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ASSAYS FOR RECOMBINANT EXPRESSION SYSTEMS

20180052173 ยท 2018-02-22

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention is a method for assessing protein expression by recombinant expression systems. The method uses mass spectrometry to quantify protein expression. The method has particular application in potency testing of vaccine compositions.

    Claims

    1. A method of potency testing a viral vector vaccine composition encoding a recombinant protein or a nucleic acid vaccine composition encoding a recombinant protein, wherein the method comprises: (i) infecting a cell culture with the viral vector vaccine composition or transfecting a cell culture with the nucleic acid vaccine composition; and (ii) quantifying the recombinant protein encoded by the viral vector vaccine composition or the nucleic acid vaccine composition as expressed in the cell culture using a mass spectrometry analysis.

    2. The method of claim 1, wherein the cell culture is lysed to form a cell lysate that is analysed by mass spectrometry to quantify the recombinant protein, optionally wherein the cell lysate is a clarified cell lysate.

    3. The method according to claim 1, wherein the viral vector vaccine composition comprises an adenovirus vector vaccine component encoding the recombinant protein, such as wherein the viral vector vaccine composition encodes at least two recombinant proteins.

    4. The method according to claim 3, wherein the viral vector vaccine composition comprises (i) an adenovirus vector vaccine component encoding at least two recombinant proteins and/or (ii) multiple different adenovirus vector vaccine components encoding different recombinant proteins.

    5. The method according to claim 1, wherein the viral vector vaccine composition comprises a poxvirus vector vaccine component encoding the recombinant protein.

    6. The method according to claim 1, wherein: (i) two or more recombinant proteins are quantified using mass spectrometry; (ii) another protein in the cell culture has at least 85% identity to the recombinant protein, such as wherein the other protein is also encoded by the viral vector vaccine composition or the nucleic acid vaccine composition, or is a protein from the cells in the cell culture infected by the viral vector vaccine composition or the nucleic acid vaccine composition; and/or (iii) the quantifying is determining the relative or the absolute expression level of the recombinant protein.

    7. The method of claim 6(iii), wherein the determining is: (i) determining the relative expression level of the recombinant protein comprising the steps of: (a) identifying proteins in a sample by mass spectrometry analysis; and (b) determining the relative expression level of each identified protein, including the recombinant protein, optionally wherein the relative determination is performed by determining expression of the recombinant protein relative to a cellular marker protein; or (ii) determining the absolute expression level of the recombinant protein comprising the steps of: (a) identifying proteins in a sample by mass spectrometry analysis; (b) determining the relative expression level of each identified protein, including the recombinant protein; (c) detecting a proteotypic peptide of the recombinant protein; and (d) determining the absolute expression level of the recombinant protein by comparing the detected amount of the proteotypic peptide to a known standard.

    8. A method of determining infectivity of a viral vector vaccine composition comprising: (i) infecting a cell culture with a viral vector vaccine composition; and (ii) quantifying the intracellular level of a viral protein encoded by the viral vector vaccine composition in the cell culture using a mass spectrometry analysis.

    9. The method of claim 8, wherein the cell culture is lysed to form a cell lysate that is analysed by mass spectrometry to quantify the protein of a viral vector component, optionally wherein the cell lysate is a clarified cell lysate.

    10. The method according to claim 8, wherein the viral vector vaccine component is an adenovirus vector vaccine component.

    11. The method according to claim 10, wherein the viral vector vaccine composition comprises (i) an adenovirus vector vaccine component encoding at least two recombinant proteins and/or (ii) multiple different adenovirus vector vaccine components encoding different recombinant proteins.

    12. The method according to claim 8, wherein the viral vector vaccine component is a poxvirus vector vaccine component.

    13. The method according to claim 8, wherein: (i) two or more proteins are quantified using mass spectrometry; (ii) another protein in the cell culture has at least 85% identity to the protein of a viral vector vaccine component, such as wherein the other protein is a protein of another viral vector vaccine component of the viral vector vaccine composition, is encoded by the viral vector vaccine composition, or is a protein from the cells in the cell culture infected by the viral vector vaccine composition; and/or (iii) the quantifying is determining the relative or the absolute expression level of the protein of a viral vector vaccine component.

