypeptide molecule, which is a multi-copy complex composed of n (2≤n≤10) target peptide monomers.

[0090] In some embodiments, the number of amino acids in the peptide monomer ≤ 2000.

[0091] In some embodiments, the peptide monomers are directly linked or linked by a short peptide, the length of the short peptide is preferably 0≤m≤100, and the short peptide is preferably a flexible linker peptide.

[0092] In some embodiments, the peptide monomer is insulin C peptide or procalcitonin.

[0093] In the present disclosure, the preparation of the polypeptide molecule includes inserting the nucleic acid sequence repeatedly for many times into the expression region of the genome of an engineered bacterium. If the peptide monomer itself is relatively large, a flexible linker peptide sequence can be optionally added between each copy, which is beneficial to the formation of a composite structure of the expressed multi-copy protein. After fermentation, separation and purification, the polypeptide molecule is obtained.

[0094] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modification and change can be made without departing from the spirit of the present invention.

[0095] Examples

[0096] Example 1:

[0097] 1. Obtain multi-copy C peptide:

[0098] 1.1 The C peptide has the amino acid sequence shown in SEQ ID NO:1.

[0099] 1.2 The coding nucleic acid sequence of the C peptide is shown in SEQ ID NO:2. For simplicity, this sequence is represented by CP hereafter.

[0100] 1.3 The above nucleic acid sequence was repeated 4 times to obtain a 4-copy sequence (Suoxin Biology), as shown in SEQ ID NO:4.

[0101] In addition, the nucleic acid sequence described in 1.2 was repeated 15 times to obtain a 15-copy sequence: CP-CP-CP-CP-CP-CP-CP-CP-CP-CP-CP-CP-CP-CP-CP, and the nucleotide sequence is shown in SEQ ID NO:6.

[0102] For convenient purification, His tags were added to the above sequences and then inserted into the expression region of Pichia pastoris.

[0103] 1.4 After positive clones were selected, they were cultured on a large scale and induced with methanol.

[0104] 1.5 After harvesting the bacteria, they were lysed with a homogenizer. After centrifugation of the lysate, the supernatant was taken, filtered, loaded onto a nickel column, washed, eluted with imidazole, and dialyzed into PBS buffer. C-peptide with 4 copies (amino acid sequence as shown in SEQ ID NO:3) and C-peptide with 15 copies (amino acid sequence as shown in SEQ ID NO:5) were obtained for comparison.

[0105] 2. Detection of multi-copy C-peptide:

[0106] 2.1 Preparation of buffer: 1% BSA was added to PBS buffer and the pH was adjusted to 7.4. Using the said buffer, multi-copy C-peptides (including C-peptides with 4 copies and 15 copies) and the C-peptide national standard product (National Institutes for Food and Drug Control, No.: 150553, C-peptide pure product, containing C-peptide monomer) reconstituted with purified water after lyophilization were both diluted to 10 ng / mL.

[0107] 2.2 Both the 10 ng / mL multi-copy C-peptide and the C-peptide pure product were aliquoted into 0.5 mL per vial. Five replicates were made respectively (i.e., tests 1 - 5), and the difference in signal values was detected using the C-peptide detection reagent from Kemi Company (C-peptide (CP) detection kit (photochemiluminescence method), Shanghai Medical Device Registration No. 20152400268). The results are shown in Table 1.

[0108] Table 1: Comparison of antigenicity between multi-copy C-peptide (liquid state) and C-peptide pure product (lyophilized and reconstituted)

[0109]

[0110] It can be seen therefrom that the signal values of multi-copy C-peptides (including 4 copies and 15 copies) are basically the same as those of the C-peptide pure product at the same concentration. There is no difference in antigen reactivity.

[0111] 2.3 The 10 ng / mL C-peptide pure product (5 replicate samples, i.e., tests 1 - 5) was lyophilized. After lyophilization and reconstitution, it was detected by the detection method described in 2.1. The results are shown in Table 2.

