WO2017050825A1 - Procédé pour quantifier le bévacizumab - Google Patents

Procédé pour quantifier le bévacizumab Download PDF

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Publication number
WO2017050825A1
WO2017050825A1 PCT/EP2016/072421 EP2016072421W WO2017050825A1 WO 2017050825 A1 WO2017050825 A1 WO 2017050825A1 EP 2016072421 W EP2016072421 W EP 2016072421W WO 2017050825 A1 WO2017050825 A1 WO 2017050825A1
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bevacizumab
seq
peptide
proteolysis
internal standard
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PCT/EP2016/072421
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English (en)
Inventor
Rachel LEGERON
Fabien XUEREB
Dominique BREILH
Alain Pierre GADEAU
Jean-Michel Boiron
Jean-Marie Schmitter
Jean-William DUPUY
Stéphane CHAIGNEPAIN
Original Assignee
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Université De Bordeaux
Chu De Bordeaux
Centre National De La Recherche Scientifique
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Publication of WO2017050825A1 publication Critical patent/WO2017050825A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to the field of antibody quantification. It more precisely relates to the quantification of Bevacizumab in a biological sample.
  • Monoclonal antibodies constitute a therapeutic class which knows the strongest current rate of development in the field of pharmaceutical biotechnology. There are to date more than 25 mAbs marketed in various fields such as oncology, immunology, ophthalmology and cardiology. Among them, Bevacizumab (Avastin ® ) is an IgGl monoclonal antibody directed against the vascular endothelial growth factor (VEGF) and neutralizes the biological activity of VEGF, resulting in anti-angiogenic activity (Presta, L.G., et al., Cancer Res, 1997. 57(20): p. 4593-9.).
  • VEGF vascular endothelial growth factor
  • Bevacizumab is also used in the first line treatment of metastatic breast cancer, advanced or metastatic non- small cell lung cancer and advanced or metastatic kidney cancer always in combination with chemotherapy.
  • Bevacizumab significantly improved the therapeutic management of mCRC, it is characterized by inter-individual variability in clinical response (efficacy and safety) as well as other mAbs.
  • 11(1-2): p. 81-8 could be a cause of response variability to treatment.
  • Evaluation of mAb concentrations in biological fluids is an essential prerequisite for the determination of pharmacokinetic parameters to assess the relationship between response and drug exposure. In this context, it is necessary to develop quantification methods sufficiently sensitive and specific to ensure accurate and reliable measurement of expected therapeutic concentrations in plasma between 75 ⁇ g/ml and 270 ⁇ .
  • ELISA techniques can be affected by cross-reactions with other molecules present in biological matrices and can generate false negatives or false positives (van den Broek, I., W.M. Niessen, and W.D. van Dongen,. J Chromatogr B Analyt Technol Biomed Life Sci, 2013. 929: p. 161-79).
  • Bevacizumab can be found in three different forms in the human plasma: free, bound and partially bound to its target, VEGF.
  • LC-MS/MS allows more reliability analysis than immunoassay technique while maintaining a sufficient sensitivity for antibody quantification in human plasma.
  • LC-MS/MS which is already widely used for the quantification of small molecules is a growing analytical tool for analysis of therapeutic proteins and proteomic biomarkers.
  • the specificity of LC-MS/MS detection is achieved through the analysis in multiple reaction monitoring (MRM) mode of intact protein or of a surrogate peptide of the protein of interest obtained after enzymatic proteolysis of samples (Duan, X., et al., J Chromatogr A, 2012. 1251: p. 63-73; Dubois, M., et al.,. Anal Chem, 2008. 80(5): p.
  • MRM multiple reaction monitoring
  • SIL of signature peptides are often used to correct variability resulting from ions suppression phenomenon and also chemical degradation of peptide.
  • the alternative to synthesize the stable isotope labeled of the intact protein is difficult to implement given the complexity of the chemical structure of protein, and recourse to production in a same way of the analyte is very expensive and time consuming.
  • the present invention relates to a method for quantifying Bevacizumab in a biological sample.
  • This invention notably concerns a method for quantifying Bevacizumab in a sample originating from an individual that has been administered the Bevacizumab antibody.
  • the Bevacizumab quantification method of the invention is aimed notably at assessing the pharmacokinetic profile of this therapeutic antibody in individuals in need thereof.
  • the quantification method described throughout the present specification fully complies with the relevant FDA guidelines for bioanalytical methods validation and allows assessing plasmatic concentration of a total fraction of bevacizumab, which includes free bevacizumab and bevazicumab bound to plasma proteins, such as bevacizumab bound to its target VEGF.
  • a labeled surrogate peptide of bevacizumab selected in a group comprising the peptides of SEQ ID NO 1 (FTFSLDTSK), SEQ ID NO 2 (STAYLQMNSLR), and SEQ ID NO 3 (VLIYFTSSLHSGVPSR), or - a monoclonal antibody which generates upon trypsin proteolysis one or more surrogate peptides selected in a group comprising the peptides of SEQ ID NO 4 (FTISADTSK), SEQ ID NO 5 (DTYIHWVR) and SEQ ID NO 6 (NTA YLQMNSLR) .
  • the said Internal Standard compound is used in a quantification method of Bevacizumab in a sample to improve accuracy and precision of the method comprising the steps of :
  • proteolysis peptides derived from Bevacizumab which proteolysis peptides comprise surrogate proteolysis peptides derived from Bevacizumab
  • the Internal Standard compound is a monoclonal antibody
  • the said antibody preferably consists of Trastuzumab.
  • step b) of the quantification method above (i) the said surrogate proteolysis peptide derived from Bevacizumab is a peptide of SEQ ID N° 1 and (ii-a) the surrogate peptide used as an Internal Standard is the labeled peptide of SEQ ID N° l .
  • step b) of the quantification method above (i) the said surrogate proteolysis peptide derived from Bevacizumab is a peptide of SEQ ID N° 1 and (ii-b) the surrogate proteolysis peptide from Internal Standard monoclonal antibody is a peptide of SEQ ID N° 5.
  • trypsin is incubated during a time period ranging from 7 to 9 hours, and is preferably of about 8 hours.
  • FIG. 2 LC-MS/MS chromatogram obtained after analysis of human serum sample spiked with 40( g/ml of bevacizumab (QC3) and with SIL peptide just before proteolysis ( Figures 2A, 2B, 2C) or mAbIS before any step of treatment ( Figure 2A, 2B, 2D). Analysis was conducted on the LC-ESI-QqQ system under chromatogram and mass spectrometry conditions described in experimental section.
  • Figure 3 Assessment of bevacizumab and mAbIS trypsin proteolysis as function of trypsin/total protein ratio through measurement of mean peak area (AUC) corresponding to the two surrogate peptides of bevacizumab (Figure 3a), ( Figure 3b) and the surrogate peptide of mAbIS ( Figure 3c).
  • AUC mean peak area
  • Figure 3a The two surrogate peptides of bevacizumab
  • Figure 3c the surrogate peptide of mAbIS
  • Figure 4 Assessment of bevacizumab and mAbIS trypsin proteolysis as function of trypsin/total protein ratio through measurement of mean peak area (AUC) corresponding to the two sur
  • This invention provides for a quantification method of Bevacizumab in a sample.