    14. The method of claim 13(iii), wherein the determining is: (i) determining the relative expression level of the protein of a viral vector vaccine component comprising the steps of: (a) identifying proteins in a sample by mass spectrometry analysis; and (b) determining the relative expression level of each identified protein, including the protein of a viral vector vaccine component, optionally wherein the relative determination is performed by determining expression of the recombinant protein relative to a cellular marker protein; or (ii) determining the absolute expression level of the protein of a viral vector vaccine component comprising the steps of: (a) identifying proteins in a sample by mass spectrometry analysis; (b) determining the relative expression level of each identified protein, including the protein of a viral vector vaccine component; (c) detecting a proteotypic peptide of the protein of a viral vector vaccine component; and (d) determining the absolute expression level of the protein of a viral vector vaccine component by comparing the detected amount of the proteotypic peptide to a known standard.

    15. A method of testing the potency and infectivity of a viral vector vaccine composition, comprising (a) determining the potency by a method comprising: (i) infecting a cell culture with the viral vector vaccine composition; and (ii) quantifying a recombinant protein encoded by the viral vector vaccine composition as expressed in the cell culture using a mass spectrometry analysis, and (b) determining the infectivity by a method comprising: (i) infecting a cell culture with the viral vector vaccine composition; and (ii) quantifying the intracellular level of a viral protein encoded by the viral vector vaccine composition in the cell culture using a mass spectrometry analysis, optionally using the same mass spectrometry analysis as that used in step (a)(ii).

    16. A method of manufacturing a viral vector vaccine composition encoding a recombinant protein or a nucleic acid vaccine composition encoding a recombinant protein, comprising at least one of (a) determining potency of the viral vector or nucleic acid vaccine composition using a method according to claim 1, (b) determining infectivity of the viral vector vaccine composition using a method comprising: (i) infecting a cell culture with the viral vector vaccine composition; and (ii) quantifying the intracellular level of a viral protein encoded by the viral vector vaccine composition in the cell culture using a mass spectrometry analysis, optionally using the same mass spectrometry analysis as that used in claim 1(ii); and the method further comprising (c) producing the viral vector vaccine composition or the nucleic acid vaccine composition.

    17. A method of manufacturing vaccine doses of a viral vector vaccine composition encoding a recombinant protein or a nucleic acid vaccine composition encoding a recombinant protein, comprising: (i) manufacturing a bulk of vaccine composition comprising the viral vector vaccine composition or the nucleic acid vaccine composition using the method of claim 16; and, if the results of step (i) indicate at least one of an acceptable potency of the viral vector or nucleic acid vaccine composition and infectivity of the viral vector vaccine composition, (ii) dispensing the bulk vaccine into doses.

    18. A vaccine prepared according to the method of claim 16.

    19. A vaccine dose prepared according to the method of claim 17.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0134] FIG. 1 illustrates the typical method steps of the invention.

    [0135] FIG. 2 shows the mass spectra data of a trivalent adenovirus-based HIV vaccine drug product expressed in A549 cells. PSMs corresponds to the MS/MS spectra; proteins corresponds to the amount of protein.

    [0136] FIG. 3 shows the three recombinant proteins from an HIV viral vector vaccine composition (Env, Mos1GagPol and Mos2GagPol) in the context of the A549 proteome. Actin from A549 cells is ranked no. 1 at 7712 ppm; Env is ranked no. 338 at 802 ppm; Mos1GagPol is ranked no. 867 at 304 ppm; Mos2GagPol is ranked 902 at 289 ppm; and Hexon from the adenovirus vector (Ad26) is ranked no. 1557 at 102 ppm.

    [0137] FIG. 4 shows the relative expression of the three recombinant proteins from a HIV viral vector vaccine composition (Env, Mos1GagPol and Mos2GagPol).

    [0138] FIG. 5 shows the normalised relative expression of the three recombinant proteins from the data in FIG. 4. The expression is normalised to Mos1GagPol expression.

    [0139] FIG. 6 shows the recombinant protein from an Ebola virus vector vaccine composition (EboTransgene) in the context of the A549 proteome. EboTrasgene is ranked 247 at 1027 ppm.

    [0140] FIG. 7 compares the protein expression levels of the Ebola virus recombinant protein by different batches of the virus vector vaccine composition.

