METHOD FOR THE DETERMINATION OF SEQUENCE VARIANTS OF POLYPEPTIDES

Abstract

The invention is directed to a method for determining amino acid sequence mutations in a produced polypeptide, comprising the following steps of a) providing a sample of a produced polypeptide, b) incubating the polypeptide in the sample with a protease, c) performing a two dimensional analysis using reversed phase chromatography coupled with a high resolution mass spectroscopy (FT-ICR/FT-orbitrap) and MS/MS analysis of the amino acid sequence fragments of the peptides, d) data evaluation by comparing the LC-MS data sets obtained for the samples side by side with the data set of a reference sample, by searching for differences in the signal intensities at given retention times and by evaluation of differential signals with respect to amino acid sequence mutations. The reference sample for data evaluation (d) can be either a well characterized standard or one of the samples to be analyzed.

Claims

1. A method for determining a polypeptide with a mutant amino acid sequence, characterized in comprising: a) providing at least two samples of the polypeptide; b) incubating the samples each with the same protease; c) Analyzing the incubated samples by a two dimensional data analysis using a combination of a reversed phase liquid chromatography separation and a mass spectrometry analysis and/or MS/MS analysis; and d) defining a data set obtained with one sample in c) as reference sample and comparing data sets obtained with the other samples in c) with the data set of the reference sample, whereby every amino acid sequence difference with a ratio of more than 3 of the intensity of the sample mass spectrum signal to the intensity of the reference mass spectrum signal is an amino acid sequence mutation of the polypeptide, and thereby determining the polypeptide with a mutant amino acid sequence.

2. The method according to claim 1, characterized in that the analyzing is performed using a m/z frame width of 1.6 or more for pattern comparison.

3. The method according to claim 1, further comprising: e) determining the identity and position of the amino acid mutations in the amino acid sequence by MS/MS analysis.

4. The method according to claim 1, characterized in that a further sample is provided which comprises the polypeptide spiked with the polypeptide with a known amino acid sequence mutation and the further sample is incubated and analyzed and compared in addition to the provided samples.

5. The method according to claim 1, characterized in that the analyzing is performed at a pH value of less than pH 8.0 and at a temperature of less than 40° C.

6. The method according to claim 1, characterized in that the samples are provided in a tris (hydroxymethyl) aminomethane buffer.

7. The method according to claim 1, characterized in that the incubating of the samples with a protease is a cleavage of the polypeptide by the protease in amino acid sequence fragments of from 3 to 60 amino acid residues in length.

8. The method according to claim 1, characterized in that the comparing in step d) is performed with the data of one or some or all MS charge states.

9. The method according to claim 1, characterized in that the polypeptide is an immunoglobulin, immunoglobulin fragment or immunoglobulin conjugate.

10. The method according to claim 1, characterized in that the samples are incubated for 16 hours to 18 hours with the protease and thereafter formic acid or trifluoro acetic acid are added.

11. The method according to claim 1, characterized in that the samples are incubated for 4 hours with the protease and thereafter formic acid or trifluoro acetic acid are added.

12. The method according to claim 1, characterized in that the comparing comprises overlaying of the mass spectrometric total ion chromatogram (MS-TIC) of the reference sample and each of the other samples to be analyzed, whereby the intensity ratio of all overlapped and aligned masses is calculated, whereby peaks with a ratio of more than 10 are evaluated for being an amino acid sequence mutation.

13. The method according to claim 1, characterized in that the comparing comprises in addition comparing the DNA translated proteolytic fragment peptide pattern of the theoretical amino acid sequence and the total mass spectrometry ion chromatogram (MS-TIC) of the sample to be analyzed, and amino acid sequence mutations are identified by adding to or substracting from, respectively, each calculated theoretical mass of the theoretical amino acid sequence the mass differences resulting from a nucleic acid mutation, deletion, or insertion in a base triplet (codon) with an amino acid change.

14. A method for producing a polypeptide comprising the following step: selecting a cell producing a polypeptide, whereby the polypeptide comprises the smallest number and the smallest ratio, respectively, of amino acid sequence mutations, determined with the method according to claim 1 with respect to a reference sample of the polypeptide or predetermined amino acid sequence of the polypeptide.

