Formulation for Anti-alpha4beta7 antibody
Inactive Publication Date: 2012-11-08
MILLENNIUM PHARMA INC
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AI-Extracted Technical Summary
Problems solved by technology
Because proteins are larger and more complex than traditional organic and inorganic drugs (i.e. possessing multiple functional groups in addition to complex three-dimensional structures), the formulation of such proteins poses special problems.
Proteins may suffer from a lack of stability, and monoclonal and polyclonal antibodies in particular may be relatively unstable (See e.g., Wang et al., J.
A large number of formulation options are available, and not one approach or system is suitable for all proteins.
In fact, even in the case of purified antibodies, the antibody structures may be heterogeneous, which further complicates the formulation of such systems.
Physical instability can result from denaturation, ...
Method used
[0138]In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[0140]In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
[0142]The glycosylation machinery of different cell types can produce antibodies with different glycosylation composition than in another cell type, or no glycosylation, as with bacterial cells. In one aspect, cell types for production of the anti-α4β7 antibody are mammalian cells, such as NS0 or CHO cells. In one aspect, the mammalian cells can comprise the deletion of an enzyme involved in cell metabolism and the exogenous gene of interest can be operably linked to a replacement enzyme, e.g., in a construct or vector for introduction into the cells, e.g., by transformation or transfection. The construct or vector with the exogenous gene confers to the cells which host the construct or vector a selection advantage to encourage production of the polypeptide encoded by the exogenous gene. In one embodiment, CHO cells are DG44 cells (Chasin and Urlaub (1980) PNAS USA 77:4216), comprising the deletion or inactivation of the dihydrofolate reductase gene. In another embodiment, CHO cells are CHO K1 cells comprising the deletion or inactivation of the glutamine synthase gene (see, e.g., U.S. Pat. No. 5,122,464 or 5,827,739).
[0155]Vial size can be selected based on the surface area which will be exposed to the shelf and to the vacuum during lyophilization. Drying time is directly proportional to cake height, thus the vial size may be chosen based upon what is determined to be a reasonable cake height. A vial with a large diameter relative to volume can provide a high amount of contact with the shelf for efficient heat transfer during the lyophilization cycle. A dilute antibody solution in a high volume of liquid will require more time for drying. A balance in vial size versus formulation volume needs to be struck, because larger vials can be more expensive to store and ship and have a larger headspace to formulation ratio and may expose a high proportion of the formulation to the degradative effects of moisture during long term storage. For a 300 mg dose, anti-α4β7 antibody formulation can have a volume of 3 ml, 5 ml, 6 ml, 10 ml, 20 ml, 50 ml or 100 ml prior to lyophilization. In one aspect, the vial size is 20 ml for a 60 mg/ml solution in a 300 mg dose.
[0167]Pancreatitis and insulin-dependent diabetes mellitus are other diseases which can be treated using the formulations of the invention. It has been reported that MAdCAM (e.g., MAdCAM-1) is expressed by some vessels in the exocrine pancreas from NOD (nonobese diabetic) mice, as well...
Benefits of technology
[0009]The invention relates to the identification of a non-reducing sugar and at least one amino acid, as useful excipients for formulating anti-α4β7 antibody formulations whose instability mak...
Abstract
Antibody formulations are described comprising a mixture of a non-reducing sugar, an anti-α4β7 antibody and at least one amino acid. The disclosed formulations have improved stability, reduced aggregate formation, and may retard degradation of the anti-α4β7 antibody therein or exhibit any combinations thereof. The present invention further provides a safe dosing regimen of these antibody formulations that is easy to follow, and which results in a therapeutically effective amount of the anti-α4β7 antibody in vivo.