    [0141] FIG. 8 shows the expression of HIV transgenes Mos1.Env (left graph) as analyzed using peptide IEPLGVAPTK (SEQ ID NO:25) based on SEQ ID NO:1, and Mos2S.Env (right graph) as analyzed using peptide TTLF[C]ASDAK (SEQ ID NO:27) based on SEQ ID NO:26 ([C] means cysteine was carbamidomethylated after synthesis).

    [0142] FIG. 9 shows the expression of Mos1.Env transgene (SEQ ID NO:1) as a function of cell seeding density (see legend, in number of cells per well in a 24-well cell plate) and viral load (expressed in MOI as based on the cell density 72 h post seeding of 1.5e4 cells per well). MOI is expressed in number of viral particles per cell.

    EXAMPLES

    Example 1: Simultaneous Analysis of Recombinant Protein Expression from a Trivalent Adenovirus Based HIV Vaccine by Mass Spectrometry

    [0143] The HIV vaccine drug product (Ad26.HIV DP) consists of a blend of three different Ad26 adenoviruses each encoding a different transgene designed to be expressed in human cells upon administration and subsequently induces an immune response. In this example a blend of Ad26.Env, Ad26.Mos1GagPol and Ad26.Mos2GagPol were present in the final Ad26.HIV DP in a ratio of 2:1:1, based on the amount of virus particles. The sequences of the recombinant proteins expressed from these transgenes are provided in the sequence listing: Env (SEQ ID NO: 1), Mos1GagPol (SEQ ID NO: 2) and Mos2GagPol (SEQ ID NO: 3). Both Mos1GagPol (SEQ ID NO: 2) and Mos2GagPol (SEQ ID NO: 3) contain a mosaic of epitopes based on the HIV Gag and Pol proteins, and Env (SEQ ID NO: 1) contains a mosaic of epitopes based on the HIV Env [5].

    [0144] Obtaining specific antibodies for the recombinant proteins encoded by either the Mos1GagPol or the Mos2GagPol transgenes (i.e. antibodies recognising one protein but not the other) proved very difficult due to their high similarity. These two recombinant proteins are 89% identical in sequence and the differences are spread throughout the entire sequence as single amino acid changes. These sequence differences mean that distinctive epitopes are unlikely to be present for raising antibodies that are specific and selective for each protein, and hence no antibody to distinguish the two recombinant proteins could be obtained.

    Sample Preparation

    [0145] A549 cells (adenocarcinomic human alveolar basal epithelial cells) grown to a density of 4.510.sup.6 were infected with Ad26.HIV DP at an MOI of 50,000. The cells were harvested at 48 h post infection. The experiment was performed in duplicate. The adherent cells were washed 3 times with ice cold PBS and lysed in 8M urea in 50 mM ammonium bicarbonate and Roche complete mini protease inhibitor cocktail. The lysate was sonicated and centrifuged. The supernatant was collected and the clarified cell lysate (250 g of total protein) was subjected to in-solution digestion with Lys-C (1:100 w/w) at 37 C. for 4 h, followed by a 4-dilution to 2M urea and addition of trypsin (1:100 w/w). The resulting peptide mixture was desalted on a C18 Seppak column and dried in vacuo.

    Setup of the LC-MS/MS

    [0146] An Ultimate3000 nanoLC (Thermo Scientific), equipped with a Nano Trap pre-Column filled with Acclaim PepMap100 C18 (3 m, 100 ; Thermo Scientific) and an EASY-Spray Column (PepMap RSLC, C18, 2 m, 100 , 75 m50 cm), was directly coupled to a qExactive Orbitrap mass spectrometer (Thermo Scientific). 500 ng of each full cell digest mentioned above was loaded on the pre-column using 100% buffer A (0.1% formic acid) at a flow rate of 5 L/min for 10 min. Subsequently, peptides were eluted from the pre-column onto the analytical EASY-Spray column using the following gradient setup on the nanoLC operating at 200 nL/min: 0-15 min 1% buffer (80% acetonitrile, 0.1% FA), between 15-120 min a gradient from 8-19.2% buffer B. 120-165 min 19.2-40% buffer B; 165-168 min 40-80% buffer B, 168-169 at 80% buffer B, 169-170 min 80-1% buffer B, and 170-185 min at 1% buffer B. This was adapted from Reference 76. Each digest was analysed on the LC-MS/MS setup in duplicate.

