IMMUNOGLOBULIN GLYCOSYLATION PATTERN ANALYSIS

Abstract

The current invention is directed to a method for the determination of the glycosylation pattern of a human immunoglobulin of the subclass IgG1 or IgG4 or of a murine immunoglobulin of the subclass IgG2a or IgG3 comprising the following steps: a) cleaving said immunoglobulin into fragments by enzymatic digestion with the enzyme IdeS, b) separating the fragments of said immunoglobulin obtained by the enzymatic digestion by reversed phase high performance liquid chromatography, c) subjecting the separated fragments of said immunoglobulin obtained in step b) to a mass spectrometric analysis, and d) determining the glycosylation pattern of said immunoglobulin from the mass spectrometric data obtained in step c).

Claims

1. A method for the determination of the glycosylation pattern of an immunoglobulin comprising the following steps: a) cleaving said immunoglobulin into fragments by enzymatic digestion with the enzyme IdeS, b) separating the fragments of said immunoglobulin obtained by the enzymatic digestion by reversed phase high performance liquid chromatography, c) subjecting the separated fragments of said immunoglobulin obtained in step b) to a mass spectrometric analysis, and d) determining the glycosylation pattern of said immunoglobulin from the mass spectrometric data obtained in step c); wherein said immunoglobulin is a human immunoglobulin selected from subclass IgG1 subclass IgG2 subclass IgG4 a variant of subclass IgG1 a variant of subclass IgG2 and a variant of subclass IgG4; or wherein said immunoglobulin is a humanized immunoglobulin selected from subclass IgG1 subclass IgG2 subclass IgG4 a variant of subclass IgG1 a variant of subclass IgG2, and a variant of subclass IgG4; or wherein said immunuoglobulin is a murine immunoglobulin selected from subclass IgG2a subclass IgG2b subclass IgG3 a variant of subclass IgG2a a variant of subclass IgG2b and a variant of subclass IgG3.

2. A method for the determination of the glycosylation pattern of the Fab.sub.2-fragment of an immunoglobulin comprising the following steps: a) cleaving said immunoglobulin into fragments by enzymatic digestion with the enzyme IdeS, b) treating said enzymatically digested immunoglobulin of step a) with formic acid and a reducing agent, c) separating the obtained fragments of said immunoglobulin by reversed phase high performance liquid chromatography or a size exclusion chromatography, d) subjecting the separated fragments of said immunoglobulin obtained in step c) to a mass spectrometric analysis by direct infusion into a mass spectrometer, and e) determining the glycosylation pattern of said Fab.sub.2-fragment from the mass spectrometric data obtained in step d) wherein said immunoglobulin is a human immunoglobulin selected from subclass IgG1 subclass IgG2, and subclass IgG4; or wherein said immunoglobulin is a humanized immunoglobulin selected from subclass IgG1, subclass IgG2, and subclass IgG4; or wherein said immunuoglobulin is a murine immunoglobulin selected from subclass IgG2a, subclass IgG2b, and subclass IgG3.

3. A method for the production of a Fab.sub.2-fragment, of an immunoglobulin wherein the method comprises the following steps: a) providing an immunoglobulin from which the Fab.sub.2-fragment is to be obtained, b) cleaving said immunoglobulin into the Fab.sub.2-fragment and the HC-FC-fragment by enzymatic digestion with the enzyme IdeS, and c) producing said Fab.sub.2-fragment by chromatography with a protein A chromatographical resin of the fragments obtained in b), wherein said immunoglobulin is a human immunoglobulin selected from subclass IgG1, subclass IgG2, and subclass IgG4; or wherein said immunoglobulin is a humanized immunoglobulin selected from subclass IgG1, subclass IgG2, and subclass IgG4; or wherein said immunuoglobulin is a murine immunoglobulin selected from subclass IgG2a, subclass IgG2b, and subclass IgG3.

