TNF-Binding Protein Isolation via Affinity Chromatography
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Solution Overview
Problem
Current methods for isolating and characterizing TNF-binding proteins (TNF-BP) face challenges such as inactivation during reductive cleavage and heterogeneity in starting materials, leading to insoluble, membrane-bound TNF-BP that are difficult to separate and purify effectively.
Innovation Solution
Development of homogenous, insoluble TNF-binding proteins and their soluble or insoluble fragments capable of binding TNF, using specific amino acid sequences and DNA sequences encoding these proteins, along with a process for their isolation involving immune affinity chromatography, ligand affinity chromatography, and HPLC, to produce recombinant proteins and antibodies for TNF binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If reductive cleavage is used to separate TNF-BP from TNF complex, then TNF-BP can be isolated, but the binding protein becomes inactivated
Solution Approach 1:
The patent extracts and isolates soluble TNF-BP from membrane extracts using affinity chromatography columns, separating the binding protein from the membrane matrix and TNF complex without requiring reductive cleavage that would inactivate the protein
Solution Approach 2:
The patent uses affinity chromatography media as an intermediary to selectively bind and separate TNF-BP from the complex mixture of membrane proteins and TNF, enabling purification without harsh chemical treatment
2Reliability
If membrane-bound TNF-BP is isolated from urine, then TNF binding activity is obtained, but the material becomes heterogeneous and difficult to purify
Solution Approach 1:
The patent focuses on isolating soluble TNF-BP with specific molecular weight characteristics (55-75 kD) that differ from membrane-bound forms, creating a homogeneous population with consistent binding properties
Solution Approach 2:
The patent changes the physical state parameter from membrane-bound (insoluble) to soluble form, enabling effective purification through chromatography and resulting in homogeneous material with consistent TNF binding activity
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 enables the production of high-purity, TNF-binding proteins that maintain activity, allowing for effective isolation and characterization, and can be used in pharmaceutical applications to treat conditions mediated by TNF.
Implementation Method 1
capable of binding tumor necrosis factor-(TNF)... containing amino acid sequences of FIG. 1 or FIG. 4... proteins containing fragments of these sequences
Implementation Method 2
a process for the isolation of an insoluble homogenous protein capable of binding TNF... immune affinity chromatography
Implementation Method 3
TNF-BP have already been characterized... which was used as an antigen preparation for the production of monoclonal antibodies against TNF-BP
Implementation Method 4
which, in turn, was used as an antigen preparation for the production of monoclonal antibodies against TNF-BP... HPLC
Data Source
AI summary
The present invention is concerned with non-soluble proteins and soluble or insoluble fragments thereof, which bind TNF, in homogeneous form, as well as their physiologically compatible salts, especially those proteins having a molecular weight of about 55 or 75 kD (non-reducing SDS-PAGE conditions), a process for the isolation of such proteins, antibodies against such proteins, DNA sequences which code for non-soluble proteins and soluble or non-soluble fragments thereof, which bind TNF, as well as those which code for proteins comprising partly of a soluble fragment, which binds TNF, and partly of all domains except the first of the constant region of the heavy chain of human immunoglobulins and the recombinant proteins coded thereby as well as a process for their manufacture using transformed pro- and eukaryotic host cells.