Title: Process for fractionating polyethylene glycol (PEG) --protein adducts and an adduct of PEG and granulocyt-macrophage colony stimulating factor
United States Patent: 6,384,195
Inventors: Delgado; Cristina (London, GB); Francis; Gillian Elizabeth (London, GB); Fisher; Derek (London, GB)Assignee: PolyMASC Pharmaceuticals plc. (London, GB)
Appl. No.: 234493Filed: January 21, 1999
Foreign Application Priority Data: Oct 20, 1988[GB] (8824591)
The present invention provides adducts of polyethylene glycol and granulocyte-macrophage colony stimulating factor as well as methods for modifying a protein with pleiotropic activities which involve coupling polyethylene glycol to the protein.
Description of the Invention
The present invention-relates to a process for
fractionating polyethylene glycol-protein adducts.
Polyethylene glycol is a long chain, linear synthetic polymer composed of
ethylene oxide units, HO(CH2 CH2 O)n CH2
CH2 OH, in which n can vary to provide compounds with molecular
weights from 200-20,000. It is non-toxic and has been administered orally
and intravenously to humans (PEG-adenosine deaminase for severe combined
immunodeficiency disease; PEG-asparaginase for acute lymphoblastic
leukaemia; PEG-superoxide dimutase for oxygen toxicity (3-7)). PEG can be
coupled to proteins following appropriate derivatisation of the OH groups
of the PEG. The NH2 groups of lysine side chains are particularly
accessible sites and either few or many sites can be modified. Given,
adequate technology for their production, PEG-modified proteins have
numerous therapeutic and other applications. Many proteins of potential
clinical use have extremely short half lives necessitating administration
by continuous infusion (an expensive, unpleasant and potentially hazardous
procedure). PEG modification extends plasma half lives and has been used
to increase the bio-availability of enzymes (see below). Reduction of
antigenicity of proteins is also produced by PEG modification and this
will extend their clinical use allowing more protracted administration. In
addition, with proteins having pleiotropic biological effects, PEG
modification creates products with a new spectrum of activities, because
of differential loss of separate biological properties. With antibodies,
for example, PEG modification dissociates antibody binding and complement
fixing activities. PEG modification also alters biochemical and physical
properties of proteins in ways that may increase their usefulness (e.g.
increased solubility; increased resistance to proteolytic degradation;
altered kinetics, pH and/or temperature optima and changed substrate
specificity of enzymes) This covalent modification of proteins has a
number of consequences:
(i) Increased Plasma Half-life:
This has been found with numerous proteins (See Table 1 and reference
8-17) and has already been exploited clinically. Two children with
adenosine deaminase deficiency were successfully treated with PEG-modified
bovine adenosine deaminase (18). In acute lymphoblastic leukaemia, 74% of
20 patients achieved complete or partial remissions with PEG-asparaginase
(5). Increased half-life and enhanced antitumour potency was also observed
with PEG-interleukin 2 in the Meth A murine sarcoma model (19). The basis
for this increase in half-life is not understood and may include such
factors as reduction of glomerular filtration of small peptides because of
the increase in size due to PEG modification (19). The increase in
biological potency (which may relate to other phenomena in addition to the
increased half-life) is potentially very important in the use of
PEG-cytokine adducts as pharmacological agents in cancer therapy.
TABLE I
The known effects of linking PEG to proteins upon their
circulation half lives.
HALF-LIFE (HOURS)
native REFER-
PROTEIN ANIMAL protein PEG-protein ENCE
asparaginase man 20 357 8.
glutaminase-asparaginase man <0.5 72 9.
uricase man <3 8 10.
glutaminase-asparaginase mouse 2 24 11.
asparaginase mouse <6 96 12.
arginase mouse <1 12 13.
superoxide dismutase mouse 0.06 16.5 14.
lactoferrin mouse 0.05 1 14.
streptokinase mouse 0.07 0.33 15.
plasma-streptokinase mouse 0.05 0.22 15.
complex
adenosine deaminase mouse 0.5 28 16.
asparaginase rat 2.9 56 17.
ii) Altered Biochemical and Physical Properties:
These include increased solubility (20), because of the addition of hydrophilic PEG chains (useful for proteins like interleukin 2 which have limited solubility at physiological pH (19)), increased resistance to proteolytic degradation (21), changes in kinetics or pH and temperature optima or substrate specificity of enzymes (10,20,22,23)). Relevant to the present project are observations which suggest differential effect on function e.g. complement fixing activity and antigen-binding are lost and retained respectively after PEG-modification of IgG (24). PEG-ribonuclease has an altered activity for high but not low molecular weight substrates (25). To some extent, these effects can be controlled by varying the number of sites on the protein modified and the length of the PEG polymer.
