Patent Application
20190225694
The present invention is directed to a cell culture obtainable from CHO DG44 cells which are capable of being cultured under serum-free or protein-free culture conditions, and which express a biosimilar antibody for the monoclonal antibody natalizumab. The present invention is further directed to a cell of said cell culture, a method for producing said biosimilar antibody, and the use of said cell in said method.
The recombinant therapeutic monoclonal antibody natalizumab is an IgG4 full-length antibody humanized from a murine monoclonal antibody that binds to the α4β1 integrin (also known as VLA-4 or CD49d-CD29) and α4β7 integrin, and blocks the interaction of said α4 integrins with their respective receptors VCAM-1 and MadCAM-1 which are expressed on endothelial cells. See also WO 95/19790. α4-integrin is required for inflammatory lymphocytes to attach to and pass through the cell layers lining the intestine and blood-brain-barrier.
Natalizumab is marketed by Biogen Idec and Elan under the name Tysabri, and was previously named Antegren. It has FDA-approval for the treatment of multiple sclerosis and Crohn's disease, and EMEA approval for the treatment of multiple sclerosis. Recently, it was suggested that natalizumab could also be used in a combination treatment of B-cell malignancies, where it is intended to overcome the resistance to rituximab. Natalizumab is typically administered by intravenous infusion. According to the Scientific Discussion available from the EMEA, natalizumab is recombinantly produced in a NS/0 murine myeloma cell line. The antibody is then purified using Protein A affinity chromatography and hydrophobic interaction chromatography, followed by a buffer exchange and concentration by ultrafiltration/diafiltration.
Currently, cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology that originated from the 1980's, or Lonza's glutamin synthetase (GS) system. Both systems make use of a specific drug to inhibit a selectable enzyme marker essential for cellular metabolism: MTX inhibits dihydrofolate reductase (DHFR) in the MTX amplification system, and methionine sulphoximine (MSX) inhibits GS in the GS system. Upon optimisation of culture conditions, values of 2.7 g/l of monoclonal antibody have recently been reported for GS-NS0 cells in fedbatch culture (Zhou et al., Biotechnology and Bioengineering, 55(5): 783-792 (1997)). Methods for high density cell cultures of NS0 cells for, inter alia, producing natalizumab is disclosed in WO 2013/006461.
While it is generally desirable to increase product titers, therapeutic monoclonal antibodies (mAbs) that are produced in specific cell line expression systems possess inherent post-translational modification profiles which are characteristic of that host cell line. In particular N-linked glycosylation profiles can vary greatly based on the cell line expression system. Glycosylation patterns dictate the stability and functionality of the resulting glycoconjugates. Glycosylation confers functional diversity to a protein, and defective glycosylation of proteins often leads to inactive or abnormal proteins that may result in defects in cellular processes, including those in development, immune reactions, and cell signaling pathways.
The goal of biocomparability/bioequivalence (BC/BE) testing is to demonstrate that variation between different formulations or manufacturing processes do not affect the “quality, safety and efficacy of the drug product” during development or post-marketing (FDA, 2005).
Since the original CHO cell line was described in 1956, many variants of the cell line have been developed. In one strain, CHO DG44, both alleles of the DHFR locus were completely eliminated (Urlaub et al. Cell, 33: 405-412 (1983)). However, it was characterized only as being DHFR deficient and was not named (“eleven of 12 clones screened”, page 408). DG44 has been first named and characterized in Urlaub et al., Somatic Cell and Molec. Genet., 12: 555-566 (1986). These DHFR-deficient strains require glycine, hypoxanthine, and thymidine for growth.
WO 2009/009523 is directed to the prevention of disulphide bond reduction during recombinant production of polypeptides. A preferred host cell is CHO cell line DP12. Anti-human α4β7 is mentioned in a washing list of antibodies, which may be produced using the disclosed method.
WO 2011/019619 discloses the use of DHFR− CHO host cell lines in the production of monoclonal antibodies.
EP 2 202 307 A1 describes the production of antibodies using, inter alia, CHO cells harbouring an enlarged number of copies of the DNA encoding said antibody.
The object of the invention was to provide a host cell expression system for a biosimilar of natalizumab. In particular, the object was to provide an expression system providing higher production yields while maintaining quality, safety and efficacy of the drug product.
