Designed Ankyrin Repeat Domains for Heat and pH Stability
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Solution Overview
Problem
Existing designed ankyrin repeat domains with binding specificity for serum albumin lack sufficient stability, particularly during storage and under conditions of elevated temperature and pH shifts, which affects the integrity and efficacy of recombinant binding proteins.
Innovation Solution
Designed ankyrin repeat domains with specific amino acid sequences, such as SEQ ID NO: 3 or 4, lacking asparagine at position 77 and optionally glutamic acid at position 78, exhibit enhanced stability, maintaining binding specificity for serum albumin and reducing degradation products during heat treatment and pH shifts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If designed ankyrin repeat domains with binding specificity for serum albumin are used, then prolonged terminal half-life is achieved, but storage stability is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues at positions 77 and 78 of the ankyrin repeat domain sequence. By changing these critical parameters (amino acid identity), the protein achieves improved storage stability while preserving the serum albumin binding capability that provides prolonged half-life.
Solution Approach 2:
The invention applies local quality by making targeted modifications only at specific positions (77 and 78) of the ankyrin repeat domain rather than altering the entire sequence. This localized approach maintains the overall binding function while improving stability at the critical degradation-prone regions.
2Productivity
If heat treatment at elevated temperatures is applied for purification, then purification efficiency is improved, but protein degradation increases
Solution Approach 1:
The modified ankyrin repeat domain with altered residues at positions 77 and 78 exhibits enhanced thermal stability, allowing the protein to withstand heat treatment during purification without significant degradation. This parameter change enables effective purification while minimizing protein loss.
3Ease of manufacture
If storage at lower pH is performed, then purification is facilitated, but formation of lower molecular weight degradation products increases
Solution Approach 1:
The amino acid modifications at positions 77 and 78 confer resistance to acid-induced degradation, allowing the protein to maintain stability during low pH storage and purification steps. This enables easy purification via pH adjustment without excessive formation of degradation products.
Data Source
AI summary
The present disclosure relates to designed ankyrin repeat domains with binding specificity for serum albumin and, in particular, to such designed ankyrin repeat domains with improved stability. The disclosure further relates to recombinant binding proteins comprising such designed ankyrin repeat domain(s), nucleic acids encoding such designed ankyrin repeat domains or proteins, pharmaceutical compositions comprising such proteins and the use of such proteins or pharmaceutical compositions in the treatment of diseases.