15. A method for producing an immunoglobulin comprising the steps of: a) providing at least two cells comprising a nucleic acid encoding the immunoglobulin, b) single depositing and cultivating the cells, c) performing a method according to claim 1, d) selecting a cell producing an immunoglobulin, whereby the immunoglobulin comprises the smallest number of amino acid sequence mutations with respect to a reference sample, e) cultivating the cell, f) producing the polypeptide by recovering the polypeptide from the cell or the cultivation medium.

Description

DESCRIPTION OF THE FIGURES

[0114] FIG. 1 A representative temporal sequence of the scan event cycle for data dependent acquisition of MS/MS spectra; abbreviations: FT ICR—Fourier transform ion cyclotron resonance; LIT—linear ion trap; CID—collisionally induced dissociation; RP—resolution power; SID—source induced dissociation.

[0115] FIG. 2 Total ion chromatogram of tryptic peptide maps from a reference anti-CD19 antibody (reference) and a sample anti-CD19 antibody (sample) acquired on an LTQ FT ICR (Thermo Scientific).

[0116] FIG. 3 Overlay of LC-MS chromatograms aligned by retention time of prominent peaks. Data were from tryptic peptide maps of two anti-CD19 antibodies (reference and sample). A and B are mean TIC profiles of two replicates each.

[0117] FIG. 4 Scatter Plot showing the differences between sample and reference anti-CD19 antibody.

[0118] FIG. 5 Zoom of FIG. 4.

[0119] FIG. 6 Assigned MS/MS spectrum of the sample anti-CD19 antibody HC amino acid sequence fragment (y-ion series and b-ion series of collisionally induced dissociation of the doubly charged parent ion). Characteristic MS/MS fragments for the amino acid sequence variant are shown in bold.

[0120] FIG. 7 Assigned MS/MS spectrum of the sample anti-CD19 antibody LC amino acid sequence fragment (y-ion series and b-ion series of collisionally induced dissociation of the doubly charged parent ion). Characteristic MS/MS fragments for the amino acid sequence variant are shown in bold.

[0121] FIG. 8 Extracted ion chromatogram of natural amino acid sequence and the amino acid sequence of the sample anti-CD19 antibody HC amino acid sequence fragment within the LC-MS sample run. Result of quantitation: 2 wt-% of mutant amino acid sequence (variant) is present in the sample.

[0122] FIG. 9 Extracted ion chromatogram of the natural amino acid sequence and the sample anti-CD19 antibody LC amino acid sequence fragment within the LC-MS sample run. Result of quantitation: 0.5 wt-% of mutant amino acid sequence (variant) is present in the sample.

[0123] FIG. 10 Overlay of LC-MS chromatograms aligned by retention time of prominent peaks. Data were from tryptic peptide maps of two anti-CCR5 antibodies (reference and sample).

[0124] FIG. 11 Scatter Plot showing the differences between reference and sample anti-CCR5 antibody.

[0125] FIG. 12 Extracted ion chromatogram of the natural amino acid sequence and the sample anti-CCR5 antibody LC amino acid fragment within the LC-MS sample run.

[0126] FIG. 13 Extracted ion chromatogram of the natural amino acid sequence and the sample anti-CCR5 antibody HC amino acid sequence fragment within the LC-MS sample run.

EXAMPLE 1

[0127] Materials and Methods

[0128] Sample Preparation Method for the Reference and the Sample:

[0129] a) Reduction and Alkylation:

[0130] 250 μg of immunoglobulin in a volume of maximal 100 μl were diluted with denaturation buffer (0.4 M Tris-HCl, 8.0 M guanidinium-hydrochloride, pH 8) to a final volume of 240 μl. 20 μl of dithiothreitol (240 mM in denaturation buffer) were added to the solution and the mixture was incubated at 37° C.±2° C. for 60 minutes. Afterwards 20 μl of an iodoacetic acid solution (0.6 M in purified water) were added, vigorously mixed and incubated for 15 min. at room temperature in the dark. The alkylation reaction was stopped by the addition of 30 μl of a dithiothreitol solution (240 mM in denaturation buffer).

[0131] b) Buffer Exchange:

[0132] The buffer of 300 μl (approximately 250 μg or 3.2 nmol) of a solution comprising the denatured, reduced, carboxymethylated immunoglobulin was exchanged using a NAP™ 5 Sephadex™ G-25 desalting column. Briefly, the column was equilibrated with 10 ml of a buffer solution comprising 50 mM Tris-HCl, pH 7.5, the sample was applied to the column, the column was washed with 350 μl of the previous buffer solution and the sample was recovered in approximately 480 μl. Between each step (column equilibration, sample application, washing and elution) the solution was allowed to enter the packed column bed completely.