Application Domain
AntipyreticAnalgesics +7
Technology Topic
Reducing sugarDosing regimen +4
Image
Examples
- Experimental program(10)
Example
Example 1
Comparative Data for Varying % Sugar and Amino Acids in Lyophilized Formulations
[0204]A design of experiments approach was performed to look at the effect of varying the molar ratio of sugar (sucrose and mannitol) to protein, the molar ratio of arginine to protein, and the molar amount of histidine buffer. Histidine and arginine are known not to crystallize during the lyophilization process, making them potential cryo or lyo protectants. 1.5 mL of formulation was filled into 5 mL vials lyophilized with Primary Drying at −30° C., 150 mT and Secondary Drying at 20° C., 150 mT. The stability of the lyophilized formulations reconstituted to 1.5 ml after different storage conditions is shown in Tables 1-3 (compiling 60 mg/ml results from two experiments). FIG. 6A shows the predictive models for changes in Percent Monomer, Percent Aggregates, and Percent Major Isoform when stored at 40° C. when pH and the molar ratio of sugar and arginine was varied. The stability of the formulation was best at low pH and high molar ratio of (sugar+arginine) to protein. At the histidine molar amounts examined, histidine did not affect the stability of the formulation. All formulations had 1-2% moisture during storage.
TABLE 1 Change in Percent Monomer when stored at 5° C., 25° C./60% RH, and 40° C./75% RH for 3 months. Percent Monomer was measured using Size Exclusion Chromatography (SEC). % Monomer by SEC 25° C. 40° C. Formulation 5° C. 60% RH 75% RH 60 mg/mL vedolizumab + t = 0 3 mo 3 mo 3 mo 25 mM histidine, 75 mM arginine, 98.1 98.1 97.8 96.5 2% sucrose, 0.05% polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 98.0 98.2 98.0 97.5 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM 98.0 98.3 98.1 97.4 arginine, 2% sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine, 125 mM 98.0 98.3 98.1 97.4 arginine, 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM 98.7 98.4 98.4 98.1 arginine, 6% sucrose, 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mM histidine, 125 mM 98.7 98.3 98.1 98.3 arginine, 9% sucrose, 0.06% polysorbate 80, pH 6.3
TABLE 2 Change in Percent Aggregates when stored 5° C., 25° C./60% RH, and 40° C./75% RH for 3 months. Percent Monomer was measured using Size Exclusion Chromatography (SEC). % Aggregates by SEC 25° C. 40° C. Formulation 5° C. 60% RH 75% RH 60 mg/mL vedolizumab + t = 0 3 mo 3 mo 3 mo 25 mM histidine, 75 mM arginine, 0.42 0.53 0.89 1.99 2% sucrose, 0.05% polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 0.41 0.51 0.62 1.15 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM 0.42 0.47 0.60 1.23 arginine, 2% sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine, 125 mM 0.36 0.44 0.52 0.82 arginine, 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM 0.53 0.49 0.51 0.56 arginine, 6% sucrose, 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mM histidine, 125 mM 0.51 0.51 0.59 0.56 arginine, 9% sucrose, 0.06% polysorbate 80, pH 6.3
TABLE 3 Change in Percent Major Isoform when stored at 5° C., 25° C./60% RH, and 40° C./75% RH for 3 months. Major Isoform was measured using Cation Exchange Chromatography (CEX). % Major Isoform by CEX 25° C. 40° C. Formulation 5° C. 60% RH 75% RH 60 mg/mL vedolizumab + t = 0 3 mo 3 mo 3 mo 25 mM histidine, 75 mM arginine, 70.5 68.8 67.4 66.3 2% sucrose, 0.05% polysorbate 80, pH 6.3 25 mM histidine, 75 mM arginine, 70.8 98.9 68.0 67.7 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM 70.5 68.9 67.8 66.5 arginine, 2% sucrose, 0.05% polysorbate 80, pH 6.7 50 mM histidine, 125 mM 70.6 68.9 68.0 67.4 arginine, 4% sucrose, 0.05% polysorbate 80, pH 6.9 50 mM histidine, 125 mM 69.6 69.5 69.3 67.4 arginine, 6% sucrose, 1.5% mannitol, 0.06% polysorbate 80, pH 6.3 50 mM histidine, 125 mM 69.5 69.3 69.2 68.1 arginine, 9% sucrose, 0.06% polysorbate 80, pH 6.3
[0205]FIG. 6A shows the predicted models based on the statistical analysis of 40C data from Tables 1-3. The model for change in percent monomer per month at 40° C. by SEC analysis is −3.10+(0.386)*pH+0.000516*((moles of sugar+moles arginine)/moles of protein)). The model for change in percent aggregate per month at 40° C. by SEC analysis is 2.43−(0.263)*pH−0.000787*((moles of sugar+moles arginine)/moles of protein)). The model for change in percent major isoform per month at 40° C. by CEX analysis is −2.54+(0.109)*pH−0.00130*((moles of sugar+moles arginine)/moles of protein)). The center line shows the results for the predictive models and the outer lines show the 95% confidence limit for the predictive models.
