Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2001-09-24
2004-12-14
Gelsa, Bennett (Department: 1639)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C435S006120, C435S007100
Reexamination Certificate
active
06831161
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
2. Discussion of the Related Art
The present invention relates to affinity separation and to ligands for use therein.
Affinity separations generally take place on an affinity chromatography column and take advantage of the potentially highly specific nature of interactions involving biomolecules, especially proteins. Other possible separation methods include membrane filtration, two-phase extraction, fluidised beds, expanded beds and magnetic bead separation (Scopes, R. K. Protein Purification, Principles and Practice, 3rd Ed. ISBN 0-387-94072-3). Interactions include protein-protein, protein-nucleic acid, enzyme-substrate, receptor-ligand (e.g. hormone) protein-carbohydrate and protein-metal interactions. Generally, affinity separation is based on the biologically important binding interactions that occur on protein surfaces, such as that between an enzyme and its substrate, between nucleic acid and a DNA binding protein or between antigen and antibody. Either one of a pair of binding partners can be immobilised, e.g. by covalent bonding to an insoluble matrix in an affinity column in order to assay for or purify its binding partner. The very selective nature of interactions between or involving affinity binding molecules, particularly proteins makes them ideal for purification/separation techniques and for many applications, affinity chromatography involving an immobilised protein ligand is preferred over ion-exchange or gel-filtration chromatography.
If a sample is added to a column which carries immobilised on a solid matrix a specific binding partner to a target molecule in the sample, the target molecule will be retained in the column while the majority of the non-target molecules will simply run through. A solution, typically containing a gradually decreasing pH can be added to the column to wash through firstly any non-specifically bound molecules and finally to elute the target molecule. The target molecule, having the highest affinity for the immobilised ligand, will be the last molecule to be washed off the column and it is therefore the final fractions which will contain the highest concentration of the target molecule and can then be used in a further purification/concentration step or be assayed directly or indirectly for the presence of the target molecule.
Medicine and research in the biochemical and biotechnological fields generally, has created a demand for ever purer samples of organic and biological molecules and thus for strategies which can provide samples of high purity as quickly and cheaply as possible.
As well as satisfying a need to obtain pure samples of proteins and other molecules, affinity separation is important in assaying samples quantitatively and qualitatively for a target molecule. This may be important, for example, when blood or urine samples are being assayed for molecules indicative of a disease such as a metabolic disorder or even the presence of non-naturally occurring substances, narcotics, steroid derivatives etc.
An affinity chromatography column, complete with its solid affinity matrix, can be expensive and clearly it is desirable to be able to re-use the column several times, i.e., to complete a number of runs before it has to be discarded due to reduced ability of the immobilised ligand to bind a target molecule or to a reduced specificity of binding. In order to achieve a set of results which can be directly compared and to clear the column of non-specific and specifically bound molecules before performing another run, it is necessary to clean the column. The recognised standard for cleaning and sanitizing separation media and systems is NaOH, often in combination with NaCl. An applied 0.1-1.0 M NaOH solution is able to remove viruses, bacteria, nucleic acid, proteins, yeasts, endotoxins, prions and other contaminating agents. The NaOH contact time may vary, between 30 minutes and 1 hour is typical, and removal from the system is monitored by simple in-line pH and conductivity measurements.
However, the ability of the separation media to withstand these rather harsh sanitizing conditions depends on the functional groups of the attached ligand (binding partner), attachment chemistries, and the stability of the base matrices to alkaline conditions. Proteins are sensitive to extreme pH, such as is experienced during NaOH cleaning and, generally speaking, this will adversely affect the effectiveness of protein-based affinity media. Thus, although protein based affinity separation has advantages over ion exchange and gel-filtration chromatography due to its good specificity, these other less specific techniques are not adversely affected by standard cleaning methods.
The sensitivity of proteins to alkaline pH is primarily due to deamidation of asparagine and glutamine residues, particularly asparagine residues. Deamidation of asparagine results in the formation, via a cyclic imide intermediate, of isoaspartate and aspartate, usually in the ratio of 3:1 to 4:1. This reaction does take place at physiological pH but is far faster at alkaline pH such as present in a chromatography column which is being cleaned by an NaOH solution. The isoaspartyl form is characterised by an atypical amide bond between the &bgr;-carboxyl of aspartate and the &agr;-nitrogen of the C-flanking amino acid. This results in an extra —CH
2
— in the backbone of the protein as well as a free &agr;-carboxyl group. Cleavage of the peptide backbone may occur as a result of the deamidation and the protein may lose its activity due to a structural change in the whole protein, or merely a small change in a sensitive region such as the active or binding site. The susceptibility of asparagine residues to deamidation is sequence and conformation dependent, Asn residues at Asn-Gly and Asn-Ser sites being particularly vulnerable.
It is a serious problem when an affinity chromatography column has been set up with a protein immobilised on an insoluble support within it and the necessary cleaning process between runs results in reduced efficacy of the system. As well as hastening the end of the absolute useful life of the column, if successive washes decrease the ability of the immobilised protein to capture its binding partner from a sample, comparisons between runs where the concentration of an analyte in a series of samples is measured become meaningless. There is therefore a need for a method of affinity separation wherein the immobilised protein is less susceptible to standard cleaning methods, particularly to alkaline pH.
OBJECTS AND SUMMARY OF THE INVENTION
Thus, in one aspect, the present invention provides a method of affinity separation wherein the affinity ligand is an immobilised proteinaceous ligand wherein one or more of its asparagine residues has been modified.
The term “modified” includes deletion of the asparagine residue or replacement of it with a less alkaline-sensitive amino acid, or wherein the asparagine residue has been modified by substitution (i.e. chemical substitution of one or more groups) or other chemical derivitisation, e.g. by a protecting group. Replacement of one or more asparagine residues with a less alkaline-sensitive amino acid is preferred.
By ‘affinity separation’ is meant any purification or assaying technique which involves the addition of a sample containing a target analyte to a solid which carries on it a specific binding partner to the analyte.
Gravity or other means allows the sample to pass through or across the solid, and the interaction between the analyte and its specific binding partner immobilised on the solid means that the analyte will be retained on the solid while the rest, or most of the rest, of the sample passes through the system. The separation may conveniently be carried out on an affinity chromatography column. The solid is preferably arranged in a column, so that the sample can be added to the top and the non-target part of the sample runs off. An eluant such as a salt solution or a change of pH can be used to displace the specifically bound analyte which c
Hober Sophia
Uhlen Mathias
Affibody AB
Epperson Jon D.
Garabedian Todd E.
Gelsa Bennett
Wiggin and Dana LLP
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