  • the invention's quantification method of Bevacizumab may be especially useful for determining the pharmacokinetic profile of Bevacizumab in individuals that are administered with this specific therapeutic antibody. Determination of pharmacokinetic profile of bevacizumab might be necessary in pharmacokinetic/pharmacodynamic studies exploring inter and intra-individual variability in clinical response, in comparative studies of administration way or also in pharmacokinetics studies of biosmiliars.
  • the invention thus relates to the use of an Internal Standard compound in a method for quantifying Bevacizumab in a sample by mass spectrometry, wherein the said Internal Standard compound is selected in a group comprising:
  • the invention also relates to a method for quantifying Bevacizumab in a sample by mass spectrometry, said method comprising the use of an Internal Standard compound selected in a group comprising:
  • the method for quantifying Bevacizumab in a sample comprises the steps of :
  • said Internal Standard compound is a Stable Isotope Labeled (SIL) peptide selected in a group comprising the peptides of SEQ ID N° 1, SEQ ID N°2, and SEQ ID N°3.
  • SIL Stable Isotope Labeled
  • said Internal Standard compound consists of a monoclonal antibody such as Trastuzumab.
  • the said surrogate proteolysis peptide derived from Bevacizumab is selected in a group comprising the peptides of SEQ ID N° 1, SEQ ID N°2, and SEQ ID N°3.
  • the selected proteolysis surrogate peptide derived from Bevacizumab is a peptide of SEQ ID N° 1.
  • the selected proteolysis surrogate peptide derived from Bevacizumab is a peptide of SEQ ID N° 1 and (ii) the surrogate peptide of Internal Standard is a labeled peptide of SEQ ID N° l .
  • trypsin is added at step a) of the method at a trypsin / total protein molar ratio ranging from 1/100 to 1/10, advantageously from 1/75 to 1/25 and preferably froml/60 to 1/40.
  • trypsin is incubated during a time period ranging from 7 to 9 hours, and is preferably of about 8 hours.
  • the said sample is selected in a group comprising whole blood, plasma and serum, or a sample derived therefrom.
  • the said sample consists of a sample derived from a human organism.
  • the inventors have shown that a precise quantification of Bevacizumab in a sample, which may be also termed "test sample” herein, may be allowed through the design of a method wherein the amount of Bevacizumab, if present, in the said sample is determined by a mass spectrometry method after:
  • the accuracy and precision of the Bevacizumab quantification method described herein may also be provided by a combination of various parameters, which include (i) the kind of method steps, (ii) the order in which the successive method steps are carried out and (ii) the specific reagents that are used, which encompasses the specific Internal Standard compounds used.
  • Internal Standard compounds that may be used for improving accuracy and precision of the absolute quantification of Bevacizumab in a sample by a method wherein quantification is performed by mass spectrometry, (i) a monoclonal antibody, especially Trastuzumab and (ii) labeled peptides of the same amino acid sequence as non-labeled surrogate peptides generated by trypsin proteolysis of Bevacizumab.
  • the present invention pertains to the use of an Internal Standard compound in a method for improving accuracy and precision of the absolute quantification of Bevacizumab in a sample by mass spectrometry, wherein the said Internal Standard compound is selected in a group comprising:
  • a labeled surrogate peptide of bevacizumab selected in a group comprising the peptides of SEQ ID N° 1 (FTFSLDTSK), SEQ ID N°2 (STA YLQMNSLR) , and SEQ ID N°3 (VLIYFTSSLHSGVPSR), and
  • a monoclonal antibody which generates upon trypsin proteolysis one or more surrogate peptides selected in a group comprising the peptides of SEQ ID N° 4 (FTISADTSK), SEQ ID N°5 (DTYIHWVR) and SEQ ID N° 6 (NTA YLQMNSLR).
  • the said monoclonal antibody is Trastuzumab.
  • Bevacizumab is a monoclonal antibody that has been widely described in the art.
  • Bevacizumab is contained in the pharmaceutical composition marketed under the name of Avastin .
  • Bevacizumab may be used for various therapeutic purposes, including for treating colorectal cancer, breast cancer, lung cancer, renal cancer, brain cancer, gynecological cancers (e.g. ovarian cancer) as well as eye diseases such as Age- related Macular Degeneration (AMD) and diabetic retinopathy.
  • Bevacizumab is a monoclonal antibody directed against VEGF comprising:
  • this invention provides for specific Internal Standard compounds allowing an accurate and precise quantification of Bevacizumab in a sample by mass spectrometry.
  • the one skilled in the art may perform any of the protein quantification methods by mass spectrometry which are known in the art, and especially any of the monoclonal antibody quantification methods which are known in the art, which include those which are referred to in the present specification, including in the description of the prior art methods.
  • the one or more Internal Standard compounds that are described herein are used in monoclonal antibody quantification methods by mass spectrometry where a known amount of one of the two kinds of Internal Standard compounds is added at a step preceding the step of quantification by mass spectrometry.
  • the amount of Bevacizumab in the initial sample is finally determined against a calibration curve obtained by a method comprising the steps of :
  • FTFSLDTSK FTFSLDTSK
  • SEQ ID NO 2 STAYLQMNSLR
  • VLIYFTSSLHSGVPSR VLIYFTSSLHSGVPSR
  • a monoclonal antibody which generates upon trypsin proteolysis one or more surrogate peptides selected in a group comprising the peptides of SEQ ID NO 4 (FTISADTSK), SEQ ID NO 5 (DTYIHWVR) and SEQ ID NO 6
  • test sample or “sample tested” encompasses material that originates from a body fluid that was previously collected from an individual, especially from a human individual.
  • a test sample encompasses any material originating from an individual to which Bevacizumab has been administered one or more times.
  • a test sample originates from a body fluid selected in a group comprising whole blood, plasma and serum.
  • a test sample derives from a body fluid after dilution in an aqueous solution, e.g. a saline solution or a buffer solution.
  • an aqueous solution e.g. a saline solution or a buffer solution.
  • a test sample is not subjected to any pretreatment, and particularly is used without being subjecting to a dilution step, for performing the Bevacizumab quantification method described in the present specification.
  • Two kinds of Internal Standard compounds are provided herein for improving accuracy and precision of the absolute quantification of Bevacizumab, labeled surrogate peptide of bevacizumab and monoclonal antibodies, respectively.
  • the high specificity against endogenous plasmatic proteins and the high physico-chemical homology between (i) these Internal Standard compounds or proteolysis peptides generated therefrom, and (ii) Bevacizumab or proteolysis peptides generated therefrom, allows a highly precise quantification of Bevacizumab in a sample.
  • the peptides of SEQ ID NO 1, 2 and 3 consist of peptides that are generated after subjecting Bevacizumab to proteolysis by trypsin. These peptides have been shown herein to be unique and specific to Bevacizumab. Notably, it is shown herein that the peptides of any of SEQ ID NO 1, 2 or 3 are not found in a collection of tryptic peptides obtained after having subjected a composition of human polyvalent IgG antibodies to proteolysis by trypsin.
  • trypsin proteolysis of Bevacizumab generates in silico a total of 54 distinct tryptic peptides without missed cleavage by trypsin. Among them 25 tryptic peptides without missed cleavage were identified after experimental analysis.
