REHEARSAL

Description

46 REHEARSAL Separated from BR112021001372-5, deposited on 07 / 08 / 2019 RELATED ORDERS

[001] This application claims priority and benefit of the Application U.S. Provisional Order No. 62 / 715973, filed August 8, 2018, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[002] The snapshot request contains a sequence listing that was sent in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The said ASCII copy, created on July 29, 2019, is named “REGE-015001WO_SeqList_ST25.txt” and is 50,295 bytes in size. BACKGROUND OF THE INVENTION

[003] C1q is an important, drug-amenable protein involved in the complement system of the innate immune system. Currently, immunological methods exist to determine the concentration of C1q in biological samples derived from humans. However, there are limited immunoreagents to assess the abundance of C1q in samples derived from non-human primates, an important model organism in research and preclinical trials. Thus, there is a need in the art for methods and compositions targeted at determining the concentration of C1q in samples derived from humans, non-human primates, and other model organisms that are rapid, specific, and accurate and do not require the expensive and time-consuming development of immunoreagents. This disclosure addresses these needs. BRIEF DESCRIPTION OF THE INVENTION

[004] The present disclosure provides an assay comprising: (1) placing a biological sample in contact with at least one enzyme Petition 870260049114, dated 05 / 22 / 2026, page 13 / 84 / 46 proteolytic to produce at least one peptide fragment of the C1q protein present in the biological sample; and (2) perform selected reaction monitoring mass spectrometry (SRM-MS) to measure the abundance of at least one C1q peptide fragment, wherein the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[005] The previous assay may further comprise, between step (1) and step (2), adding to the biological sample at least one labeled synthetic C1q peptide fragment comprising an amino acid sequence identical to the amino acid sequence of at least one C1q peptide fragment.

[006] Measure the abundance of at least one peptide fragment C1q in the previous assay may comprise comparing a signal corresponding to at least one C1q peptide generated by SRM-MS with a standard curve.

[007] The present disclosure provides an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample; (2) performing selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment; and (3) determining the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, wherein the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[008] The preceding assay may further comprise, between step (1) and step (2), adding to the biological sample at least one labeled synthetic peptide fragment comprising an amino acid sequence identical to the amino acid sequence of at least one C1q peptide fragment, and between step (2) and step (3), performing SRM-MS to generate Petition 870260049114, dated 05 / 22 / 2026, page 14 / 84 / 46 a signal corresponding to at least one labeled synthetic peptide.

[009] The biological sample can be a blood sample. The biological sample can be a human sample. The biological sample can be a non-human primate sample.

[0010] At least one peptide fragment may comprise at least 5 amino acids. At least one peptide fragment may comprise a peptide selected from Table 2.

[0011] At least one peptide fragment may comprise SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36). At least one peptide fragment may comprise at least two of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36). At least one peptide fragment may comprise each of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) and QTHQPPAPNSLIR (SEQ ID NO: 36).

[0012] The selected reaction monitoring mass spectrometry can be LC-SRM-MS / MS.

[0013] At least one proteolytic enzyme may be trypsin.

[0014] A standard curve can be produced using a method comprising: (a) preparing at least two C1q concentration standards by mixing known amounts of purified C1q protein and C1q-depleted serum; (b) adding to the at least two C1q concentration standards at least one synthetic peptide fragment labeled with an amino acid sequence identical to the at least one C1q peptide fragment expected to be produced upon contact of the C1q concentration standard with a proteolytic enzyme; (c) bringing the at least two labeled C1q concentration standards into contact with a proteolytic enzyme to produce at least one C1q peptide fragment; (d) performing mass spectrometry to monitor a selected reaction. Petition 870260049114, dated 05 / 22 / 2026, page 15 / 84 / 46 to determine the signal strength corresponding to at least one C1q peptide fragment and the signal strength corresponding to at least one labeled synthetic peptide fragment in each of at least two labeled C1q concentration standards; and (e) determine a standard curve using the signals and known amounts of C1q protein.

[0015] This disclosure provides a composition comprising at least one isolated synthetic peptide, said composition comprising at least one isolated synthetic peptide with an amino acid sequence selected from the C1q protein.

[0016] Composition comprising at least one isolated synthetic peptide, said composition characterized by comprising at least one isolated synthetic peptide with an amino acid sequence selected from the C1q protein, wherein the amino acid sequence selected from the C1q protein is the sequence of a C1q peptide fragment generated by the contact of C1q with at least one proteolytic enzyme.

[0017] The C1q protein may be from a human. The C1q protein may be from a non-human primate.

[0018] At least one isolated synthetic peptide may comprise at least 5 amino acids.

[0019] The selected amino acid sequence of the C1q protein may be the sequence of a C1q peptide fragment generated by contact of C1q with at least one proteolytic enzyme. The at least one proteolytic enzyme may be trypsin.

[0020] At least one isolated synthetic peptide can be labeled.

[0021] At least one isolated synthetic peptide may comprise a peptide selected from Table 2.

[0022] At least one isolated synthetic peptide may comprise SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or Petition 870260049114, dated 05 / 22 / 2026, page 16 / 84 / 46 QTHQPPAPNSLIR (SEQ ID NO: 36). The cysteine ​​in the synthetic peptide SLGFCDTTNK (SEQ ID NO: 26) can be modified. The modification can be carbamidomethylation.

[0023] The present disclosure provides a composition comprising at least one pair of transition ions, said composition comprising at least one pair of transition ions of the C1q protein, wherein the at least one pair of transition ions consists of a precursor ion with a corresponding m / z and an ion fragment with a corresponding m / z ion.

[0024] The C1q protein may be from a human. The C1q protein may be from a non-human primate.

[0025] This disclosure provides a composition comprising at least one transition ion pair, said composition comprising at least one transition ion pair of the C1q protein, wherein the at least one transition ion pair consists of a precursor ion with a corresponding m / z and an ion fragment with a corresponding ion m / z, and wherein the transition ion pair is selected from the precursor SLGFC(Cam)DTTNK (SEQ ID NO: 41) transition pair 571.8-942.3, precursor IAFSATR (SEQ ID NO: 29) transition pair 383.1-581.1 and precursor QTHQPPAPNSLIR (SEQ ID NO: 36) transition pair 487.0-350.3. Any of the above aspects may be combined with any other aspect.

[0026] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by someone skilled in the subject matter to which this disclosure pertains. In descriptive reporting, singular forms also include the plural, unless the context clearly indicates otherwise; for example, the terms a, an, and the are understood as singular or plural, and the term or is considered inclusive. For example, “a Petition 870260049114, dated 05 / 22 / 2026, page 17 / 84 / 46 “element” means one or more elements. Throughout the descriptive report, the word comprising or variations such as comprises or that includes will be understood as implying the inclusion of a declared element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Approximately may be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the declared value. Unless otherwise clear in the context, all numerical values ​​provided in this document are modified by the term approximately.

[0027] Although methods and materials similar or equivalent to those described in this document may be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All applications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. The references cited in this document are not admitted as being from the prior art to the claimed invention. In case of conflict, this Descriptive Report, including the definitions, shall prevail. Furthermore, the materials, methods and examples are for illustrative purposes only and are not intended to be limiting. Other features and advantages of the disclosure will be evident from the following detailed description and claim. BRIEF DESCRIPTION OF THE FIGURES

[0028] The above and other features will be more clearly appreciated from the following detailed description when considered together with the attached figures.

[0029] Figure 1 is the amino acid sequence alignment of the C1q A subunit from human, monkey, mouse, and rat.

[0030] Figure 2 is the amino acid sequence alignment of the C1q B subunit from human, monkey, mouse, and rat. Petition 870260049114, dated 05 / 22 / 2026, page 18 / 84 / 46

[0031] Figure 3 is the amino acid sequence alignment of the C subunit of C1q from human, monkey, mouse and rat.

[0032] Figure 4 is a calibration curve generated using the methods of this disclosure and a peptide derived from the A subunit of the C1q protein.

[0033] Figure 5 is a calibration curve generated using the methods of this disclosure and peptide derived from the B subunit of the C1q protein.

[0034] Figure 6 is a calibration curve generated using the methods of this disclosure and a peptide derived from the C subunit of the C1q protein.

[0035] Figure 7 is a series of LC-SRM-MS / MS chromatograms of selected peptides derived from the A, B, and C subunits of C1q in blank, double blank, blank, and Lower Limit of Quantification (LLOQ) preparation samples.

[0036] Figure 8 is a series of LC-SRM-MS / MS chromatograms of selected peptides derived from C1q subunits A, B, and C at the Limit of Detection (LOD) and LLOQ samples showing the signal-to-noise and response values ​​for the highlighted peaks.

[0037] Figure 9 is a series of LC-SRM-MS / MS chromatograms of selected peptides derived from the A, B, and C subunits of C1q in blank preparation samples before and after analyzing a ULOQ sample.

[0038] Figure 10 shows the LC-SRM-MS / MS chromatograms of selected peptides derived from the A, B, and C subunits of C1q in standard solutions in Double Blank (L00) supplemented with 2000 pg / mL of bispecific antibody, 20 pg / mL of bispecific antibody, or no bispecific antibody and a sample of LLOQ.

[0039] Figure 11 is a series of graphs that show the response Petition 870260049114, dated 05 / 22 / 2026, page 19 / 84 / 46 relating to C1q in samples incubated with bispecific antibody measured using the methods of this disclosure.

[0040] Figure 12 is a series of graphs showing the measured relative response for endogenous C1q in samples diluted by different dilution factors in different diluents using the methods of the present disclosure.

[0041] Figure 13 is a series of graphs showing the measured relative response for C1q in samples diluted by different dilution factors in different diluents using the methods of the present disclosure.

[0042] Figure 14 is a series of graphs showing the accuracy of the measured concentration for C1q in samples subjected to three freeze-thaw cycles or stored in an autosampler for 48 hours using the methods of this disclosure.

[0043] Figure 15 shows a graph representing the concentration of C1q over time in blood samples from measured monkeys, determined using the methods of this disclosure and a selected peptide derived from the A subunit of C1q.

[0044] Figure 16 shows a graph representing the concentration of C1q over time in blood samples from measured monkeys, determined using the methods of this disclosure and a selected peptide derived from the B subunit of C1q.

