METHOD FOR SELECTING ANTIBODIES WITH MODIFIED FCRN INTERACTION

Abstract

Herein is reported a method for selecting a full length antibody comprising the steps of a) generating from a parent full length antibody a plurality of full length antibodies by randomizing one or more amino acid residues selected from the amino acid residues at positions 1-23 in the heavy chain variable domain (numbering according to Kabat), at positions 55-83 in the light chain variable domain (numbering according to Kabat), at positions 145-174 in the first heavy chain constant domain (numbering according to EU index) and at positions 180-97 in the first heavy chain constant domain (numbering according to EU index), b) determining the binding strength of each of the full length antibodies from the 10 plurality of antibodies to the human neonatal Fc receptor (FcRn), and c) selecting a full length antibody from the plurality of full length antibodies that has a different binding strength to the FcRn than the parent full length antibody.

Claims

1. A method for selecting a full length antibody comprising the following steps: a) generating from a parent full length antibody a plurality of full length antibodies by randomizing one or more amino acid residues selected from the amino acid residues at positions 1-23 in the heavy chain variable domain (numbering according to Kabat), at positions 55-83 in the light chain variable domain (numbering according to Kabat), at positions 145-174 in the first heavy chain constant domain (numbering according to EU index) and at positions 180-197 in the first heavy chain constant domain (numbering according to EU index), b) determining the binding strength of each of the full length antibodies from the plurality of antibodies to the human neonatal Fc receptor (FcRn), and c) selecting a full length antibody from the plurality of full length antibodies that has a different binding strength to the FcRn than the parent full length antibody.

2. A plurality of full length antibodies generated from a single full length antibody by randomizing one or more amino acid residues selected from the amino acid residues at positions 1-23 in the heavy chain variable domain (numbering according to Kabat), at positions 55-83 in the light chain variable domain (numbering according to Kabat), at positions 145-174 in the first heavy chain constant domain (numbering according to EU index) and at positions 180-197 in the first heavy chain constant domain (numbering according to EU index).

3. Use of one or more amino acid mutations at positions selected from the group of positions comprising positions 1-23 in the heavy chain variable domain (numbering according to Kabat), positions 55-83 in the light chain variable domain (numbering according to Kabat), positions 145-174 in the first heavy chain constant domain (numbering according to EU index) and positions 180-197 in the first heavy chain constant domain (numbering according to EU index) for changing the in vivo half-life of a full length antibody.

4. A variant full length antibody comprising two light chain polypeptides and two heavy chain polypeptides, wherein the variant antibody is derived from a parent full length antibody by introducing amino acid mutations at one or more positions selected from the group of positions comprising positions 1-23 in the heavy chain variable domain (numbering according to Kabat), positions 55-83 in the light chain variable domain (numbering according to Kabat), positions 145-174 in the first heavy chain constant domain (numbering according to EU index) and positions 180-197 in the first heavy chain constant domain (numbering according to EU index), and wherein the variant antibody has a different affinity for the human neonatal Fc receptor than the parent full length antibody.

5. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 5-18 in the heavy chain variable domain (numbering according to Kabat).

6. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 145-174 in the first heavy chain constant domain (numbering according to EU index).

7. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 161-174 in the first heavy chain constant domain (numbering according to EU index).

8. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 181-196 in the first heavy chain constant domain (numbering according to EU index).

9. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 182-197 in the first heavy chain constant domain (numbering according to EU index).

10. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 55-83 in the light chain variable domain (numbering according to Kabat).

11. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 55-73 in the light chain variable domain (numbering according to Kabat).

12. The antibody according to claim 4, wherein the one or more amino acid residues are selected from the amino acid residues at positions 57-70 in the light chain variable domain (numbering according to Kabat).

13. The antibody according to claim 4, wherein the antibody is a full length IgG antibody.

14. The antibody according to claim 13, wherein the antibody is a full length IgG1 antibody or a full length IgG4 antibody.

15. The antibody according to claim 4, wherein the mutation is a mutation from the amino acid residue to a different amino acid residue from the same group of amino acid residues.

16. The antibody according to claim 4, wherein one or more of the following mutations are introduced (numbering according to Kabat variable domain numbering and Kabat EU index numbering scheme, respectively) heavy chain E6Q, heavy chain A162D, heavy chain A162E, heavy chain T164D, heavy chain T164E, heavy chain S165D, heavy chain S165E, heavy chain S191D, heavy chain S191E, heavy chain G194D, heavy chain G194E, heavy chain T195D, heavy chain T195E, heavy chain Q196D, heavy chain Q196E, light chain G57K, light chain G57R, light chain S60K, light chain S60R.