[0112] Table 2: Comparison of C-peptide pure product before and after lyophilization

[0113] Signal value Before lyophilization After lyophilization Test 1 155748 129624 Test 2 149892 122864 Test 3 146946 121578 Test 4 155696 133313 Test 5 150865 118408 Mean value 151829 125157 Deviation before and after - -17.0％

[0114] It can be seen from this that the signal value of the lyophilized pure C-peptide decreased by 17% compared with that before lyophilization, indicating that the lyophilization process has a great impact on the activity of C-peptide. Nevertheless, in the prior art, pure C-peptide is often stored by lyophilization because the stability of pure C-peptide (liquid state) is very poor. As detected by the detection method described in 2.1, for three groups of replicates of 10 ng / mL pure C-peptide, the signal values before storage were 150893, 149325, and 150276, and the signal values decreased to the background level after being stored in the liquid state for 3 days.

[0115] 2.4 Place the lyophilized C-peptide and multi-copy C-peptides (4-copy and 15-copy) at 2-8 °C, with 5 replicates (tubes 1-5) respectively, and conduct long-term stability monitoring using the detection method described in 2.1. The results are shown in Table 3-5 and Figure 1-2 。

[0116] Table 3: Long-term stability monitoring of pure C-peptide (lyophilized)

[0117] Signal value Initial value February April June August October December 14th month Tube 1 129624 133583 125467 124405 111879 113689 108275 108092 Tube 2 122864 118435 127062 125467 119671 120405 113085 124645 Tube 3 121578 117773 126278 121527 116735 119842 124281 120030 Tube 4 133313 133398 114713 122177 125388 125074 111678 120302 Tube 5 118408 128754 128156 116261 126937 117292 119310 110663 Mean value 125157 126389 124335 121967 120122 119260 115326 116746 Signal value deviation / 1.0％ -0.7％ -2.5％ -4.0％ -4.7％ -7.9％ -6.7％

[0118] Table 4: Long-term stability monitoring of 4-copy C-peptide (liquid state)

[0119] Signal value Initial value February April June August October December 14th month Tube 1 155852 158291 150727 143054 142679 141047 146985 135998 Tube 2 146077 152393 157782 153885 146192 142880 148010 136211 Tube 3 148346 148334 155974 154053 148062 139577 142444 148527 Tube 4 156947 157158 146237 156084 151343 149644 144339 149815 Tube 5 146831 148907 155350 145476 146668 150809 144274 142906 Mean value 150811 153017 153214 150510 146989 144791 145210 142691 Signal value deviation / 1.5％ 1.6％ -0.2％ -2.5％ -4.0％ -3.7％ -5.4％

[0120] Table 5: Long-term stability monitoring of 15-copy C-peptide (liquid state)

[0121] Signal value Initial value February April June August October December 14th month Tube 1 146319 150302 136471 131304 133661 118698 118651 116560 Tube 2 155280 139447 139638 133305 128242 126582 121141 107418 Tube 3 153360 151111 136549 128863 134152 118795 114991 111936 Tube 4 142821 150126 144883 140078 129506 123675 119801 112857 Tube 5 152497 139679 135453 138842 124947 125706 116823 109325 Mean value 150055 146133 138599 134478 130102 122691 118281 111619 Signal value deviation / -3％ -8％ -10％ -13％ -18％ -21％ -26％

[0122] From the long-term stability results, the stability of 4-copy C-peptide under liquid storage conditions is basically the same as that of the lyophilized pure C-peptide, while the stability of 15-copy C-peptide under liquid storage conditions is inferior to that of the lyophilized pure C-peptide.

[0123] 3. Conclusion: The stability of multi-copy C-peptide under liquid storage conditions is basically the same as that under lyophilization treatment. At the same time, it avoids the influence of the lyophilization process on the antigen activity. By using the multi-copy stabilization technology, the stability of C-peptide stored in the liquid state has been successfully improved to the level of the lyophilized powder. However, the stability of 15-copy C-peptide under liquid storage conditions is relatively poor compared with that under lyophilization treatment. An excessive number of copies does not necessarily improve stability.