[0206]FIG. 6B shows alternative models based on the statistical analysis of 40° C. data from Tables 1-3 when the input factors are pH, sugar:protein molar ratio, and arginine:protein molar ratio. The model for change in percent monomer per month at 40° C. by SEC analysis is −3.02+(0.370)*pH+0.000482*((moles of sugar)/(moles of protein)+0.000657*((moles of arginine/moles of protein). The model for change in percent aggregate per month at 40° C. by SEC analysis is 2.35−(0.244)*pH−0.000727*((moles of sugar)/(moles of protein)−0.00102*((moles of arginine)/(moles of protein)). The model for change in percent major isoform per month at 40° C. by CEX analysis is −2.92+(0.210)*pH+0.00164*((moles of sugar)/)/(moles of protein)−0.000220*((moles of arginine)/(moles of protein)). The center line shows the results for the predictive models and the outer lines show the 95% confidence limit for the predictive models.
Example
Example 2
Stability Data
[0207]Three primary stability batches of the formulation (Batch A, B, and C) were tested for stability after storage at the prescribed storage condition (5 and 25° C./60% RH for up to 24 months). All three batches contain the same liquid formulation that was lyophilized: 60 mg/mL anti-α4β7 antibody, 50 mM histidine, 125 mM arginine, 10% sucrose, 0.06% polysorbate 80, pH 6.3. For Batch A, 3.5 mL of solution was filled into 20 mL vials and lyophilized, for Batches B and C, 5.52 mL of solution was filled into 20 mL vials and lyophilized.
[0208]In a separate study, a single drug formulation of 60 mg/ml anti-α4β7 antibody, 50 mM histidine, 125 mM arginine, 10% sucrose, 0.06% polysorbate 80, pH 6.3 was lyophilized in two volumes, 3.5 ml and 9.5 ml, respectively, to yield Batches R and S for stability samples, which were analyzed over 38 months. Blanks are NT (not tested).
[0209]The data (Tables 4-19) showed that the antibody formulations remained stable when stored for up to 24 months at 5° C. and 25° C./60% RH. All product attributes remained within the specifications through the 24 month time point.