4. A method for monitoring a sample comprising an immunoglobulin comprising the following steps: a) storing the sample for a time period, b) cleaving said immunoglobulin contained in the sample into fragments by enzymatic digestion with the enzyme IdeS, c) separating the fragments of said immunoglobulin obtained by the enzymatic digestion by reversed phase high performance liquid chromatography, d) treating said enzymatically cleaved immunoglobulin of step c) with formic acid and a reducing agent, e) separating the obtained fragments of said immunoglobulin by reversed phase high performance liquid chromatography or a size exclusion chromatography, and f) determining the presence of degradation products by a shift of the retention time of the fragments compared to a reference sample treated with steps b) to e).

5. The method according to claim 1, wherein said separating step is: desalting the obtained fragments of said immunoglobulin by a size exclusion chromatography.

6. The method according to claim 1, wherein said cleaving step further comprises: treating said immunoglobulin with carboxypeptidase B.

7. (canceled)

8. The method according to claim 1, wherein said cleaving is performed in a pH range between pH 5.5. and pH 8.5.

9. The method according to claim 1, wherein the cleaving is performed for two hours.

10. The method according to claim 2, wherein said reducing agent is tris-(2-carboxyethyl)-phosphine.

11. The method according to claim 1, wherein the molar ratio of IdeS to the immunoglobulin molecule is between 1:25 and 1:2500.

12. A method for the production of a Fab-fragment of an immunoglobulin, wherein the method comprises the following steps: a) providing an immunoglobulin from which the Fab-fragment is to be obtained, b) cleaving said immunoglobulin into an Fab.sub.2-fragment and an HC-FC-fragment by enzymatic digestion with the enzyme IdeS, c) treating said enzymatically cleaved immunoglobulin of step b) with formic acid and a reducing agent, and d) producing said Fab-fragment by chromatography with a protein A chromatographical resin of the fragments obtained in c) wherein said immunoglobulin is a human immunoglobulin selected from subclass IgG1, subclass IgG2, and subclass IgG4; or wherein said immunoglobulin is a humanized immunoglobulin selected from subclass IgG1, subclass IgG2, and subclass IgG4; or wherein said immunuoglobulin is a murine immunoglobulin selected from subclass IgG2a, subclass IgG2b, and subclass IgG3.

Description

DESCRIPTION OF THE FIGURES

[0105] FIG. 1 Schematic representation of the IdeS digestion of IgG1 immunoglobulins.

[0106] FIG. 2 Mass spectrum of IdeS cleaved human IgG1 analyzed by SEC and direct infusion into mass spectrometer. The spectrum shows only the glycosylated Fc fragment.

[0107] FIG. 3 Mass spectrum of IdeS cleaved human IgG1 analyzed by SEC and direct infusion into mass spectrometer. The spectrum shows the glycosylated Fc-fragment and the Fab.sub.2-fragment.

[0108] FIG. 4 Chromatogram of IdeS digested and reduced murine IgG3 immunoglobulin.

[0109] FIG. 5 Zoom mass spectrum of heavy chain Fab-fragment with O-glycosylation in the hinge region of murine IgG3 immunoglobulin.

[0110] FIG. 6 Zoom mass spectrum of heavy chain Fc-fragment with N-glycosylation of murine IgG3 immunoglobulin.

[0111] FIG. 7 Deconvoluted mass spectrum of the O-Glycosylated tryptic peptide (IPKPSTPPGSSCPPGNILGGPSVFIFPPKPK (HC=heavy chain, amino acid (aa) 217-247; S and T: possible O-glycosylation sites) as obtained from LC-MS.

[0112] FIG. 8 Chromatogram of IdeS digested humanized IgG4 immunoglobulin.

[0113] FIG. 9 Zoom mass spectrum of heavy chain Fc-fragment with N-glycosylation of humanized IgG4 immunoglobulin.

[0114] FIG. 10 Chromatogram of IdeS digested humanized IgG1 immunoglobulin.

[0115] FIG. 11 Zoom mass spectrum of heavy chain Fc-fragment with N-glycosylation of humanized IgG1 immunoglobulin.

[0116] FIG. 12A Overlay of chromatograms of IdeS digested humanized IgG1 immunoglobulin taken from the supernatant of a cultivation sample.

[0117] FIG. 12B Overlay of chromatograms of IdeS digested humanized IgG1 immunoglobulin taken from the supernatant of a protein A purified sample.