    Bioinformatics

    [0147] This resulted in 8 datasets which were analysed for recombinant protein expression. In each dataset, the MS data was used to identify the proteins present in the sample by a database search algorithm (e.g. Sequest [7]). Typical q-Exactive search parameters were used in terms of accuracy in MS and MS/MS mode to search the data against the entire human proteome, supplemented with the adenovirus proteins and the sequences of the three different recombinant proteins. False discovery rates were also estimated by a suitable algorithm (e.g. Percolator [9]). The data was filtered to a suitable level of false positives, here 1% at the identified MS/MS spectrum level.

    [0148] To obtain expression levels of each protein present in the dataset, MS data characteristics were converted to represent semi-absolute expression levels. The ratio of identified MS/MS spectra for a protein over its molecular weight was calculated. These ratios were subsequently normalised to the sum of all ratios in the dataset and expressed in ppm.

    Results

    [0149] The results from the duplicate LC-MS/MS-run of each sample were combined to increase accuracy on the MS-based quantitation. This resulted in the two datasets presented in FIG. 2 (Runs 1 and 2). These two datasets proved highly comparable in terms of the amount of proteins and MS/MS spectra (PSMs) that were identified. The observed small variation is typical for the approach and differences were seen close to the detection limit of the method.

    [0150] The three recombinant proteins were readily observed in each run (FIGS. 3 and 4) and the normalised expression ratios of the transgenes were in close agreement between the 2 experiments (normalization to Ad26.Mos1GagPol expression, FIG. 5). Normalizing the expression ratios revealed that recombinant protein expression of the Env, Mos1GagPol and Mos2GagPol were in close agreement with the blending ratio in the Ad26.HIV DP which was 2:1:1.

    [0151] Furthermore, the level of a protein from the adenovirus vector, e.g. Hexon (see FIG. 3), especially when compared with the level of a cellular protein, provides an indication of the infection efficiency of the adenovirus vector. Besides providing a relative expression ratio, the data presented above could also be mined for proteotypic peptides of each recombinant protein. These can be developed into a more accurate quantitative assay based on single reaction monitoring (SRM).

    Example 2: Batch to Batch Comparison of Potency by Mass Spectrometry Based Recombinant Transgene Expression Analysis

    [0152] The potency of several batches of a monovalent Ebola (Ad26.ZEBOV) vaccine encoding a single recombinant protein from an Ebola strain in an adenovirus 26 virus was tested.

    Methods

    [0153] A549 cells were infected with 5 different batches of the Ad26.ZEBOV vaccine at an MOI of 25,000, in triplicate. Cells were harvested 48 h post-infection and lysed according to the methodology described in Example 1. Sample work-up towards the q-Exactive Orbitrap was executed identical to Example 1. The peptide mixtures were analysed using the same protocols as in Example 1. The Orbitrap LC-MS/MS data was searched against the human database, supplemented with three versions of the recombinant protein from the different Ebola strains.

    Results

    [0154] After database searching, the Ebola recombinant protein encoded by the Ad26 virus could readily be identified by several unique peptide sequences, without interfering identifications of the other, similar Ebola proteins, indicating the specificity of the approach. Subsequently, spectral count based relative quantitation was used to estimate the expression level of the Ebola transgene in the context of the cellular lysate (FIGS. 6 and 7). On average, all combined data indicated an expression level of 1027 ppm and ranked the Ebola recombinant protein among the 300th most abundantly expressed proteins in the A549 cell lysate (Rank #247).

    [0155] When inspecting the five triplicate experiments separately, small variations within triplicates were observed (i.e. relative standard deviations (RSD) of 4.9-10.2%). These variations reflect variation occurring throughout sample preparation and LC-MS/MS analysis.

    Example 3: Identifying Proteotypic Peptides Useful for Absolute Quantitation of Proteins by Mass Spectrometry

    [0156] The aim of this example was to identify proteotypic peptides that are useful for synthesis of stable isotope labelled analogues as internal references for absolute quantitation of the proteins by MS.