The present inventors have surprisingly found that CHO DG44 cells can be used for producing a monoclonal antibody which is a biosimilar to the therapeutic antibody natalizumab. In particular, it was surprisingly found that similar glycosylation patterns can be obtained when using this CHO cell strain. Glycosylation plays a predominant role in determining the function, pharmacokinetics, pharmacodynamics, stability, and immunogenicity of biotherapeutics. There are many physical functions of N-linked glycosylation in a mAb such as affecting its solubility and stability, protease resistance, binding to Fc receptors, cellular transport and circulatory half-life in vivo. At the same time, the production strain shows a high a peak viable cell concentration and achieves a productivity which is higher than the productivity reported for NS0 expression systems.
Accordingly, the present invention provides a cell culture obtainable from CHO DG44 cells which are capable of being cultured under serum-free or protein-free culture conditions, and which express a polypeptide comprising amino acids 19 to 231 of SEQ ID NO: 2 and a polypeptide comprising amino acids 19 to 468 of SEQ ID NO: 4. In a preferred embodiment, the cells express a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 4; more preferably the cells express a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 and a polypeptide consisting of SEQ ID NO: 4.
The expressed polypeptides have a N-glycan content comprising:
In an alternative preferred embodiment, said expressed polypeptides have a N-glycan content comprising
The cell culture of this disclosure has a peak viable cell concentration of more than 1.2×106 cells/ml, and achieves a productivity of more than 3.0 g/l.
In addition, the present invention provides a method for producing a therapeutic monoclonal antibody, in particular natalizumab, comprising the steps of
Finally, the present invention also provides a cell of the cell culture of the invention, and the use of said cell of the cell culture of the invention in the production of a therapeutic antibody, in particular wherein the therapeutic antibody is natalizumab.
More specifically, the present disclosure provides a cell culture obtainable from CHO DG44 cells which are capable of being cultured under serum-free or protein-free culture conditions, and which express a polypeptide comprising amino acids 19 to 231 of SEQ ID NO: 2 and a polypeptide comprising amino acids 19 to 468 of SEQ ID NO: 4.
The cell culture of the disclosure is obtainable from CHO DG44 cells, e.g. by using a suitable screening and subculturing approach as reported in Example 1 herein. CHO DG44 cells are commercially available, and characterized in that they are DHFR negative. The cells of the cell culture of the present disclosure have been adapted to serum-free and protein-free cell culture conditions. In the present case, this was achieved by gradually reducing serum concentrations from 10% to 2% to 0.5% to 0.1% and to 0%. Cells which have been adapted to serum-free conditions can also be cultured in protein-free media. Suitable media for protein-free cell culture of CHO cells are also commercially available. In a preferred embodiment, the cells express a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, i.e. the expressed polypeptide comprises the signal sequence shown in positions 1-18 of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Generally, the antibody may comprise a further tag or fusion, which can alleviate purification of the antibody. However, since any such tag could increase the antigenicity of the antibody, it is more preferred that the cells express a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, and a polypeptide consisting of SEQ ID NO: 4.