[0045] Figure 17 shows a graph representing the concentration of C1q over time in blood samples from measured monkeys, determined using the methods of this disclosure and a selected peptide derived from the C subunit of C1q. DETAILED DESCRIPTION OF THE INVENTION

[0046] This disclosure provides methods and compositions for determining the abundance and / or concentration of protein biomarkers in a biological sample. In some respects, this protein biomarker is the Petition 870260049114, dated 05 / 22 / 2026, page 20 / 84 / 46 C1q protein. In some respects, the methods of this disclosure comprise liquid chromatography-selected reaction monitoring mass spectrometry analysis (LC-SRM-MS).

[0047] Complement component 1q (C1q) is a protein complex involved in the complement system, which is part of the innate immune system. C1q, along with C1r and C1s, forms the C1 complex. C1q is a 400 kDa protein consisting of 18 polypeptide subunits: six A subunits, six B subunits, and six C subunits. Complement inhibitors have been successfully used in the treatment of various diseases. Monoclonal antibodies targeting C1q have potential as a therapy for autoimmune diseases involving the classical complement pathway. The development of treatment approaches targeting C1q requires methods to determine the concentration levels of C1q in biological samples during laboratory research and clinical trials. To date, determining the abundance of C1q in human samples requires the use of immunoassays, such as ELISA.Furthermore, there are limited immunoreagents for the C1q assay in non-human primate samples, which is an important aspect of preclinical research and trials. Thus, there is a need for an improved assay to determine the concentration of C1q in biological samples derived from humans, non-human primates, and other model organisms.

[0048] Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM-MS) methods are highly desirable because LC-SRM-MS methods provide absolute structural specificity for the target protein and relative or absolute measurement of target protein concentration when suitable internal standards are used. METHODS OF THE PRESENT DISCLOSURE

[0049] Several methods of the present disclosure are described in full detail in this document. Petition 870260049114, dated 05 / 22 / 2026, page 21 / 84 / 46

[0050] This disclosure provides a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample; and (2) performing selected reaction monitoring mass spectrometry (SRM-MS) to measure the abundance of at least one C1q peptide fragment, wherein the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0051] In some respects, the foregoing method may further comprise, between step (1) and step (2), adding to the biological sample at least one labeled synthetic C1q peptide fragment comprising an amino acid sequence identical to the amino acid sequence of at least one C1q peptide fragment.

[0052] This disclosure also provides a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample; (2) performing selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment; and (3) determining the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, wherein the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0053] In some respects, the foregoing method may further comprise, between step (1) and step (2), adding to the biological sample at least one labeled synthetic peptide fragment comprising an amino acid sequence identical to the amino acid sequence of at least one C1q peptide fragment, and between step (2) and step (3), performing SRM-MS to generate a signal corresponding to at least one peptide Petition 870260049114, dated 05 / 22 / 2026, page 22 / 84 / 46 synthetic marked.

[0054] In some respects, the biological sample may be a blood sample. In preferred respects, the biological sample may be a serum sample. In some respects, the biological sample may be a human sample. Alternatively, the biological sample may be a non-human primate sample. The non-human primate may be Macaca fascicularis or Macaca mulatta.

[0055] In some respects, the C1q protein may be the human C1q protein. In other respects, the C1q protein is the Macaca fascicularis C1q protein. In yet another respect, the C1q protein may be the Macaca mulatta C1q protein. The C1q protein may comprise any of the sequences shown in Table 1. TABLE 1. C1Q PROTEIN SEQUENCES Espécies Subunidade n° de ref NCBI Sequência SEQ ID NO Humano (Homo sapiens) A NP_057075 MEGPRGWLVLCVLAISLASMVTEDLCRAPDGK KGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQG LKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPG IKGTKGSPGNIKDQPRPAFSAIRRNPPMGGNVVI FDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQV LSQWEICLSIVSSSRGQVRRSLGFCDTTNKGLFQ VVSGGMVLQLQQGDQVWVEKDPKKGHIYQGS EADSVFSGFLIFPSA 1 B NP000482 _ MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTG PPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLA GDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGG PGAPGAPGPKGESGDYKATQKIAFSATRTINVP LRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPG LYYFTYHASSRGNLCVNLMRGRERAQKVVTFC DYAYNTFQVTTGGMVLKLEQGENVFLQATDK NSLLGMEGANSIFSGFLLFPDMEA 2 C NTD 7^20^7 NP_75895 7 MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCY GIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIP GIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGV PGPMGIPGEPGEEGRYKQKFQSVFTVTRQTHQP PAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPG LYYFVYHASHTANLCVLLYRSGVKVVTFCGHT SKTNQVNSGGVLLRLQVGEEVWLAVNDYYDM VGIQGSDSVFSGFLLFPD 3 Macaco Cynomolgus (Macaca fascicularis) A XP_015296582 MEGPQGWLVVCVLAISLASIVTQNVCRAPDGKNGVAGRPGRPGRPGLKGERGEPGAPGIRTGIQG LKGDQGEPGPSGNPGKVGYPGPSGPLGDRGIPG IKGIKGNPGNIKDQPRPAFSAIRRNPPMGGNVVI FDMVITNQEEPYQNHSGRFVCTVPGYYYFTFQ VVSEREICLSIVSSSRGQVRRSLGFCDTTNKGLF QVVSGGMVLQLQRGDQVWVEKDPRKGNIYQG LEADSVFSGFLIFPSS 4 Petition 870260049114, dated 05 / 22 / 2026, page 23 / 84 / 46 Espécies Subunidade n° de ref NCBI Sequência SEQ ID NO B XP_005544557 MMMKILWGSIPVLMLLLLLGLLDVSWAQGSCT GPPAIPGTPGIPGTPGSDGQPGTPGIKGEKGLPG LAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPK GGPGAPGAPGPKGESGDYKATQKIAFSATRTV NTPLRRDQTIRFDHVITNMNNNYEPRSGKFTCR VPGLYYFTYHASSRGNLCVKLMRGRERPQKVV TFCDYAYNTFQVTTGGMVLKLEQGENVFLQAT DKNSLLGMEGANSIFSGFLLFPDVEA 5 C XP_015296579 MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCY GIPGMPGLPGAPGKDGHDGLPGPKGEPGIPAIP GTRGPKGQKGEPGTPGHPGKNGPMGPPGMPG VPGPMGIPGEPGEEGRYKQKYQSVFTVARQTH QPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKV PGLYYFVYHASHTANLCVLLYRGGVKVVTFCG HTSQANQVNSGGVLLRLQVGEEVWLGVNDYY DMVGIQGSD SVFSGFLLF PD 6 Macaco rhesus (Macaca mulata) A XP_014985904 MEGPQGWLVVCVLAISLASIVTQNVCRAPDGK NGVAGRPGRPGRPGLKGERGEPGAPGIRTGIQG LKGDQGEPGPSGNPGKVGYPGPSGPLGDRGIPG IKGIKGNPGNIKDQPRPAFSAIRRNPPMGGNVVI FDMVITNQEEPYQNHSGRFVCTVPGYYYFTFQ VVSEREICLSIVSSSRGQVRRSLGFCDTTNKGLF QVVSGGMVLQLQRGDQVWVEKDPRKGNIYQG LEADSVFSGFLIFPST 7 B XP_014985910 MMMKILWGSIPVLMLLLLLGLLDVSWAQGSCT GPPAIPGTPGIPGTPGSDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPK GGPGAPGAPGPKGESGDYKATQKIAFSATRTIN TPLRRDQTIRFDHVITNMNNNYEPRSGKFTCRV PGLYYFTYHASSRGNLCVKLMRGRERPQKVVT FCDYAYNTFQVTTGGMVLKLEQGENVFLQAT DKNSLLGMEGANSIFSGFLLFPDVEA 8 C NP_001253737 MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCY GIPGMPGLPGAPGKDGHDGLPGPKGEPGIPAIP GTRGPKGQKGEPGTPGHPGKNGPMGPPGMPG VPGPMGIPGEPGEEGRYKQKYQSVFTVARQTH QPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKV PGLYYFVYHASHTANLCVLLYRGGVKVVTFCG HTSQANQVNSGGVLLRLQVGEEVWLGVNDYY DMVGIQGSD SVFSGFLLF PD 9 Camundongo (Mus musculus) A NP_031598 METSQGWLVACVLTMTLVWTVAEDVCRAPNG KDGAPGNPGRPGRPGLKGERGEPGAAGIRTGIR GFKGDPGESGPPGKPGNVGLPGPSGPLGDSGPQ GLKGVKGNPGNIRDQPRPAFSAIRQNPMTLGN VVIFDKVLTNQESPYQNHTGRFICAVPGFYYFN FQVISKWDLCLFIKSSSGGQPRDSLSFSNTNNKG LFQVLAGGTVLQLRRGDEVWIEKDPAKGRIYQ GTEADSIFSGFLIFPSA 10 B NP_033907 MKTQWGEVWTHLLLLLLGFLHVSWAQSSCTG PPGIPGIPGVPGVPGSDGQPGTPGIKGEKGLPGL AGDLGEFGEKGDPGIPGTPGKVGPKGPVGPKG TPGPSGPRGPKGDSGDYGATQKVAFSALRTINS PLRPNQVIRFEKVITNANENYEPRNGKFTCKVP GLYYFTYHASSRGNLCVNLVRGRDRDSMQKVVTFCDYAQNTFQVTTGGVVLKLEQEEVVHLQA TDKNSLLGIEGANSIFTGFLLFPDMDA 11 Petition 870260049114, dated 05 / 22 / 2026, page 24 / 84 / 46 Espécies Subunidade n° de ref NCBI Sequência SEQ ID NO C NP_031600 MVVGPSCQPPCGLCLLLLFLLALPLRSQASAGC YGIPGMPGMPGAPGKDGHDGLQGPKGEPGIPA VPGTRGPKGQKGEPGMPGHRGKNGPRGTSGLP GDPGPRGPPGEPGVEGRYKQKHQSVFTVTRQT TQYPEANALVRFNSVVTNPQGHYNPSTGKFTC EVPGLYYFVYYTSHTANLCVHLNLNLARVASF CDHMFNSKQVSSGGVLLRLQRGDEVWLSVND YNGMVGIEGSNSVFSGFLLFPD 12 Rato (Rattus norvegicus) A NP_001008515 METSQGWLVACVLAVTLVWTVAEDVCRAPNG KDGVAGIPGRPGRPGLKGERGEPGAAGIRTGIR GLKGDMGESGPPGKPGNVGFPGPTGPLGNSGP QGLKGVKGNPGNIRDQPRPAFSAIRQNPPTYGN VVVFDKVLTNQENPYQNRTGHFICAVPGFYYF TFQVISKWDLCLSIVSSSRGQPRNSLGFCDTNSK GLFQVLAGGTVLQLQRGDEVWIEKDPAKGRIY QGTEADSIFSGFLIFPSA 13 B NP_062135 MKTQWSEILTPLLLLLLGLLHVSWAQSSCTGSP GIPGVPGIPGVPGSDGKPGTPGIKGEKGLPGLAG DHGELGEKGDAGIPGIPGKVGPKGPVGPKGAP GPPGPRGPKGGSGDYKATQKVAFSALRTVNSA LRPNQAIRFEKVITNVNDNYEPRSGKFTCKVPG LYYFTYHASSRGNLCVNIVRGRDRDRMQKVLT FCDYAQNTFQVTTGGVVLKLEQEEVVHLQATD KNSLLGVEGANSIFTGFLLFPDMDV 14 C NP_001008524 MVVGTSCQPQHGLYLLLLLLALPLRSQANAGC YGIPGMPGLPGTPGKDGHDGLQGPKGEPGIPAIPGTQGPKGQKGEPGMPGHRGKNGPMGTSGSP GDPGPRGPPGEPGEEGRYKQKHQSVFTVTRQT AQYPAANGLVKFNSAITNPQGDYNTNTGKFTC KVPGLYYFVHHTSQTANLCVQLLLNNAKVTSF CDHMSNSKQVSSGGVLLRLQRGDEVWLAVND YNGMVGTEGSDSVFSGFLLFPD 15 Cão (Canis lupus familiaris) A XP_535367 MEAPWGWLALCVLATSLASAVTQDVCRALDG RDGAAGTPGRPGRPGLKGEQGEPGAPGMRTGI RGLKGDQGDPGPPGNPGNMGFPGPSGLMGLPG IPGRRGPKGNPGNIRDQPRPAFSAIRRNPPTGGN VVIFDTVITNQEGPYQNHSGRFICAVPGYYYFTF QVVSKWDICLSIVSSGRAQIRRSLGFCDTNSKGI FQVVSGGMALQLQQGDQVWIEKDPIKGRIYQG PEADSIFSGFLIFPSL 16 B XP 544507 _ MKTPRGGILALLLPLLLGLLEVSWAQSCTGHPA IPGIPGIPGAPGTDGTPGTPGTKGEKGLPGLAGD HGEFGEKGDPGIPGTPGKVGPKGPVGPKGSPGP PGARGAKGESGDYKATQKIAFSAMRTINIPLRR DQTIRFDHIVTNENRNYEPRSGKFTCNVPGIYYF AYHASSRGNLCVNVMRGRERMQKVVTFCDYV QNTFQVTTGSVVLKLSQGENVYLQATDKNSLL GMEGANSIF SGFLLFPDAEA 17 C XP_003433793 MDTGPSSWPHLGLNLLLLLLALPLGGQASTGC YGIPGMPGLPGAPGKDGHDGLPGPKGEPGIPAI PGTRGPKGQKGEPGTPGYPGKNGPMGTPGIPG VPGPVGPPGEPGEEGRYKQKHQSVFTVTRQTA QYPLANNLVKFNTVITNPQGDYDTSTGKFTCK VPGLYYFVYHTSLTSNLCVHLYRSGTRVTTFCDHMSNSKQVSSGGVLLRLQMGEQVWLAVNDYN GMVGTEGSDSVFSGFLLFPD 18 Petition 870260049114, dated 05 / 22 / 2026, page 25 / 84 / 46 Species Subunit NCBI ref. no. Sequence SEQ ID NO Zebrafish (Danio rerio) A ACN62221 MQPSAFFAFLWAGALFPFSFCQDECVKHGRNG ADGPNGRDGLPGPKGEKGEPALQVKLSCIALEE LKGDMGVRGPPGEPGLEGLMGAIGPRGPLGPA GPRGSSVGADGAKASEKPAFSVLRNEASQAQY KQPVTFNDKLSDANDDFQIKTGYFTCKVPGVY YFVFHASSEGRLCLRLKSTSAPPVSLSFCDFNSK SVSLVVSGGAVLTLLKGDKVWIEPFAGDGGVG QMPKRLYAVFNGFLIYRNAE 19 B ACN62222 MLFALMSAHVVPQLAIMLLLVTSSMSETCAGN KGFPGTPGIPGVPGTDGKDGAKGEGDPGENE VQMTGPKGDPGKPGLPGRPGVKGPEGPQGPPG PPGPKGQRGVLSGKVAPDQYFVFSYKKSQKLE KILQDKLVVFDVPLITGIDGVLDGEGYFDVTITG MYIISYQISFQQSACLKIQIGAEEKVKFCDSPKL ILGTAASVVLKLNKGDKVSVQSTGESTVFSRDT DCTFTGFMLFPIK 20 C ACN62223 MFGGHLILVSLLSASLCLCLASADTCPAGAMPG LPGIPGFPGRDGRQGMKGEKGDLGIPIKPGDPTV KKGERGAFGLKGPPGKRGPHGIMGPPGPP GEPGEAGLVDVSGSQLQSAFSVSRHTRIPPDAN KVIRFSKVITNPQGHFSTDESKFVCKIPGTYYFV LHASSHDKKLCVILVHDDKNLVSFCDHTQRGS QQVSSGGLSLYLKENEKVWLMTNALNGMYAT ADRADSVFSGFLIHAH 21