TABLE 4 Change in Percent Monomer by SEC when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 99.8 99.8 99.8 98.9 98.8 1 99.8 99.1 99.2 98.8 99.2 3 99.8 99.1 99.1 98.8 98.8 6 99.8 99.8 99.8 98.9 99.0 9 99.1 99.2 99.2 99.2 99.1 12 99.4 99.0 99.0 98.8 98.9 15 99.4 99.1 99.1 18 99.5 99.4 99.4 98.9 98.9 24 99.4 99.2 99.2 99.0 99.0 30 99.2 99.2 38 99.3 99.3
TABLE 5 Change in Percent Aggregates by SEC when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 0.1 0.1 0.1 0.2 0.2 1 0.1 0.2 0.2 0.2 0.1 3 0.1 0.2 0.2 0.2 0.2 6 0.2 0.2 0.2 0.2 0.2 9 0.1 0.2 0.2 0.2 0.2 12 0.2 0.2 0.2 0.2 0.2 15 0.2 0.2 0.2 18 0.2 0.2 0.2 0.2 0.2 24 0.2 0.2 0.2 0.2 0.2 30 0.2 0.2 38 0.2 0.2
TABLE 6 Change in Percent Major Isoform by CEX when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 68.6 69.9 69.5 71.7 71.6 1 67.5 68.9 68.8 71.2 72.0 3 68.7 68.8 68.7 70.4 70.3 6 67.7 68.2 68.2 71.9 71.9 9 70.0 68.3 67.8 69.2 69.7 12 67.8 68.3 68.1 70.8 70.9 15 66.9 67.5 67.5 18 67.4 67.0 66.7 71.0 70.8 24 68.1 69.6 69.1 71.3 70.9 30 68.5 68.6 38 73.6 73.1
TABLE 7 Change in Percent Acidic Isoforms by CEX when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 22.8 20.8 21.4 20.3 20.6 1 21.9 21.7 22.3 21.6 20.3 3 21.7 22.2 22.8 22.0 22.0 6 22.9 23.1 23.6 21.1 21.4 9 19.8 22.2 22.9 21.8 21.8 12 22.9 21.3 22.1 21.2 21.2 15 22.7 22.3 22.8 18 22.8 22.3 22.6 21.1 21.5 24 21.7 22.1 22.9 20.6 20.7 30 22.8 23.2 38 18.9 19.1
TABLE 8 Change in Percent Basic Isoforms by CEX when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 8.5 9.3 9.1 8.1 7.8 1 10.7 9.4 8.9 7.3 7.7 3 9.7 9.0 8.5 7.6 7.8 6 9.5 8.7 8.2 7.0 6.7 9 10.2 9.6 9.3 9.0 8.4 12 9.3 10.3 9.9 8.0 7.9 15 10.4 10.1 9.7 18 9.8 10.7 10.7 7.9 7.7 24 10.2 8.3 8.1 8.1 8.3 30 8.7 8.2 38 7.5 7.7
TABLE 9 Change in % (H + L) by Reduced-SDS Page when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 98 98 98 96 96 1 98 94 98 98 98 3 98 98 98 98 98 6 98 97 97 97 97 9 97 97 97 98 98 12 98 96 97 98 98 15 97 98 97 18 98 97 97 99 99 24 98 98 98 99 99 30 97 97 38 99 99
TABLE 10 Change in Binding Efficacy when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 107 106 105 93 102 1 106 106 103 103 111 3 101 109 108 91 98 6 97 106 105 114 121 9 100 93 88 102 102 12 103 101 87 119 116 15 105 90 94 18 86 101 96 95 104 24 92 82 95 81 101 30 87 94 38 89 91
TABLE 11 Change in % Moisture by KF when stored at 5° C. Time (months) Batch A Batch B Batch C Batch R Batch S 0 0.5 0.6 0.6 0.8 1.0 1 0.5 0.4 0.6 3 0.5 0.6 0.6 6 0.6 0.7 0.5 0.8 1.3 12 0.6 0.6 0.7 0.9 0.9 24 0.5 0.7 0.7 0.9 0.9 30 0.7 0.7
TABLE 12 Change in Percent Monomer by SEC when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 99.8 99.8 99.8 98.9 98.8 1 99.8 99.1 99.2 98.7 98.7 3 99.8 99.0 99.0 98.6 98.5 6 99.8 99.7 99.7 98.9 98.9 9 99.0 99.1 99.1 99.1 99.1 12 99.3 98.9 98.9 98.8 98.9 15 99.3 99.0 99.0 18 99.4 99.3 99.3 98.7 98.9 24 99.2 99.1 99.1 98.9 98.9 30 99.0 99.0
