DIRECT AFFINITY MEASUREMENT OF HUMAN IGG1 BINDING MULTIMERIC ANTIGENS

Abstract

Herein is reported a method for determining the binding affinity of the binding sites of a bivalent full length antibody of the human IgG1 subclass to a homo-multimeric antigen comprising the steps of i) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of Porphyromonas gingivalis at a pH of from pH 7.5 to pH 8.5, in the presence of a reducing agent, at a temperature of from 30° C. to 42° C., for time of from 10 min. to 240 min. to cleave the antibody into Fabs and Fc-region, and ii) determining the binding affinity of the Fabs of the antibody for its antigen using a surface plasmon resonance method by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method and therewith determining the binding affinity of the binding sites of the bivalent full length antibody of the human IgG1 subclass.

Claims

1. A method for determining the binding affinity of the binding sites of a bivalent full length antibody of the human IgG1 subclass to a homo-multimeric antigen comprising the following steps: incubating a mixture comprising the antibody and lysine-gingipain of Porphyromonas gingivalis or an enzymatically active fragment thereof at a pH of 7.5 to 8.5, in the presence of a reducing agent, at a temperature of 30° C. to 42° C., for a time of 10 minutes to 240 minutes to cleave the antibody into Fabs and Fc-region, and determining the binding affinity of the Fabs of the antibody for its antigen using surface plasmon resonance by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method, thereby determining the binding affinity of the binding sites of the bivalent full length antibody of the human IgG1 subclass.

2. The method according to claim 1, wherein the lysine-gingipain of Porphyromonas gingivalis has the amino acid sequence of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04 or is a functional variant thereof.

3. The method according to claim 1, wherein the enzymatically active fragment of lysine-gingipain of Porphyromonas gingivalis has an amino acid sequence that comprises at least residues 230 to 739 of SEQ ID NO: 01.

4. The method according to claim 1, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

5. The method according to claim 4, wherein the reducing agent is cysteine.

6. The method according to claim 1, wherein the reducing agent is cysteine at a concentration of 0.5 mM to 10 mM.

7. The method according to claim 1, wherein the pH value is about pH 8.

8. The method according to claim 1, wherein the incubating is at a temperature of 35° C. to 38° C.

9. The method according to claim 1, wherein the incubating is for a time of about 60 minutes.

10. The method according to claim 1, wherein the antibody comprises in the Fc-region the mutations P329G, L234A and L235A in both heavy chain polypeptides.

11. A method for selecting an antibody specifically binding to a homo-multimeric antigen comprising the following steps: providing a plurality of bivalent full length antibodies of the human IgG1 subclass binding to the same homo-multimeric antigen, determining the binding affinity of each of the antibodies of the plurality of antibodies to its antigen with a method according to claim 1, and selecting one or more antibodies based on the binding affinity determined in the previous step.

12. The method according claim 1, wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

13. The method according to claim 2, wherein the antibody comprises in the Fc-region the mutations P329G, L234A and L235A in both heavy chain polypeptides.

14. The method according to claim 13, wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification.

15. The method according to claim 14, wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol.

16. The method according to claim 15, wherein the reducing agent is cysteine.

17. The method according to claim 15, wherein the reducing agent is cysteine at a concentration of 0.5 mM to 10 mM.

18. The method according to claim 15, wherein the pH value is about pH 8.

19. The method according to claim 18, wherein the incubating is at a temperature of 35° C. to 38° C.

20. The method according to claim 19, wherein the incubating is for a time of about 60 minutes.

Description

DESCRIPTION OF THE FIGURES

[0160] FIG. 1 UHR ESI-QTOF mass spectrometry of bevacizumab digested with lysine-gingipain of Porphyromonas gingivalis for one hour at 37° C. Only Fab and Fc fragments were detected.

[0161] FIG. 2 UHR ESI-QTOF mass spectrometry of bevacizumab digested with papain for 1.5 h at 37° C. Beside Fab and Fc fragments, several Fab- and antibody fragments were detected.

[0162] FIG. 3 UHR ESI-QTOF mass spectrometry of bevacizumab digested with papain for 2 h at 37° C. Several antibody fragments were detected. Fab and Fc fragment could not be identified.

[0163] FIG. 4A Surface plasmon resonance sensorgrams of bevacizumab.

[0164] FIG. 4B Surface plasmon resonance sensorgrams of a recombinant bevacizumab Fab.

[0165] FIG. 4C Surface plasmon resonance sensorgrams of bevacizumab digested with lysine-gingipain of Porphyromonas gingivalis.

[0166] FIG. 4D Surface plasmon resonance sensorgrams of bevacizumab, bevacizumab digested with papain without termination of the digest, and bevacizumab digested with papain with termination of the digest.

EXAMPLES

[0167] Bevacizumab was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Papain was obtained as suspension with a concentration of 10 mg/mL from Sigma-Aldrich/Roche Diagnostics GmbH. Lysine-gingipain of Porphyromonas gingivalis was obtained under the trade name GingisKHAN from Genovis (Lund, Sweden). GingisKHAN was reconstituted in 200 μL double distilled water (ddH2O) resulting in 2000 U/200 μL, and the 10× reducing agent was freshly prepared in 50 μL ddH2O (final concentration: 20 mM cysteine) prior to each digestion.