[0056] In some aspects of the previous methods, at least one peptide fragment of the C1q protein comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 20 amino acids.

[0057] In some aspects of the previous methods, at least one peptide fragment of the C1q protein comprises a peptide selected from Table 2. In other aspects, at least one peptide fragment comprises a tryptic peptide of the C1q protein. TABLE 2. C1Q PEPTIDE SEQUENCES C1q Subunit Peptide Sequence SEQ ID NO VGYPGPSGPLGAR A 22 DQPRPAFSAIR* A 23 NPPMGGNVVIFDTVITNQEEPYQNHSGR A 24 FVCTVPGYYYFTFQVLSQWEICLSIVSSSR A 25 SLGFCDTTNK* A 26 GLFVVSGGMVLQLQQGDQVWVEKDPK A 27 GHIYQGSEADSVFSGFLIFPSA A 28 IAFSATR* B 29 TINVPLRR B 30 FDHVITNMNNNYEPR* B 31 VPGLYYFTYHASSR* B 32 GNLCVNLMR B 33 LEQGENVFLQATDK* B 34 FQSVFTVTR C 35 QTHQPPAPNSLIR* C 36 Petition 870260049114, dated 05 / 22 / 2026, page 26 / 84 / 46 Peptide Sequence C1q Subunit SEQ ID NO FNAVLTNPQGDYDTSTGK* C 37 VPGLYYFVYHASHTANLCVLLYR* C 38 VVTFCGHTSK C 39 TNQVNSGGVLLR C 40 * denotes the common human / monkey C1q peptide.

[0058] In some aspects of the previous methods, at least one peptide fragment of the C1q protein comprises SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36). In other aspects, at least one peptide fragment comprises at least two of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36). In still other aspects, at least one peptide fragment comprises each of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) and QTHQPPAPNSLIR (SEQ ID NO: 36).

[0059] Thus, the present disclosure covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises each of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) and QTHQPPAPNSLIR (SEQ ID NO: 36); (2) perform SRM-MS to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising each of the precursor SLGFCDTTNK (SEQ ID NO: 26) transition pair 571.8-942.3, precursor IAFSATR (SEQ ID NO: 29) transition pair 383.1-581.1 and precursor QTHQPPAPNSLIR (SEQ ID NO: 36) transition pair 487.0 -350.3; and (3) determine the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, where the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0060] This disclosure also covers a method that Petition 870260049114, dated 05 / 22 / 2026, page 27 / 84 / 46 comprises an assay comprising: (1) placing a biological sample in contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises each of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) and QTHQPPAPNSLIR (SEQ ID NO: 36); (2) adding to the biological sample at least one labeled synthetic peptide fragment comprising an amino acid sequence identical to the amino acid sequence of each of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) and QTHQPPAPNSLIR (SEQ ID NO: 36); (3) perform SRM-MS to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising each of the precursor SLGFCDTTNK (SEQ ID NO: 26) transition pair 571.8-942.3, precursor IAFSATR (SEQ ID NO: 29) transition pair 383.1-581.1 and precursor QTHQPPAPNSLIR (SEQ ID NO: 36) transition pair 487.0-350.3, and a signal corresponding to at least one labeled synthetic peptide; and (4) determine the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, where the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0061] This disclosure also covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises SLGFCDTTNK (SEQ ID No.: 26); (2) performing selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising Petition 870260049114, dated 05 / 22 / 2026, page 28 / 84 / 46 the precursor SLGFC(Cam)DTTNK (SEQ ID NO: 41) transition pair 571,8942,3; and (3) determine the abundance of at least one C1q peptide fragment by comparing the normalized signal to a standard curve, where the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0062] This disclosure also covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises SLGFCDTTNK (SEQ ID No: 26); (2) adding to the biological sample at least one labeled synthetic peptide fragment comprising an amino acid sequence identical to the amino acid sequence of SLGFCDTTNK (SEQ ID No: 26); (3) performing selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising the precursor SLGFC(Cam)DTTNK (SEQ ID No: 41) transition pair 571.8942.3, and a signal corresponding to at least one labeled synthetic peptide; and (4) determine the abundance of at least one C1q peptide fragment by comparing the normalized signal to a standard curve, where the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0063] This disclosure also covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises IAFSATR (SEQ ID NO: 29); (2) performing reaction monitoring mass spectrometry Petition 870260049114, dated 05 / 22 / 2026, page 29 / 84 / 46 selected (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising the precursor IAFSATR (SEQ ID NO: 29) transition pair 383.1-581.1; and (3) determine the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, wherein the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0064] This disclosure also covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises IAFSATR (SEQ ID NO: 29); (2) adding to the biological sample at least one labeled synthetic peptide fragment comprising an amino acid sequence identical to the amino acid sequence of IAFSATR (SEQ ID NO: 29); (3) performing selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising the precursor IAFSATR (SEQ ID NO: 29) transition pair 383.1-581.1, and a signal corresponding to at least one labeled synthetic peptide; and (4) determine the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, where the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0065] This disclosure also covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises Petition 870260049114, dated 05 / 22 / 2026, page 30 / 84 / 46 (1) perform selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals are in accordance with transition ion pairs comprising the precursor QTHQPPAPNSLIR (SEQ ID NO: 36) transition pair 487.0-350.3; and (2) determine the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, wherein the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0066] This disclosure also covers a method comprising an assay comprising: (1) bringing a biological sample into contact with at least one proteolytic enzyme to produce at least one peptide fragment of the C1q protein present in the biological sample, wherein the at least one peptide fragment comprises QTHQPPAPNSLIR (SEQ ID NO: 36); (2) adding to the biological sample at least one labeled synthetic peptide fragment comprising an amino acid sequence identical to the amino acid sequence of QTHQPPAPNSLIR (SEQ ID NO: 36); (3) performing selected reaction monitoring mass spectrometry (SRM-MS) to generate a signal corresponding to at least one C1q peptide fragment, wherein the SRM-MS signals correspond to transition ion pairs comprising the precursor QTHQPPAPNSLIR (SEQ ID NO: 36) transition pair 487.0-350.3, and a signal corresponding to at least one labeled synthetic peptide;and (4) determine the abundance of at least one C1q peptide fragment by comparing the signal to a standard curve, where the abundance of at least one C1q peptide fragment determines the concentration of C1q in the biological sample.