[0133] c) Enzymatic Digestion:

[0134] 48 μl of a trypsin solution (0.2 g/l in Tris-HCl, pH 7.5) were added to the buffer exchanged immunoglobulin solution and incubated at 37° C. for about 16 hours at room temperature. The digestion was stopped by the addition of 20 μl 10% (v/v) trifluoro acetic acid (TFA) solution.

[0135] LC-MS/MS Data Acquisition Method:

[0136] The LC-MS/MS analysis was performed by chromatographic separation (LC) of the hydrolytic peptides obtained in the tryptic digestion steps followed by MS and MS/MS detection, respectively, using a nano ESI ion source from Advion BioSciences as an interface between HPLC and mass spectrometer.

[0137] The chromatography was carried out with the following parameters: [0138] HPLC: Dionex Ultimate 3000 [0139] Flow rate: 40 μl/min [0140] UV detection wavelength: 220 nm and 280 nm [0141] Temperature of the column oven: 35° C. [0142] Sample loop: 10 μl [0143] Column: Dionex pep Map C18, 3 μm, 100 Å, 1×150 mm [0144] Injection volume: 10 μl [0145] Eluent A=HPLC gradient grade water containing 0.1% formic acid [0146] Eluent B=HPLC gradient grade acetonitrile containing 0.1% formic acid

[0147] The applied gradient was from 5 vol % Eluent B to 100 vol % Eluent B in 75 minutes.

[0148] The nano ESI MS or MS/MS was carried out with the following parameters: [0149] Nano ESI source: Triversa NanoMate (Advion) [0150] Flow into mass spectrometer ion source: approx. 200 nl/min. managed by a flow splitter [0151] Gas pressure: 0.1-0.5 psi [0152] Voltage to apply: 1.1-1.7 kV [0153] Positive ion: selected

[0154] The mass spectrometric detection was carried out with the following parameters: [0155] Instrument: ESI LTQ-FT ICR (Thermo Scientific) [0156] Capillary temperature: 175° C. [0157] Ion trap collision energy for MS/MS: 40% [0158] Tube lens voltage: 100 V [0159] Dynamic exclusion feature: enabled (Repeat Count: 1, Exclusion Duration: 8 sec, Exclusion mass width: 3 ppm).

[0160] A representative temporal sequence of the scan event cycle for data dependent acquisition of MS/MS spectra is shown in FIG. 1.

[0161] The data acquisition range was 350-2000 m/z for MS spectra. M/Z range for MS/MS spectra was used according to standard instrument settings. The number of MS/MS spectra per high resolution FT scan can vary between 3 to 5. The SID scan is not mandatory for this type of analysis.

EXAMPLE 2

[0162] Analysis of an Anti-CD19 Antibody Reference and Sample

[0163] Data Generation:

[0164] The reference and the sample of the anti-CD19 antibody have been treated according to the Materials and Methods section. MS data has been acquired according to the Materials and Methods section.

[0165] Data Analysis:

[0166] a) Detection of Differences Between Reference and Sample Using LC-MS Data Sets

[0167] For comparison of mass profiles obtained for the reference and the sample (total ion chromatograms (TICs), see FIG. 2) SIEVE™ software package (version 1.1.0 from Thermo Scientific) has been used. Briefly, the TIC data sets of the reference and the sample were aligned by retention time (FIG. 3) and compared for mass peak intensities within a preset retention time window down to a preset threshold (FIGS. 4 and 5).

[0168] Mass signals different in reference and sample according to the parameters predefined for evaluation are listed in Table 1. Mass signals present at identical intensities in the reference and the sample appear at a ratio of 1. Mass signals with higher intensity in the sample than in the reference (e.g. amino acid point mutations) appear with ratios larger than 1. The ratios were calculated by dividing the overall signal intensity in the given m/z versus retention time frame of the sample by the corresponding intensity in the reference. If the overall signal intensity in the given m/z versus retention time frame of the reference is zero (e.g. no background signal due to active noise reduction during data acquisition), the ratio is equal to the overall signal intensity of the sample in the corresponding frame (e.g. hits 2-10 in Table 1).