Example 1

Transient Fab Expression and Purification

[0168] The antibody light chain and heavy chain Fd-fragments were ordered as gene syntheses and cloned via unique restriction sites using standard cloning procedures into separate expression vectors for each chain enabling secretory expression in HEK cells growing in suspension. Transfection (1:1 plasmid ratios) into HEK293-F cells (Invitrogen, Cat. No. 510029) was performed according to the cell supplier's instructions using Maxiprep (Qiagen, Cat. No. 12163) preparations of the antibody vectors, Opti-MEM I medium (Invitrogen, Cat. No. 31985) 293fectin (Invitrogen, Cat. No. 31985070), and an initial cell density of 1-2×10E+06 viable cells/mL in serum-free FreeStyle 293 expression medium (Invitrogen, Cat. No. 12338018). Antibody containing cell culture supernatants were harvested after 7 days of cultivation in shake flasks by centrifugation at 14,000×g for 30 min. and filtered through a 0.22 μm sterile filter (Thermo Scientific, Cat. No. 566-0020). The antibodies were purified directly from the supernatant, or the supernatant was stored at −80° C. until purification. The quality of the purified Fab was analyzed by SEC and BioAnalyzer.

Example 2

[0169] Enzymatic Cleavage of Bevacizumab with Papain

Without Purification:

[0170] The antibody was diluted in 20 mM Histidine, 140 mM NaCl, pH 6.0 to a final concentration of 1 mg/mL, added 2 μL 250 mM L-cysteine (Sigma-Aldrich, Schnelldorf, Germany) and 10.9 μL diluted papain (7.34 U/mL in 20 mM Histidine, 140 mM NaCl, pH 6.0), and incubated 1 h at 37° C.

With Purification:

[0171] The antibody was incubated with Papain (0.8 U/mg mAb; Sigma-Aldrich/Roche) in presence of 5 mM Cystein for 170 minutes at 37° C. To isolate the Fab from non-cleaved antibodies, Fc-fragments and Papain, the mixture was applied to a CaptureSelect IgG-CH1 and MabSelectSuRe affinity chromatography (GE Healthcare) according to manufacturer protocol. Finally, a size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) was performed using 140 mM NaCl, 20 mM histidine (pH 6.0) as running buffer. Protein concentration of the Fab was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. The purity was analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.

Example 3

[0172] Enzymatic Cleavage of Bevacizumab with Lysine-Gingipain of Porphyromonas Gingivalis

[0173] GingisKHAN was reconstituted in 200 μL ddH2O resulting in 2000 U/200 μL, and the 10× reducing agent was freshly prepared in 50 μL ddH2O (final concentration: 20 mM Cysteine) prior to each digestion. 100 μg antibody was diluted to a final concentration of 1 mg/mL in 100 mM Tris, pH 8.0 and subsequently digested with 10 μL GingisKHAN and 11 μL of freshly prepared 10× reducing agent at 37° C. for 1 hour.

Example 4

ESI-QTOF Mass Spectrometry

[0174] Samples were desalted by HPLC on a Sephadex G25 column (Kronlab, 5×250 mm, TAC05/250G0-SR) using 40% acetonitrile with 2% formic acid (v/v). The total mass was determined via ESI-QTOF MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion). Calibration was performed with sodium iodide (Waters ToF G2-Sample Kit 2 Part: 700008892-1). For the recombinant and purified Fabs, data acquisition was done at 900-2600 m/z (ISCID: 0.0 eV), for the hIgG1s or digested hIgG1s, data acquisition was done at 900-4000 m/z (ISCID: 0.0 eV). The raw mass spectra were evaluated and transformed into individual relative molar masses using an in-house developed Roche software tool. For visualization of the results, the same in-house developed software was used to generate deconvoluted mass spectra.

Example 5

Surface Plasmon Resonance

[0175] Binding affinities and kinetics were investigated by surface plasmon resonance using a BIAcore T200 instrument (GE Healthcare). All experiments were performed at 25° C. using PBS-T (10 mM Na2HPO4, 140 mM NaCl, 0.05% Tween 20, pH 7.4) as running and dilution buffer. An anti-His-tag (GE Healthcare, #28995056) or an anti-human Fab antibody (GE Healthcare, #28958325) was immobilized on a Series S CMS Sensor Chip (GE Healthcare, #29104988) using standard amine coupling chemistry. Histidine-tagged human VEGF or full length IgG/Fabs were captured on the surface leading to a response between 10 and 50 RU. The analytes were injected for 180 s at concentrations from 2.2 nM up to 1800 nM onto the surface (association phase) at a flow rate of 30 μL/min. The dissociation phase was monitored for up to 3600 sec. by washing with running buffer. The surface was regenerated by injecting 10 mM Glycine pH 1.5 for 60 sec. at a flow rate of 5 μL/min. Bulk refractive index differences were corrected by subtracting the response obtained from a mock surface and by subtracting blank injections (double referencing). The derived curves were fitted to a 1:1 Langmuir binding model using the BIAevaluation software.