[0067] In some aspects of the methods of the present disclosure, the selected reaction monitoring mass spectrometry is LC-SRMPetition 870260049114, dated 05 / 22 / 2026, page 31 / 84 / 46 MS / MS.

[0068] In some aspects of the methods of the present disclosure, at least one proteolytic enzyme is trypsin. Other suitable proteolytic enzymes will be known to those skilled in the art, including, but not limited to, Glu-C protease, Lys-N protease, Lys-C protease, Asp-N protease, or chymotrypsin.

[0069] In some aspects of the methods of the present disclosure, a standard curve can be produced using a method comprising: (a) preparing at least two C1q concentration standards by mixing known amounts of purified C1q protein and C1q-depleted serum; (b) adding to the at least two C1q concentration standards at least one synthetic peptide fragment labeled with an amino acid sequence identical to at least one C1q peptide fragment expected to be produced upon contact of the C1q concentration standard with a proteolytic enzyme; (c) bringing the at least two labeled C1q concentration standards into contact with a proteolytic enzyme to produce at least one C1q peptide fragment;(d) perform selected reaction monitoring mass spectrometry to determine the signal strength corresponding to at least one C1q peptide fragment and the signal strength corresponding to at least one labeled synthetic peptide fragment in each of at least two labeled C1q concentration standards; and (e) determine a standard curve using the signals and known amounts of C1q protein.

[0070] In some respects, a standard curve can be produced using at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten C1q concentration standards. In some respects, the preparation of a C1q concentration standard may comprise diluting or serially diluting purified C1q protein in C1q-depleted serum, wherein the Petition 870260049114, dated 05 / 22 / 2026, page 32 / 84 / 46 dilution factor may be 1:1, 1:1.5, or 1:2, or 1:2.5, or 1:3, or 1:3.5, or 1:4, or 1:5, or 1:6, or 1:7, or 1:8, or 1:9, or 1:10, or 1:100, or 1:1000, or any dilution factor within the range of 1:1 to 1:10000.

[0071] In some aspects of the methods of the present disclosure, at least one labeled synthetic peptide fragment may be added to a biological sample before bringing the biological sample into contact with a proteolytic enzyme.

[0072] In some aspects of the present disclosure, at least one labeled synthetic peptide fragment can be used to solve the methods of the present disclosure.

[0073] In some aspects of the present disclosure, the signal corresponding to at least one labeled synthetic peptide fragment can be used to normalize the signal of at least one peptide fragment of the C1q protein to which the labeled synthetic peptide fragment corresponds.

[0074] In some aspects of the methods of the present disclosure, a C1q standard curve can be used to measure the abundance of C1q in biological samples. The abundance of C1q peptides in predetermined standard samples can be defined and the results compared to the LC-SRM-MS results of a corresponding C1q peptide found in a biological sample. This allows the calculation of the abundance of the peptide in the biological sample. Thus, knowing the abundance of a peptide in a sample, the abundance of the protein to which it corresponds is determined. COMPOSITIONS OF THIS PUBLICATION

[0075] Several compositions of the present disclosure are described in detail in this document.

[0076] This disclosure provides a composition comprising at least one isolated synthetic peptide, said composition comprising at least one isolated synthetic peptide with an amino acid sequence selected from the C1q protein. Petition 870260049114, dated 05 / 22 / 2026, pages 33 / 84 / 46

[0077] Synthetic peptides can be generated using any method known in the art. These methods may include recombinant expression techniques, such as expression in bacteria or in vitro expression in eukaryotic cell lysate. These methods may also include solid-phase synthesis.

[0078] Synthetic peptides can be isotopically labeled. Isotopes with which they can be labeled include 13C, 2H, 15N, and 18O. A labeled peptide may comprise at least one 13C-labeled and / or 15N-labeled lysine residue, or at least one 13C-labeled and / or 15N-labeled arginine residue. Peptides may also include a polar solvent. Polar solvents may include water and mixtures of ethanol and water.

[0079] In some aspects of the compositions of the present disclosure, the C1q protein may be the human C1q protein. In other aspects, the C1q protein is the C1q protein of Macaca fascicularis. In yet another aspect, the C1q protein may be from Macaca mulatta. The C1q protein may comprise any of the sequences shown in Table 1.

[0080] In some aspects of a composition of the present disclosure, the at least one isolated synthetic peptide comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 20 amino acids.

[0081] In some aspects of a composition of the present disclosure, an isolated synthetic peptide comprises a sequence of a C1q peptide fragment generated by contact of C1q with a proteolytic enzyme. In preferred aspects, the proteolytic enzyme is trypsin. Thus, in preferred aspects, an isolated synthetic peptide is a C1q tryptic peptide.

[0082] In some aspects of a composition of the present disclosure, an isolated synthetic peptide is labeled. Isolated synthetic peptides may be isotopically labeled. The isotopes with which Petition 870260049114, dated 05 / 22 / 2026, page 34 / 84 / 46, which may be labeled, include, but are not limited to, 13C, 2H, 15N and 18O. Peptides may also include a polar solvent. Polar solvents may include water, mixtures of ethanol and water, and acetonitrile.

[0083] In some aspects of a composition of the present disclosure, the isolated synthetic peptide comprises a peptide selected from Table 2. In other aspects, the composition comprises any two peptides described in Table 2. In other aspects, the composition includes any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 peptides described in Table 2.

[0084] In a preferred aspect, a composition of the present disclosure may comprise at least one isolated synthetic peptide comprising SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36). A composition may comprise at least one isolated synthetic peptide comprising at least two of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36). In still other aspects, a composition may comprise at least one isolated synthetic peptide comprising each of SLGFCDTTNK (SEQ ID NO: 26), IAFSATR (SEQ ID NO: 29) or QTHQPPAPNSLIR (SEQ ID NO: 36).

[0085] In some aspects of the compositions of the present disclosure, the cysteine ​​in the synthetic peptide SLGFCDTTNK (SEQ ID NO: 26) may be modified. The modification may be carbamidomethylation.

[0086] The present disclosure provides a composition comprising at least one pair of transition ions, said composition comprising at least one pair of transition ions of the C1q protein, wherein the at least one pair of transition ions consists of a precursor ion with a corresponding m / z and an ion fragment with a corresponding m / z ion.

[0087] In some aspects of the compositions of this publication, the Petition 870260049114, dated 05 / 22 / 2026, page 35 / 84 / 46: The C1q protein may be the human C1q protein. In other respects, the C1q protein is the C1q protein of Macaca fascicularis. In yet another respect, the C1q protein may be from Macaca mulatta. The C1q protein may comprise any of the sequences shown in Table 1.

[0088] This disclosure provides a composition comprising at least one transition ion pair, said composition comprising at least one transition ion pair of the C1q protein, wherein the at least one transition ion pair consists of a precursor ion with a corresponding m / z and an ion fragment with a corresponding ion m / z, and wherein the transition ion pair is selected from the precursor SLGFC(Cam)DTTNK (SEQ ID NO: 41) transition pair 571.8-942.3, precursor IAFSATR (SEQ ID NO: 29) transition pair 383.1-581.1 and precursor QTHQPPAPNSLIR (SEQ ID NO: 36) transition pair 487.0-350.3. DEFINITIONS

[0089] As used in this document, m / z indicates the mass-to-charge ratio of an ion.

[0090] As used in this document, MS / MS stands for tandem mass spectrometry, which is a type of mass spectrometry involving multiple stages of mass analysis with some form of fragmentation occurring between the stages.

[0091] As used in this document, LC-SRM-MS is an acronym for selected reaction monitoring and may be used interchangeably with LC-MRM-MS or LC-SRM-MS / MS.

[0092] LC-SRM-MS is a highly selective tandem mass spectrometry method that has the potential to effectively filter out all molecules and contaminants except the desired analyte(s). This is particularly beneficial if the sample being analyzed is a complex mixture that may comprise several isobaric species within Petition 870260049114, dated 05 / 22 / 2026, p. 36 / 84 / 46 a defined analytical window. LC-SRM-MS methods can use a triple quadrupole mass spectrometer which, as is known in the art, includes three sets of quadrupole rods. A first mass selection stage is performed in the first quadrupole rod set, and the selectively transmitted ions are fragmented in the second quadrupole rod set. The resulting transition (product) ions are transported to the third quadrupole rod set, which performs a second mass selection stage. The product ions transmitted through the third quadrupole rod set are measured by a detector, which generates a signal representative of the number of selectively transmitted product ions.The RF and DC potentials applied to the first and third quadrupoles are adjusted to select (respectively) precursor and product ions that have m / z values ​​within specified narrow ranges. By specifying the appropriate transitions (m / z values ​​of precursor and product ions), a peptide corresponding to a targeted protein can be measured with high degrees of sensitivity and selectivity. The signal-to-noise ratio in LC-SRM-MS is often higher than conventional tandem mass spectrometry (MS / MS) experiments, which do not selectively target (filter) specific analytes but aim to search for all analytes in the sample.