TABLE-US-00002 TABLE 1 Data for all differential peaks with a ratio >50. m/z m/z time time manual # start stop start stop ratio interpretation 1 1061.19 1062.79 20.7385 21.0385 140043 mutation 1; z = 1 2 1441.94 1443.54 36.043 36.343 68943 background, not peptide related, no MS/MS triggered 3 1989.61 1991.21 36.0909 36.3909 59552 background, not peptide related, no MS/MS triggered 4 1864.12 1865.72 36.0909 36.3909 55943 background, not peptide related, no MS/MS triggered 5 890.645 892.245 36.0909 36.3909 52340 background, not peptide related, no MS/MS triggered 6 1718.96 1720.56 36.0909 36.3909 52235 background, not peptide related, no MS/MS triggered 7 1347.59 1349.19 36.0909 36.3909 45291 background, not peptide related, no MS/MS triggered 8 1110.76 1112.36 36.0909 36.3909 45107 background, not peptide related, no MS/MS triggered 9 1235.23 1236.83 44.5509 44.8509 36205 background, not peptide related, no MS/MS triggered 10 965.611 967.211 36.0909 36.3909 33871 background, not peptide related, no MS/MS triggered 11 606.874 608.474 44.0258 44.3258 798 mutation 2; z = 3 12 607.21 608.81 43.8741 44.1741 571 mutation 2; z = 3 13 606.875 608.475 44.2013 44.5013 261 mutation 2; z = 3 14 910.713 912.313 44.0258 44.3258 194 mutation 2; z = 2 15 607.879 609.479 44.0258 44.3258 143 mutation 2; z = 3 16 910.218 911.818 43.8741 44.1741 108 mutation 2;, z = 2 17 601.538 603.138 45.6276 45.9276 96 not identified 18 735.059 736.659 53.367 53.667 42 present in both LC-MS runs but with only slightly different intensity 19 1022.27 1023.87 6.84557 7.14557 40 present in both LC-MS runs but with only slightly different intensity

[0169] Parameters used for comparison of reference and sample LC-MS data with SIEVE were as follows: [0170] Frame m/z width: 1.6 [0171] Frame time width: 0.3 min. [0172] Intensity threshold: 10000 [0173] M/z start: 350 [0174] M/z end: 2000 [0175] Search peak width: 30% [0176] Retention time start: 5 min. [0177] Retention time stop: 60 min.

[0178] These parameters have been optimized for distinguishing amino acid sequence difference related hits from false positive, i.e. non-amino acid sequence difference related, hits. They only need to be adjusted according to the actual parameters of the LC-MS data as they are: [0179] i) chromatographic resolution (frame time width), and [0180] ii) the sensitivity of the instrument used and the background noise (intensity threshold).

[0181] Further the required sensitivity of the method, e.g. for the detection of very low abundant mutant amino acid sequence (variants), determines the intensity threshold to be set. Using e.g. a LTQ FT ICR instrument mutant sequences (variants) down to 0.2% absolute frequency have been identified.

[0182] b) Identification of Differences Between Reference and Sample Using MS/MS Data

[0183] For identification of the differences found using the procedure as described in the previous paragraph first of all the isotope peak cluster was checked for being typical for peptides. Then, it was checked whether the respective m/z signal was selected for generation of MS/MS fragmentation and whether it was identified by the Mascot error tolerant search (Mascot ETS). Each tentative sequence variant identified by Mascot ETS was checked and confirmed manually using the obtained MS/MS fragment ion spectra (see FIG. 6 and FIG. 7).

[0184] Alternatively, if MS/MS data have been recorded and Mascot ETS did not propose a sequence de novo sequencing was applied either manually or by using software.