    [0157] Certain peptides have been selected from the MS analysis in Example 1 to be useful proteotypic peptides in consistently identifying the proteins of interest. Tables 1 and 2 show the characteristics of these proteotypic peptides in multiple reaction monitoring (MRM)-transitions for the negative ion mode and the positive ion mode, respectively. The average molecular weight of the peptide (MW), the precursor mass in the first quadrupole (Q.sub.1; m/z), the predicted fragment mass in the third quadrupole (Q.sub.3; m/z) are depicted. To optimize the fragmentation behaviour of each peptide in the the quadrupole ion trap to be able to monitor the best transition(s) (Q1-Q3 combinations) with the highest specificity and sensitivity, several parameters were optimized using synthetic versions of each proteotypic peptide identified in example 1 above. The retention time (Rt; min), the differential pulse voltammetry (DPV), Collission Energy (CE) and CXP were monitored/optimized in both negative (Table 1) and positive (Table 2) ion mode, respectively.

    TABLE-US-00002 TABLE1 CharacteristicsofproteotypicpeptidesinMRM-transitionsforthenegativeionmode. Q1 Q3 Peptide Average Mass Mass Rt Name Aminoacidsequence MW (m/z) (m/z) (min) DPV CE CXP Transgenepeptides Env4 AIEAQQHLLQLTVWGIK 1948.3 973.0 950.7 4.3 -66 -48 -29 (SEQIDNO:4) Mos1.3 IGPENPYNTPVFAIK 1659.9 828.8 806.9 3.7 -63 -36 -25 (SEQIDNO:5) Mos2.1 IGPENPYNTPIFAIK 1673.9 835.8 814 3.9 -40 -38 -27 (SEQIDNO:6) Mos2.2 YTAFTIPSINNETPGIR 1894.1 945.8 923.9 3.9 -105 -36 -27 (SEQIDNO:7) Housekeepingpeptides Act1 SYELPDGQVITIGNER 1790.9 894.3 885.2 3.5 -95 -38 -27 (SEQIDNO:8) G3P1 LISWYDNEFGYSNR 1763.9 880.7 871.8 3.7 -90 -36 -29 (SEQIDNO:9) G3P3a AVGKVIPEELNGK 1353.6 675.8 666.9 2.5 -90 -30 -19 (SEQIDNO:10) Viralpeptide VII1 TTVDDVIDSVVADAR 1575.7 786.7 777.5 4.2 -86 -32 -25 (SEQIDNO:11)

    TABLE-US-00003 TABLE2 CharacteristicsofproteotypicpeptidesinMRM-transitionsforthepositiveionmode. Q1 Q3 Peptide Average Mass Mass Rt Name Aminoacidsequence MW (m/z) (m/z) (min) DPV CE CXP Transgenepeptides Env1 EATTTLFZASDAK 1414.5 708.0 798.6 2.7 70 31 23 (SEQIDNO:12) Env2 VSFEPIPIHYZAPAGFAILK 2230.6 744.4 884.7 4.5 75 31 30 (SEQIDNO:13) Env3 AFYTAGDIIGDIR 1411.6 706.6 858.7 3.9 75 29 30 (SEQIDNO:14) Env3a AFYTAGDIIGDIR 1411.6 706.6 1030.7 3.9 75 33 36 (SEQIDNO:14) Mos1.1 FAVNPGLLETSEGZR 1649.8 825.7 1218.7 3.4 56 37 38 (SEQIDNO:15) Mos1.2 SLYNTVATLYZVHQR 1825.1 913.3 1147.5 3.9 65 49 28 (SEQIDNO:16) Mos1.4 IVSLTETTNQK 1233.4 617.5 1021.7 2.3 54 27 32 (SEQIDNO:17) Mos2.3 IATESIVIWGK 1216.4 609.0 600 3.6 61 25 20 (SEQIDNO:18) Mos2.3a IATESIVIWGK 1216.4 609.0 1032.8 3.6 61 27 34 (SEQIDNO:18) Mos2.4 VVSLTDTTNQK 1205.3 603.5 1007.7 2.1 66 27 32 (SEQIDNO:19) Mos2.5 FALNPGLLETSEGZK 1635.8 818.7 1190.8 3.7 50 37 42 (SEQIDNO:20) Housekeepingpeptides G3P2 VIPELNGK 869 435.5 657.3 2.4 43 19 20 (SEQIDNO:21) G3P3a AVGKVIPEELNGK 1353.6 452.1 592.6 2.5 50 19 20 (SEQIDNO:22) Viralpeptides Hex1 ATDTYFSLGNK 1216.3 608.9 665.5 2.9 66 29 22 (SEQIDNO:23) IX1 LLALLAELEALSR 1411.7 706.6 888.7 5.8 90 31 28 (SEQIDNO:24)

    Preparation of Calibration Samples

    [0158] Synthetic peptides from Tables 1 and 2 were obtained from JPT Peptide Technologies (Berlin, Germany). The peptides were dissolved in 10% formic acid to obtain stock concentrations of 0.1 mg/mL. Combined working solution in 10% formic acid were prepared for all peptides with an estimated starting concentration of 1000 ng/mL. Calibration curves ranging from 0.1-1000 ng/mL were prepared.