[0093] LC-SRM-MS mass spectrometry involves gas-phase ion fragmentation and occurs between different stages of mass analysis. There are many methods used to fragment ions, and these can result in different types of fragmentation and therefore different information about the structure and composition of the molecule. The transition ions observed in an LC-SRM-MS spectrum result from several different factors, which include, but are not limited to, primary sequence, the amount of internal energy, the means of energy introduction, and the charge state. Transitions must carry at least one charge to be Petition 870260049114, dated 05 / 22 / 2026, page 37 / 84 / 46 detected. An ion is categorized as a, b, or c if the charge is in a transition comprising the original N-terminal of the peptide, while the ion is categorized as x, y, or z if the charge is in a transition comprising the original C-terminal of the peptide. A subscript indicates the position of the residues in the transition (e.g., first peptide residue at xi of the C-terminal, second peptide residues at y2 of the C-terminal, and third peptide residues at z3 of the C-terminal, etc.).

[0094] In a generic peptide repeating unit represented -NC(O)-C-, an x- ion and an ab ion result from the cleavage of the carbonyl-carbon bond (i.e., C(O)-C). The x- ion is an acylium ion and the ab ion is an iminium ion. A y- ion and an ab ion result from the cleavage of the carbonyl-nitrogen bond (i.e., C(O)-N, also known as an amide bond). In this case, the y- ion is an ammonium ion and the b- ion is an acylium ion. Finally, the az- ion and the ac- ion result from the cleavage of the nitrogen-carbon bond (i.e., CN-). The z- ion is a carbocation and the ab ion is an ammonium ion.

[0095] Superscripts are sometimes used to indicate neutral losses in addition to main chain fragmentation, for example, * for ammonia loss and ° for water loss. In addition to protons, β- and γ- ions can abstract an additional proton from the precursor peptide. In electrospray ionization, tryptic peptides can carry more than one charge.

[0096] Internal transitions arise from the cleavage of the double main chain. These can be formed by a combination of β-type cleavage and γ-type cleavage (i.e., cleavage producing βγγ ions). Internal cleavage ions can also be formed by a combination of αβ-type cleavage and γ-type cleavage. An internal transition with a single side chain formed by a combination of αβ-type cleavage and γ-type cleavage is called an iminium ion (sometimes also known as an immonium or imonium ion). These ions are labeled with a one-letter code for the corresponding amino acid.

[0097] Low-energy CID (i.e., dissociation induced by Petition 870260049114, dated 05 / 22 / 2026, page 38 / 84 / 46 collision in a triple quadrupole or an ion trap) involves the fragmentation of a peptide carrying a positive charge, mainly along its structure, to generate primarily α, β and γ ions.

[0098] One or more liquid chromatography (LC) purification steps are performed before a subsequent LC-SRM-MS analysis step. Traditional LC analysis relies on chemical interactions between sample components and column packing materials, where laminar flow of the sample through the column is the basis for the separation of the analyte of interest from the test sample. A person skilled in the art will understand that separation in such columns is a diffusion process. A variety of column packing materials are available for the chromatographic separation of samples, and the selection of an appropriate separation protocol is an empirical process that depends on the characteristics of the sample, the analyte of interest, the interfering substances present and their characteristics, etc. Various packing chemicals can be used depending on the needs (e.g., structure, polarity, and solubility of the compounds being purified).In many respects, columns are C2, C-8, C-18 polar, ion-exchange (cation and anion), hydrophobic interaction, phenyl, polar-coated porous polymer, or other commercially available types. During chromatography, the separation of materials is affected by variables such as eluent choice (also known as mobile phase), elution gradient choice and gradient conditions, temperature, etc. In certain respects, an analyte can be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these respects, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove it. Petition 870260049114, dated 05 / 22 / 2026, page 39 / 84 / 46, the material retained from the column, since the non-retained materials are washed away. Alternatively, an analyte can be purified by applying a sample to a column under mobile phase conditions, where the analyte of interest elutes at a differential rate compared to one or more other materials. As discussed above, such procedures can enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

[0099] The following parameters are used to specify a test. LC-SRM-MS of a protein under a particular LC-SRM-MS system: (1) a tryptic peptide of the protein; (2) the retention time (TR) of the peptide; (3) the m / z value of the peptide precursor ion; (4) the decompression potential used to ionize the precursor ion; (5) the m / z value of an ion fragment generated from the peptide precursor ion; and (6) the collision energy (EC) used to fragment the peptide precursor ion that is optimized for the specific peptide.

[00100] As used herein, PPI refers to internal pattern peptides.

[00101] To facilitate the precise quantification of peptide transitions by the methods disclosed herein, a set of isotopically labeled synthetic versions of the peptides of interest may be added in known quantities to the sample for use as internal standards. Since isotopically labeled peptides have identical physical and chemical properties to the corresponding surrogate peptide, they co-elute from the chromatographic column and are easily identifiable in the resulting mass spectrum. The addition of the labeled standards may occur before or after proteolytic digestion. The methods of synthesizing isotopically labeled peptides will be known to those skilled in the art. Thus, in some respects, experimental samples contain internal standard peptides. Other respects may use external standards or other expedients. Petition 870260049114, dated 05 / 22 / 2026, pages 40 / 84 / 46 for peptide quantification.

[00102] As used herein, a tryptic peptide refers to the peptide that is formed by treating a protein with trypsin.

[00103] As used in this document, the term standard curve may be used interchangeably with the term calibration curve.

[00104] As used in this Specific Report and the accompanying claims, the singular forms a and the include plural referents, unless the context clearly dictates otherwise.

[00105] Unless specifically stated or obvious from the context, as used in this document, the term "or" is understood to be inclusive and covers both "or" and "and".

[00106] Unless specifically stated or obvious from the context, as used in this document, the term "approximately" is understood to mean within a normal tolerance range in the technique, for example, within 2 standard deviations of the mean. "Approximately" may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​given in this document are modified by the term "approximately".

[00107] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by someone skilled in the art to which the invention pertains. Although other probes, compositions, methods, and kits similar or equivalent to those described in this document may be used in the practice of the present disclosure, the preferred materials and methods are described in this document. It should be understood that the terminology used in this document is for the purpose of describing particular aspects only and is not intended to be limiting. EXAMPLES: Petition 870260049114, dated 05 / 22 / 2026, p. 41 / 84 / 46 EXAMPLE 1 - DETERMINING A STANDARD CONCENTRATION CURVE FOR C1Q USING THE METHODS OF THIS DISCLOSURE

[00108] The methods of this disclosure were used to generate standard curves, also called calibration curves, using a set of C1q reference solutions of known C1q concentrations, including C1q standard and quality control (QC) samples. The sensitivity and accuracy of the methods of this disclosure were also tested.

[00109] Calibration curves were generated using standard C1q samples that were prepared and applied using the following guidelines: • The purified C1q protein was diluted in the same biological matrix as the experimental samples; • The set of C1Q reference solutions tested consisted of a double blank, a blank, and at least 6 zero-free concentrations of C1q; • The lower limit of quantification (LLOQ) is defined as the concentration of the C1q protein at which the measured response of the LLOQ sample is at least 5 times greater compared to the response of the blank sample, such that the precision is within 25% of the nominal concentration and the coefficient of variation is less than 25%; • The upper limit of quantification (ULOQ) is defined such that the precision is within 20% of the nominal concentration and the coefficient of variation is less than 20%.

[00110] Calibration curves were generated using C1q QC samples that were prepared and applied using the following guidelines: • at least 3 concentrations of QC samples were prepared and Petition 870260049114, dated 05 / 22 / 2026, pages 42 / 84 / 46 used for calibration; • Each QC sample was prepared in duplicate; • The QC samples covered the low, medium, and high quantification range of the assay, and the low QC (LQC) sample was within 3 times the concentration of LLOQ; • At least 67% of the QC samples had an accuracy within 20% of the nominal concentration; • The accuracy of at least 50% of the QC samples at each level was within 20% of the nominal concentration; • The minimum number of QC samples was equal to the greater of at least 5% of the number of unknown samples or 6 total QC samples; • The QC samples were prepared with a C1q stock solution that was separated from the stock solution for the preparation of the C1q reference solutions. MATERIALS: • Purified Human Complement Protein C1q (Quidel, item # A400) • Human Complement C1q Depleted Serum (Sigma-Aldrich, Cat# 234401-1ML) • SLGFC(Cam)DTTNK (SEQ ID NO: 41) (New England Peptide) • IAFSATR (SEQ ID NO: 29) (New England Peptide) • QTHQPPAPNSLIR (SEQ ID NO: 36) (New England Peptide) • Bispecific Monoclonal Antibody Drug Candidate • Sequencing-grade modified trypsin supplied with resuspension buffer (Promega, Cat# V5117) • UltraPure 1.0 M Tris-HCl pH 7.5 (Invitrogen, Cat# 15567-027) • UltraPure 1.0 M Tris-HCl pH 8.0 (Invitrogen, Cat# 15568-025) • UltraPure 1.0 M Tris-HCl pH 8.5 (Alfa Aesar, Cat# J61038) Petition 870260049114, dated 05 / 22 / 2026, p. 43 / 84 / 46 • Urea (Sigma Aldrich, Cat# U5128-100G) • TCEP HCl (Thermo Scientific; Cat# 20491) • Iodoacetamide (Sigma-Aldrich; Cat# A3221-10VL) • Formic acid (Thermo Scientific; Cat# 28905) • Acetonitrile (Fisher Chemical, Cat# A955-4) • C18 BEH130 ACQUITY UPLC column, 1.7 gm, 2.1 mm x 50 mm (Águas, Part# 1860035554) • 96-well plate, 0.5 mL, polypropylene (Agilent, Part# 50421386) • 25 mL disposable reagent reservoir (VistaLab, Part# 3054-1004) TripleQuad Mass Spectrometer (Agilent, Model # 6495) • Infinity II LC 1290 System (Agilent, Model # 1290) SAMPLE PREPARATION

[00111] A 200 pg / mL C1q stock solution was prepared in C1q-depleted human serum and assay dilution buffer (ADB) containing 20 pg / mL of the bispecific monoclonal antibody drug candidate. The C1q stock solution was serially diluted 1 to 3, six times to prepare six C1q standard solutions (L6-L1). The LLOQ (lower limit of quantification), QC (lower limit of quantification), QC (medium limit of quantification), and ULOQ (upper limit of quantification) samples were prepared in ADB independently of the C1q stock solution. An aliquot of ADB was reserved as an L0 (blank) sample. The concentrations of the C1q standard and QC solutions are listed in Table 3. TABLE 3. CONCENTRATIONS OF THE C1Q STANDARD AND THE QC SOLUTIONS. Level / QC Concentration (μg / mL) L1 / LLOQ 0.27 L2 0.82 L3 2.47 L4 7.41 L5 22.22 L6 / ULOQ 66.67 Petition 870260049114, dated 05 / 22 / 2026, page 44 / 84 / 46 Level / QC Concentration (μg / mL) LQC 0.78 MQC 6.25 HQC 50

[00112] The following isotope-labeled internal standard peptides (IPSPs) were reconstituted to 6-12 mM in 30% acetonitrile in 0.1% formic acid to create isotope-labeled IPSP solutions: SLGFC(Cam)DTTNK (SEQ ID NO: 41), IAFSATR (SEQ ID NO: 29) and QTHQPPAPNSLIR (SEQ ID NO: 36).

