# US 10,767,213 B2 · Quantification of vaccine compositions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,213 B2 - Document ID: US10767213B2 - Status: Granted - Kind: B2 - Application number: 15/456,013 - Filed: 2017-03-10 - Published: 2017-09-07 - Granted: 2020-09-08 - Earliest claimed: 2013-03-01 - Assignees: Boehringer Ingelheim Animal Health USA Inc. - Applicants: Boehringer Ingelheim Animal Health USA Inc. - Inventors: Marc Allan Eichmeyer; Michael B. Roof; Merrill Lynn Schaeffer; Eric Martin Vaughn; Kun Yang; Jeremy Richard Rush; Daniel John Murfin - Web page: https://patentgrep.com/patent/US10767213B2 ## CPC - C12Q 1/37: involving peptidase or proteinase - C07K 7/06: having 5 to 11 amino acids - C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68) - C07K 14/001: {by chemical synthesis} - C07K 14/005: from viruses - G01N 33/56983: {Viruses} - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 2333/01: DNA viruses - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The invention provides methods and mass-labeled peptides for use in said methods for quantifying the presence of a one or more viral proteins in a sample of a preparation containing agents which bind to said viral protein, using mass-spectroscopic analyses of the sample and standards containing known amounts of labeled and unlabeled signature peptides, in particular wherein said viral proteins are antigens in a vaccine for porcine circovirus. ## Claims ### Claim 1 A method of quantifying the presence of one or more porcine circovirus type 2 (PCV2) viral proteins forming a virus like particle in a sample, the method comprising: a) adding a known quantity of one or more stable-isotope-labeled signature peptides specific to the one or more PCV2 viral proteins in the sample, wherein the one or more stable-isotope-labeled signature peptides are selected from the group consisting of: i) (SEQ ID NO: 14) ANALTYDPYVNYSSR; ii) (SEQ ID NO: 15) HTITQPFSYHSR; ii) (SEQ ID NO: 18) LQTTGNVDHVGLGTAFENSIYDQDYNIR; iv) (SEQ ID NO: 19) ITMYVQFR; v) (SEQ ID NO: 22) YFTPK PVLDR; and xviii) (SEQ ID NO: 23) EFNLK DPPLNPK; b) digesting the sample with a protease; c) running mass-spectroscopic analyses of the sample; and d) determining an amount of the one or more PCV2 viral proteins in the sample, wherein the one or more PCV2 viral proteins are capable of forming a virus like particle and/or wherein the sample comprises virus like particles composed of a plurality of the one or more PCV2 viral proteins comprising PCV subtype 2a (PCV2a) open reading frame 2(ORF2) and/or PCV subtype 2b (PCV2b) ORF2; wherein the sample is removed from one of: an animal material selected from a body fluid and a tissue, a preparation of the animal material, and a vaccine preparation. ### Claim 2 (depends on 1) The method of claim 1, wherein determining the amount of the one or more PCV2 viral proteins in the sample comprises comparing one or more signals resulting from the mass-spectroscopic analyses of the sample, wherein a first signal from each of the one or more stable-isotope-labeled signature peptides is compared with a second signal from each of the one or more peptides produced by the protease digestion of the one or more PCV2 viral proteins in the sample, wherein each amino acid sequence of the one or more signature peptides corresponds to an amino acid sequence of one peptide produced by the protease digestion of the one or more PCV2 viral proteins in the sample. ### Claim 3 (depends on 1) The method of claim 1, wherein the signature peptides are preselected by determining that they are specific to the protease digest of the viral protein to be quantified and/or that they are specifically absent from the protease digest of the sample in the absence of the viral protein. ### Claim 4 (depends on 1) The method of claim 1, further comprising: e) determining the amount of viral protein in the sample by comparing the results of the sample mass-spectroscopic analysis with a calibration standard curve; and/or f) running mass-spectroscopic analyses of standards containing known amounts of labeled and/or unlabeled signature peptides; and determining the amount of viral protein in the sample by comparing the results of the sample mass-spectroscopic analysis with the results of the standards, wherein the calibration standard curve is generated with the results of the standards and compared with the results of the sample mass-spectroscopic analysis. ### Claim 5 (depends on 1) The method of claim 1, wherein the preparation contains agents which bind to said viral protein. ### Claim 6 (depends on 1) The method of claim 1, further comprising, before protease digestion, immunopurifying the viral protein. ### Claim 7 (depends on 6) The method of claim 6, wherein the immunopurification comprises: i) contacting the viral proteins with antibodies that bind specifically to the viral proteins that correspond to the sequence in the signature peptides, wherein said antibodies are fixed to a substrate, and whereby substrate/antibody-protein complexes are formed; ii) washing the substrate with an eluent that does not cause the antibody-protein complexes to dissociate; iii) contacting the antibody-protein complexes with a protease; and iv) eluting the peptides generated by the protease digest with a eluent that causes the antibody-peptide complexes to dissociate; wherein the peptides obtained thereby are used in subsequent steps a)-d). ### Claim 8 (depends on 1) The method of claim 1, further comprising, before protease digestion, passing the preparation over a size-exclusion chromatographic column and selecting the fractions eluted from the column containing viral protein from fractions containing other compounds in the preparation, on the basis of molecular size. ### Claim 9 (depends on 8) The method of claim 8, wherein the fractions containing intact virus-like particles (VLPs) are selected. ### Claim 10 (depends on 1) The method of claim 1, further comprising, after protease digestion, immunopurifying the viral protein digest. ### Claim 11 (depends on 1) The method of claim 1, wherein at least two stable-isotope-labeled signature peptides are used, and wherein a first signature peptide is used for quantitation of the viral peptide in the preparation and a second signature peptide is used for qualitative determination of the stability of the peptide in the preparation. ### Claim 12 (depends on 11) The method of claim 11, wherein the qualitative determination measures whether degraded viral protein is present in the sample. ### Claim 13 (depends on 1) The method of claim 1, wherein the running mass-spectroscopic analyses includes: a. selecting an ion of interest related to the signature peptide; b. filtering a plurality of ions generated by an ionization of particles within the sample; and c. analyzing fragment ions corresponding to the ion of interest. ### Claim 14 (depends on 1) The method of claim 1, wherein the running of the mass-spectroscopic analyses comprises: a. ionizing the sample; b. separating a plurality of ions according to their mass-to-charge ratios; and c. detecting at least one ion corresponding to the viral protein. ### Claim 15 (depends on 1) The method of claim 1, wherein the protease is selected from the group consisting of trypsin, chymotrypsin, pepsin, thrombin, papain, bromelain, thermolysin, subtilisin, Factor Xa, Staphylococcus aureus protease, carboxypeptidase A, and combinations thereof. ### Claim 16 (depends on 1) The method of claim 1, wherein the animal material is selected from blood, blood serum, blood plasma, urine, colostrum, tissue sections, and tissue biopsies. ### Claim 17 (depends on 1) A method of diagnosis or monitoring of a virus infection comprising the method of claim 1. ### Claim 18 (depends on 17) The method of claim 17, wherein the virus infection is an infection with PCV2 wherein the PCV2 comprises PCV2a and/or PCV2b, and/or wherein the animal is a pig.