    Sample Preparation and MS Analysis

    [0159] Using the SRM parameters determined for each proteotypic peptide above, a first estimation of transgene expression can be performed. A549 cells were transfected with samples by different batches of the HIV vaccine drug product Ad26.HIV DP (see Example 1; Samples 1 and 2), separately, as described in Example 1. A549 cells transfected with an empty adenovirus vector (i.e. homologues adenovirus vectors that do not encode any recombinant proteins), Ad26.DE3.5ORF6 using the same protocol (Control 1) and an uninfected A549 cell extract (Control 2) were used as negative controls. Adenovirus vectors encoding Env only, Mos1GagPol only and Mos2GagPol only were also used as controls (Controls 3-5, respectively) in order to demonstrate that the method could quantify specifically and precisely highly similar recombinant proteins.

    [0160] The cells were harvested and the cell lysates were prepared for MS analysis as described in Example 1. The samples were diluted by and 1/10 in 10% formic acid.

    MS Analysis

    [0161] The samples were injected into a triple quadrupole mass spectrometer API-6500 (AB Sciex), and the data were analysed using the Turbo-Ionspray Interface (AB Sciex) in negative and positive-ion modes. The SRM settings depicted in Table 1 were used.

    Results

    [0162] The amounts of the proteotypic peptides for the recombinant proteins (ENV, Mos1GagPol and Mos2GagPol), of the cellular proteins and of the viral vector proteins (the capsid proteins of the viral particle) were determined, and the results are shown in Tables 3-7.

    TABLE-US-00004 TABLE 3 Results for the proteotypic peptides for the transgene ENV. (ND = not detected; results in Italics are above quantitation limit and need further optimization of the transitions to be used; (+) = positive ion mode was used; () = negative ion mode was used) ENV1 (+) ENV2 (+) ENV3 (+) ENV4 () Sample nM 1/10 diluted samples Control 1 ND ND ND ND Control 2 ND ND ND ND Control 3 242 2815 672 402 Control 4 ND ND ND ND Control 5 ND ND ND ND Sample 1 83 995 341 227 Sample 2 83 883 343 267 diluted samples Control 1 ND ND ND ND Control 2 ND ND ND ND Control 3 256 2421 384 215 Control 4 ND ND traces ND Control 5 ND ND ND ND Sample 1 91 893 227 136 Sample 2 79 753 191 147

    TABLE-US-00005 TABLE 4 Results for 4 proteotypic peptides for the transgene Mos1GagPol. (ND = not detected; BQL = below quantitation limit; (+) = positive ion mode was used; () = negative ion mode was used) Mos1.1 Mos1.3 (+) Mos1.2 (+) () Mos1.4 (+) Sample nM 1/10 diluted samples Control 1 ND ND ND ND Control 2 ND ND ND ND Control 3 ND ND ND ND Control 4 184 274 57 30 Control 5 ND ND ND ND Sample 1 21 BQL 8 2 Sample 2 21 BQL 8 2 diluted samples Control 1 ND ND ND ND Control 2 ND ND ND ND Control 3 ND ND ND ND Control 4 160 238 34 45 Control 5 ND ND ND ND Sample 1 18 36 5 2 Sample 2 17 24 5 2

    TABLE-US-00006 TABLE 5 Results for 5 proteotypic peptides for the transgene Mos2GagPol. (ND = not detected; BQL = below quantitation limit; (+) = positive ion mode was used; () = negative ion mode was used) Mos2.1 Mos2.2 Mos2.3 Mos2.4 Mos2.5 () () (+) (+) (+) Sample nM 1/10 diluted samples Control 1 ND ND ND ND ND Control 2 ND ND ND ND ND Control 3 ND ND ND ND ND Control 4 ND ND ND ND ND Control 5 131 458 86 38 147 Sample 1 19 BQL BQL 1 BQL Sample 2 16 BQL BQL 2 BQL diluted samples Control 1 ND ND ND ND ND Control 2 ND ND ND ND ND Control 3 ND ND ND ND ND Control 4 ND ND ND ND ND Control 5 70 211 68 34 105 Sample 1 11 58 6 1 8 Sample 2 8 BQL 5 1 8