[00113] Each C1q sample was diluted 50 times in 100 mM Tris-HCl, pH 7.5, and 20 pg / mL of the bispecific antibody. 5 pL of each diluted C1q sample were then denatured and reduced in 20 pL of 8 M urea and 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 56 °C with stirring for 30 minutes. 5 pL of 50 mM iodoacetamide were then added to each sample, and the samples were then incubated in the dark at 25 °C with stirring for 30 minutes. 10 pL of the appropriate isotope-labeled PPI solution were then added to each sample. After the addition of the PPIs, 100 pL of 0.01 pg / pL trypsin were also added to each sample. The samples were then incubated at 37 °C in the dark with shaking for 4 hours. 5 µL of 20% formic acid were added to the samples to quench the tryptic digestion reaction. The samples were mixed and centrifuged at 4680 rpm for 5 minutes before being analyzed by LC-SRM-MS / MS. LC-SRM-MS ANALYSIS

[00114] LC-SRM-MS analysis was performed on a TripleQuad mass spectrometer (Agilent, model 6495) with an LC 1290 Infinity II system (Agilent, model #1290). The LC gradient used is described in Table 4, where Buffer A consisted of 0.1% formic acid in water and Buffer B consisted of 0.1% formic acid in acetonitrile. TABLE 4. LC GRADIENT Time (minutes) Buffer A (%) Buffer B (%) Flow (mL / minute) 0.5 97 3 0.4 8.0 75 25 0.4 8.1 10 90 0.4 10.5 10 90 0.4 Petition 870260049114, dated 05 / 22 / 2026, pages 45 / 84 / 46 Time (minutes) Buffer A (%) Buffer B (%) Flow (mL / minute) 10.6 97 3 0.4 13.0 97 3 0.4

[00115] SRM-MS analysis simultaneously monitored native C1q peptide fragments and isotope-labeled peptides in the samples. Peak areas for two transitions (native and heavy marker) were collected and reported for both native and isotope-labeled C1q peptides. For each C1q standard and QC samples, the data output for the C1q protein analyzed by LC-SRM-MS yielded six measurements consisting of two transition measurements (native and heavy marker) of each of the three selected peptides presented in Table 5 below. TABLE 5. M / ZEA TRANSITION COLLISION ENERGY OF C1Q TARGET PEPTIDES._______.________.___________.______________. C1q Subunit Peptide Sequence SEQ ID NO m / z Transition Collision Energy (V) A SLGFC(Cam)DTTNK 41 571.8 > 942.3 18 B IAFSATR 29 383.1 > 581.1 10 C QTHQPPAPNSLIR 36 487.0 > 350.3 13

[00116] Each of the three peptides in Table 5 is from a different C1q subunit. The peptide fragment derived from subunit B was used as the quantification peptide (referred to in this document as the subunit B peptide), and the peptide fragments derived from subunits A (referred to in this document as the subunit A peptide) and C (referred to in this document as the subunit C peptide) were used as confirmation peptides. The isotope-labeled PPIs have amino acid sequences that are identical to each of the three selected peptides and are referred to in this document as the isotope-labeled subunit A control peptide, the isotope-labeled subunit B control peptide, and the isotope-labeled subunit C control peptide.

[00117] The three peptides listed in Table 5 were selected based on previous results that showed these peptides were the best peptides for quantifying C1q concentration in an LCSRM-MS / MS assay. The selection of these peptides was partly based on Petition 870260049114, dated 05 / 22 / 2026, page 46 / 84 / 46 conservation of peptide sequence between humans and macaques (Macaca fascicularis). Figure 1 shows a sequence alignment of the C1q A subunit from human, macaque (Macaca fascicularis), mouse, and rat highlighting the A subunit peptide. Figure 2 shows a sequence alignment of the C1q B subunit from human, macaque (Macaca fascicularis), mouse, and rat highlighting the B subunit peptide. Figure 3 shows a sequence alignment of the C1q C subunit from human, macaque (Macaca fascicularis), mouse, and rat highlighting the C subunit peptide. Several tryptic peptides from each of the C1q A, B, and C subunits were initially tested. The tested peptides are listed in Table 2. RESULTS

[00118] Each standard C1q sample and each QC C1 sample were analyzed using LC-SRM-MS / MS. For each sample, 6 signals were recorded: the signal corresponding to the native peptide of subunit A, the signal corresponding to the native peptide of subunit B, the signal corresponding to the native peptide of subunit C, the signal corresponding to the isotope-labeled control peptide of subunit A, the signal corresponding to the isotope-labeled control peptide of subunit B, and the signal corresponding to the isotope-labeled control peptide of subunit C. The data for isotope-labeled peptides were used as internal controls for assay performance troubleshooting purposes.

[00119] For each of the three selected peptides (the subunit A peptide, the subunit B peptide, and the subunit C peptide), a calibration curve was generated by plotting the normalized LC-SRM-MS signal recorded from standard C1q samples against the corresponding nominal concentrations of those samples. Figure 4 shows the calibration curve generated using the signal corresponding to the peptide of Petition 870260049114, dated 05 / 22 / 2026, page 47 / 84 / 46 subunit A. Figure 5 shows the calibration curve generated using the signal corresponding to the peptide of subunit B. Figure 6 shows the calibration curve generated using the signal corresponding to the peptide C of the subunit. For Figures 4 to 6, the black dots represent standard samples of C1q at concentrations of L1-L6. The blue triangles represent QC samples of C1q at concentrations of LLOQ, LQC, MQC, HQC, and ULOQ.

[00120] After generating the calibration curve, the concentrations of the QC samples were then determined by comparing the LC-SRM-MS / MS signal of each of the three target peptides in the QC samples with the corresponding calibration curve. The accuracy of the assay was evaluated by comparing the determined concentrations with the nominal concentrations of the QC samples. The results of the comparison are shown in Tables 6-8. TABLE 6. ACCURACY OF THE ASSAY USING THE TARGET PEPTIDE SLGFC(CAM)DTTNK (SEQ ID NO: 41) DERIVED FROM SUBUNIT A DE C1Q. Standards L1 L2 L3 L4 L5 L6 Nominal Concentration (ng / mL) 0.27 0.82 2.47 7.41 22.22 66.67 Precision (%) 10³ 91 10⁴ 100 10⁴ 99 QC (N=3) LLOQ LQC MQC HQC ULOQ Nominal Concentration (ng / mL) 0.27 0.78 6.25 50 66.67 Precision (%) - Set 1 96 99 97 95 97 Precision (%) - Set 2 10² 94 100 96 10² Precision (%) - Set 3 71 10¹ 10¹ 97 10⁴ % of RSD (N=3) 19% 4% 2% 1% 4% TABLE 7. ASSAY ACCURACY USING THE TARGET PEPTIDE IAFSATR (SEQ ID NO: 29) DERIVED FROM SUBUNIT B OF C1Q. Standard L1 L2 L3 L4 L5 L6 Nominal Concentration (ng / mL) 0.27 0.82 2.47 7.41 22.22 66.67 Precision (%) 101 95 106 98 99 100 QC (N=3) LLOQ LQC MQC HQC ULOQ Nominal Concentration (ng / mL) 0.27 0.78 6.25 50 66.67 Precision (%) - Set 1 98 95 97 105 96 Precision (%) - Set 2 94 94 100 106 111 Precision (%) - Set 3 99 94 95 109 113 % of RSD (N=3) 3% 1% 3% 2% 9% TABLE 8. CALIBRATION OF THE C1Q TEST BASED ON TARGET PEPTIDE QTHQPPAPNSLIR (SEQ ID NO: 36) DERIVED FROM Petition 870260049114, dated 05 / 22 / 2026, page 48 / 84 / 46 C1Q SUBUNIT C. Standard L1 L2 L3 L4 L5 L6 Nominal Concentration (ng / mL) 0.27 0.82 2.47 7.41 22.22 66.67 Precision (%) 101 95 103 100 101 100 QC (N=3) LLOQ LQC MQC HQC ULOQ Nominal Concentration (μg / mL) 0.27 0.78 6.25 50 66.67 Precision (%) - Set 1 98 98 99 106 99 Precision (%) - Set 2 104 101 103 105 110 Precision (%) - Set 3 93 97 102 107 111 % of RSD (N=3) 6% 2% 2% 1% 6%

[00121] These results demonstrate that the assay is accurate and sensitive. They also demonstrate that the use of the IAFSATR peptide (SEQ ID NO: 29) derived from the B subunit of C1q provided the best results, as there was a high response recorded by LC-SRM-MS / MS and the signal was free from background interference. LLOQ AND LIMIT OF DETECTION (LOD)

[00122] As shown in the left and middle panels of Figure 7, the mass chromatogram recorded at the LLOQ concentration (0.27 pg / mL) for the subunit A peptide and the subunit B peptide exhibited little or no background peak located at the same acquisition time as the peptide peaks in the blank and double-blank samples. However, as shown in the right panel of Figure 7, the mass chromatogram recorded for the subunit C peptide exhibited background peaks in both the blank and double-blank samples. The area under the curve for the background peak in the blank sample was approximately 5% of the area under the curve for the sample peak. Overall, as shown in Figure 8, the signal-to-noise ratios for the subunit A peptide, the subunit B peptide, and the subunit C peptide at the LLOQ concentration were 51, 36, and 94, respectively, and at the limited detection concentration (LOD) were 7, 9, and 6, respectively. EXAMPLE 2 - TRANSPORT OF TEST INSTRUMENTS FOR THE METHODS OF THIS DISCLOSURE

[00123] How the methods of this disclosure can be used Petition 870260049114, dated 05 / 22 / 2026, page 49 / 84 / 46: To perform consecutive experiments on the same instrument, it is important to ensure that transporting the last sample will not interfere with the test for the next sample. Instrument transport during the practice of the methods in this disclosure was measured.