As demonstrated in Examples 3 and 4 below, said expressed polypeptides have a N-glycan content comprising:
In a preferred embodiment, labeling is carried out with 2AB using the following protocol:
The labeled 2-AB-glycan composition is then separated and determined by HILIC-UPLC measurement. The chromatographic separation is carried out by Cation Exchange High Performance Liquid Chromatography on UPLC H-Class Bio System using fluorescent detection (excitation at 330 nm and emission at 420 nm) under Empower™ Software control. The Waters BEH Glycan (1.7 μm, 4.6 mm i.d.×150 mm) is used applying eluents: A: Acetonitrile, and B: 0.1M Ammonium formate adjusted to pH 4.4 with formic acid. The glycans are separated using a linear gradient from 22% B to 44.1% B in 38.5 min with flow rate 0.7 ml/min and the column temperature is 60° C. Gradient is followed by a washing step of 100% eluent B in 2 min and re-equilibration with 78% solvent A. The total run time is 48 min. Data is evaluated using Waters Empower 3 software. The peak assignment is performed by retention time. The sample composition was determined by detecting peaks based on their retention time and the relative proportions of each peak were calculated from the peak areas, as also shown in
Preferably, 36.5-60% of the asialo-, agalactosylated-biantennary type has a core substituted with fucose (G0F); more preferably 40-58%; even more preferably 45-56%; and most preferably 47-54%. It is also preferred that 0.3-0.9% of the asialo-, agalactosylated-biantennary type has a core substituted with fucose and has a bisecting N-acetylglucosamin (G0FB); more preferably 0.35-0.85%; even more preferably 0.45-0.8%; and most preferably 0.4-0.75%. Further, it is preferred that 0.05-0.48 of the asialo-, mono-galactosylated-biantennary type which has a core substituted with fucose has a bisecting N-acetylglucosamin (G1FB); more preferably 0.1-0.47%; even more preferably 0.15-0.46%; and most preferably 0.17-0.45%. Likewise, it is preferred that 4.9-11% of the asialo-, galactosylated-biantennary type has a core substituted with fucose (G2F); more preferably 5-9%; even more preferably 5.1-8.5%; and most preferably 5.2-8.2%. Preferably, 0.05-0.5% of the asialo-, galactosylated-biantennary type has a core substituted with fucose and has a bisecting N-acetylglucosamin (G2FB); more preferably 0.1-0.45%; even more preferably 0.15-0.4%; and most preferably 0.17-0.35%. In addition, said expressed polypeptides are characterized in that they have a N-glycan content comprising 0.5-3.1 of the oligomannose 5 type; preferably 0.6-2.9%; more preferably 0.7-2.5%; most preferably 0.9-2.0%; and/or 0.1-0.35 of the oligomannose 6 type; preferably 0.11-0.3%; more preferably 0.12-0.25%; most preferably 0.13-0.2%.
Alternatively, or in addition, said expressed polypeptides have a N-glycan content comprising
Interestingly, the cell culture of the present disclosure is not only characterized in that the produced antibody has a similar glycosylation pattern, but at the same time the cell culture exhibits a high peak viable cell concentration, and an improved productivity as compared to the NS0 expression system.
As used herein, the term “the peak viable cell concentration” is intended to mean the peak viable cell concentration as determined using trypan blue staining. In a preferred embodiment, a Vi-Cell XR Viability Analyzer is used for this determination. The Vi-CELL XR Cell Viability Analyzer is a video imaging system for analyzing yeast, insect and mammalian cells in culture media or in suspension. It automates the trypan blue dye exclusion protocol and is designed to analyze a wide variety of cell types. The software includes features to monitor bioreactors and other cell culture processes and is designed to comply with the US Food and Drug Administration's (FDA) regulations on electronic records and electronic signatures (21 CFR Part 11). Vi-CELL XR Cell Viability Analyzer works in concentration range of 50,000 to 10,000,000 cells per mL and the cell size range of 2 μm to 70 μm. The measurement of overall health of cell cultures requires accurate measurements of both cell concentration and percentage of viable or live cells.
The trypan blue dye exclusion method is a generally known method for cell viability determination. When cells die, their membranes become permeable allowing for the uptake of the trypan blue dye. As a result, the dead or non-viable cells become darker than the viable cells. This contrast is measured in order to determine viability. The Beckman Coulter Vi-CELL XR automates the Trypan Blue Dye Exclusion Method. Utilizing video capture technology and sample handling, the Vi-CELL XR takes the cell sample and delivers it to a flow cell and camera for imaging. The Vi-CELL XR will then capture up to 100 images for its determination of cellular viability. The software determines which cells have absorbed trypan blue dye and those that have not. Cells absorbing the trypan blue dye appear darker hence have lower gray scale values. Cells with higher gray scale values are considered viable. Briefly, the protocol includes the following steps:
Preferably, the peak viable cell concentration is more than 1.2×106 cells/ml, such as more than 1.3×106 cells/ml, more preferably more than 1.4×106 cells/ml, more preferably more than 1.5×106 cells/ml, more preferably more than 1.6×106 cells/ml, more preferably more than 1.7×106 cells/ml, and most preferably more than 1.8×106 cells/ml.
