Streptomyces koganeiensis Hyaluronidase Purification Process

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Solution Overview

Problem

The industrial-scale production and purification of bacterial or animal hyaluronidase are challenging due to enzyme instability in aqueous solutions and loss of activity during purification, limiting its therapeutic and veterinary applications.

Innovation Solution

A process involving weak cation-exchange, strong anion-exchange, strong cation-exchange, and strong anion-exchange chromatography steps is used to isolate and purify hyaluronidase from Streptomyces koganeiensis, resulting in a stable enzyme with high activity and purity, suitable for pharmaceutical and veterinary compositions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional purification methods (dialysis and ion exchange chromatography) are used to obtain hyaluronidase, then the enzyme can be isolated from culture supernatant, but the enzyme becomes unstable in aqueous solution and loses activity upon purification

Engineering Contradiction:

Improveenzyme purityVSAvoidenzyme stability

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying the purification protocol to include specific pH control during chromatography steps and using particular buffer conditions that maintain enzyme stability. The process uses weak cation-exchange at pH 4.0 followed by strong anion-exchange at pH 8.0, with each step optimized to preserve hyaluronidase activity while achieving high purity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent uses specific buffer solutions as intermediaries during the purification process. These buffers (sodium acetate at pH 4.0 for cation-exchange and Tris-HCl at pH 8.0 for anion-exchange) act as mediators that maintain the enzyme's stable state throughout the purification steps, preventing denaturation and activity loss

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If multiple chromatography steps are performed to achieve high purity hyaluronidase, then manufacturing precision improves, but the process complexity and time increase

Engineering Contradiction:

Improveenzyme purityVSAvoidpurification process complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent segments the purification process into two distinct chromatography steps with different mechanisms: weak cation-exchange chromatography followed by strong anion-exchange chromatography. Each step targets different protein contaminants, and the segmentation allows for optimized conditions at each stage, achieving high purity while keeping the process manageable

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent employs periodic action by alternating between two different chromatography modalities (cation-exchange and anion-exchange) in sequence. This periodic alternation of charge-based separation mechanisms enables comprehensive purification while maintaining a systematic and reproducible workflow

Inventive Principle:

Principle #19Periodic action

3Quantity of substance

If conventional purification methods are used, then some hyaluronidase activity can be recovered, but the majority of enzyme activity is lost during the purification process

Engineering Contradiction:

Improveenzyme activityVSAvoidenzyme activity loss

Core Design Contradiction:

Quantity of substanceVSLoss of substance

Solution Approach 1:

The patent applies beforehand cushioning by optimizing buffer conditions and pH levels prior to each chromatography step to create a protective environment for the enzyme. The use of mild buffer systems and controlled pH transitions cushions the enzyme against denaturation and activity loss during the purification process

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

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 purified hyaluronidase exhibits high stability in aqueous solutions, enabling its use in various therapeutic and veterinary applications, including edema treatment, inflammatory conditions, and cancer therapy, with enhanced bioavailability and effectiveness.

Implementation Method 1

subjecting the supernatant obtained from fermentation of Streptomyces koganeiensis ATCC 31394 to weak cation-exchange chromatography and isolating the protein fraction with hyaluronidase activity

Methodology Applied

Scientific EffectIon exchange: Ion Exchange

Implementation Method 2

subjecting the protein fraction with hyaluronidase activity obtained in step a) to diafiltration and strong anion-exchange chromatography and isolating the protein fraction with hyaluronidase activity

Methodology Applied

Scientific EffectIon exchange: Ion Exchange

Implementation Method 3

subjecting the protein fraction with hyaluronidase activity obtained in step b) to strong cation-exchange chromatography and isolating the protein fraction with hyaluronidase activity

Methodology Applied

Scientific EffectIon exchange: Ion Exchange

Implementation Method 4

subjecting the protein fraction with hyaluronidase activity obtained in step c) to strong anion-exchange chromatography and isolating the protein fraction with hyaluronidase activity

Methodology Applied

Scientific EffectIon exchange: Ion Exchange

Data Source

PatentUS10208300B2Extracellular hyaluronidase from <i>Streptomyces koganeiensis </i>

Publication Date: 2019.02.19 FIDIA FARM SPA

AI summary

A process for preparing Streptomyces koganeiensis ATCC 31394 hyaluronidase by obtaining hyaluronidase having molecular weight of 21.6 kDalton, which has hyaluronidase activity and stability markedly higher than those of the hyaluronidase obtained from such microorganism to date.