[0118] FIG. 13A Overlay of mass spectra of IdeS digested humanized IgG1 immunoglobulin taken from the supernatant of a cultivation sample (retention time: 16.8 minutes).

[0119] FIG. 13B Overlay of mass spectra of IdeS digested humanized IgG1 immunoglobulin taken from the supernatant of a protein A purified sample (retention time: 16.8 minutes).

[0120] FIG. 14A Zoom overlay of mass spectra of IdeS digested humanized IgG1 immunoglobulin taken from the supernatant of a cultivation sample (retention time: 16.8 minutes).

[0121] FIG. 14B Zoom overlay of mass spectra of IdeS digested humanized IgG1 immunoglobulin taken from the supernatant of protein A purified sample (retention time: 16.8 minutes).

[0122] FIG. 15 Analytical size exclusion chromatogram overlay of the complete immunoglobulin, the Fab.sub.2-fragment and the standard.

EXAMPLE 1

[0123] Determination of the Antibody Concentration:

[0124] The antibody concentration was determined by means of an absorption measurement at 280 nm on a spectral photometer of the type UVIKON XL (Goebel Company). The extinction coefficient of the antibody that was used was 1.55 ml/(mg*cm) and was calculated according to the method of Pace, C. N., et al., (Protein Sci. 4 (1995) 2411-2423).

EXAMPLE 2

[0125] General Method A (for IgG1, IgG3)

[0126] IdeS Digestion for Analysis of the Glycosylation Pattern in the Fc-Fragment:

[0127] 100 g (0.66 nmol) immunoglobulin are diluted to a final concentration of 1 mg/ml in 50 mM TRIS/HCl buffer pH 8.0 and 2 l (c=1 mg/ml, 0.06 nmol) of Immunoglobulin degrading enzyme (IdeS, MW 345890 Da) are added to give an enzyme to immunoglobulin ratio of 1:50 by weight. The solution is incubated for 2 to 5 h at 37 C. depending on the immunoglobulin used. For analysis with size exclusion chromatography and direct infusion into mass spectrometer the enzyme activity is stopped by addition of an equal volume of 1% formic acid to the solution.

[0128] Reduction of IdeS Digested Immunoglobulin for Additional Analysis of Glycosylation Pattern in the Fab-Fragment:

[0129] For reduction half of the sample is diluted with 64 l of 100 mM potassium phosphate buffer pH 7.0 to give a final volume of 115 l. Then, 60 l of 0.5 M TCEP (tris (2-carboxyethyl) phosphine, Pierce) dissolved in a volume of 4 M guanidine hydrochloride and 50 ml of 8 M guanidine hydrochloride are added. Afterwards the sample is incubated for 30 minutes at 37 C. The reaction is stopped by addition of 5 l of 20% (v/v) formic acid.

EXAMPLE 3

[0130] General Method B (for IgG1, IgG3, IgG4)

[0131] Combined IdeS and CpB Digest:

[0132] 100 g (0.66 nmol) immunoglobulin are diluted to a final concentration of 1 mg/ml in 50 mM TRIS/HCl buffer pH 8.0 and 2 l (c=1 mg/ml, 0.06 nmol) of Immunoglobulin degrading enzyme (IdeS, MW 345890 Da) are added to give an enzyme to immunoglobulin ratio of 1:50 by weight. The solution is incubated for 2 h to 5 h at 37 C. depending on the immunoglobulin used. 1 l (1 mg/ml) of Carboxypeptidase B (CpB, Roche Diagnostics GmbH, Mannheim, Germany) is added to the solution 30 minutes before the end of the IdeS incubation time to give an enzyme to immunoglobulin ratio of 1:25 by weight.

EXAMPLE 4

[0133] General Method C (for IgG with Complex Glycosylation Pattern)

[0134] Combined IdeS and EndoH Digest:

[0135] 25 g (0.17 nmol) immunoglobulin are diluted to a final concentration of 0.5 mg/ml in sodium phosphate buffer at pH 6.0 and 2.5 l (c=2.5 U/500 l) of Endoglycosidase H (Roche Diagnostics GmbH, Mannheim, Germany) are added and incubated for 18 h at 37 C. to cleave the oligosaccharide structures. Subsequently, the pH is adjusted to 8.0 by adding 25 l of 0.1 M TRIS/HCl buffer pH 8.0. Cleavage of the immunoglobulin is achieved by adding 0.5 l of (c=1 mg/ml, 0.02 nmol) of Immunoglobulin degrading enzyme (IdeS, MW 345890 Da) and incubation for 2 h at 37 C.