It is understood by the artisan that an extended fermentation time may increase the productivity of a cell line in terms of the amount of the target protein which is obtained per volume of the cell culture, while a shorter fermentation time will typically result in a reduced yield. For sake of clarity, the term “productvity” as used herein is intended to mean the productivity of a 13 days process as determined using protein A chromatography. More specifically, UPLC measurements (UPLC system bio H-class) for the quantification of natalizumab in cell culture supernatants are performed using Protein-A HPLC. Briefly, cell culture supernatants were loaded onto a Protein A column (POROS A 20 2.1×30 mm, Applied Biosystems) with 50 mM sodium phosphate buffer 0.15 M NaCl pH 7.5 (mobile phase A) and bound Natalizumab was eluted by a shift to 50 mM sodium phosphate buffer 0.15 M NaCl pH 2.5 (mobile phase B). Wash and purge solution is 50 mM sodium phosphate buffer pH 7.5. Gradient started with pre-equilibration of 100% buffer A in 0.8 min, then 30% buffer B in 0.1 min was achieved. Elution linear gradient started from 30% to 100% of buffer B in 3.1 min. After elution the column was washed with 100% solvent B for 2 min and re-equilibrated with 100% solvent A. The total run time is 12 min. The flow rate was 0.5 ml/min, and the injection volume is 2 μl. The column temperature was 25° C. and elution is monitored at 280 nm. Product concentrations are determined by comparison with a standard curve, which is generated with reference material (natalizumab). The quantification method has a variation of about +10%.
The cell culture of the present disclosure achieves a productivity of more than 2.7 g/l; preferably more than 3.0 g/l; more preferably more than 3.5 g/l; more preferably more than 4.0 g/l, more preferably more than 4.1 g/l, more preferably more than 4.2 g/l; more preferably more than 4.3 g/l; more preferably more than 4.4 g/l; more preferably more than 4.5 g/l; more preferably more than 4.6 g/l; more preferably more than 4.7 g/l; and most preferably more than 4.8 g/l.
Moreover, the present disclosure provides a method for producing a therapeutic monoclonal antibody, in particular natalizumab, comprising the steps of
Step (b) can be carried out using standard techniques as known in the field, including Protein A affinity chromatography, as described herein in further detail.
Likewise, the present disclosure further provides the use of a cell of the cell culture according to the present disclosure in the production of a therapeutic antibody, in particular natalizumab.
In a final aspect, the present disclosure also provides a cell of the cell culture according to the present disclosure.
The invention is further described by the following embodiments.
In the following, the present invention as defined in the claims is further illustrated by the following figures and examples, which are not intended to limit the scope of the present invention. All references cited herein are explicitly incorporated by reference.
The start and stop codons are indicated in bold, the signal sequence is underlined.
Atgaagtgggtgaccttcatctccctgctgtttctgttctcctccgcctac
tccgacatccagatgacccagtccccctccagcctgtccgcctccgtgggc
MKWVTFISLLFLFSSAYSDIQMTQSPSSLSASVGDRVTITCKTSQDINKYM
atgaagtgggtgaccttcatctccctgctgtttctgttctccagcgcctac
tcccaggtgcagctggtgcagtctggcgccgaagtgaagaaacctggcgcc
MKWVTFISLLFLESSAYSQVQLVQSGAEVKKPGASVKVSCKASGFNIKDTY
An expression construct was generated based on a standard expression vector. The vector comprises two expression cassettes encoding the light and heavy chain of natalizumab, respectively. See also SEQ ID NO: 2 and SEQ ID NO: 4 above. The plasmid further contains a dihydrofolate reductase gene as a selection marker. Cloning of the expression vector was performed using molecular biological standard techniques. Plasmid DNA was prepared and verified by transforming competent E. coli cells and preparation of mini prep DNA (PureLink HiPure Plasmid Filter Maxiprep Kit) from a correct clone which was obtained during the molecular cloning procedure. Verification was by both restriction analysis and sequencing.
This expression construct was linearized, purified and concentrated by isopropanol precipitation, and used to transfect CHO DG44 host cell line using routine electroporation techniques.