TABLE 13 Change in Percent Aggregates by SEC when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 0.1 0.1 0.1 0.2 0.2 1 0.2 0.2 0.2 0.2 0.2 3 0.2 0.3 0.2 0.3 0.3 6 0.2 0.3 0.3 0.2 0.2 9 0.2 0.3 0.3 0.2 0.2 12 0.2 0.2 0.2 0.3 0.3 15 0.3 0.3 0.3 18 0.3 0.3 0.3 0.3 0.2 24 0.3 0.3 0.3 0.3 0.2 30 0.4 0.3
TABLE 14 Change in Percent Major Isoform by CEX when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 68.6 69.9 69.5 71.7 71.6 1 67.2 68.4 68.6 71.2 71.0 3 68.1 68.6 68.2 70.3 70.3 6 65.9 67.8 67.8 71.5 71.1 9 69.3 67.5 66.3 68.6 69.0 12 66.7 67.5 67.4 70.1 70.2 15 66.2 66.6 66.8 18 66.1 65.8 64.9 70.0 70.3 24 66.7 68.4 68.2 70.6 70.1 30 67.2 67.2
TABLE 15 Change in Percent Acidic Isoforms by CEX when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 22.8 20.8 21.4 20.3 20.6 1 21.9 21.8 22.2 21.4 21.6 3 21.7 22.2 22.8 21.8 22.0 6 22.6 22.9 23.5 21.1 21.4 9 19.9 22.1 23.1 21.8 21.8 12 23.0 21.4 22.0 21.3 21.3 15 22.5 22.1 22.7 18 22.6 22.1 22.6 21.3 21.5 24 21.7 21.9 22.6 20.7 20.7 30 22.7 23.2
TABLE 16 Change in Percent Basic Isoforms by CEX when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 8.5 9.3 9.1 8.1 7.8 1 10.8 9.8 9.2 7.4 7.3 3 10.3 9.3 9.0 7.8 7.7 6 11.5 9.3 8.7 7.4 7.5 9 10.8 10.4 10.6 9.7 9.3 12 10.3 11.1 10.7 8.7 8.5 15 11.3 11.2 10.6 18 11.2 12.1 12.5 8.7 8.2 24 11.6 9.7 9.1 8.7 9.2 30 10.2 9.6
TABLE 17 Change in % (H + L) by Reduced-SDS Page when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 98 98 98 96 96 1 98 98 98 98 98 3 97 98 98 98 98 6 97 97 97 97 97 9 97 97 97 98 98 12 98 96 96 98 98 15 97 97 97 18 98 97 97 99 99 24 98 97 98 99 99 30 97 98
TABLE 18 Change in Binding Efficacy when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 107 106 105 93 102 1 115 103 109 3 92 113 100 96 94 6 109 89 97 101 114 9 97 89 85 97 102 12 83 91 123 15 96 91 96 18 106 123 87 92 102 24 103 82 90 98 94 30 84 114
TABLE 19 Change in % Moisture by KF when stored at 25° C./60% RH Time (months) Batch A Batch B Batch C Batch R Batch S 0 0.5 0.6 0.6 0.8 1.0 1 0.5 0.6 0.5 3 0.5 0.7 0.6 6 0.5 0.7 0.7 1.3 1.2 12 0.6 0.8 0.6 0.9 1.0 24 0.7 0.8 0.6 1.1 1.0 30 0.8 0.7
[0210]Cation Exchange Chromatography (CEX)
[0211]A phosphate/sodium chloride gradient on a weak cation exchange column is used in a high performance liquid chromatography system to separate charged species in anti-α4β7 antibody formulations and determine the charge composition of the antibody species. Acidic Isoforms elute before the Major Isoform and Basic Isoforms elute after the Major Isoform.
[0212]Stability data for all vedolizumab batches generated using a CEX assay is presented in Tables 3, 6-8 and 14-16. The Tables show, that at these storage conditions, there was no trend of reducing the % Major Isoform below 55.0%.