Description

DESCRIPTION OF THE FIGURES

[0466] FIG. 1 HDX-MS peptide map of the anti-digoxygenin antibody. Peptic peptides from which HDX data could be obtained are shown as white bars for the HC (A) and LC (B). Open big boxes denote peptides with reduced deuterium content upon FcRn binding. Solid boxes indicate targeted regions where ETD of deuterated peptic peptides was used to resolve differential deuterium labeling upon FcRn binding to individual sites. Antibody residues implicated in FcRn binding in crystal structures of rat Fc-FcRn and human FcRn-Fc-YTE are indicated with asterisks (see Martin, W. L., et al., Mol. Cell. 7 (2001) 867-877, Oganesyan, V., et al., J. Biol. Chem. 289 (2014) 7812-7824).

[0467] FIG. 2 HDX profiles of anti-digoxygenin antibody regions in the presence and absence of human FcRn. HDX plots are shown for light chain peptide 55-71 and heavy chain peptides 1-18, 57-79, 159-174 and 180-197. Upper curves display the HDX of the antibody alone and lower curves display HDX of HC and LC peptides, respectively, in the presence of FcRn. For HC 57-79 with FcRn is shown as a dashed black curve. The deuterium incorporation was monitored in triplicates at 1 min, 1 h, 2.5 h and 5 h. The full deuterium level measured in control experiments (at 90% D2O) is shown in black at the 5 h time point.

[0468] FIG. 3 Alignment of the amino acid sequence of the heavy chain variable domains of the anti-digoxigenin antibody, Briakinumab and Ustekinumab. Amino acid residue stretches in which for all three antibodies protection from HDX has been found are boxed.

[0469] FIG. 4 Alignment of the amino acid sequence of the light chain variable domains of the anti-digoxigenin antibody, Briakinumab and Ustekinumab. Amino acid residue stretches in which for all three antibodies protection from HDX has been found are boxed.

[0470] The following Examples, Sequences and Figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES

Materials and Methods

Recombinant DNA Techniques

[0471] Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene and Oligonucleotide Synthesis

[0472] Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany)

Reagents

[0473] All commercial chemicals, antibodies and kits were used as provided according to the manufacturer's protocol if not stated otherwise.

Example 1

Generation of Recombinant Expression Vectors for the Anti-Digoxygenin Antibody

Generation of Vector for the Expression of the Anti-Digoxygenin Antibody Heavy Chain

[0474] The heavy chain encoding fusion gene comprising the human IgG1 constant region (CH1, hinge, CH2, CH3) and the anti-digoxygenin antibody heavy chain variable domain was assembled by fusing a DNA fragment coding for the respective anti-digoxygenin antibody heavy chain variable domain to a sequence element coding the human IgG1 constant region.

[0475] The anti-digoxygenin antibody heavy chain variable domain has the following amino acid sequence:

TABLE-US-00003 (SEQIDNO:01) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYAMSWIRQA PGKGLEWVSSINIGATYIYYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTAVYYCARPGSPYEYDKAYYSMAYWGQGTT VTVSS.

[0476] The human IgG1 constant region has the following amino acid sequence:

TABLE-US-00004 (SEQIDNO:02) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK.

[0477] The expression vector also comprised an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene, which confers ampicillin resistance in E. coli.

[0478] The transcription unit of the antibody heavy chain comprises the following functional elements in 5 to 3 direction: [0479] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, [0480] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0481] a murine immunoglobulin heavy chain signal sequence, [0482] a heavy chain variable (VH) domain encoding nucleic acid, [0483] a human IgG1 constant region encoding nucleic acid, and [0484] the bovine growth hormone polyadenylation sequence (BGH pA).

Generation of Vector for the Expression of the Anti-Digoxygenin Antibody Light Chain

[0485] The kappa light chain encoding fusion gene comprising the human Ig-kappa constant region (CL-kappa) and the anti-digoxygenin antibody light chain variable domain of the kappa isotype was assembled by fusing a DNA fragment coding for the respective anti-digoxygenin antibody light chain variable domain to a sequence element coding for the human Ig-kappa constant region.

[0486] The anti-digoxygenin antibody light chain variable domain has the following amino acid sequence:

TABLE-US-00005 (SEQIDNO:03) DIQMTQSPSSLSASVGDRVTITCRASQDIKNYLNWYQQKP GKAPKLLIYYSSTLLSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQSITLPPTFGGGTKVEIKR.

[0487] The human Ig-kappa constant region has the following amino acid sequence:

TABLE-US-00006 (SEQIDNO:04) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC.