[00124] An LC-SRM-MS chromatogram was first recorded for a blank preparation sample. Immediately afterwards, a mass chromatogram for a QC sample of C1q at the ULOQ concentration (66.7 pg / mL) was recorded on the same instrument. Finally, a second blank preparation sample was analyzed on the same instrument after the analysis of the ULOQ sample. As shown in Figure 9, there was no significant change in the blank preparation chromatograms before or after analyzing the ULOQ sample for the peptide subunit A, subunit B, or subunit C. These results demonstrate that instrument portability for the methods of the present disclosure is minimal. EXAMPLE 3 - TESTING THE DRUG TOLERANCE OF THE METHODS IN THIS DISCLOSURE

[00125] To examine whether the presence of antibody drugs interferes with the methods of this disclosure, different concentrations (0.20 pg / mL or 2000 pg / mL) of a bispecific antibody were added to C1q reference samples at the Double Blank concentration (blank matrix only, no internal standard; L00). As shown in Figure 10, the addition of the bispecific antibody did not produce any significant changes in the recorded mass chromatogram.

[00126] Reference C1q samples at LQC, MQC, and HQC concentrations (0.8 pg / mL, 6.3 pg / mL, and 50.0 pg / mL, respectively) were incubated in the absence of the bispecific antibody or with 0 pg / mL, 20 pg / mL, 40 pg / mL, or 2000 pg / mL of the bispecific antibody and analyzed using LCSRM-MS / MS. These bispecific antibody concentrations in the assay of Petition 870260049114, dated 05 / 22 / 2026, page 50 / 84 / 46 C1q corresponded to 1 mg / mL, 2 mg / mL, or 100 mg / mL of the bispecific antibody in pure serum. To place these concentrations in the context of pharmacokinetics, when administered at a dosage of 50 mg / kg, the maximum serum concentration (Cmax) of the bispecific antibody is 1.25–1.5 mg / mL. As shown in Figure 11, the addition of the bispecific antibody actually enhances the signal recovery for the subunit A, subunit B, and subunit C peptides. In Figure 11, the first pink bar in each group corresponds to samples incubated in the absence of the bispecific antibody; the yellow bar, or the second bar in each group, corresponds to samples incubated with 20 pg / mL of the bispecific antibody; the green bar, or the third bar in each group, corresponds to samples incubated with 40 pg / mL of the bispecific antibody; The blue bar, or the fourth bar in each group, corresponds to samples incubated with 2000 pg / mL of the bispecific antibody. EXAMPLE 4 - TESTING THE DILUTION RECOVERY AND DILUTION LINEARITY OF THE METHODS IN THIS DISCLOSURE

[00127] The recovery of the endogenous C1q signal in the methods of this disclosure was also tested in samples diluted in different diluents and with different dilution factors. The diluents tested included 2% depleted human serum incubated with 20 pg / mL of the bispecific antibody, 2% depleted human serum, 0.1% BSA, and a Tris-HCl solution. These samples were diluted 20x, 50x, and 100x, and LC-SRM-MS / MS was used to analyze the dilutions. As shown in Figure 12, the addition of the bispecific antibody enhances the recovery of peptide signals from subunit A, subunit B, and subunit C, even at higher dilution factors. There was less signal recovery in the samples diluted with 0.1% BSA and Tris-HCl. In Figure 12, the blue or first bar in each group corresponds to samples diluted with 2% depleted human serum. Petition 870260049114, dated 05 / 22 / 2026, p. 51 / 84 / 46 incubated with 20 pg / mL of the bispecific antibody; the orange bar or second in each group corresponds to samples diluted with 2% depleted human serum; the green bar or third in each group corresponds to samples diluted with 0.1% BSA; the purple bar or fourth in each group corresponds to samples diluted with Tris-HCl.

[00128] The recovery of C1q reference pattern signals using different diluents was also tested. C1q reference samples at LQC, MQC, and HQC concentrations (0.8 pg / mL, 6.3 pg / mL, and 50.0 pg / mL, respectively) were diluted with 2% depleted human serum incubated with 20 pg / mL of the bispecific antibody, 2% depleted human serum, or 0.1% BSA. As shown in Figure 13, the bispecific antibody improved the recovery of peptide signals from subunit A, subunit B, and subunit C. In Figure 13, the blue or first bar in each group corresponds to samples diluted with 2% depleted human serum incubated with 20 pg / mL of the bispecific antibody; the orange or second bar in each group corresponds to samples diluted with 2% depleted human serum; The green bar, or the third bar in each group, corresponds to samples diluted with 0.1% BSA.

[00129] To test the dilution linearity of the methods in this disclosure, pooled samples of human serum, male monkey serum, and female macaque serum were diluted 20-fold, 50-fold, and 100-fold. The concentrations of endogenous C1q in these diluted samples were determined using the methods in this disclosure. The results of this test are shown in Table 9 below. TABLE 9. DILUTION LINEARITY TEST WITH C1Q ENDOGENOUS.________________________________________ Sample Dilution factor By C1Q-A By C1Q-B By C1Q-C Cal. Conc. (ug / mL) % RSD Cal. Conc. (ug / mL) % RSD Cal. Conc. (ug / mL) % RSD Serum 100X 89 80 78 Human 50X 70 12.4% 72 5.3% 70 6.0% Pooled 20X 87 74 74 Petition 870260049114, dated 05 / 22 / 2026, page 52 / 84 / 46 Sample Dilution Factor By C1Q-A By C1Q-B By C1Q-C Cal. Conc. (ug / mL) % of RSD Cal. Conc. (ug / mL) % of RSD Cal. Conc. (ug / mL) % of RSD Male Monkey 100X 50X 20X 87 81 96 8.7% 59 58 58 0.8% 61 60 58 2.7% Female Monkey 100X 50X 20X 70 67 69 2.1% 56 49 54 6.2% 55 50 59 8.7% EXAMPLE 5 - REPEATABILITY OF SAMPLE PREPARATION AND SAMPLE STABILITY IN PRESENT METHODS DISCLOSURE

[00130] The repeatability of sample preparation of the methods in this disclosure was tested using QC samples of C1q at LLOQ, LQC, MQC, HQC, and ULOQ concentrations. For injection repeatability, aliquots of the same QC sample were injected into the assay instrument on the same day (intraday) or on different days (interday). For sample preparation repeatability, samples were prepared from QC solutions on the same day (intraday) or on different days (interday). Three samples for each condition were tested, and the relative standard deviation of the three samples for each condition is shown in Table 10 below. TABLE 10. REPEATABILITY OF INJECTION AND PREPARATION OF SAMPLE OF THE METHODS OF THE PRESENT DISCLOSURE C1Q QC (N=3) % of RSD Intraday Injector Interday Injector Intraday Sample Preparation Interday Sample Preparation LLOQ 0.6 10.2 3.0 14.0 LQC 7.8 7.1 9.2 13.7 C1Q-A MQC 1.2 5.4 2.7 4.3 HQC 1.2 1.3 0.9 1.3 ULOQ 2.5 2.0 2.0 2.0 LLOQ 4.8 5.0 0.8 6.0 LQC 4.4 9.3 2.0 4.6 C1Q-B MQC 1.8 6.2 1.5 3.9 HQC 0.5 2.0 1.2 1.8 ULOQ 0.4 0.8 1.2 1.7 LLOQ 6.8 6.2 4.6 5.6 LQC 1.3 1.6 3.3 5.6 C1Q-C MQC 2.3 2.0 3.2 2.9 HQC 1.3 2.2 1.6 2.5 ULOQ 1.0 1.9 1.9 2.7 Petition 870260049114, dated 05 / 22 / 2026, pages 53 / 84 / 46

[00131] For sample stability, QC samples of C1q at LLOQ, LQC, MQC, HQC, and ULOQ concentrations (0.3 μg / mL, 0.8 pg / mL, 6.3 μg / mL, 50.0 μg / mL, and 66.7 pg / mL, respectively) were subjected to three freeze-thaw cycles or stored in an autosampler for 48 hours before their C1q concentrations were determined by the C1q assay. As shown in Figure 14, the accuracy of the methods in this disclosure was not significantly affected by three freeze-thaw cycles or by 48 hours of storage in an autosampler. In the top row of the graphs in Figure 14, the first bar in each group corresponds to samples that were recently analyzed before freezing; the second bar in each group corresponds to samples that were subjected to three freeze-thaw cycles.In the bottom row of the graphs in Figure 14, the first bar in each group corresponds to samples that were recently analyzed; the second bar in each group corresponds to samples that were analyzed after 48 hours of storage in an autosampler.

[00132] In separate experiments, 72 hours of storage were also tested, and no degradation or loss of the sample was observed. EXAMPLE 6 - ASSAY VARIATION RELATED TO INTERNAL STANDARD PEPTIDES IN METHODS OF THE PRESENT DISCLOSURE.

[00133] Peptides labeled with heavy isotopes; called internal standard peptides (IPSs) have amino acid sequences identical to the peptides of subunit A, subunit B and subunit C. QC samples of C1q at concentrations of LLOQ, LQC, MQC, HQC and ULOQ (0.3 μg / mL, 0.8 pg / mL, 6.3 μg / mL, 50.0 μg / mL and 66.7 pg / mL, respectively) were analyzed in the presence and absence of each IPS. The results of this analysis are shown in Table 11. The inclusion of IPSs did not interfere with the analysis. Petition 870260049114, dated 05 / 22 / 2026, page 54 / 84 / 46 Therefore, isotope-labeled peptides were used for retention time confirmation, instrument performance calibration, and troubleshooting. TABLE 11. VARIATION OF THE C1Q ASSAY WITH OR WITHOUT ISOTOPE-LABELED INTERNAL STANDARD PEPTIDES QC (N=3) % RSD of QC (n=3) C1QA - PPI C1QB + PPI - PPI C1QC + PPI - PPI + PPI LLOQ 8% 12% 4% 2% 2% 8% LQC 11% 11% 4% 2% 2% 1% MQC 2% 6% 3% 5% 2% 4% HQC 2% 6% 1% 4% 0% 8% ULOQ 2% 3% 1% 1% 2% 7% EXAMPLE 6 - QUANTIFICATION OF C1Q IN BLOOD SAMPLES FROM MONKEYS TREATED USING THE METHODS OF THIS DISCLOSURE.