[0124] Example 2:

[0125] 1. Obtain multi-copy PCT:

[0126] 1.1 Procalcitonin (PCT) is a polypeptide composed of 116 amino acids with a molecular weight of approximately 14.5 kDa, and its amino acid sequence is shown in SEQ ID No: 12.

[0127] 1.2 The coding nucleic acid sequence of procalcitonin is shown in SEQ ID NO:13. For simplicity, it will be replaced by PCT hereinafter. Since PCT is relatively large, in order not to affect the formation of stable structure, a flexible linker peptide (G4S)2 is inserted between each copy. Finally, a 5-copy amino acid sequence: PCT-(G4S)2-PCT-(G4S)2-PCT-(G4S)2-PCT-(G4S)2-PCT is obtained, as shown in SEQ ID NO:14. Then its coding nucleic acid sequence (such as SEQ ID NO:15) is inserted into the yeast expression region.

[0128] 1.3 After fermentation, separation and purification, 5-copy liquid PCT is obtained.

[0129] 2. Detection of multi-copy PCT:

[0130] 2.1 Both 5-copy PCT and the pure PCT product (purchased from Medix, pure PCT product, containing PCT monomer) reconstituted with purified water after lyophilization are diluted to 10 pg / mL, and the difference in signal values is detected using the PCT detection kit from Kemei Company (Procalcitonin Detection Kit (Photoactivated Chemiluminescence Method), Shanghai Medical Device Registration No. 20202400124). The results are shown in Table 6.

[0131] Table 6: Comparison of antigenicity between 5-copy PCT (liquid) and pure PCT product (lyophilized and reconstituted)

[0132]

[0133] It can be seen that the signal values of 5-copy PCT and pure PCT product are basically the same at the same concentration. There is no difference in antigen reactivity.

[0134] 2.2 Both multi-copy PCT and pure PCT product at 10 pg / mL are dispensed into vials at 0.5 mL / vial.

[0135] 2.3 Five replicates of 10 pg / mL pure PCT product are lyophilized. After lyophilization and reconstitution, they are detected according to the detection method described in 2.1. The results are shown in Table 7.

[0136] Table 7: Comparison of pure PCT product before and after lyophilization

[0137] Signal value Before lyophilization After lyophilization Test 1 135830 136695 Test 2 139553 141523 Test 3 129071 129761 Test 4 131765 134685 Test 5 141256 134782 Mean value 135495 135489 Deviation before and after - 0.0％

[0138] It can be seen that the lyophilization process has basically no effect on the activity of PCT.

[0139] 2.4 The lyophilized PCT and 5-copy PCT are placed in an environment of 2 - 8 °C, and five replicates (tube 1 - tube 5) are respectively made for long-term stability monitoring according to the monitoring method described in 2.1. The results are shown in Table 8, Table 9 and Figure 3 .

[0140] Table 8: Long-term Stability Monitoring of PCT Pure Product (Freeze-dried and Reconstituted)

[0141] Signal value Initial value February April June August October December 14th month Tube 1 136695 138560 127888 137419 130932 122133 125689 128564 Tube 2 141523 134893 131217 131763 125543 124424 121754 127571 Tube 3 129761 133804 135079 133439 125886 134779 127646 118092 Tube 4 134685 141572 134013 126840 125453 133927 122820 121483 Tube 5 134782 137704 140192 131731 132745 122848 122103 119732 Mean value 135489 137307 133678 132238 128112 127622 124002 123088 Signal value deviation 1％ -1％ -2％ -5％ -6％ -8％ -9％

[0142] Table 9: Long-term Stability Monitoring of 5-copy PCT (Liquid)

[0143] Signal value Initial value February April June August October December 14th month Tube 1 140441 139970 135880 136907 134655 128531 124563 128860 Tube 2 134479 137320 130315 135698 125685 123880 129790 121842 Tube 3 138013 130384 131762 137536 135352 130138 121024 120395 Tube 4 137153 134946 127539 131239 131267 123962 125049 121080 Tube 5 132981 133535 139548 124755 128346 128238 127563 124728 Mean value 136613 135231 133009 133227 131061 126950 125598 123381 Signal value deviation -1％ -3％ -2％ -4％ -7％ -8％ -10％

[0144] From the long-term stability results, the stability of 5-copy PCT under liquid storage conditions is almost the same as that of the freeze-dried PCT pure product.

