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Stabilized liquid and lyophilized formulations of adamts13

A liquid preparation and preparation technology, which can be applied in the directions of enzyme stabilization, freeze-dried delivery, liquid delivery, etc., and can solve the problems of reducing ADAMTS13 activity and other issues

Active Publication Date: 2014-10-15
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

UL-vWF multimers accumulate over time as the persistence of the inhibitor (anti-ADAMTS13 Ab) reduces ADAMTS13 activity

Method used

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  • Stabilized liquid and lyophilized formulations of adamts13
  • Stabilized liquid and lyophilized formulations of adamts13
  • Stabilized liquid and lyophilized formulations of adamts13

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0344] A. Example 1: Expression of recombinant ADAMTS13 (rA13)

[0345] Chemostat cell cultures of recombinant CHO cell line #640-2 expressing human ADAMTS13 were grown in chemically defined BACD-A13 medium supplemented with additional zinc and vitamin B3. The 10 L culture was maintained for 53 days, and the production of rA13 protein and activity was monitored over time.

[0346] Recombinant CHO cells expressing human ADAMTS13 were adapted to a chemically defined proprietary medium (BCS medium). Thaw DWCB and prepare cell inoculum in BCS medium. Cells propagated from A13 expressing clone #640-2 were transferred to a 10L bioreactor with a Rushton-type impeller, utilizing the proprietary BACD-A13 medium at an online controlled pH of 7.15-7.20, 37°C and 20°C. The cells were cultured in repeated batch cultures at a dissolved oxygen concentration of % air saturation. Two batch cultures were grown to a final working volume of 10 L and the bioreactor was switched to continuous me...

Embodiment 2

[0351] B. Example 2: Formation of purified recombinant ADAMTS13 (rA13)

[0352] Recombinant ADAMTS13 was expressed in recombinant CHO cells and purified by anion exchange chromatography. Purified rA13 had a final concentration of about 750 μg / ml and a specific activity of about 850 mU / μg. In a buffer containing 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, at pH 7.0, with 20 mM selected from (1) histidine, (2) phosphate buffer or (3) sodium citrate Buffer preparation of rA13. Then, the samples were divided evenly and half of the samples were lyophilized.

[0353] Lyophilized samples were reconstituted with sterile water to a final volume equal to that of the previously lyophilized formulation. Individual aliquots of each liquid formulation and lyophilized formulation were then characterized by gel filtration by loading the samples onto a Superose 6GL column (GE Healthcare). Such as image 3 As can be seen in , all formulations yielded ADAMTS13 samples running as a single...

Embodiment 3

[0354] C. Example 3: Characterization of activity retention of rA13 formulations stored at 4°C and 37°C

[0355] rA13 samples formulated and aliquoted as in Example 2 were stored at 4°C or 37°C for periods of up to 6 months. Solution formulations were analyzed for rA13 protein concentration by ELISA assay and rA13 activity by FRETS-VWF73 assay at 0, 1, 2, 3, 12 and 24 weeks ( figure 1 ). The lyophilized samples were reconstituted with sterile water to a final volume equal to that of the pre-lyophilized formulation and analyzed similarly at 0, 2, 4, 8, 12 and 24 weeks ( figure 2 ).

[0356] Liquid formulations of rA13 stored at 4°C showed no loss in antigen content (ie, protein concentration) or FRETS-VWF73 activity at approximately the 6 month time point. Such as figure 1 As can be seen in , this is the case for all 3 formulations buffered with histidine, phosphate and sodium citrate, respectively. In contrast, liquid formulations buffered with histidine or sodium citrat...

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Abstract

The present invention relates to formulations of ADAMTS13 with improved or desirable properties. Accordingly, the present invention provides liquid and lyophilized formulations of ADAMTS13 suitable for pharmaceutical administration. Among other aspects, the present invention provides methods of treating various diseases and conditions associated with VWF and / or ADAMTS13 dysfunction in a subject. Also provided herein are kits comprising formulations of ADAMTS13 useful in the treatment of various diseases and conditions.

Description

[0001] Cross Reference Related Applications [0002] This application claims the benefit of US Provisional Application No. 61 / 244,353, filed September 21, 2009, which is hereby incorporated by reference in its entirety for all purposes. [0003] Statement Regarding Rights to Inventions Made Under Federally Sponsored Research or Development [0004] Not applicable [0005] Reference to "Sequence Listing", Table or Computer Program Listing Appendix filed on CD-ROM [0006] Not applicable Background of the invention [0007] ADAMTS (disintegrin and metalloprotease with thrombospondin type I motif) protein is a structure containing many conserved domains (including zinc-dependent catalytic domain, cysteine-rich domain, disintegrin-like domain (disintegrin-like domain) and at least one (in most cases multiple) thrombospondin type I repeat family of metalloproteases (for review, see Nicholson et al., BMC Evol Biol. 2005 Feb 4; 5(1): 11). Such proteins (Jones GC, Curr Pharm Biote...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96A61K38/43A61K38/48A61K9/00A61K9/10
CPCA61K9/08A61K47/183A61K38/4886A61K47/02A61K47/26A61K47/10A61K9/19A61K9/0019C12Y304/24087A61P7/02A61P9/10Y02A50/30A61K47/22
Inventor H·P·马蒂森P·L·图雷切克H·P·施瓦茨
Owner TAKEDA PHARMA CO LTD
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