N-terminal modified interferon-alpha reducing non-natural disulfide bonds

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

The production of mature human IFN-α2b proteins in E. coli often results in substantial structural isoforms due to non-natural disulfide bonds, which are either inactive or immunogenic, complicating their therapeutic applications.

Innovation Solution

Adding a proline residue to the N-terminus cysteine (Cys 1) of mature IFN-α2b reduces the formation of non-natural disulfide bonds, forming a polypeptide linked via a peptide bond, thereby minimizing isoform contamination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If mature IFN-α2b is prepared by expressing its encoding cDNA in E. coli, then production efficiency is improved, but structural isoforms with non-natural disulfide bonds are formed

Engineering Contradiction:

Improveproduction efficiencyVSAvoidstructural purity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent applies preliminary action by adding a proline residue to the N-terminus of the IFN-α2b protein before the formation of disulfide bonds occurs during refolding. This pre-modification prevents the cysteine at position 1 from forming non-natural disulfide bonds, thereby eliminating isoform formation at the source rather than attempting to correct it afterward.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention applies local quality by making a specific localized modification only at the N-terminus position 1 of the IFN-α2b molecule. The proline residue is added only to this specific location, leaving the rest of the protein structure unchanged, thereby preventing non-natural disulfide bond formation without affecting other functional regions.

Inventive Principle:

Principle #3Local quality

2Quantity of substance

If non-natural disulfide bonds form during protein expression, then structural isoforms are created, but therapeutic value is lost due to inactivity or immunogenicity

Engineering Contradiction:

Improveprotein yieldVSAvoidtherapeutic efficacy

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent applies preliminary anti-action by introducing a proline residue that actively prevents the formation of harmful non-natural disulfide bonds. This preemptive measure counteracts the tendency of cysteine residues to form incorrect disulfide bonds during refolding, thereby protecting the therapeutic efficacy of the produced protein.

Inventive Principle:

Principle #9Preliminary anti-action

3Manufacturing precision

If additional amino acid residues are added to Cys 1 of mature IFN-α2b, then formation of non-natural disulfide bonds is reduced, but protein structure is modified

Engineering Contradiction:

Improveisoform reductionVSAvoidprotein structure

Core Design Contradiction:

Manufacturing precisionVSShape

Solution Approach 1:

The invention applies parameter changes by modifying the N-terminal amino acid sequence parameter of IFN-α2b. Specifically, a proline residue is added to position 1, changing the sequence from Cys-Pro-... to Pro-Cys-.... This parameter change prevents non-natural disulfide bond formation while maintaining the essential disulfide bonds required for protein activity.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentEP2195338B1N-terminal modified interferon-alpha

Publication Date: 2013.12.25 PHARMAESSENTIA CORP

Figure 1
Figure 2
Figure 3

AI summary

A method of reducing formation of non-natural disulfide bonds in a mature IFN-a by adding one or more amino acid residues to its N-terminus cystein. Also disclosed herein is the IFN-a thus modified.