[0145] 3. Conclusion: The stability of 5-copy PCT under liquid storage conditions is the same as that of freeze-drying treatment. By using the multi-copy preparation method, a standard solution with good stability can also be obtained.

[0146] Example 3:

[0147] 1. Obtain 4-copy C-peptide: The steps are shown in Example 1

[0148] 2. Prepare the calibrator buffer: Add 1% BSA to the PBS buffer and adjust the pH to 7.4.

[0149] 3. Dilute the multi-copy C-peptide obtained in step 1 with the calibrator buffer described in step 2 to a final concentration of 0.5, 1, 5, 12, 30 ng / mL, and dispense them into a set of calibrators 2 - 6.

[0150] 4. Stability monitoring (compared with the calibrator of the freeze-drying process).

[0151] Place the freeze-dried calibrator of CP pure product and the liquid calibrator prepared from 4-copy CP in an environment of 2 - 8°C for long-term stability monitoring. The results are shown in Tables 10 - 13 and Figures 4-8 .

[0152] Table 10: Long-term Stability Monitoring of the Freeze-dried Calibrator of CP Pure Product

[0153]

[0154] Table 11: Deviation of Long-term Stability Signal Values of the Freeze-dried Calibrator of CP Pure Product

[0155] Signal value deviation February April June August October December 14th month Pure product lyophilized calibrator 2 -1％ -2％ -5％ -4％ -6％ -8％ -7％ Pure product lyophilized calibrator 3 0％ -1％ -2％ -6％ -5％ -7％ -10％ Pure product lyophilized calibrator 4 1％ -3％ -2％ -3％ -4％ -6％ -8％ Pure product lyophilized calibrator 5 -2％ -3％ -2％ -5％ -5％ -7％ -7％ Pure product lyophilized calibrator 6 -2％ 0％ -4％ -2％ -5％ -7％ -7％

[0156] Table 12: Long-term Stability Monitoring of the Liquid Calibrator Prepared from 4-copy CP

[0157] Signal value Initial value February April June August October December 14th month Calibrator 2 702 685 676 679 685 674 650 646 Calibrator 3 1491 1472 1461 1442 1403 1415 1423 1359 Calibrator 4 47307 47288 47113 45107 45641 45667 44427 43477 Calibrator 5 243630 242579 239255 232925 231334 228906 227701 225983 Calibrator 6 848777 849137 819647 813755 818590 796419 809713 778638

[0158] Table 13: Deviation of Long-term Stability Signal Values of the Liquid Calibrator Prepared from 4-copy CP

[0159] Signal value deviation February April June August October December 14th month Multi-copy C-peptide calibrator 2 -2％ -4％ -3％ -2％ -4％ -7％ -8％ Multi-copy C-peptide calibrator 3 -1％ -2％ -3％ -6％ -5％ -5％ -9％ Multi-copy C-peptide calibrator 4 0％ 0％ -5％ -4％ -3％ -6％ -8％ Multi-copy C-peptide calibrator 5 0％ -2％ -4％ -5％ -6％ -7％ -7％ Multi-copy C-peptide calibrator 6 0％ -3％ -4％ -4％ -6％ -5％ -8％

[0160] From the perspective of long-term stability results, for the 4-copy CP, within the concentration range of 0.5 - 30 ng / mL, the stability under liquid storage conditions is almost the same as that of the lyophilized CP pure product.

[0161] 5. Conclusion: The application of multi-copy CP in the CP reagent calibrator has excellent effects. The liquid calibrator prepared using multi-copy C-peptide can be used clinically with good effects.

