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Stabilized liquid and lyophilized ADAMTS13 formulations

A stabilization and preparation technology, applied in the field of stabilized liquid and lyophilized ADAMTS13 preparations, which can solve problems such as reducing the activity of ADAMTS13

Active Publication Date: 2012-07-11
TAKEDA PHARMACEUTICA 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

Method used

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  • Stabilized liquid and lyophilized ADAMTS13 formulations
  • Stabilized liquid and lyophilized ADAMTS13 formulations
  • Stabilized liquid and lyophilized ADAMTS13 formulations

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

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

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

The recombinant CHO cells expressing human ADAMTS13 are adapted to a patented medium (BCS medium) with a defined chemical composition. Thaw DWCB and prepare cell inoculum in BCS medium. The cells propagated from the A13 expression clone #640-2 were transferred to a 10L bioreactor with a Rushton impeller, and the patented BACD-A13 medium was used to control the pH of 7.15-7.20, 37℃ and 20 The cells were cultured in a repeated batch culture under a dissolved oxygen concentration of% air saturation. The 2 batch cultures were grown to a final working volume of 10 L, the bioreactor was switched to continuous medium feed on da...

Example Embodiment

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

Recombinant ADAMTS13 was expressed in recombinant CHO cells and purified by anion exchange chromatography. The purified rA13 has 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, and 0.05% polysorbate 80, at pH 7.0, use 20 mM selected from (1) histidine, (2) phosphate buffer or (3) sodium citrate Buffer preparation rA13. Then, the samples were divided equally and half of the samples were lyophilized.

The lyophilized sample is reconstituted with sterile water to a final volume equal to the final volume of the pre-lyophilized formulation. Then, a single aliquot of each liquid formulation and lyophilized formulation was characterized by gel filtration by loading the sample onto a Superose 6GL column (GE Healthcare). Such as image 3 It can be seen that all the formulations obtained ADAMTS13 samples running as a single peak correspon...

Example Embodiment

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

The rA13 samples prepared and aliquoted as in Example 2 were stored at 4°C or 37°C for up to 6 months. At 0, 1, 2, 3, 12 and 24 weeks, the rA13 protein concentration of the solution formulation was analyzed by ELISA assay and its rA13 activity was analyzed by FRETS-VWF73 test ( figure 1 ). The lyophilized sample was reconstituted with sterile water to a final volume equal to the final volume of the pre-lyophilized formulation and similar analysis was performed at 0, 2, 4, 8, 12, and 24 weeks ( figure 2 ).

The rA13 liquid formulation stored at 4°C showed no loss in antigen content (ie protein concentration) or FRETS-VWF73 activity at a time point close to 6 months. Such as figure 1 It can be seen that 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 enhanced or desirable properties. As such, the invention provides liquid and lyophilized formulations of ADAMTS13 that are suitable for pharmaceutical administration. Among other aspects, the present invention also provides methods of treating various diseases and conditions related to VWF and/or ADAMTS13 dysfunction in a subject. Also provided herein are kits comprising ADAMTS13 formulations useful for 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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IPC IPC(8): A61K9/00A61K9/10A61K38/43A61K38/48
CPCA61K47/183A61K9/08A61K47/26A61K47/10A61K47/02A61K38/4886A61K9/19A61K9/0019C12Y304/24087A61P7/02A61P9/10Y02A50/30A61K47/22
Inventor H·P·马蒂森P·L·图雷切克H·P·施瓦茨
Owner TAKEDA PHARMACEUTICA CO LTD
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