Collagenase Purification for Neutral Protease Removal

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Solution Overview

Problem

Existing collagenase manufacturing processes result in atypical degradation due to the presence of neutral protease, which is highly active in the presence of certain metals, leading to purity failures and safety concerns for medical applications.

Innovation Solution

Implementing process controls to manage metal levels during fermentation and purification, specifically maintaining zinc levels below 3 ppm and nickel levels below 0.5 ppm, and incorporating neutral protease elimination steps to ensure collagenase compositions are essentially free of neutral protease, using methods such as SDS-PAGE and Zymography to reject impure fractions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If metal levels are maintained during fermentation and purification, then collagenase stability and reproducibility are improved, but process complexity increases

Engineering Contradiction:

Improvecollagenase reproducibility and stabilityVSAvoidprocess control complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies preliminary action by controlling metal levels (zinc below 3 ppm, nickel below 0.5 ppm) during the fermentation and purification stages before collagenase production. This preventive approach ensures that neutral protease remains inactive throughout the process, avoiding degradation issues later and improving overall process reliability without requiring complex intervention steps downstream.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent implements parameter changes by establishing specific metal concentration thresholds (zinc < 3 ppm, nickel < 0.5 ppm) as critical process parameters. By monitoring and controlling these chemical parameters throughout fermentation and purification, the process achieves consistent collagenase quality and stability while managing complexity through defined parameter specifications rather than complex procedural interventions.

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If neutral protease elimination steps are implemented, then collagenase purity is improved, but manufacturing time and cost increase

Engineering Contradiction:

Improvecollagenase purityVSAvoidmanufacturing cycle time

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The patent applies the extraction principle by removing neutral protease through specific elimination steps during the purification process. By actively separating and eliminating this contaminating enzyme, the process achieves high collagenase purity (>95%) and prevents degradation, with the trade-off of additional processing steps that increase manufacturing time and complexity.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent employs analytical detection methods (SDS-PAGE and Zymography) to replace extensive manual purification steps. These detection techniques allow for precise identification of neutral protease contamination, enabling targeted elimination and achieving high purity levels more efficiently than traditional mechanical separation methods alone.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Object-affected harmful factors

If metal levels are strictly controlled below threshold values, then neutral protease activity is suppressed, but process monitoring complexity increases

Engineering Contradiction:

Improveneutral protease activityVSAvoidmetal level monitoring

Core Design Contradiction:

Object-affected harmful factorsVSDifficulty of detecting and measuring

Solution Approach 1:

The patent implements feedback control by establishing clear metal level thresholds (zinc < 3 ppm, nickel < 0.5 ppm) and monitoring these parameters throughout the process. This feedback mechanism allows operators to adjust conditions in real-time to maintain suppressive metal levels, preventing neutral protease activation while managing monitoring complexity through defined target values and ranges.

Inventive Principle:

Principle #23Feedback

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

The solution achieves highly pure collagenase products with improved reproducibility and stability, ensuring safety and efficacy for medical treatments by minimizing degradation and maintaining purity above 95% as measured by RP-HPLC.

Implementation Method 1

using methods such as SDS-PAGE and Zymography to reject impure fractions

Methodology Applied

Scientific EffectElectrophoresis: Electrophoresis

Implementation Method 2

using methods such as SDS-PAGE and Zymography to reject impure fractions

Methodology Applied

Scientific EffectEnzyme: Enzyme

Implementation Method 3

purity and stability of collagenase I and collagenase II compositions, wherein the compositions are pure to at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC)

Methodology Applied

Scientific EffectChromatography: Chromatography

Data Source

PatentUS12448615B2Method of purifying and isolating collagenase

Publication Date: 2025.10.21 ENDO OPERATIONS LTD

AI summary

The present invention relates to the fields of collagenase production and collagenase products, and particularly to improving the reproducibility, purity, and stability of collagenase I and collagenase II compositions, where the compositions are pure to at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC) and essentially free of neutral protease.