    TABLE-US-00007 TABLE 6 Results for proteotypic peptides for the cellular proteins: Actin and G3P. (ND = not detected; results in Italics are above quantitation limit; (+) = positive ion mode was used; () = negative ion mode was used) Act () G3P1 () G3P2 (+) Sample nM 1/10 diluted samples Control 1 3392 913 162 Control 2 9075 2041 672 Control 3 6817 1621 452 Control 4 3084 839 131 Control 5 8007 1996 598 Sample 1 5066 1327 252 Sample 2 4196 1191 211 diluted samples Control 1 2269 635 174 Control 2 4692 1118 550 Control 3 4141 1020 437 Control 4 2132 607 153 Control 5 4383 1191 513 Sample 1 3304 934 276 Sample 2 2511 717 195

    TABLE-US-00008 TABLE 7 Results for proteotypic peptides for proteins of the viral vector: Hex, VII and IX1. (ND = not detected; results in Italics are above quantitation limit; (+) = positive ion mode was used; () = negative ion mode was used) Hex (+) IX1 (+) VII () Sample nM 1/10 diluted samples Control 1 13.4 6305 307 Control 2 52.5 20259 717 Control 3 7.0 1665 265 Control 4 3.5 273 154 Control 5 9.3 275 291 Sample 1 4.0 BQL 194 Sample 2 3.5 BQL 204 diluted samples Control 1 15.8 7027 164 Control 2 41.9 14167 307 Control 3 7.4 1238 155 Control 4 3.8 252 99 Control 5 8.6 179 132 Sample 1 4.4 58 103 Sample 2 3.2 56 101

    Discussion and Conclusions

    [0163] This example demonstrates that the transitions of many selected proteotypic peptides were highly specific for identifying, and quantifying, the proteins of interest. In particular, each specific recombinant protein was detected in the transfected cells, while no recombinant proteins were detected in the viral empty vector construct transfected cells. Controls 3-5 across Tables 3-5 show specific detection of the relevant recombinant proteins based on their proteotypic peptides when individually expressed. The viral proteins were detected in all transfected cells.

    [0164] The amounts determined for the diluted samples ( and 1/10) showed a good correlation for most peptides analysed in the positive ion, whereas currently much less correlation was observed for some of the peptides analysed in the negative ion mode.

    [0165] The cellular proteins were detected at high concentration levels in all samples, but the concentration levels varied by a factor of 3 between the samples. This could be attributed to the inaccuracy of the calibration curve because no detailed information on the purity or the solubility of the synthetic peptides was provided. It was also noted that variation in the samples may arise because neither the peptidase digestion efficiency nor the stability of the individual peptides during the digestion process have been evaluated at this stage. These parameters can be controlled and optimised in future experiments.

    [0166] For a number of peptides, the calibration range was sub-optimal, so the readings were either above the quantitation limit or below the quantitation limit. For example, some samples showed concentration levels above the upper limit of quantification at the 1/10 dilution (Env2, Act, G3P, IX1). To improve the readings, the calibration range can be extended in future experiments, as well as the optimal dilution at which to measure these peptides.

    [0167] It was also noted that, since the calibration samples were prepared synthetically, the matrix of the calibration samples was different from the matrix of the study samples. To evaluate potential matrix effects, a peptidase digest of a blank cell lysate (i.e. the cell was not infected with any viral vectors) was used (Control 2). The accuracy was acceptable for most peptides.

    [0168] From the results of this example, the following proteotypic peptides are considered to be useful for the synthesis of stable isotope labelled analogues for absolute quantitation of the proteins by MS analysis (as explained in Stage 2 of the MS analysis above): [0169] Env: Env1 and Env2. [0170] Mos1GagPol: Mos1.4, and Mos1.2 or Mos1.3. [0171] Mos2GagPol: Mos2.4, and Mos2.5 or Mos2.3. [0172] Viral vector protein: Hex1. [0173] Cellular protein: Act and G3P2.