[0488] The expression vector also comprised an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

[0489] The transcription unit of the antibody kappa light chain comprises the following functional elements in 5 to 3 direction: [0490] the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A, [0491] a human heavy chain immunoglobulin 5-untranslated region (5UTR), [0492] a murine immunoglobulin heavy chain signal sequence, [0493] a light chain variable (VL) domain encoding nucleic acid, [0494] a human Ig-kappa constant region encoding nucleic acid, and [0495] the bovine growth hormone polyadenylation sequence (BGH pA).

Example 2

Recombinant Production of the Anti-Digoxygenin Antibody

[0496] The antibody was produced in transiently transfected HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in F17 Medium (Invitrogen Corp.). For transfection of the respective vectors as described in Example 1 the 293-Free Transfection Reagent (Novagen) was used. The antibody was expressed from individual expression plasmids. Transfections were performed as specified in the manufacturer's instructions. Recombinant antibody-containing cell culture supernatants were harvested three to seven days after transfection. Supernatants were stored at reduced temperature (e.g. 80 C.) until purification.

[0497] General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example 3

Purification of Recombinant Anti-Digoxygenin Antibody

[0498] The antibody-containing culture supernatants were filtered and purified by two chromatographic steps.

[0499] The antibody was captured by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH.sub.2PO.sub.4, 10 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and the antibody was recovered with 25 mM citrate buffer, pH 3.1, which was immediately after elution adjusted to pH 6.0 with 1 M Tris-base, pH 9.0.

[0500] Size exclusion chromatography on Superdex 200 (GE Healthcare) was used as second purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody containing solutions were concentrated with an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, Mass., USA) and stored at 80 C.

Example 4

Generation of Recombinant Expression Vectors for Ustekinumab and Briakinumab

[0501] Ustekinumab: CNTO 1275, Stelara, CAS Registry Number 815610-63-0

[0502] Briakinumab: ABT 874, J 695, Ozespa, SEQ ID NO: 36, WO2001/014162

[0503] For the expression of the above antibodies expression plasmids for transient expression (e.g. in HEK293-F) based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were used.

[0504] Beside the antibody expression cassette the plasmids contained: [0505] an origin of replication which allows replication of this plasmid in E. coli, [0506] a -lactamase gene which confers ampicillin resistance in E. coli, and [0507] the dihydrofolate reductase gene from Mus musculus as a selectable marker in eukaryotic cells.

[0508] The transcription unit of the antibody gene was composed of the following elements: [0509] unique restriction site(s) at the 5 end [0510] the immediate early enhancer and promoter from the human cytomegalovirus, [0511] followed by the Intron A sequence in the case of the cDNA organization, [0512] a 5-untranslated region of a human antibody gene, [0513] an immunoglobulin heavy chain signal sequence, [0514] the human antibody chain either as cDNA or as genomic organization with the immunoglobulin exon-intron organization [0515] a 3 non-translated region with a polyadenylation signal sequence, and [0516] unique restriction site(s) at the 3 end.

[0517] The fusion genes comprising the antibody chains were generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective plasmids. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

Example 5

Recombinant Production of Ustekinumab and Briakinumab

[0518] Standard cell culture techniques were used as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc.

[0519] The antibodies were generated by transient transfection with the respective plasmids (e.g. encoding the heavy chain, as well as the corresponding light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293fectin or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1*10.sup.6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO.sub.2. The day after the cells were transfected at a cell density of ca. 1.5*10.sup.6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 g total plasmid DNA (1 g/mL) encoding the heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM+1.2 mL 293 fectin or fectin (2 L/mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the supernatant or the supernatant was frozen and stored.

Example 6

Purification of Recombinant Ustekinumab and Briakinumab

[0520] The antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl-Sepharose (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.

[0521] Briefly, sterile filtered cell culture supernatants were captured on a MabSelectSuRe resin equilibrated with PBS buffer (10 mM Na.sub.2HPO.sub.4, 1 mM KH.sub.2PO.sub.4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate at pH 3.0. The eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0. The antibody pools were prepared for hydrophobic interaction chromatography by adding 1.6 M ammonium sulfate solution to a final concentration of 0.8 M ammonium sulfate and the pH adjusted to pH 5.0 using acetic acid. After equilibration of the butyl-Sepharose resin with 35 mM sodium acetate, 0.8 M ammonium sulfate, pH 5.0, the antibodies were applied to the resin, washed with equilibration buffer and eluted with a linear gradient to 35 mM sodium acetate pH 5.0. The antibody containing fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0. The antibody containing fractions were pooled, concentrated to the required concentration using Vivaspin ultrafiltration devices (Sartorius Stedim Biotech S.A., France) and stored at 80 C.