  • the selected peptides of SEQ ID NO 1, 2 and 3 are the Bevacizumab-derived surrogate peptides having no cystein in their sequence with the lowest Bit score from the in silico sequence alignment.
  • MRM multiple reaction monitoring
  • the peptides of SEQ ID NO 1, 2 or 3 may be labelled according to various methods known in the art, provided that the labelled peptide is discriminated from its non- labelled counterpart in a mass spectrometry analysis method.
  • the peptides of SEQ ID NO 1, 2 or 3 are labelled with one or more stable isotopes.
  • Stable isotopes may be selected in a group comprising 2 H, 13 C, 15 N and
  • stable isotopes are selected in a group comprising 1 i 3 J C and 1 1 5 J N.
  • a Stable Isotope Labelled peptide due to a sufficient mass increment relative to the same but unlabeled peptide, is thus discriminated from the said unlabeled peptide by mass spectrometry analysis. Otherwise said, a Stable Isotope Labelled peptide selected in a group comprising the surrogate peptides of SEQ ID NO 1, 2 and 3 is discriminated by mass spectrometry analysis, from the non-labelled surrogate peptides of the same respective amino acid sequences that are generated upon trypsin treatment of Bevacizumab.
  • Stable Isotope Labelled peptides of SEQ ID NO 1, 2 and 3 may also be termed "labelled surrogate peptides of bevacizumab" in the present specification.
  • any of the surrogate peptides of SEQ ID NO 1, 2 and 3, when used as Internal Standard compounds in a Bevacizumab quantification method by mass spectrometry, may be labelled by both the 13 C and 15 N isotopes.
  • Stable Isotope Labelled (SIL) peptides may be synthesized according to techniques well known in the art. Illustratively, the SIL peptides may be obtained from JPT
  • the proteolysis surrogate peptide derived from Bevacizumab is a peptide of SEQ ID N° 1.
  • step b) of the method (i) the selected proteolysis surrogate peptide derived from Bevacizumab is a peptide of SEQ ID NO: 1
  • the Internal Standard compound which is used may be a monoclonal antibody, and preferably a humanized IgG monoclonal antibody, which shall not be present in combination with Bevacizumab in the starting sample used for Bevacizumab quantification, e.g. which shall not be present initially in a plasma sample of a patient who received Bevacizumab chemotherapy.
  • Trastuzumab was preferably selected as a monoclonal antibody Internal Standard compound. As shown in the examples herein, proteolysis by trypsin of the selected monoclonal antibody Trastuzumab notably generates the three peptides of SEQ ID NO 4, 5 and 6.
  • the selected peptides of SEQ ID NO 4, 5 and 6 are the monoclonal antibody-derived peptides, and especially the Trastuzumab-derived surrogate peptides having no cystein in their sequence with the lowest Bit score from the in silico sequence alignment.
  • the particularity of the SEQ ID NO 5 is the most sensitive mass spectrometry signal in MRM acquisition mode.
  • the peptides of SEQ ID NO 4, 5 and 6 may also be termed "surrogate peptides of trastuzumab" in the present specification.
  • a specific surrogate peptide is selected in a group comprising the peptides of SEQ ID NO 4, 5 and 6.
  • Trastuzumab is a monoclonal antibody directed against HER-2/Neu and is widely known in the art. Trastuzumab is used as an active ingredient for treating breast and gastric cancers. Trastuzumab is contained in the pharmaceutical composition marketed under the name of Herceptin .
  • Trastuzumab is a monoclonal antibody comprising : - a first light chain comprising the amino acid sequence of the following SEQ ID NO 9 : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSV FIFPP
  • Bevacizumab is performed by reporting the ratio value calculated for the sample tested, or test sample, to a calibration curve of ratio values generated with known amounts of
  • CS serial or set of calibration samples
  • each calibration sample contains a known amount of Bevacizumab
  • each calibration sample contains a fixed and known amount of a selected Internal Standard compound
  • the serial or set of calibration samples are prepared so as to cover an amount range of Bevacizumab encompassing at least the amount range of Bevacizumab which is expected to be contained in a test sample.
  • each calibration sample comprises the same fixed and known amount of the selected Internal Standard compound.
  • the amount range of Bevacizumab which is covered by the serial or set of calibration samples, when expressed as a final concentration in the calibration samples may range from 6.25 ⁇ g/mL to 1,000 ⁇ g/mL.
  • a serial or set of calibration samples may comprise eight calibration samples comprising Bevacizumab at respective final concentrations of 6.25 ⁇ g/mL, 12.5 ⁇ g/mL, 25 ⁇ g/mL, 50 ⁇ g/mL, 100 ⁇ g/mL, 250 ⁇ g/mL, 500 ⁇ g/mL and 1000 ⁇ g/mL.
  • the given amount of the selected monoclonal antibody used as the Internal Standard compound is preferably an amount which generates mass spectrometry signal of the same order of magnitude as a mid-range calibration standard of bevacizumab in order to limit the difference in mass spectrometry signal intensity generated by the respective amounts (i) of surrogate peptides derived from trypsin proteolysis of the said monoclonal antibody used as the Internal Standard compound and (ii) of the tryptic peptides derived from Bevacizumab.
  • the given amount of a SIL-peptide is preferably an amount which generates mass spectrometry signal of the same order of magnitude as a mid-range calibration standard of bevacizumab .
  • the amount of bevacizumab that may be found in a test sample, especially in a test sample consisting of a human serum sample originating from a patient treated by Bevacizumab may vary, depending of (i) the amount of Bevacizumab which has been administered to the said patient, (ii) the time period when the serum sample as been collected since the starting time period of the treatment, (ii) the time period of collection of the serum sample since the last administration of Bevacizumab, and (iv) physiological parameters which may be specific to the said patient, such as the rate of clearance of Bevacizumab from the blood.
  • the given amount of the selected monoclonal antibody used as the Internal Standard compound, especially of Trastuzumab, may be of a final concentration in a calibration sample of about 2 ⁇ g/mL, as shown in the examples herein.
  • the given amount of the selected Stable Isotope labelled surrogate peptide used as the Internal Standard compound may be of a final concentration of about 0.2 ⁇ g/mL.
  • the serial or set of calibration samples may further comprise one or more control calibration samples which do not contain Bevacizumab.
  • a calibration sample is prepared starting from a body fluid sample initially exempt of Bevacizumab or of the selected Internal Standard compound, and preferably serum or plasma from a non-human mammal or from a human individual.
  • each of the calibration sample is subjected to the same purification, concentration and trypsin treatment as that which is described for the test samples elsewhere in the present specification, so as to provide a serial or a set of calibration assay samples (CAS).
  • CAS calibration assay samples
  • each calibration assay sample is subjected to spectrometric analysis, in the same conditions as those described for the test samples elsewhere in the present specification and the values of the spectrometry signals generated by (i) a selected surrogate peptide generated by trypsin proteolysis of Bevacizumab and (ii) by the selected Stable Isotope Labelled surrogate peptide (also termed "labelled surrogate peptide of bevacizumab") used as the Internal Standard compound or by the selected peptide (also termed "surrogate peptide of monoclonal antibody”) generated by trypsin proteolysis of the monoclonal antibody, especially by Trastuzumab, used as the Internal Standard compound, are then measured.