    [0174] Thus, the proteotypic peptides for Env, Mos1GagPol and Mos2GagPol can provide an absolute quantitation of the level of each of the recombinant proteins in the cell lysate. This provides information on how effectively the vaccine composition causes expression of the recombinant proteins encoded by its viral vector components, and hence the in vitro potency of the vaccine composition.

    [0175] The proteotypic peptide for the viral vector protein can provide an absolute quantitation of the level of the protein in the cell lysate. This provides information on the infectivity of the adenovirus vector of the vaccine composition.

    Example 4: Proteotypic Peptides for Absolute Quantitation of Env Proteins

    [0176] Determination of transgene expression is achieved by the methodology described in example 3.

    [0177] In this example, the level of two HIV vaccine transgenes expressed by two different adenovirus vectors was determined in A549 cells in which the adenovirus vectors cannot replicate. The A549 cells were seeded on 24 well cell growing plates and left to grow to confluency for 72 h. Cells were then infected with the HIV drug product blend consisting of the two different adenovirus vectors, each containing a different HIV transgene based on the envelope proteins; Mos1.Env (SEQ ID NO:1) and Mos2S.Env (SEQ ID NO:26). The blending ratio was 1:1 based on virus particles per mL. Each transgene was represented by a single proteotypic peptide (SEQ ID NO:25 for Mos1.Env, and SEQ ID NO:27 for Mos2S.Env).

    [0178] Different multiplicities of infection (MOI) were used, ranging from 70,000 down to 3250 virus particles per cell. Each MOI was performed in triplicate. 48 h post infection, the cells were washed three times with ice cold PBS. Cells were then lysed in 8M urea. The lysate was transferred to a 96 well plate which was sonicated to improve solubility of each protein in the lysate. After reduction in DTT and alkylation using iodoacetamide, samples were digested in 96 well plate format using LysC (4 h) and trypsin (18 h) sequentially. The resulting peptide mixtures were spiked with the heavy labelled internal standard proteotypic peptides and subsequently desalted, dried in vacuo and stored at 20 C. until LC-MRM-MS analysis on an AB-Sciex Q-Trap 6500.

    [0179] Each peptide was quantified using one transition (Table 2). Run to run equilibration was performed using the spiked heavy labelled proteotypic peptide. After equilibration, the area under the curve of the light peptide transition was quantified using a standard curve of a synthesized version of the light peptide. The results for both Mos1.Env and Mos2S.Env transgene expression were analyzed from the same MS runs and the expression levels (in pmol/well of cells) are depicted in FIG. 8. It can be concluded that both transgenes (FIG. 8 left graph for Mos1.Env and FIG. 8 right graph for Mos2S.Env) show merely equal expression levels for each MOI. Also, the correlation between MOI and expression is linear within the range tested. The error bars represent the triplicates at cell infection level. This shows that the expression of several transgenes in a single vaccine composition can be determined in a broad range with high selectivity and precision.

    Example 5: Optimizing Cell Density and MOI for an Adenoviral Vector Vaccine Component

    [0180] In this example, the methodology described in example 3 and 4 is amended to further investigate the impact of cell density on the 24-well plate and the multiplicity of infection of the adenovirus viral vaccine composition comprising an adenoviral vector vaccine component carrying the HIV transgene Mos1.Env (SEQ ID NO:1). In this experiment, cells were seeded at three different densities, i.e. 7500/well, 15,000/well or 30,000 per well, in a 24 well plate. After seeding, cells were left to grow for 72 h and were then infected with three different MOIs, i.e. 30,000 viral particles (VP)/cell, 50,000 VP/cell or 75,000 VP/cell (as based on the cell count achieved in the 15,000 cells/well after 72 h). In other words, cells were infected with an equal amount of virus particles/well, regardless of cell density. Similar to example 4, these experiments were also performed in triplicate at the level of cell infection. After following the same sample preparation as described in example 4, the spiked peptide mixtures from each well were analyzed using the same MS setup as used in example 4. The results are depicted in FIG. 9. The expression levels are expressed in pmol transgene per well (sample).

    [0181] These data indicate that cell density has a distinct impact on transgene expression level. From these experiments we concluded that higher cell densities give higher expression when exposed to identical numbers of virus particles in the well. Such experiments are useful in setting up the optimal cell seeding and infection conditions for a relative potency experiment in which a test article is compared to a reference batch.

    [0182] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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