TABLE-US-00007 TABLE Yields of the antibodies. Final purified Final purified product Sample product [mg] concentration [mg/mL] Briakinumab 23.50 2.36 Ustekinumab 12.55 2.67

[0522] Purity and antibody integrity were analyzed after each purification step by CE-SDS using microfluidic Labchip technology (Caliper Life Science, USA). Five l of protein solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.

Example 7

Expression of FcRn in HEK293 Cells

[0523] FcRn was transiently expressed by transfection of HEK293 cells with two plasmids containing the coding sequence of FcRn (SEQ ID NO: 09) and of beta-2-microglobulin (SEQ ID NO: 10). The transfected cells were cultured in shaker flasks at 36.5 C., 120 rpm (shaker amplitude 5 cm), 80% humidity and 7% CO.sub.2. The cells were diluted every 2-3 days to a density of 3 to 4*10.sup.5 cells/ml.

[0524] For transient expression, a 14 l stainless steel bioreactor was started with a culture volume of 81 at 36.5 C., pH 7.00.2, pO.sub.2 35% (gassing with N.sub.2 and air, total gas flow 200 ml min.sup.1) and a stirrer speed of 100-400 rpm. When the cell density reached 20*10.sup.5 cells/ml, 10 mg plasmid DNA (equimolar amounts of both plasmids) was diluted in 400 ml Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to this mixture, which was then incubated for 15 minutes at room temperature and subsequently transferred into the fermenter. From the next day on, the cells were supplied with nutrients in continuous mode: a feed solution was added at a rate of 500 ml per day and glucose as needed to keep the level above 2 g/l. The supernatant was harvested 7 days after transfection using a swing head centrifuge with 1 l buckets: 4000 rpm for 90 minutes. The supernatant (13 L) was cleared by a Sartobran P filter (0.45 m+0.2 m, Sartorius) and the FcRn beta-2-microglobulin complex was purified therefrom.

Example 8

Characterization of Monoclonal Antibodies and FcRn by MS

[0525] Reduced intact mass spectrometry was performed on 50 g monoclonal antibody or FcRn which was reduced and denatured using 0.5 M TCEP (Perbio, Bonn, Germany) in 4 M guanidinium hydrochloride solution at 37 C. for 30 minutes. Samples were desalted by size exclusion chromatography (Sephadex G-25, isocratic elution with 40% acetonitrile with 2% formic acid (v/v)). ESI Mass spectra were recorded on a Q-TOF instrument (MaXis, Bruker, Germany) equipped with a Triversa NanoMate (Advion, Ithaca, USA). For data evaluation, in house developed software was used.

[0526] Peptide mapping of monoclonal antibody and FcRn (250 g) was done by denaturing the compounds by addition of 0.4 M Tris, 8 M guanidinium hydrochloride, pH 8 and 0.24 M DTT for one hour at 37 C. and alkylated by addition of 0.6 M iodoacetic acid in water for 15 minutes at room temperature in the dark. The samples were buffer exchanged to 50 mM Tris/HCl, pH 7.5 using NAP 5 Sephadex G-25 DNA grade columns (GE Healthcare, Munich, Germany). Digestion was performed with trypsin (Promega, Mannheim, Germany) for 5 hours at 37 C. (enzyme to substrate ratio of 1:37). The peptide mixture obtained was injected and separated without pretreatment using reversed phase U-HPLC (NanoAcquity, Waters GmbH, Eschborn, Germany). An Acquity UPLC BEH C18 column (1150 mm, 1.7 m particle diameter, 300 pore size) from Waters was used for separation. The solvents were 0.1% (v/v) formic acid in water (A) and in acetonitrile (B) (Sigma Aldrich, Munich, Germany). A linear gradient of 60 l/min. from 1 to 40% B was run over 120 min. at 50 C. Mass analysis was performed by coupling the UPLC system to a LTQ Orbitrap XL tandem mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany) operating in positive ion mode through a Triversa NanoMate interface (Advion, Ithaca, USA).

Example 9

Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

[0527] HDX-MS experiments were performed using the following sample setup:

[0528] The monoclonal antibody (73 pmol/l) with and without FcRn (112 pmol/l) were mixed and diluted in with D.sub.2O (99.9 atom % deuterium) containing 50 mM sodium phosphate, 50 mMNaCl, pH 6.5 to a final deuterium content of 90% and concentrations of antibody of 1.2 pmol/l and FcRn of 8.96 pmol/l (84% antibody bound, 1:2 IgG1:FcRn binding ratio).