  • a selected surrogate peptide generated by trypsin proteolysis of Bevacizumab and (ii) by the selected Stable Isotope Labelled surrogate peptide also termed "labelled surrogate peptide of bevacizumab” used
  • a spectrometric signal value may consist of the peak area of specific SRM (Selected Reaction Monitoring), or more precisely of the sum of the peak areas of specific SRM, generated by a selected peptide of interest, typically by a selected surrogate tryptic peptide derived from Bevacizumab or by a surrogate peptide derived from Internal Standard described herein.
  • SRM Select Reaction Monitoring
  • a serial or a set of ratio values each ratio value being calculated from a calibration assay sample obtained from a starting calibration sample comprising known amounts, e;g. known final concentrations, of Bevacizumab and fixed and known amount of the Internal Standard compound.
  • a calibration curve may then be generated by plotting the serial or set of calculated ratio values versus the corresponding theoretical Bevacizumab amounts, e;g. versus the corresponding known final concentrations of Bevacizumab.
  • a "final" concentration of Bevacizumab is the concentration of Bevacizumab in an initial Calibration Sample (CS), which CS comprises a known added amount of Bevacizumab.
  • CS Calibration Sample
  • the present invention concerns a method for quantifying Bevacizumab in a sample comprising the steps of:
  • proteolysis peptides derived from Bevacizumab which proteolysis peptides comprise surrogate peptides derived from Bevacizumab
  • surrogate peptide encompasses:
  • the said labelled peptide is added just before proteolysis step without being cleaved by trypsin during the step of treatment with trypsin.
  • a surrogate peptide is the labeled surrogate peptide of bevacizumab used as the Internal Standard compound itsef; iii) in the embodiments of the method wherein the Internal Standard compound which is used is a monoclonal antibody, a surrogate peptide is a specific peptide generated by trypsin proteolysis of the said internal standard monoclonal antibody, especially of Trastuzumab, discriminating it from other plasma endogenous proteins
  • the said monoclonal antibody is cleaved in a plurality of tryptic peptides during the step of treatment with trypsin.
  • the internal standard monoclonal antibody is Trastuzumab
  • the said monoclonal antibody is cleaved in a plurality of tryptic peptides, including each of the surrogate peptides of SEQ ID NO 4, 5 and 6.
  • the present invention also pertains to a specific embodiment of the quantification method above, which specific embodiment of the method for quantifying Bevacizumab in a sample, which may also be termed a test sample, comprises the steps of : i) enriching the said sample in IgG antibodies, whereby an IgG-enriched composition is provided,
  • the known amount of the Internal Standard compound is added before step i) in the embodiments wherein the Internal Standard compound is a monoclonal antibody, such as Trastuzumab, or
  • proteolysis peptides derived from Bevacizumab which proteolysis peptides comprise bevacizumab surrogate peptides
  • - peptides derived from Internal Standard compound including surrogate peptides of Internal Standard selected in a group comprising (ii-a) the labeled selected surrogate peptide of bevacizumab and (ii-b) proteolysis surrogate peptides derived from the said monoclonal antibody
  • steps iii), iv) and v) are identical to steps a), b) and c) of the Bevacizumab quantification method described previously.
  • steps i) and ii) precede step a) of the method previously described.
  • step a) may be preceded by steps identical to steps i) and ii) of the Bevacizumab quantification method above.
  • the sample which is used in the quantification method originates from a whole blood sample that has been previously collected from an individual.
  • the blood cells, and especially erythrocytes, are removed by centrifugation so as to obtain a plasma sample.
  • coagulation of the whole blood sample is allowed to occur and a serum sample is obtained.
  • the sample which is used in the quantification method may consist of other extracellular fluids such as lymphatic fluid (endolymph or perilymph) and interstitial fluid.
  • lymphatic fluid endolymph or perilymph
  • interstitial fluid preferably, at least for determining the pharmacokinetic profile of Bevacizumab in an individual, the said sample is a blood plasma sample or a blood serum sample, or a sample derived from blood plasma or blood serum.
  • the initial sample is subjected to dilution, e;g. in an aqueous medium such as in a saline solution or in a buffer solution, before being used as the assay sample in the Bevacizumab quantification method according to the invention.
  • the initial sample such as a plasma sample or a serum sample
  • the initial sample is used without any pre-treatment and in particular is used as such undiluted.
  • Internal Standard compound is a monoclonal antibody, especially Trastuzumab, the sample is added with a known amount of the selected Internal Standard compound at that step..
  • the said sample comprises only one Internal
  • the Internal Standard compound is subjected to each of the further steps of the Bevacizumab quantification method described herein.
  • the sample optionally comprising the Internal Standard compound
  • the sample is enriched in IgG antibodies. It is reminded that the sample contains the Internal Standard Compound in embodiments wherein the said Internal Standard Compound consists of amonoclonal antibody
  • This step precedes step a) of the general Bevacizumab quantification method and is step i) of the specific embodiment thereof.
  • enrichment in IgG antibodies may be performed by ammonium sulfate precipitation, by using methods well known in the art, so as to obtain an IgG-enriched composition.
  • enrichment in IgG antibodies may be performed by affinity chromatography, which includes the use of chromatography substrates onto which have been immobilized relevant ligands such as protein A, protein G or alternatively antibodies binding to the Fc portion of IgG antibodies, as well as nucleic acid or peptide aptamers that bind to the Fc portion of IgG antibodies.
  • the step of enrichment in IgG antibodies allows separating antibodies from other abundant plasma proteins and thus contributes to improve sensitivity and reproducibility of the Bevacizumab quantification method.
  • IgG enrichment by methods of protein depletion e.g. methods of protein depletion by ion exchange or affinity chromatography
  • albumin and optionally other abundant plasma proteins also causes a partial depletion in IgG antibodies which is a concern for reproducibility of a monoclonal antibody quantification method.
  • the inventors practical experience is that these kind of enrichment methods do not allow removing the majority of plasma proteins and simultaneously do not allow preserving the content in IgG antibodies.
  • IgG enrichment by subjecting the sample to protein A or protein G chromatography allows depletion of almost the whole plasma proteins while retaining the whole IgG antibodies initially contained therein, which includes the whole Bevacizumab antibodies initially contained therein.
  • enrichment in IgG antibodies is performed by protein A chromatography.
  • recovery of Bevacizumab is of about at least 85 %.
  • elution of the retained IgG antibodies is conventionally performed at an acidic pH, generally at a pH in the range of 2-3, preferably at a pH of 2.8, and the fraction containing the most part of the IgG antibodies is collected in a buffer at basic pH so as to provide an IgG-enriched composition having a pH value ranging from neutral pH to slightly basic pH, advantageously at a pH in the range of 8.0 to 9.0, preferably at a pH range of 8.5 to 9.5, and most preferably at a pH of about 8.0, so as to allow optimal trypsin proteolysis in a further step of the method.
  • Internal Standard compound is a monoclonal antibody, especially Trastuzumab and wherein the said Internal Standard compound has not already been added to the starting sample before IgG enrichment, then the IgG-enriched composition may be added with a known amount of the selected Internal Standard compound at this step.
  • an IgG-enriched composition containing a known amount of the Internal Standard compound and an unknown amount of Bevacizumab.
