Trehalose-Stabilized Factor IX Lyophilization
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Solution Overview
Problem
Existing formulations of Factor IX lack effective stabilization during freezing, lyophilization, and storage, leading to denaturation and loss of calcium binding ability and biological activity.
Innovation Solution
Inclusion of trehalose in the Factor IX composition during freezing, lyophilization, and storage, which acts as a cryoprotectant and stabilizer, preserving more than 90% of the calcium binding property and biological activity of Factor IX.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If Factor IX is formulated without cryoprotectant or stabilizer, then the formulation is simpler, but the protein is unprotected from denaturing effects during freezing, lyophilization and storage
Solution Approach 1:
Trehalose acts as an intermediary substance that mediates between the harsh conditions of freezing/lyophilization/storage and the sensitive Factor IX protein. It forms a protective matrix that physically shields the protein from denaturing effects, allowing stability without complex formulation components
Solution Approach 2:
The invention changes the physical-chemical parameters of the formulation by incorporating trehalose, which alters the glass transition temperature and creates a protective amorphous matrix. This parameter change enables the protein to withstand stress conditions that would otherwise cause denaturation
2Temperature
If Factor IX is stored at higher temperatures, then storage conditions are more relaxed, but the calcium binding ability and biological activity are lost
Solution Approach 1:
Trehalose provides beforehand cushioning by creating a protective environment that anticipates and prevents thermal denaturation. The cryoprotectant forms a stable matrix before stress occurs, cushioning the protein against temperature-induced conformational changes that would destroy calcium binding ability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Trehalose significantly improves the stability of Factor IX, maintaining over 90% of its calcium binding ability and biological activity during storage for 6 months at 25°C, and retards the loss of potency and calcium binding ability.
Implementation Method 1
inclusion of trehalose in the Factor IX composition during freezing, lyophilization and storage stabilized the calcium binding ability of Factor IX
Implementation Method 2
stabilization of protein structure and activity during lyophilization and storage
Implementation Method 3
stabilized the calcium binding ability of Factor IX and maintained biological activity of the purified protein
Data Source
AI summary
Methods of preparing lyophilized preparations of Factor IX which preserve more than 90% of the calcium binding property of Factor IX are disclosed. Factor IX formulated with trehalose shows a superior stability profile after 12 weeks storage at 25° C./60% relative humidity (RH) and 40° C./75% RH relative to Factor IX formulated without trehalose. The data suggest that the inclusion of trehalose in the formulation could allow for temperature excursions or even long-term room temperature storage of a Factor IX lyophilized product. The formulations tested contained 10 mM histidine pH 6.8, 3% mannitol, 66 mM sodium chloride, 0.0075% Polysorbate 80, with and without 1% trehalose. Upon storage at 40° C./75% RH or 25° C./60% RH over 12 weeks the trehalose-containing formulation was comparable to product stored at 2-8° C. while the formulation without trehalose was found to undergo significant aggregation and loss of activity. The two formulations demonstrated comparable stability over 26 weeks of real time storage at −20° C. and 2-8° C.