The cells were subjected to selection and methotrexate (MTX) amplification procedures employing large pools (LPs), and mini pools (MPs). Briefly, following transfection, cells were cultivated in host cell growth medium for 2 days. Subsequently, they were transferred into selective medium and subcultivated in the same medium every 3-4 day until viability recovered and cells started to grow. Growing cells were transferred into selective medium+5 nm MTX, and subcultivated in the same medium every 3-4 day until viability recovered and cells started to grow. Subsequently, pools were transferred into selective medium+30 nM MTX to induce the amplification process and expanded up to shake flask level.
Subsequently, single cell clones were isolated from the best available cell pools by FACS sorting. Briefly, 3×106 cells were centrifuged and stained with fluorescence conjugated Protein A. Subsequently, cells were washed, resuspended in PBS, filtered through a FACS tube with cell strainer cap and analyzed by flow cytometry. The top 3-5% population with regard to fluorescence was selected and single cells were sorted into 384 well flat bottom plates containing.
The best pools were chosen based on similarity of their glycan profile with the originator molecule, which were analysed from pool fed-batch supernatants, as further described in Examples 3 and 4 below. Subsequently, growing clones were pre-selected according to productivity as well as monoclonality and expanded up to shake flask level.
The best 40 high-expressing clones were chosen and evaluated in a standard fed-batch process with regard to productivity and process characteristics. The five best performing clones regarding product quality with the productivity between 2.1-4.4 g/L in 10 days process, were chosen as preferential production cell lines. For each of these clones, research cell banks consisting of 20 vials each were prepared and stored in the gas phase of liquid nitrogen. Subsequently, one vial of each clone was thawed and subjected to a stability study for 7 weeks including two fed-batch runs starting at different points in time of the study. The obtained data indicate that all clones are phenotypically stable. One of the clones, has a duration of 13 days, leads to peak viable cell concentrations of approximately 18.4×106/mL and product concentrations of about 4.9 g/L.
Chromatographic analysis by Protein A affinity chromatography was carried out on UPLC H-Class Bio System using UV detection under Empower 3 Software control. The Applied Biosystems Poros® Protein A column (20 μm, 2.1 mm i.d.×30 mm) was used for testing applying a two steps of linear gradient of buffer A (50 mM sodium phosphate buffer pH 7.5, 0.15 M NaCl) and buffer B (50 mM sodium phosphate buffer pH 2.5, 0.15 M NaCl). Gradient started with pre-equilibration of 100% buffer A in 0.8 min, then 30% buffer B in 0.1 min was achieved. Elution linear gradient started from 30% to 100% of buffer B in 3.1 min. After elution the column was washed with 100% solvent B for 2 min and re-equilibrated with 100% solvent A. The total run time is 12 min. The flow rate was 0.5 ml/min. The column temperature was 25° C. and elution is monitored at 280 nm. Exemplary chromatogram of test solution is presented on
Mab concentration calculation based on linear standard curve was determined by Empower 3 Software. Exemplary results of mAb concentration for reference product (range based on 9 batches testing) and tested product from clone selection step are presented in Table 1.
Clones 1 and 2 showed antibody titers of more than 5.6 mg/ml=5.6 g/L.
Monoclonal antibodies are subject to post-translational modifications or degradation at several independent sites. Such modifications may result in the presence of many different species in the final product. Monoclonal antibodies therefore display considerable heterogeneity that can be characterized by ion exchange liquid chromatography (IEX-LC).
The separation was carried out by Cation Exchange High Performance Liquid Chromatography on UPLC H-Class Bio System using UV detection under Empower™ Software control. The Waters Protein-Pak Hi Res SP (7 μm, 4.6 mm i.d.×100 mm) was used for testing applying a linear gradient of NaCl. Eluents were: buffer A (10 mM NaPi buffer pH 6.0) and buffer B (10 mM NaPi buffer pH 6.0, 0.125 M NaCl). Gradient starts with pre-equilibration of 100% buffer A in 5 min. Elution gradient starts from 10% to 30% of buffer B in 25 min min, followed by a washing step for 5 min at 30% B and re-equilibration with 90% solvent A. The total run time is 45 min. The flow rate was 0.7 ml/min. The column temperature was 40° C. and elution is monitored at 220 nm.