  • the said IgG-enriched composition comprises only one Internal Standard compound.
  • the IgG- enriched composition is obtained by a step of chromatography wherein sample dilution is susceptible to occur, the said composition is then subjected to a concentration step, so as to provide a concentrated IgG-enriched composition.
  • This step precedes step a) of the general Bevacizumab quantification method and is step ii) of the specific embodiment thereof.
  • the concentration step may be performed by any method known in the art, including dialysis and filtration, e.g. microfiltration or ultrafiltration.
  • the concentration step is an ultrafiltration step wherein a filter membrane of a relevant cut-off value is used.
  • the ultrafiltration step does not cause a significant loss in IgG antibodies. Consequently, in contrast to what was expected, the antibodies contained in the IgG-enriched composition do not significantly adsorb onto the ultrafiltration membrane.
  • the ultrafiltration step may be performed by using an ultrafiltration membrane having a cut-off value of about 100 kDa.
  • a buffer exchange is performed during the ultrafiltration step so as to optimize the conditions of the further steps of the method are conducted.
  • the buffer exchange that may be performed during the ultrafiltration step allows obtaining a concentrated IgG-enriched composition in which the subsequent step of proteolysis by trypsin will be optimally realized.
  • step a) of the general Bevacizumab quantification method precedes step iii) of the specific embodiment thereof.
  • the Internal Standard compound is a selected Stable Isotope Labelled peptide
  • concentrated IgG-enriched composition obtained at the previous method step is added with a known and fixed amount of the selected Internal Standard compound. Indeed, according to these embodiments, no Internal Standard compound was added in any previous step of the method. There is thus provided a pre-proteolysis mixture containing a known amount of an Internal Standard compound and an unknown amount of Bevacizumab, irrespective of the step in which the said Internal Standard compound was added.
  • the said pre-proteolysis mixture comprises only one Internal Standard compound.
  • the said pre-proteolysis mixture comprises the labelled peptide of SEQ ID NO 1 or the labelled peptide of SEQ ID NO 2 as the Internal Standard compound.
  • the labelled peptide of SEQ ID NO 1 and the labelled peptide of SEQ ID NO 2 may also be termed "surrogate peptides" herein.
  • the said pre-proteolysis mixture comprises Trastuzumab as the monoclonal antibody used as the Internal Standard compound.
  • This step is step a) of the general Bevacizumab quantification method and is step iv) of the specific embodiment thereof.
  • trypsin is added to the pre-proteolysis mixture, so as to generate tryptic peptides from Bevacizumab and, in the embodiments wherein the Internal Standard compound is a monoclonal antibody, especially Trastuzumab, also tryptic peptides generated by trypsin proteolysis of the said internal standard monoclonal antibody.
  • the specific tryptic peptides derived from the internal standard monoclonal antibody may also be termed "surrogate peptides" herein.
  • the inventors have determined that the proteolysis step shall be performed in conditions that are optimal for :
  • trypsin autolysis avoiding trypsin autolysis.
  • it is used a purified trypsin having a low ability to autolysis.
  • it may be used a trypsin termed Trypsin Gold which is marketed by the company Promega (Madison, WI, United States).
  • the optimal proteolysis conditions are reached by using a trypsin / total protein molar ratio ranging from 1/60 to 1/40, e;g. a trypsin / total protein molar ratio of about 1/50.
  • a trypsin / total protein molar ratio of 1/50 or close thereto is particularly suitable for an optimal tryptic cleavage of both Bevacizumab and Trastuzumab.
  • the proteolysis step is performed in non- denaturing conditions, i.e. in conditions which do not cause protein denaturation.
  • the proteolysis step is performed in the absence of a protein denaturation agent such as urea.
  • proteolysis in the presence of trypsin is performed during a period of time that may be optimally adapted by the one skilled in the art.
  • proteolysis is performed during a time period ranging from 7 to 9 hours, and is preferably of about 8 hours.
  • proteolysis is performed at 37 °C during a period of time ranging from 4 hours to 16 hours, preferably from 6 hours to 10 hours, and most preferably ranging from 7 hours to 9 hours, the time period being ideally around 8 hours.
  • the surrogate peptide of Bevacizumab is the peptide of SEQ ID NO 1
  • a 8 hour incubation time with trypsin at 37°C allows to reach the maximum concentration of the said tryptic peptide of SEQ ID NO 1 which is used as the signature peptide for Bevacizumab quantification.
  • reduction of the pre-proteolysis mixture is performed before addition of trypsin, e.g. by adding a reducing agent such as dithiothreitol (DTT).
  • a reducing agent such as dithiothreitol (DTT).
  • prevention of oxidation of free cysteine residues and prevention of reformation of disulfide bridges may be performed by addition of an alkylation agent such as Iodoacetamide.
  • the overall Bevacizumab recovery of the quantification method, from the initial sample used to the composition obtained after trypsin proteolysis which is used for mass spectrometry quantification is of about 39.4 %.
  • This step is step d) of the general Bevacizumab quantification method and is step v) of the specific embodiment thereof.
  • the step of quantification of Bevacizumab is performed by mass spectrometry, according to techniques of protein quantification by mass spectrometry that are known in the art.
  • the step of quantification is performed according to the method of Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS), as it is shown in the examples herein.
  • LC-MS/MS Liquid Chromatography coupled to tandem Mass Spectrometry
  • QqQ triple quadrupole
  • MRM multiple reaction monitoring
  • Liquid Chromatography is performed with a reverse phase chromatography substrate.
  • the most abundant state of charge of selected surrogate tryptic peptides derived from Bevacizumab, internal standard monoclonal antibody and labelled surrogate peptide observed preferably between 200 m/z and 2000 m/z in ESI ionization source and are selected and fragmented.
  • SRM Selected Reaction Monitoring
  • SRM transitions of tryptic peptides from Bevacizumab, of tryptic peptides from the internal standard monoclonal antibody and of the labelled peptides are preferably established after comparing the fragmentation spectra obtained from pure solutions of each of these peptides, with in silico fragmentation spectra generated with a relevant available software tool, such as MS-product query available with the bioinformatics tool protein prospector.
  • quantification of Bevacizumab is based on the ratio of the sum of the peak areas of specific SRM of Bevacizumab and the sum of the peak areas of the internal standard selected surrogate peptide.
  • the amount of Bevacizumab in the sample tested e.g. the concentration of Bevacizumab in the test sample, is determined by reporting the ratio value that is calculated for the said test sample to a calibration cure that was generated as previously described elsewhere in the present specification.
  • the quantification described herein allows linearity between peak area ratios and Bevacizumab sample concentration, and especially between peak area ratios and Bevacizumab plasma concentration.
  • Quantifying Bevacizumab with the quantification method described herein allows a high quantification precision, a high quantification repeatability, as well as Bevacizumab quantification over a wide range of amounts.
  • the accuracy of the Bevacizumab quantification method according to the invention ranges from 101.7 % to 110.6 , as shown in the examples. In this embodiment, the precision of the Bevacizumab quantification method according to the invention ranges from 7 % to 10 , as shown in the examples herein.
  • the Bevacizumab quantification method according to the invention allows a linearity of the quantification measure from 12.5 ⁇ g/mL or less to 500 ⁇ g/mL or more.