[0185] c) Quantitation of Identified Mutant Amino Acid Sequences (Variants)

[0186] The identified mutant amino acid sequence (variant) was quantified at the level of the (tryptic) amino acid sequence fragment containing the mutation (variation) and in relation to the original, non-mutated amino acid sequence fragment within the same sample (see FIG. 8 and FIG. 9). Two extracted ion chromatograms (EIC) were generated from the LC-MS data of the sample, one for the sample and one for the reference sample. The extracted ion chromatograms (EICs) include all charge states and all isotopic peaks of the respective peptide. The peaks generated by the respective EICs were integrated using the instrument software (XCalibur) and the areas calculated hereby are used in the following formula:

[00003]$\mathrm{Percentage}\left(\mathrm{sequence}\mathrm{variation}\right)=\frac{100}{\mathrm{natural}\mathrm{amino}\mathrm{acid}\mathrm{sequence}+\mathrm{mutant}\mathrm{amino}\mathrm{acid}\mathrm{sequence}}*\mathrm{mutant}\mathrm{amino}\mathrm{acid}\mathrm{sequence}$

[0187] Wherein the peak area of the extracted ion chromatogram taking into account all charge states and all isotopic peaks is used.

EXAMPLE 3

[0188] Analysis of an Anti-CCR5 Antibody Reference and Sample

[0189] For the determination of a single amino acid change (mutation) in the variable domains, i.e. light chain variable domain and heavy chain variable domain, an anti-CCR5 antibody has been employed. The first or reference anti-CCR5 antibody has a variable heavy chain and variable light chain domain amino acid sequence selected from the pairs of SEQ ID NO: 01 and 02, SEQ ID NO: 03 and 04, and SEQ ID NO: 05 and 06. The second or sample anti-CCR5 antibody has the following amino acid mutations (changes): in the heavy chain variable domain (VH) the isoleucine residue at amino acid position 109 is mutated (changed) to threonine (VH-I109T), in the light chain variable domain (VL) the valine residue at amino acid position 52 is mutated (changed) to isoleucine (VL-V52I).

[0190] The sample anti-CCR5 antibody has been spiked at 1 wt-% to the reference anti-CCR5 antibody.

[0191] a) Detection of Differences Between Reference and Sample Amino Acid Sequences Using LC-MS Data Sets

[0192] For comparison of mass profiles obtained for the reference and the sample (total ion chromatograms (TICs)) SIEVE™ software package (version 1.1.0 from Thermo Scientific) has been used. Briefly, the data sets of the reference and the sample were aligned chromatographically by retention time (FIG. 10) and compared for mass peak intensities within a preset retention time window down to a preset threshold (FIG. 11).

[0193] Mass signals differences in reference and sample according to the parameters predefined for evaluation are listed in Table 2. Mass signals present at identical intensities in the reference and the sample appear at a ratio of 1. Mass signals with higher intensity in the sample than in the reference (e.g. amino acid point mutations) appear with ratios larger than 1. The ratios were calculated by dividing the overall signal intensity in the given m/z versus retention time frame of the sample by the corresponding intensity in the reference. If the overall signal intensity in the given m/z versus retention time frame of the reference is zero (e.g. no background signal due to active noise reduction during data acquisition), the ratio is equal to the overall signal intensity of the sample in the corresponding frame.

TABLE-US-00003 TABLE 2 Data for differential peaks. m/z m/z time time manual # start stop start stop ratio interpretation 1 947.913 947.933 39.6552 42.1552 429464 mutation 2 948.414 948.434 39.6873 42.1873 312.837 mutation

[0194] b) Identification of Differences Between Reference and Sample Using MS/MS Data

[0195] For identification of the differences found using the procedure as described in the previous paragraph first of all the isotope peak cluster was checked for being typical for peptides. Then, it was checked whether the respective m/z signal was selected for generation of MS/MS fragmentation and whether it was identified by the Mascot error tolerant search (Mascot ETS). Each tentative amino acid sequence mutation (variant) identified by Mascot ETS was checked and confirmed manually using the obtained MS/MS fragment ion spectra.

[0196] Alternatively, if MS/MS data have been recorded and Mascot ETS did not propose a sequence de novo sequencing was applied either manually or by using software.

[0197] c) Quantitation of Identified Sequence Variants

[0198] The identified mutant amino acid sequence (variant) was quantified at the level of the (tryptic) amino acid sequence fragment containing the mutation (variation) and in relation to the original, non-mutated peptide within the same sample (see FIG. 12 and FIG. 13). Two extracted ion chromatograms (EIC) were generated from the LC-MS data of the sample, one for the mutant peptide (variant) and one for the native peptide. The extracted ion chromatograms (EICs) include all charge states and all isotopic peaks of the respective peptide. The peaks generated by the respective EICs were integrated using the instrument software (XCalibur) and the areas calculated hereby according to the formula as shown in Example 1.