[0162] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A polypeptide molecule, characterized in that, Comprising n copies of peptide monomers, where n is any integer from 2 to 14.

2. The polypeptide molecule according to claim 1, wherein The peptide monomers have less than 4000 amino acid residues, preferably less than 1000 amino acid residues, and more preferably less than 200 amino acid residues.

3. The polypeptide molecule according to claim 1, wherein Two adjacent peptide monomers are directly linked or linked via a linking peptide; Preferably, the linking peptide is a flexible linking peptide.

4. The polypeptide molecule according to claim 1, wherein The peptide monomer has less than 50 amino acid residues, and two adjacent peptide monomers are directly linked; or, The peptide monomer has ≥50 amino acid residues, and two adjacent peptide monomers are linked via the linking peptide.

5. The polypeptide molecule according to claim 3, wherein The linking peptide has 0 to 100 amino acid residues; Preferably, the amino acid sequence of the linker peptide is selected from one or more of the amino acid sequences shown by (G m S) n , (G) n or (EA m K) n , where n and m are each independently selected from integers of 0 to 5.

6. The polypeptide molecule according to claim 1, wherein The peptide monomer is selected from one or more of hormones, cytokines, proteases, receptor molecules, monoclonal antibodies or fragments thereof, Preferably, the peptide monomer is insulin C peptide or procalcitonin; Preferably, the polypeptide molecule is 2 to 14 copies of insulin C peptide or procalcitonin; Preferably, the insulin C peptide has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity thereto; Preferably, the polypeptide molecule has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 85% sequence identity thereto; Preferably, the procalcitonin has the amino acid sequence shown in SEQ ID NO:12, or an amino acid sequence having at least 85% sequence identity thereto; Preferably, the polypeptide molecule has the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 85% sequence identity thereto; Preferably, the peptide monomer or the polypeptide molecule can be modified by one or more of polyethylene glycol modification, glycosylation modification, hydroxyethylation modification, or removal of O-glycosylation.

7. A nucleic acid molecule encoding the polypeptide molecule according to any one of claims 1 to 6; Preferably, the nucleic acid molecule encodes 2 to 14 copies of insulin C peptide or procalcitonin; Preferably, the insulin C peptide has the nucleotide sequence shown in SEQ ID NO:2, or a nucleotide sequence having at least 85% sequence identity thereto; Preferably, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence having at least 85% sequence identity thereto; Preferably, the procalcitonin has the nucleotide sequence shown in SEQ ID NO:13, or a nucleotide sequence having at least 85% sequence identity thereto; Preferably, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:15, or a nucleotide sequence having at least 85% sequence identity thereto.

8. A liquid quality control substance containing the polypeptide molecule according to any one of claims 1 to 6, Preferably, the liquid mass control substance further comprises: PBS buffer and serum albumin; Preferably, the liquid quality control substance contains 0.5% to 5% serum albumin, preferably 1% serum albumin; Preferably, the serum albumin is selected from one or more of bovine serum albumin, horse serum albumin, porcine serum albumin, rabbit serum albumin, goat serum albumin or chicken serum; Preferably, the bovine serum albumin is selected from fetal bovine serum albumin, newborn bovine serum albumin, calf serum albumin or adult bovine serum albumin; Preferably, the pH of the liquid quality control substance is selected from any value of 7 to 8.5, more preferably 7.

4.

9. Use of the polypeptide molecule according to any one of claims 1 to 6 or the liquid quality control substance according to claim 8 in detection; Preferably, the detection is an immunoassay; Preferably, the immunoassay includes, but is not limited to, affinity chromatography, liquid chromatography, colorimetry, immunochemistry; Preferably, the immunoassay includes, but is not limited to, colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA) or chemiluminescent immunoassay (CLIA).

10. A method for preparing a polypeptide molecule according to any one of claims 1 to 6, the method comprising: In a single expression region of the genome of a host cell, multiple copies of the target sequence are repeatedly inserted for expression to obtain a complex composed of multiple peptide monomers linked together.

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