  • the Bevacizumab quantification method according to the invention is at the same time sufficiently sensitive and reproducible to quantify bevacizumab in human plasma samples.
  • the present invention is further illustrated, without being limited thereto.
  • Bevacizumab was obtained from an aqueous 25 mg/mL solution (Avastin ® , Roche, Basel, Switzerland) and the mAbIS trastuzumab was obtained from lyophilised powder (Herceptin ® , Roche, Basel, Switzerland) reconstituted at a 21 mg/mL solution in water for injection.
  • the solution of human IgG pooled from several healthy donors used to confirm the uniqueness of the surrogate peptide was from LFB-biomedicaments (Tegeline ® , Courtaboeuf, France).
  • Stable isotope labeled (SIL) of surrogate peptide of Bevacizumab used as IS in the second intern calibration approach was synthesized by JPT Peptide
  • Trypsin Gold mass spectrometry was purchased from Promega (Madison, WI, USA).
  • DTT DL-Dithiotreitol
  • IAA iodoacetamide
  • Synthetic surrogate peptide of Bevacizumab and mAbIS were purchased from Thermo Fisher Scientific GmbH (Ulm, Germany). Protein A high capacity kit were purchased from Pierce biotechnology (Nab Spin Kits, Rockford, IL, USA).
  • Ultra centrifugal filters with a cut off of 100-kDa were obtained from Merck Millipore (Amicon-Ultra ® 100-kDa, Billerica, Massachusetts, USA).
  • HPLC-MS grade acetonitrile was from Scharlau (Barcelona, Spain) and Ultrapure water for HPLC was produced using a filtration system (Aquadem ® EFP 310, Veolia water STI, Antony, France).
  • Human plasma was used as a biological matrix for the development of the assay and the preparation of the calibration standards and the quality controls. Plasma was obtained from blood of several anonymous healthy volunteers by Etableau Francais du Sang Aquitaine-Limousin (EFSAL), Bordeaux, France. A.2. Plasma sample preparation
  • Sets of calibration standards were obtained by diluting Bevacizumab solution for injection in pooled human plasma to 12.5, 25, 50, 100, 250 a 500 ⁇ g/mL to cover the expected concentration range in therapeutics.
  • the 500 ⁇ g/mL calibration point was prepared by diluting in 720 ⁇ _, of human plasma, 80 ⁇ _, of 5 mg/mL Bevacizumab plasmatic solution obtained previously from the Bevacizumab aqueous stock solution for injection diluted to 1/5 in human plasma.
  • the other calibration points were then obtained by serial dilutions in human plasma from the same plasmatic stock solution at 5mg/ml. Blank plasma samples without Bevacizumab were prepared. Sets of calibration points were used for calibration of quantitative analysis and linearity tests.
  • trastuzumab solution was added to 65 ⁇ _, of each plasma sample (standard, quality control or plasma patient samples) before any treatment sample steps.
  • SIL surrogate peptide 5 ⁇ SIL surrogate peptide solution was added to 65 ⁇ _, of each standard, quality control or plasma patient samples just before proteolysis step as internal standard.
  • IgG IgG elution buffer
  • Purified sample was introduced on Amicon Ultra ® 100-kDa and the device was centrifuged for 10 min at 14,000xg. After discarding the filtrate, 415 ⁇ _, of 100 mM ammonium bicarbonate proteolysis buffer (pH 8) was introduced into the device and a second ultrafiltration by centrifugation was performed during 10 min at 14,000xg. To recover the concentrated IgG sample, the Amicon ® Ultra 100-kDa filter device was placed upside down in a clean microcentrifuge tube and centrifuged 1 min at l,000xg. This step allows concentrating (x 15) the purified sample in the proteolysis buffer.
  • proteolysis buffer 100 mM ammonium bicarbonate buffer, pH 8) was added to each concentrated sample.
  • a reduction step was performed by addition of 5.5 ⁇ ⁇ of DTT 100 mM to each sample (final concentration of 10 mM) and incubation at 55 °C during 20 min.
  • an alkylation steps was performed by addition of 6 ⁇ ⁇ IAA 100 mM to each sample (final concentration of 10 mM).
  • This LC-MS/MS system used consisted of a U-3000 Ultimate nano LC system coupled to a nanospray LTQ- OrbiTrap XL mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an ESI source.
  • Ten microliters of each protein digest were separated on an analytical 75- ⁇ - inner diameter x 15-cm CI 8 PepMapTM column (LC Packings) with a 5-40% linear gradient of solvent B (water/acetonitril 20:80 (v/v) containing 0.1% formic acid (v/v)) for 70 min.
  • the separation flow rate was set at 200 nL/min.
  • Tryptic peptides were validated using the following criteria: DeltaCN > 0.1, Xcorr > 1.5 (single charge), 2.0 (double charge), 2.5 (triple charge), 3.0 (Quadruple charge), and Peptide Probability ⁇ 0.001. Proteins were validated as soon as two different specific peptides were identified.
  • Bevacizumab quantification was performed on a LC-MS/MS system consisted of 1100 Series HPLC from Agilent (Santa Clara, CA, Schl-Unis) connected to a API3200 triple quadrupole (QqQ) mass spectrometer from AB Sciex (Foster City, CA, USA) equipped with an ESI source operating in positive ion mode and using multiple reaction monitoring (MRM) mode for quantification.
  • the quantification LC-MS/MS system was controlled with Analyst 1.5.1 software.
  • SRM transitions specific of Bevacizumab and mAbIS were researched and monitored for the protein identification. SRM transitions were determined after analysis of fragmentation spectra obtained for each solutions of synthesized signature peptide of Bevacizumab (included SIL surrogate peptide) and internal standard mAbIS relative to the in silico fragmentation spectra generated from MS-product query of Protein Prospector online bioinformatics tool v 5.12 .2 (University of California, San Francisco, USA). In order to increase the signal-to-noise ratio and the accuracy, the most intense SRM transitions with the less variability were selected and summed for protein quantification.
  • Bevacizumab quantification was based on the ratio of the sum of the peak areas of specific SRM of Bevacizumab and the sum of the peak areas of internal standard (surrogate peptide of mAbIS or SIL peptide).
  • the different ionization and fragmentation parameters were optimized using the automatic tool of Analyst software (Analyst 1.5.1 software, AB Sciex) from each solution of synthesized surrogate peptide of Bevacizumab, mAbIS and SIL internal standard.
  • the analytical validation is a prerequisite to Bevacizumab quantification.
  • the assay was validated according to the Food and Drug Administration (FDA) guidelines for bioanalytical method validation by determining linearity, accuracy, precision, specificity, sensibility and recovery.
  • Calibration curve was constructed from the peak area ratios (Bevacizumab surrogate peptide/internal standard SRM sum) obtained after LC-MS/MS analysis of the six Bevacizumab calibrations standard of known concentration. Linearity between peak area ratios and Bevacizumab plasmatic concentration was assessed by determining equation and coefficient of correlation of calibration curve. Precision and accuracy were determined after intraday and interday variability study by the analysis of six replicates of each of the three QC samples each day, and for four days.