[0213]Size Exclusion Chromatography (SEC)
[0214]SEC is performed using an analytical SEC column (Tosoh Bioscience, LLC, King of Prussia, Pa.). The mobile phase is a phosphate-buffered saline solution and the absorbance is monitored at 280 nm.
[0215]Stability data generated using an SEC assay is presented in Tables 1, 2, 4, 5, 12 and 13. The Tables show that none of the listed storage conditions resulted in lowering the % Monomer below 96.0%. Similarly, the % Aggregates remained≦2.5% for all batches at all listed storage conditions.
[0216]SDS-PAGE Assay
[0217]SDS-PAGE is performed using an Invitrogen (Carlsbad, Calif.) Tris-Glycine gel, 4-20% for reducing condition and 4-12% for non-reducing condition. The reconstituted antibody formulation sample is diluted in liquid formulation buffer then diluted one to two with Tris-Glycine SDS Sample Buffer (2×, Invitrogen) either with 10% 2-mercaptoethanol (reducing sample buffer) or without 2-mercaptoethanol (non-reducing sample buffer). Samples are briefly heated and loaded in comparison with a molecular weight marker (Invitrogen). The gels are stained with colloidal coomassie blue (Invitrogen) according to the manufacturer's instruction. Protein bands are analyzed by densitometry to identify the % heavy and light chain for reduced gels and % IgG for non-reduced gels.
[0218]Stability data generated using a Reduced SDS-PAGE assay are presented in Tables 9 and 17. No noticeable changes were observed for the % Heavy+Light (H+L) chains at all storage conditions listed for all stability lots. The banding pattern was similar to that of the reference standard and % (H+L) remained at a level ≧90%.
[0219]Binding Efficacy
[0220]HuT78 cells (human T cell lymphoma cells, American Type Culture Collection, Manassas, Va.) suspended in 1% BSA in PBS, 0.01% sodium azide are contacted with serial dilutions of primary test antibody. After incubation on ice, the cells are washed and treated with fluorescently labeled secondary antibody. After a further wash, the cells are fixed and suspended in FACS reagent for analysis by flow cytometry (Becton Dickinson Franklin Lakes, N.J.); also see U.S. Pat. No. 7,147,851.
[0221]Binding efficacy of vedolizumab was measured relative to the Reference Standard and reported as % Reference Standard and EC50. Stability data is presented in Tables 10 and 18. Data for the % Reference Standard showed variability but remained within the specification limits at all storage conditions. No evaluated lots of vedolizumab displayed a trend of diminished binding efficacy at the storage conditions listed.
[0222]Moisture by Karl Fischer
[0223]The formulation is titrated with methanol for a coulometric Karl Fischer moisture determination. Moisture data is presented in Tables 11 and 19. All evaluated lots of vedolizumab had less than 5% moisture at all listed storage conditions.
[0224]Capillary Isoelectric Focusing (cIEF)
[0225]cIEF is performed using an iCE280 whole column detection cIEF system (Convergent Biosciences, Toronto, Ontario). Choice of ampholyte can be as recommended by the manufacturer or can be a combination of commercially available ampholytes. A useful combination is a mixture of 3-10 and 5-8 PHARMALYTE™ (GE Healthcare, Piscataway, N.J.).
Example
Example 3
Modeling the Scale-Up of the Lyophilization Process
[0226]Quality by design was used while manipulating the load in the freeze dryer and the solids content of the formulation. The load was varied from 33 to 100%. The formulation solids content was varied from 9 to 27% by including in the loads a formulation which was either 0.5×, 1.0× and 1.5× of the target formulation. These formulations had similar Tg′. With higher % solids, the primary drying time increased. In addition, at higher solids content, the product temperature increased due to larger Rp. The load also has an effect on both stages of drying (FIG. 8).
PUM
Property | Measurement | Unit |
Time | 1209600.0 | s |
Time | 3628800.0 | s |
Time | 2419200.0 | s |
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