Method for detecting multispecific antibody light chain mispairing
11486882 · 2022-11-01
Assignee
Inventors
- Michael Molhoj (Munich, DE)
- Maximiliane Koenig (Pullach, DE)
- Vincent Larraillet (Penzberg, DE)
- Katja Montan (Munich, DE)
- Bianca Gruenwalder (Bad Heilbrunn, DE)
Cpc classification
International classification
Abstract
Use of a limited digestion with a proteolytic enzyme of a multispecific antibody for the analysis of the multispecific antibody's light chain pairing.
Claims
1. A method for determining light chain pairing in a multispecific antibody comprising the following steps: a) incubating a sample comprising the multispecific antibody with a proteolytic enzyme for 35 to 45 minutes, wherein the proteolytic enzyme is Lys-C to produce fragments of the multispecific antibody, b) identifying the mass of the fragments of the multispecific antibody obtained in step a) by mass spectrometry, and c) determining from the mass of the fragments identified in step b) the light chain pairing of the multispecific antibody, thereby determining light chain pairing of the multispecific antibody.
2. The method according to claim 1, wherein the incubating is for 40 minutes.
3. The method according to claim 1, wherein the multispecific antibody that is incubated with the proteolytic enzyme is a deglycosylated multispecific antibody.
4. The method according to claim 1, wherein the weight ratio of antibody to enzyme is 1:200.
5. The method according to claim 1, wherein the multispecific antibody that is incubated with the proteolytic enzyme has a concentration of from 200 to 600 μg/mL.
6. The method according to claim 1, wherein step b) further comprises: b) desalting the incubation mixture of step a) and identifying the mass of the fragments obtained by the proteolytic digestion in step a) by mass spectrometry.
7. The method according to claim 1, wherein the multispecific antibody is a monoclonal multispecific antibody.
8. The method according to claim 1, wherein the multispecific antibody comprises at least two non-peptidically bound light chains.
9. The method according to claim 1, wherein the multispecific antibody comprises at least three non-peptidically bound polypeptides.
10. A method for determining light chain mispairing in a multispecific antibody comprising the following steps: a) incubating a sample comprising the multispecific antibody with a proteolytic enzyme for 35 to 45 minutes, wherein the proteolytic enzyme is Lys-C to produce fragments of the multispecific antibody, b) identifying the mass of the fragments of the multispecific antibody obtained in step a) by mass spectrometry, and c) determining from the mass of the fragments identified in step b) the light chain pairing of the multispecific antibody, and thereby determining light chain mispairing of the multispecific antibody.
11. The method according to claim 10, wherein the incubating is for 40 minutes.
12. The method according to claim 10, wherein the multispecific antibody that is incubated with the proteolytic enzyme is a deglycosylated multispecific antibody.
13. The method according to claim 10, wherein the multispecific antibody that is incubated with the proteolytic enzyme has a concentration of from 200 to 600 μg/mL.
14. The method according to claim 10, wherein step b) further comprises: b) desalting the incubation mixture of step a) and identifying the mass of the fragments obtained by the proteolytic digestion in step a) by mass spectrometry.
15. The method according to claim 10, wherein the multispecific antibody is a monoclonal multispecific antibody.
16. The method according to claim 10, wherein the multispecific antibody comprises at least two non-peptidically bound light chains.
17. The method according to claim 10, wherein the multispecific antibody comprises at least three non-peptidically bound polypeptides.
Description
DESCRIPTION OF THE FIGURES
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(12) The following examples 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
(13) Materials & General Methods
(14) General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of antibody chains are numbered and referred to according to the numbering systems according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) as defined above.
(15) Recombinant DNA Techniques
(16) 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.
(17) Gene Synthesis
(18) Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The 600-1800 bp long gene segments, which were flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligating oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites e.g. KpnI/SacI or AscI/Pacl into a pPCRScript (Stratagene) based pGA4 cloning vector. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany).
(19) DNA Sequence Determination
(20) DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).
(21) DNA and Protein Sequence Analysis and Sequence Data Management
(22) The GCG's (Genetics Computer Group, Madison, Wis.) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.
(23) Expression Vectors
(24) For the expression of the described antibodies, variants of expression plasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F) cells based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were applied.
(25) Beside the antibody expression cassette the vectors contained: an origin of replication which allows replication of this plasmid in E. coli, and a ß-lactamase gene which confers ampicillin resistance in E. coli.