  • Precision was assessed using the coefficient of variation (CV %) and accuracy using ratio between concentration measured on expected concentration. Specificity of the assay was assessed by analysing blank plasma samples versus Bevacizumab plasma samples spiked and not spiked with mAbIS or SIL internal standard.
  • Recovery of purification step by affinity chromatography on protein A was assessed by comparing the means peak area values obtained between two conditions: blank plasma sample spiked with Bevacizumab before affinity chromatography, and blank plasma sample spiked with Bevacizumab immediately after affinity chromatography.
  • recovery of ultrafiltration step was assessed by comparing mean peak area values obtained between two conditions: blank plasma sample spiked with Bevacizumab before ultrafiltration step, and blank plasma sample spiked with Bevacizumab immediately after ultrafiltration step.
  • the proteolysis recovery was achieved by comparing means peak area values obtained between two conditions: blank plasma samples spiked with Bevacizumab just before proteolysis, and blank plasma samples spiked with synthetized specific peptide of Bevacizumab after proteolysis at concentration corresponding to a recovery of 100%. To determine recovery of each step of the assay, all of these analyzes were conducted in triplicates on the lower and upper QCs (QC1 and QC3).
  • the detection limit corresponds to the concentration that gives an area equivalent to three times the background noise of the chromatographic signal (background noise is measured at both sides of the peak with a width equivalent to 20 times the peak width at half -height).
  • the quantification limit corresponds to the minimum concentration giving a coefficient of variation less than 20% obtained after the analysis of six samples per concentration.
  • Bevacizumab specific peptides during LC-MS/MS analysis was conducted in triplicate for each of the three QCs during twelve hours at room temperature and was assessed by comparing the mean peak area measured at different times of the analysis (0, 2, 4, 6, 8 and 12 hours) with time zero peak area.
  • Example 1 LC-MS/MS identification and quantification of Bevacizumab
  • SRM transitions of surrogate peptides of Bevacizumab, of mAbIS and SIL peptide were established after comparing the fragmentation spectra obtained from pure solutions of each of the previously selected peptides synthetized ( 68 FTFSLDTSK 76 (SEQ ID NO 1), 77 STAYLQMNSLR 87 (SEQ ID NO 2), 31 DTYIHWVR 38 (SEQ ID NO 5) and 68 FTFSLDTSK 76 * (SEQ ID NO 1)), with in silico fragmentation spectra generated with MS-product query (figure 1).
  • the retention time of the two Bevacizumab peptides were 9.8 min for the peptide 77 STAYLQMNSLR 87 "(SEQ ID NO 2), and 10.8 min for the peptide " 68 FTFSLDTSK 76 "(SEQ ID NO 1).
  • the SIL internal standard peptide 68 FTFS LDTS K 76 * (SEQ ID NO 1) was co-eluted with the non-labeled peptide at 10.8 min and mAblS surrogate peptide 31 DTYIHWVR 38 (SEQ ID NO 5) was eluted in 9.6 min (figure 2).
  • trypsin concentration and cleavage time were optimized. Trypsin ratio relative to the amount of substrate should be sufficient to ensure complete proteolysis without be introduced in excess which could lead to trypsin autolysis. Promega trypsin was chosen for its limited self-digestion and an increase ratio of trypsin was tested to find the optimal conditions for a sufficient proteolysis with a good repeatability.
  • Denaturation has the property to increase the accessibility of trypsin to its cleavage sites breaking tertiary structure of monoclonal antibody. Denaturation with urea at 6 mole/L was tested in triplicate with blank plasma samples spiked with Bevacizumab and mAbIS (QC3 samples). Mean peak area corresponding to signature peptides of Bevacizumab and mAbIS were analyzed and compared with samples without urea denaturation. Both experiments have been subjected to the same dilution factor.
  • mAbIS monoclonal antibody
  • SIL peptide stable isotope labeled peptide
  • the r 2 values were 0.9904 for 68 FTFSLDTSK 76 (SEQ ID NO 1) and 0.9660 for 77 STAYLQMNSLR 87 (SEQ ID NO 2) when mAbIS was used.
  • the r 2 values were 0.9994 for 68 FTFSLDTSK 76 (SEQ ID NO 1) and 0.9987 for STAYLQMNSLR (SEQ ID NO 2) when SIL peptide was used.
  • the intraday precision of the QCs was between 8.7% and 11.7% for the 68 FTFSLDTSK 76 (SEQ ID NO 1) and between 8.07 % and 23.9% for 77 STAYLQMNSLR 87 (SEQ ID NO 2) when mAbIS was used.
  • the intraday precision of the QCs was between 5.49% and 8.9% for 68 FTFSLDTSK 76 (SEQ ID NO 1) and between 5.39% and 10.09 % for 77 STAYLQMNSLR 87 (SEQ ID NO 2) when SIL peptide was used.
  • the internal calibration approach using SIL peptide provide less variability than mAbIS. Interday repeatability study was therefore only assessed for SIL peptide approach (see results in table 3).
  • the interday precision of the QCs was between 6.98% and 9.85% for 68 FTFSLDTSK 76 (SEQ ID NO 1) and between 11.3 % and 19.2 % for 77 STAYLQMNSLR 87 (SEQ ID NO 2).
  • Table 2 Specific tryptic peptides of bevacizumab and mAblS identified in silico with their respective highest BLAST score obtained against UniProtKB_HUMAN database.
  • VLIYFTSSLHSGVPSR (SEQ ID NO 3) 39,2 04 16)
  • NTAYLQMNSLR (SEQ ID NO 6) 36,3 04 (10/10 ⁇ 11)
  • ASODVNTAVAWYQQKP (SEQ ID NO 18) 38,8 04 18) a : Subject identity corresponds to the number of common amino acid residue between "subject sequence” / "query sequence”. The number after the arrow corresponds to the amino acid length of the sequence of interest. Table 3.