For data evaluation was used Waters Empower 3 software. The peak assignment was performed by retention time. The sample composition was determined by detecting peaks based on their retention time and the relative proportions of each peak were calculated from the peak areas. The final results were presented as a sum of acidic species, main peak and sum of basic species. Exemplary chromatograms of standard (reference) and test solution are presented on
Glycosylation plays a predominant role in determining the function, pharmacokinetics, pharmacodynamics, stability, and immunogenicity of biotherapeutics. There are many physical functions of N-linked glycosylation in a mAb such as affecting its solubility and stability, protease resistance, binding to Fc receptors, cellular transport and circulatory half-life in vivo. Therefore, it is very important to quantitate and monitor the glycosylation pattern. The most common approach to qualitative and quantitative characterization of glycans is the analysis of glycans enzymatically released from the protein. This approach leads to mixtures of oligosaccharides that are label with a fluorescent molecule (ex. 2-aminobenzamide, 2-AB; Sigma Cat. No. A89804-100G), followed by high-performance hydrophilic interaction liquid chromatography with fluorescence detection (HILIC). The above described approach let us identify 13 of N-linked glycan's. The identification of glycans is performed by the order of elution and retention times of peaks from solution for peak identification (
The labeled 2-AB-glycan composition was separated and determined by HILIC-UPLC measurement. The chromatographic separation was carried out by Cation Exchange High Performance Liquid Chromatography on UPLC H-Class Bio System using fluorescent detection (excitation at 330 nm and emission at 420 nm) under Empower™ Software control. The Waters BEH Glycan (1.7 μm, 4.6 mm i.d.×150 mm) was used for testing applying eluents: A: Acetonitrile, and B: 0.1M Ammonium formate adjusted to pH 4.4 with formic acid. The glycans were separated using a linear gradient from 22% B to 44.1% B in 38.5 min with flow rate 0.7 ml/min and the column temperature was 60° C. Gradient was followed by a washing step of 100% eluent B in 2 min and re-equilibration with 78% solvent A. The total run time was 48 min.
For data evaluation was used Waters Empower 3 software. The peak assignment was performed by retention time. The sample composition was determined by detecting peaks based on their retention time and the relative proportions of each peak were calculated from the peak areas. The final results were presented as a sum of acidic species, main peak and sum of basic species. Exemplary chromatograms of standard (reference; GlycoWorks Control Standard, Waters, Cat no. 186007033) and test solution are presented on
Antigen binding part of natalizumab (Fab) is responsible for the interaction with its antigen: α4 subunit of integrin. Mechanism of action for natalizumab involves blocking interaction of α4β1 and α4β7 integrins with their cognate receptors VCAM-1 and MadCAM-1, respectively. The comparability study is designed in a way to mimic the biological properties of natalizumab related to Fab functions.
The aim of this assay is to confirm the potency of natalizumab to bind α4β1 integrin in a dose-dependent manner.
The principle of this method is to incubate a coated constant amount of integrin α4β1 with serially diluted natalizumab samples. The amount of bound natalizumab is subsequently determined by a mouse, monoclonal anti-human IgG antibody, which is conjugated to horseradish peroxidase (HRP). HRP converts the chromogenic substrate TMB (3,3′,5,5′-tetramethylbenzidine) into a colored dye. The color reaction is measured spectrophotometrically at wavelength 450 nm.
Data are analyzed applying 4 Parameter Logistic nonlinear regression model (4PL), which is commonly used for curve-fitting analysis in bioassays or immunoassays such as ELISAs or dose-response curves. Final result is expressed as a Relative Potency (REP) of tested sample in relation to interim reference standard established at Polpharma site. The method variability was determined at the level of 7% coefficient variation (CV) of intermediate precision within the qualification exercise.
The data in Table 4 shows that clones 1 and 2 bind to α4β1 integrin.
The aim of this assay is to test the ability of natalizumab to inhibit interaction of α4β1 integrin with its cognate receptor—VCAM-1 protein in a dose-dependent manner.
Constant amount of the coated VCAM-1 protein is incubated with serial dilutions of natalizumab in the presence of HIS-tagged α4β1 integrin. Solid-phase associated VCAM-1 and soluble natalizumab now compete for binding to α4β1 integrin. The higher the natalizumab concentration the more α4β1 Integrin is inhibited from binding to VCAM-1. The highest signal result is observed when no natalizumab is present. Bound HIS-tagged α4β1 integrin is subsequently detected with a biotinylated anti-HIS-tag antibody, POD-conjugated Streptavidin and a TMB-substrate reaction at the end of the assay.