EXAMPLE 5

[0136] General Method D (for Cultivation Supernatants)

[0137] Combined IdeS and CpB Digest:

[0138] 50 g (0.33 nmol) of a cultivation supernatant of a cultivation of an immunoglobulin expressing eukaryotic cell are centrifuged for 3 minutes at 10,800 rcf (relative centrifugal force) and diluted to a final concentration of 0.7 mg/ml in 50 mM TRIS/HCl buffer pH 8.0. 1 l (c=1 mg/ml, 0.06 nmol) of Immunoglobulin degrading enzyme (IdeS, MW 345890 Da) is added to give an enzyme to immunoglobulin ratio of 1:50 by weight. The solution is incubated for 2 to 5 h at 37 C. depending on the immunoglobulin used. 1 l (1 mg/ml) of Carboxypeptidase B (CpB, Roche Diagnostics GmbH, Mannheim, Germany) is added to the solution 30 minutes before the end of the IdeS incubation time to give an enzyme to immunoglobulin ratio of 1:25 by weight.

EXAMPLE 6

[0139] RP-HPLC-MS Method

[0140] The LC-MS is performed on an Agilent Cap LC1100 coupled to QTOF II (Micromass/Waters). The chromatographic separation is performed on a Phenomenex Jupiter C18 column (5 m particle size, 300 pore size, 1250 mm column). Eluent A is 0.5% formic acid, eluent B is 70% Isopropanol, 20% acetonitrile, 9.5% Water and 0.5% formic acid. The flow is 40 l/min, the separation is performed at 75 C. and 2 g (10 l) of immunoglobulin obtained with a method according to one of the Examples 2 to 5 are injected onto the column. Following gradient is applied:

TABLE-US-00001 Time (min) % B 0 20 7 20 9 25 29 50 32 100 37 100 38 20 50 20

[0141] During the first 7 minutes the eluate is directed into the waste to prevent the mass spectrometer ion source from salt contamination. UV signal at 280 nm (reference 360 nm) is recorded. MS spectra are acquired using a capillary voltage of 2,700 V, a cone voltage of 30 V in a mass range from 600 to 2000 m/z in positive ion mode using a desolvation temperature of 120 C. and a source temperature of 80 C. MS data are acquired from 7 to 50 minutes.

EXAMPLE 7

[0142] Glycoanalysis of IdeS Cleaved Antibody by Direct Infusion

[0143] Size Exclusion Chromatography:

[0144] 45 g (90 l) of IdeS cleaved immunoglobulin obtained with a method according to one of the Examples 2 to 5 are injected onto a Sephadex G25 self packed ECO SR column (5250 mm) (KronLab) equilibrated with 2% formic acid, 40% acetonitrile at a flow rate of 0.5 ml/min for 30 minutes. The protein is desalted using an 8 minute isocratic elution with 2% formic acid, 40% acetonitrile at a flow rate of 1 ml/min. The elution of the desalted protein is recorded by UV (280 nm) and sample is collected via fraction collector into microtiter plates. The microtiter plates can be inserted into a TriversaNanoMate (Advion) system and MS spectra are recorded automatically or sample can be pipetted manually into a metal coated glass needles (Proxeon Biosystems Nano ESI-needles, cat #ES387) and sprayed into the mass spectrometer.

[0145] MS Parameter for Direct Infusion on a QTOF II Instrument (Micromass/Waters):

[0146] MS spectra are acquired using a capillary voltage of 800 V, a cone voltage of 33 V in a mass range from 600 to 2000 m/z (glycosylated Fc-fragment) in positive ion mode using a desolvation temperature of 120 C. and a source temperature of 80 C. MS data are acquired for approx. 2 minutes.