Abstract

La présente invention concerne l'utilisation d'un composé standard interne dans un procédé pour quantifier le bévacizumab dans un échantillon par spectrométrie de masse, ledit composé standard énterne étant décrit dans la description.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3457139A1 (fr) * 2017-09-19 2019-03-20 Promise Advanced Proteomics Peptides de type anticorps pour quantifier des anticorps thérapeutiques
CN111366655A (zh) * 2020-04-01 2020-07-03 上海中科新生命生物科技有限公司 一种基于免疫亲和的贝伐珠单抗生物分析的质谱检测方法
CN113748121A (zh) * 2019-04-29 2021-12-03 先正达农作物保护股份公司 用于蛋白质检测的组合物以及方法
CN113899894A (zh) * 2021-09-14 2022-01-07 上海中科新生命生物科技有限公司 一种同时检测贝伐珠单抗和曲妥珠单抗药物浓度的方法
WO2022119839A1 (fr) 2020-12-01 2022-06-09 Akouos, Inc. Constructions d'anticorps anti-vegf et procédés associés pour le traitement de symptômes associés au neurinome de l'acoustique
US11697801B2 (en) 2017-12-19 2023-07-11 Akouos, Inc. AAV-mediated delivery of therapeutic antibodies to the inner ear
WO2023150142A1 (fr) 2022-02-02 2023-08-10 Akouos, Inc. Constructions d'anticorps anti-vegf et méthodes associées pour le traitement de symptômes associés au schwannome vestibulaire

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020031773A1 (en) * 2000-06-07 2002-03-14 Ammon Daniel M. Quantitative MALDI-time of filght mass spectrometry of peptides and proteins
WO2012155019A1 (fr) * 2011-05-12 2012-11-15 Genentech, Inc. Procédé lc-ms/ms de surveillance de réactions multiples pour détecter des anticorps thérapeutiques dans des échantillons animaux à l'aide de peptides de signature d'infrastructure

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020031773A1 (en) * 2000-06-07 2002-03-14 Ammon Daniel M. Quantitative MALDI-time of filght mass spectrometry of peptides and proteins
WO2012155019A1 (fr) * 2011-05-12 2012-11-15 Genentech, Inc. Procédé lc-ms/ms de surveillance de réactions multiples pour détecter des anticorps thérapeutiques dans des échantillons animaux à l'aide de peptides de signature d'infrastructure

Non-Patent Citations (22)

* Cited by examiner, † Cited by third party
Title
DUAN, X. ET AL., J CHROMATOGR A, vol. 1251, 2012, pages 63 - 73
DUBOIS, M. ET AL., ANAL CHEM, vol. 80, no. 5, 2008, pages 1737 - 45
FERNANDEZ OCANA, M. ET AL., ANAL CHEM, vol. 84, no. 14, 2012, pages 5959 - 67
GAO, B. ET AL., J CLIN ONCOL, vol. 30, no. 32, 2012, pages 4017 - 25
GIANTONIO, B.J. ET AL., J CLIN ONCOL, vol. 25, no. 12, 2007, pages 1539 - 44
HEUDI, O. ET AL., ANAL CHEM, vol. 80, no. 11, 2008, pages 4200 - 7
HURWITZ, H. ET AL., N ENGL J MED, vol. 350, no. 23, 2004, pages 2335 - 42
LESUR, A.; E. VARESIO; G. HOPFGARTNER, J CHROMATOGR A, vol. 1217, no. 1, 2010, pages 57 - 64
LIU, H. ET AL., ANAL BIOCHEM, vol. 414, no. 1, 2011, pages 147 - 53
LU, J.F. ET AL., CANCER CHEMOTHER PHARMACOL, vol. 62, no. 5, 2008, pages 779 - 86
MATHIEU DUBOIS ET AL: "Immunopurification and Mass Spectrometric Quantification of the Active Form of a Chimeric Therapeutic Antibody in Human Serum", ANALYTICAL CHEMISTRY, vol. 80, no. 5, 1 March 2008 (2008-03-01), pages 1737 - 1745, XP055066260, ISSN: 0003-2700, DOI: 10.1021/ac7021234 *
MATTHEW F EWLES ET AL: "LC-MS/MS STRATEGIES FOR QUANTIFICATION OF THERAPEUTIC ANTIBODIES TO SUPPORT CLINICAL AND PRECLINICAL STUDIES AND PRECLINICAL STUDIES", 15 June 2015 (2015-06-15), XP055242714, Retrieved from the Internet <URL:http://bcn2015.europeanbioanalysisforum.eu/wp-content/uploads/2015/12/os-2015-D1A2_4-Matt-Ewles.pdf> [retrieved on 20160119] *
OLIVIER HEUDI ET AL: "Towards Absolute Quantification of Therapeutic Monoclonal Antibody in Serum by LC-MS/MS Using Isotope-Labeled Antibody Standard and Protein Cleavage Isotope Dilution Mass Spectrometry", ANALYTICAL CHEMISTRY, vol. 80, no. 11, 1 June 2008 (2008-06-01), pages 4200 - 4207, XP055031993, ISSN: 0003-2700, DOI: 10.1021/ac800205s *
PRESTA, L.G. ET AL., CANCER RES, vol. 57, no. 20, 1997, pages 4593 - 9
SALTZ, L.B. ET AL., J CLIN ONCOL, vol. 26, no. 12, 2008, pages 2013 - 9
STOKVIS, E.; H. ROSING; J.H. BEIJNEN, RAPID COMMUN MASS SPECTROM, vol. 19, no. 3, 2005, pages 401 - 7
TABRIZI, M.A.; C.M. TSENG; L.K. ROSKOS, DRUG DISCOV TODAY, vol. 11, no. 1-2, 2006, pages 81 - 8
VAN DEN BROEK IRENE ET AL: "Bioanalytical LC-MS/MS of protein-based biopharmaceuti", JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS, ELSEVIER, AMSTERDAM, NL, vol. 929, 26 April 2013 (2013-04-26), pages 161 - 179, XP028553925, ISSN: 1570-0232, DOI: 10.1016/J.JCHROMB.2013.04.030 *
VAN DEN BROEK, I.; W.M. NIESSEN; W.D. VAN DONGEN, J CHROMATOGR B ANALYT TECHNOL BIOMED LIFE SCI, vol. 929, 2013, pages 161 - 79
WU, J.Y. ET AL., CHIN MED J (ENGL, vol. 123, no. 7, 2010, pages 901 - 6
YANG, Z. ET AL., ANAL CHEM, vol. 79, no. 24, 2007, pages 9294 - 301
ZIPING YANG ET AL: "LC-MS/MS Approach for Quantification of Therapeutic Proteins in Plasma Using a Protein Internal Standard and 2D-Solid-Phase Extraction Cleanup", ANALYTICAL CHEMISTRY, vol. 79, no. 24, 1 December 2007 (2007-12-01), pages 9294 - 9301, XP055157355, ISSN: 0003-2700, DOI: 10.1021/ac0712502 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3457139A1 (fr) * 2017-09-19 2019-03-20 Promise Advanced Proteomics Peptides de type anticorps pour quantifier des anticorps thérapeutiques
US11053303B2 (en) 2017-09-19 2021-07-06 Promise Proteomics Antibody-like peptides for quantifying therapeutic antibodies
US11697801B2 (en) 2017-12-19 2023-07-11 Akouos, Inc. AAV-mediated delivery of therapeutic antibodies to the inner ear
CN113748121A (zh) * 2019-04-29 2021-12-03 先正达农作物保护股份公司 用于蛋白质检测的组合物以及方法
EP3962540A4 (fr) * 2019-04-29 2023-03-29 Syngenta Crop Protection AG Compositions et procédés de détection de protéine
CN111366655A (zh) * 2020-04-01 2020-07-03 上海中科新生命生物科技有限公司 一种基于免疫亲和的贝伐珠单抗生物分析的质谱检测方法
WO2022119839A1 (fr) 2020-12-01 2022-06-09 Akouos, Inc. Constructions d'anticorps anti-vegf et procédés associés pour le traitement de symptômes associés au neurinome de l'acoustique
CN113899894A (zh) * 2021-09-14 2022-01-07 上海中科新生命生物科技有限公司 一种同时检测贝伐珠单抗和曲妥珠单抗药物浓度的方法
CN113899894B (zh) * 2021-09-14 2023-11-14 上海中科新生命生物科技有限公司 一种同时检测贝伐珠单抗和曲妥珠单抗药物浓度的方法
WO2023150142A1 (fr) 2022-02-02 2023-08-10 Akouos, Inc. Constructions d'anticorps anti-vegf et méthodes associées pour le traitement de symptômes associés au schwannome vestibulaire

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