Data are analyzed with 4PL fitting model. Final result is expressed as a Relative Potency (REP) of tested sample in relation to reference standard. The method variability was determined at the level of 7% coefficient variation (CV) of intermediate precision within the qualification exercise. Additionally accuracy, linearity and specificity were tested.
Clones 1 and 2 show a similar ability of inhibiting interaction of α4β1 integrin with VCAM-1, as compared to natalizumab.
The aim of this assay is to test the ability of natalizumab to inhibit interaction of α437 integrin with its cognate receptor—MadCAM-1 protein in a dose-dependent manner.
Constant amount of the coated α4β7 integrin is incubated with serial dilutions of natalizumab in the presence of Fc-tagged MadCAM-1 receptor. natalizumab and MadCAM-1 receptor now compete for binding to solid-phase associated α4β7 integrin. The higher the natalizumab concentration the more MadCAM-1 is inhibited from binding to α4β7 integrin. The lowest signal result is observed when no natalizumab is present. Bound natalizumab is subsequently detected with a POD-conjugated anti-human IgG antibody and a TMB-substrate reaction at the end of the assay.
Data are analyzed with 4PL fitting model. Final result is expressed as a Relative Potency (REP) of tested sample in relation to reference standard. The method variability was determined at the level of 8% coefficient variation (CV) of intermediate precision within the qualification exercise. Additionally accuracy, linearity and specificity were tested.
It was decided to reproduce Example 4 using a different fluorescence labeling, the RapiFluor-MS reagent, followed by high-performance hydrophilic interaction liquid chromatography with fluorescence detection (HILIC).
The above described approach let us identify 22 of N-linked glycan's, of which the 14 major peaks are shown in Table 7b. The identification of glycans is performed by the order of elution and GU value of peaks from RapiFluor-MS Glycan Performance Test Standard solution (GPTS) and Ribonuclease B solution (
The labeled RapiFluor-MS glycan composition was separated and determined by HILIC-UPLC measurement. The chromatographic separation was carried out on UPLC H-Class Bio System using column containing Waters amide bonded, Ethylene bridged Hybrid Technology (BEH) particles and fluorescent detection (excitation at 265 nm and emission at 425 nm) under Empower™ Software control. The Waters BEH Glycan (1.7 μm, 4.6 mm i.d.×150 mm) was used for testing applying eluents: A: Acetonitrile, and B: 50 mM Ammonium formate solution with pH 4.4 (prepared from concentrate). The glycans were separated using a linear gradient from 25% B to 46% B in 35 min with a flow rate of 0.4 ml/min, and the column temperature was 60° C. Gradient was followed by a washing step of 100% eluent B in 3 min and re-equilibration with 75% solvent A. The total run time was 55 min. Waters Empower 3 software with AppexTrack algorithm and GPC Technique was used for data evaluation. The peak assignment was performed following calibration of retention times to GU values. The sample composition was determined by detecting peaks based on their GU value and the relative proportions of each peak were calculated from the peak areas. Exemplary chromatograms of standard solutions are presented in
The data of this experiment independently confirms the results shown in Example 4 with regard to glycans G0, G0F, G1F, G1F′, Man-5, and G2F.
Profiling of total glycans which are cleaved from the glycoprotein is the most common approach for characterizing protein glycosylation and allows to obtain Information about the various populations of glycans which are present on a glycoprotein (cf. General Monography of US Pharmacopeia USP38 of May 1, 2016, p. 1171). Depending on the chosen analytical method, prior derivatization/labeling may be needed to allow for the detection of certain glycans, including sialyl residues. Many protocols are available, and most of the steps in the analysis are well established. Because of the variety of available analytical techniques, a direct comparison of results obtained by different platforms is not always possible. Thus, the skilled person will not see the fact that glycans G0FB, G1FB, Man-6, G2, G2FB, G1FS1, and AF1 could not be detected using this alternative method as contradicting the results of Example 4.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16460039.7 | Jun 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/065831 | 6/27/2017 | WO | 00 |