EXAMPLE 8

[0147] Glycoanalysis of a Murine IgG3 Immunoglobulin

[0148] The method according to the invention has been exemplified in this Example with a murine IgG3 immunoglobulin. This immunoglobulin has two glycosylation sites, one in the Fab-fragment and one in the Fc-fragment.

[0149] The chromatogram of an IdeS digested and reduced murine IgG3 immunoglobulin is shown in FIG. 4. A zoom of the mass spectrum of the heavy chain Fab-fragment with the O-glycosylation in the hinge region is shown in FIG. 5, whereas a zoom of the mass spectrum of the heavy chain Fc-fragment with the N-glycosylation site is shown in FIG. 6. In FIG. 7 a deconvoluted mass spectrum of the O-glycosylated tryptic peptide is shown with the glycosylation pattern.

EXAMPLE 9

[0150] Glycoanalysis of a Humanized IgG4 Immunoglobulin

[0151] The method according to the invention has been exemplified in this Example with a humanized immunoglobulin comprising a human IgG4 immunoglobulin constant region. This immunoglobulin has one N-glycosylation site in the Fc-fragment.

[0152] The chromatogram of the IdeS digested humanized IgG4 immunoglobulin is shown in FIG. 8. A zoom of the mass spectrum of the heavy chain Fc-fragment with the N-glycosylation site is shown in FIG. 9.

EXAMPLE 10

[0153] Glycoanalysis of a Humanized IgG1 Immunoglobulin

[0154] The method according to the invention has been exemplified in this Example with a humanized immunoglobulin comprising a human IgG1 immunoglobulin constant region. This immunoglobulin has one N-glycosylation site in the Fc-fragment.

[0155] The chromatogram of the IdeS digested humanized IgG1 immunoglobulin is shown in FIG. 10. A zoom of the mass spectrum of the heavy chain Fc-fragment with the N-glycosylation site is shown in FIG. 11.

EXAMPLE 11

[0156] Glycoanalysis of a Humanized IgG1 Immunoglobulin from a Cultivation Supernatant

[0157] The method according to the invention has been exemplified in this Example with a humanized immunoglobulin comprising a human IgG1 immunoglobulin constant region whereby the sample for analysis was obtained directly from culture supernatant of a cultivation without further purification. This immunoglobulin has one N-glycosylation site in the Fc-fragment. This Example shows that it is possible to use the method according to the invention as an online tool for monitoring the glycosylation during the cultivation of a eukaryotic cell.

[0158] The chromatogram of the IdeS digested sample from the culture supernatant in comparison with a sample purified with a protein A chromatography is shown in FIG. 12. It can be seen that the heavy chain Fc-fragment is eluted at the same point of the chromatogram. The chromatogram of the protein A purified sample does not contain the light chain of the immunoglobulin as the light chain is removed during protein A chromatography.

EXAMPLE 12

[0159] Production of Fab.sub.2-Fragment of a Humanized IgG1

[0160] 10 mg immunoglobulin are diluted to a final concentration of 1 mg/ml in 50 mM TRIS/HCl buffer pH 8.0 and 18 l (c=11.3 mg/ml) of Immunoglobulin degrading enzyme (IdeS, MW 345890 Da) are added to give an enzyme to immunoglobulin ratio of 1:50 by weight. The solution is incubated for 0.5 to 2 h at 37 C. with stirring. Directly after the incubation with IdeS a protein A column chromatography was performed using a protein A HP high trap column. Buffer A was phosphate buffered saline at pH 7.4, buffer B was 100 mM sodium citrate buffer pH 2.8 with a flow rate of 1 ml/min. The Fab.sub.2-fragment is obtained from the flow through of the column whereas the Fc-fragment is obtained by elution with buffer B. The obtained fractions have a purity of from 84% to 95% when determined by size exclusion chromatography. A further purification step using a size exclusion chromatography with a Superdex 75 HighLoad 16/60 column with a volume of 120 ml can be performed. As buffer 20 mM sodium phosphate buffer with 140 mM sodium chloride, pH 6.0 was used with a flow of 1 ml/min. As can be seen from FIG. 15 the original complete antibody has been converted to its Fab.sub.2-fragment.