[00134] The methods of this disclosure were used to quantify the concentration of the C1q protein present in blood samples from monkeys treated with a bispecific antibody. The monkey group designation and dose levels are shown in Table 12. TABLE 12. DESIGNATION OF THE MONKEY GROUP AND LEVELS OF DOSE___________________________________________ Group Number of animals (males) Dose level (mg / kg) Dose concentration (mg / mL) 1 (isotype control) 3 50 25 2 (low) 3 2 1 3 (medium) 3 10 5 4 (high) 3 50 25

[00135] Group 1 was administered a diluted isotype control antibody via slow bolus intravenous injection at a dose volume of 2 mL / kg. Groups 2, 3, and 4 received a diluted bispecific antibody via slow bolus intravenous injection at a dose of 2 mL / kg. 0.5 mL blood samples were collected according to the following schedule: pre-dose sample and approximately 5 minutes after the dose sample were collected on Day 1; subsequent samples were collected 24 hours, 72 hours, and 168 hours after the dose; samples also Petition 870260049114, dated 05 / 22 / 2026, page 55 / 84 / 46 samples were collected once on day 14 after the dose, day 42 after the dose, and day 56 after the dose. Blood samples were centrifuged for 1 hour after collection, and the collected serum samples were divided into 4 aliquots of 50 pL each.

[00136] Each monkey serum sample was diluted 50 times in 100 mM Tris-HCl, pH 7.5, and 20 pg / mL of the bispecific antibody. 5 pL of each diluted monkey serum sample were then denatured and reduced in 20 pL of 8 M urea and 10 mM tris(2-carboxyethyl)phosphine (TCEP) at 56 °C with shaking for 30 minutes. 5 pL of 50 mM iodoacetamide were then added to each sample, and the samples were then incubated in the dark at 25 °C with shaking for 30 minutes. 10 pL of the appropriate isotope-labeled internal standard peptide solution (see Example 1) were added before 100 pL of 0.01 pg / pL trypsin were added to each sample. The samples were then incubated at 37 °C in the dark with shaking for 4 hours. 5 µL of 20% formic acid were added to the samples to quench the tryptic digestion reaction. The samples were mixed and centrifuged at 4680 rpm for 5 minutes before being analyzed by LCSRM-MS / MS.

[00137] For each monkey serum sample, LC-SRM-MS / MS was used to record the signal corresponding to the subunit A peptide, the subunit B peptide, and the subunit C peptide, as well as the signals corresponding to the internal pattern peptides labeled with isotopes.

[00138] The C1q concentrations in each of the measured monkey serum samples were then determined by comparing the signals of the subunit A, subunit B, and subunit C peptides with calibration curves (generated as described in Example 1). The C1q concentrations, as determined by the subunit A peptide, subunit B peptide, and subunit C peptide, are presented in Tables 1315. The post-dose time course of C1q concentrations in blood of Petition 870260049114, dated 05 / 22 / 2026, page 56 / 84 / 46 monkeys are represented in Figures 15-17. Figure 15 shows the concentration of C1q in monkey dose samples quantified using the A subunit peptide. Figure 16 shows the concentration of C1q in monkey dose samples quantified using the B subunit peptide. Figure 17 shows the concentration of C1q in monkey dose samples quantified using the C subunit peptide. In Figures 15-17, the blue line corresponds to monkeys in Group 1, the red line corresponds to monkeys in Group 2, the green line corresponds to monkeys in Group 3, and the purple line corresponds to monkeys in Group 4. TABLE 13. QUANTIFICATION OF MONKEY BLOOD SAMPLES MEASURED FOR C1Q CONCENTRATION BY THE TARGET PEPTIDE SLGFCDTTNK (SEQ ID NO: 26) DERIVED FROM THE A SUBUNIT OF C1Q. Time Point Concentration of C1q by subunit A (pg / mL) Group 1 (isotype control) Group 2 (low) Group 3 (medium) Group 4 (high) P0001 P0002 P0003 P0101 P0102 P0103 P0201 P0202 P0203 P0301 P0302 P0303 Pre-dose 124 87 102 89 93 76 114 120 90 59 92 92 5 min 102 88 94 78 81 72 90 91 74 43 70 67 24 h 118 83 108 78 89 74 84 102 66 4* 58 9* 72 h 112 76 96 80 79 64 93 96 67 5* 53 13* 168 h 116 80 96 81 86 79 106 106 81 8* 67 28 D14 105 77 99 78 100 70 120 93 79 25 71 63 D42 106 81 96 74 103 80 85 106 91 56 83 99 D56 109 91 96 79 104 74 105 117 92 53 86 94 * An estimated C1q concentration. Concentration below LLOQ (0.27 μg / mL), but above LOD (0.027 pg / mL). TABLE 14. QUANTIFICATION OF MONKEY BLOOD SAMPLES MEASURED FOR C1Q CONCENTRATION BY THE TARGET PEPTIDE IAFSATR (SEQ ID NO: 29) DERIVED FROM THE B SUBUNIT OF C1Q._________________________________ Time Point Concentration of C1q by subunit B (pg / mL) Group 1 (isotype control) Group 2 (low) Group 3 (medium) Group 4 (high) P0001 P0002 P0003 P0101 P0102 P0103 P0201 P0202 P0203 P0301 P0302 P0303 Pre-dose 99 72 88 69 79 67 90 93 67 49 75 74 5 min 82 67 80 61 69 59 71 77 58 34 58 55 24 h 90 66 82 66 75 62 65 76 51 1* 44 5* 72 h 90 63 75 59 75 59 72 73 53 2* 44 7* 168 h 88 67 74 66 77 62 82 81 64 6* 50 19 D14 85 68 75 62 77 66 85 78 63 17 60 49 Petition 870260049114, dated 05 / 22 / 2026, page 57 / 84 / 46 Time Point Concentration of C1q by subunit B (pg / mL) Group 1 (isotype control) Group 2 (low) Group 3 (medium) Group 4 (high) P0001 P0002 P0003 P0101 P0102 P0103 P0201 P0202 P0203 P0301 P0302 P0303 D42 86 65 76 61 79 67 75 80 69 46 66 76 D56 82 76 82 63 88 68 84 85 74 44 69 73 * An estimated concentration of C1q. Concentration below LLOQ (0.27 pg / mL), but above LOD (0.027 pg / mL). TABLE 15. QUANTIFICATION OF BLOOD SAMPLES OF Monkey doses for C1Q concentration by TARGET PEPTIDE QTHQPPAPNSLIR (SEQ ID NO: 36) DERIVED FROM C1Q SUBUNIT C._________________________________ Time Point Concentration of C1q by C subunit (pg / mL) Group 1 (isotype control) Group 2 (low) Group 3 (medium) Group 4 (high) P0001 P0002 P0003 P0101 P0102 P0103 P0201 P0202 P0203 P0301 P0302 P0303 Pre-dose 94 66 78 65 73 62 94 88 66 51 41 73 5 min 76 59 70 53 60 54 68 73 61 37 31 52 24 h 88 63 76 57 68 58 62 75 51 6* 23 9* 72 h 87 58 64 57 61 54 69 70 54 7* 24 12* 168 h 79 62 72 62 69 58 79 81 60 8* 28 22 53 74 62 71 76 66 45 39 77 D56 78 73 78 58 82 62 85 80 74 47 39 75 * An estimated concentration of C1q. Concentration below LLOQ (0.27 pg / mL), but above LOD (0.027 pg / mL). Petition 870260049114, dated 05 / 22 / 2026, pp. 58 / 84

Claims

1 / 3 CLAIMS 1.Assay, characterized by comprising: (1) placing a biological sample comprising C1q protein in contact with at least one proteolytic enzyme to produce C1q protein peptide fragments, wherein the C1q protein peptide fragments each comprise one of the following target peptides: i) target peptides with the amino acid sequence of IAFSATR (SEQ ID NO: 29); ii) target peptides with an amino acid sequence of SLGFCDTTNK (SEQ ID NO: 26); and iii) target peptides with an amino acid sequence of QTHQPPAPNSLIR (SEQ ID NO: 36); (2) add to the labeled biological sample, synthetic C1q peptide fragments comprising amino acid sequences identical to the amino acid sequence of each of the target peptides, and (2) perform selected reaction monitoring mass spectrometry (SRM-MS) to measure the abundance of each of the target peptides, wherein the abundance of the target peptides determines the concentration of the C1q protein in the biological sample.

2. Assay according to claim 1, characterized in that the labeled synthetic C1q peptide fragments comprise an isotope-labeled SEQ ID NO:26, an isotope-labeled SEQ ID NO:29, and an isotope-labeled SEQ ID NO:

36.

3. Assay according to claim 1 or 2, characterized in that the cysteine ​​in SEQ ID NO:26 is carbamidomethylated.

4. Assay, according to any one of claims 1 to 3, characterized by measuring the abundance of target peptides comprising comparing a signal corresponding to the target peptides generated by SRM-MS with a standard curve.

5. Assay according to claim 4, characterized in that the standard curve is produced using a method comprising: (a) preparing at least two C1q concentration standards by mixing known amounts of purified C1q protein and C1q-depleted serum; (b) adding to the at least two C1q concentration standards at least one synthetic C1q peptide labeled with an amino acid sequence identical to at least one of the target peptides; (c) bringing the at least two labeled C1q concentration standards into contact with a proteolytic enzyme to produce the at least one target peptide; (d) performing selected reaction monitoring mass spectrometry to determine the signal strength corresponding to the at least one target peptide and the signal strength corresponding to the at least one labeled synthetic peptide in each of the at least two labeled C1q concentration standards;and (e) determine a standard curve using the known signals and quantities of C1q protein; 6. Assay, according to any one of claims 1 to 5, characterized in that the biological sample is a blood sample or a serum sample.

7. An assay, according to any one of claims 1 to 6, characterized in that the biological sample is a human sample or a non-human primate sample.

8. Assay, according to any one of claims 1 to 7, characterized in that the selected reaction monitoring mass spectrometry is liquid chromatography selected reaction monitoring mass spectrometry (LC-SRM-MS / MS). Petition 870260049114, dated 05 / 22 / 2026, p. 60 / 84 3 / 3 9. Assay, according to any one of claims 1 to 8, characterized in that at least one proteolytic enzyme is trypsin.

10. An assay of any one of claims 1 to 9, characterized in that the labeled synthetic C1q peptide fragments are internal controls used for retention time confirmation, instrument performance calibration, and troubleshooting purposes. Petition 870260049114, dated 05 / 22 / 2026, pp. 61 / 84

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