(26) The transcription unit of the antibody gene was composed of the following elements: unique restriction site(s) at the 5′ end the immediate early enhancer and promoter from the human cytomegalovirus, followed by the Intron A sequence in the case of the cDNA organization, a 5′-untranslated region of a human antibody gene, an immunoglobulin heavy chain signal sequence, the human antibody chain (wildtype or with domain exchange) either as cDNA or as genomic organization with the immunoglobulin exon-intron organization a 3′ untranslated region with a polyadenylation signal sequence, and unique restriction site(s) at the 3′ end.
(27) The fusion genes comprising the antibody chains as described below 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 vectors. 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).
(28) Cell Culture Techniques
(29) 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.
(30) Multispecific antibodies were expressed by transient co-transfection of the respective expression plasmids in adherently growing HEK293-EBNA or in HEK29-F cells growing in suspension as described below.
(31) Transient Transfections in HEK293-EBNA System
(32) Multispecific antibodies were expressed by transient co-transfection of the respective expression plasmids (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified light chain) in adherently growing HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr-Virus nuclear antigen; American type culture collection deposit number ATCC #CRL-10852, Lot. 959 218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco®) supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco®), 2 mM L-Glutamine (Gibco®), and 250 μg/ml Geneticin (Gibco®). For transfection FuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was used in a ratio of FuGENE™ reagent (μl) to DNA (μg) of 4:1 (ranging from 3:1 to 6:1). Proteins were expressed from the respective plasmids using a molar ratio of (modified and wildtype) light chain and heavy chain encoding plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1, respectively. Cells were fed at day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco®]. Multispecific antibody containing cell culture supernatants were harvested from day 5 to 11 after transfection by centrifugation and stored at −20° C. 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.
(33) Transient Transfections in HEK293-F System
(34) Multispecific antibodies were generated by transient transfection with the respective plasmids (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified 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 four expression plasmids and 293Fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. The day after the cells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 μg total plasmid DNA (1 μg/mL) encoding the heavy or modified 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 μl/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.
(35) Protein Determination
(36) The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423.
(37) Antibody Concentration Determination in Supernatants
(38) The concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with Protein A Agarose-beads (Roche). 60 μL Protein A Agarose beads were washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culture supernatant was applied to the Protein A Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1 hour at room temperature the beads were washed on an Ultrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twice with 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly four times with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was eluted by addition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample was combined with NuPAGE® Sample Reducing Agent or left unreduced, respectively, and heated for 10 min at 70° C. Consequently, 5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue.
(39) The concentration of antibodies and derivatives in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind to Protein A were applied to an Applied Biosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted protein was quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.
(40) Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche) are coated with 100 μL/well biotinylated anti-human IgG capture molecule F(ab′)2<h-Fcγ>BI (Dianova) at 0.1 μg/mL for 1 hour at room temperature or alternatively overnight at 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/well of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1-2 hour on a microtiterplate shaker at room temperature. The wells were washed three times with 200 μL/well PBST and bound antibody was detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detection antibody for 1-2 hours on a micro-titerplate shaker at room temperature. Unbound detection antibody was washed away three times with 200 μL/well PBST and the bound detection antibody was detected by addition of 100 μL ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
(41) Protein Purification
(42) Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions were pooled, concentrated (if required) using e.g., a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at −20° C. or −80° C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.
(43) SDS-PAGE
(44) The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) running buffer was used.
(45) Analytical Size Exclusion Chromatography
(46) Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.
Example 1
(47) Method for the Determination of Light Chain Mispairing of a Multispecific Antibody after Limited Proteolytic Digestion
(48) The expected primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of the limited LysC digested CrossMabs. Advantageously the antibody has been deglycosylated in advance.
(49) The VH/VL CrossMabs was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/mL and an antibody: enzyme ratio of 100:1. The limited Lys-C (Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated VH/VL CrossMabs in a Tris buffer pH 8 at 37° C. for 40 min.
(50) Prior to mass spectrometry the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare).
(51) The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).
Example 2
(52) Method for the Determination of Light Chain Mispairing of a Tetravalent Bispecific Antibody
(53) The tetravalent bispecific antibody was deglycosylated with N-Glycosidase F in a phosphate or Tris or histidine buffer at 37° C. for up to 17 h at a protein concentration of 1 mg/ml and an antibody:enzyme ratio of 100:1. The limited Lys-C(Roche Diagnostics GmbH, Mannheim, Germany) digestions was performed with 100 μg deglycosylated tetravalent bispecific antibody in a 100 mM citrate buffer pH 3.7 at 37° C. for 16 hours at a protein concentration of approx. 0.9 mg/mL and an antibody:enzyme ratio of 50:1.