[0529] Theoretical calculations were based on a KD of 0.6 M for the IgG1-FcRn interaction and a final volume of 25 l following dilution with D20. Following 15 min. pre-incubation of samples, deuterium labeling was initiated at room temperature for different time intervals: 0 min., 1 min., 1 hour, 2.5 hours and 5 hours. At each time interval aliquots (25 l) of 30 pmol target protein was removed from the labeling mixture and quenched to a final pH of 2.5 in an ice cold mixture of 25 l mM Na phosphate, 50 mM NaCl, pH 6.5 and 50 l 0.5 M TCEP, 6 M Guanidinium-hydrochloride in phosphoric acid, pH 2.3 and frozen to 80 C. until LC-MS analysis. Fully deuterated samples were prepared by overnight incubation of in 6 M deuterated guanidinium-hydrochloride (final deuterium content of 90 atom %).

[0530] The quenched deuterated proteins (30 pmol) were loaded onto a refrigerated HDX-UPLC system coupled to a hybrid Q-TOF Synapt G2 mass spectrometer (Waters, Milford, USA). The UPLC system was operated 0 C. and equipped with an in-house packed pepsin column with a 60 l internal volume (IDEX, Oak Harbor, USA) containing pepsin immobilized on agarose (Thermo Scientific Pierce, Rockford, USA), a trap C18 column (ACQUITY UPLC BEH C18 1.7 m VanGuard column (Waters, Milford, USA) and an analytical C18 column (ACQUITY UPLC BEH C18 1.7 m, 1100 mm column (Waters, Milford, USA)). Proteins were digested in-line at a temperature of 20 C. and desalted on the trap column with a flow rate of 200 l mobile phase A (0.23% FA). Peptic peptides were eluted from the trap column and across the analytical column at a flow of 40 l/min and a 7 min gradient from 8% to 40% mobile phase B (ACN, 0.23% FA) and into the mass spectrometer for mass analysis. The ESI source was operated in positive ion mode the instrument was enabled for ion mobility analysis. A reference lock-spray signal of Glu-Fibrinopeptide (Sigma-Aldrich, St. Louis, USA) was acquired for internal calibration. Identification of peptides was done by a combination of MSe, HDMSe and DDA MS/MS. Peptide identifications were made through database searching in PLGS ver. 2.5 and HDX-MS data was processed in DynamX ver. 2.2.1. HDX-MS data of overlapping peptides were only used to localize deuterium uptake to smaller segments if the back-exchange of the fully deuterated antibody peptides was similar (below 7%) (Sheff, J., et al., J. Am. Soc. Mass Spectrom. 24 (2013) 1006-1015).

Example 10

[0531] Hydrogen/Deuterium Exchange Mass Spectrometry with ETD

[0532] Deuterated samples were prepared by the same procedure as in the previous Example except the injection amount was adjusted to 100 pmol antibody and a five-fold dilution into D.sub.2O buffer (to a final 80 atom % deuterium) was employed resulting in an antibody concentration of 4 pmol/l and an FcRn concentration of 14 pmol/l and 85% bound antibody during labeling. HDX-ETD was performed in a targeted manner on selected antibody peptide fragments with differential deuterium uptake between the FcRn bound and unbound state. The ESI source and source T-wave was operated at settings optimized for minimal H/D scrambling as described in Rand, K. D., et al. (J. Am. Soc. Mass Spectrom. 22 (2011) 1784-1793) with the following parameters: capillary voltage 2.8 kV, desolvation gas flow 800 L/h, cone gas flow 0 L/h, source temperature 90 C., desolvation gas temperature 300 C., sampling cone 20 V, extraction cone 2 V, T-wave Trap wave velocity 300 m/s, wave height 0.2 V. ETD was performed in the trap T-wave using 1,4-dicyanobenzene (Sigma-Aldrich, St. Louis, USA) as the ETD reagent. 1,4-dicyanobenzene was introduced into the anion source using a nitrogen makeup flow of 20 ml/min over the reagent crystals stored in a sealed container. The radical anions were generated via glow discharge with a current of 40 A and make up gas flow of 20 ml/min. ETD data was analyzed by determining the average masses of the c- and z-type ETD fragment ions. The deuterium content was calculated by subtracting the deuterium content of the unlabeled product ions from the deuterium content of the deuterated samples. The absence of H/D scrambling was verified by monitoring the loss of ammonia from the charged reduced species in ETD spectra recorded of peptic peptides from a fully deuterated antibody sample as described in Rand, K. D., et al. (Anal. Chem. 82 (2010) 9755-9762).