(iii) Reduced Antigenicity:
This includes reduced ability to react to antibodies to the unmodified protein and low immunogenicity of the PEG-proteins themselves (26).
Coupling of PEG to proteins-is usually achieved by activation of the hydroxyl groups of PEG with a suitable reagent that can be fully substituted by nucleophilic groups in the protein (mainly lysine E-amino groups) (27). Cyanuric chloride has been the most widely used agent for activation of PEG and this requires a very basic pH for the subsequent coupling step with the protein to be modified (28,27). In order to avoid these adverse conditions (particularly important when dealing with labile proteins like growth factors), alternative methods have been sought. However, 1,1'-carbonyldiimidazole requires very long times for the coupling step (14) and using phenylchloroformates does not avoid the need for basic pH (25).
Although much of this information has been available for many years, PEG-proteins are not widely available commercially.
Tresyl chloride (2,2,2,-trifluoroethane-sulphonyl chloride) has proved useful for activating agarose and other solid supports carrying hydroxyl groups so that they may be coupled to proteins. The attraction of this method is that coupling to proteins takes place quickly and under very mild conditions (28,29). We have successfully applied this approach to the activation of monomethoxyPEG (MPEG), this has a single free derivatisable OH group. We have demonstrated the subsequent coupling of MPEG to both antibodies (30) and albumin, under mild conditions (pH 7.5 phosphate buffer, at room temperature). An advantage over previous techniques is that the reaction mixture is innocuous and does not have to be removed before the PEG-protein is used. We have also developed a technique to neutralise excess tresyl-PEG after the coupling step (to prevent reaction with other proteins and/or cells) thus avoiding the need for laborious chromatography or ultrafiltration to remove it. These improvements are of importance when applying the method to labile growth factor proteins, which are notoriously sensitive to manipulations such as ultrafiltration.
Given acceptable (non-denaturing) conditions for the coupling step, there are two main variables that will affect the biological properties of the PEG-proteins and these may be controlled in the manufacturing process. One is the length of the PEG molecules attached per protein molecule and the second is the number of PEG molecules per protein.
Where proteins have several lysine groups, varying the molar ratio of activated MPEG to protein influences the degrees of substitution markedly. What is needed is a means of determining what degree of substitution gives the best outcome vis a vis the desired biological properties and then to devise a manufacturing scheme which best achieves this degree of substitution. Biochemical monitoring methods are cumbersome (2) and do not give an estimate of the variability in substitution of the population of modified protein molecules. They also do not allow recovery of materials with different degrees of substitution (the latter is difficult to control by altering molar ratios, since a wide distribution of degrees of substitution is observed at any given molar ratio, until full substitution is approached at high molar ratios. Both analytical work to determine which degree of substitution produces the optimum effect and the manufacturing process requires a means of fractionating peptides/proteins with different (and preferably precisely defined) degrees of substitution. The problem is likely to be widespread since most clinically useful proteins have several lysine residues (Table II).
TABLE II
Growth Factor Lysine Residues Total-Amino Acids
Interleukins:
Interleukin 1 19 271
Interleukin 2 10 153
Interleukin 3 9 166
Interferons:
gamma 20 146
fibroblast (beta) 11 166
leukocyte (alpha) 8 166
G-CSF 4 178
GM-CSF 6 144
Although PEG-modification of over a dozen proteins has now been described, frequently in extensive practical detail, little attention has been given to the PEG-proteins being heterogeneous in their degree of substitution with PEG (23).
The partitioning behaviour of PEG-protein adducts in PEG-containing aqueous biphasic systems has not been previously defined, nor has the relationship between degree of PEG substitution and partitioning coefficient. On investigating the partitioning behaviour in such systems we have surprisingly discovered that PEG-containing aqueous biphasic systems are uniquely tailored to separating PEG-proteins sensitively and can thus be used to monitor the effect of degree of modification on biological properties and, on a bulk scale to prepare PEG proteins 6f specified degrees of substitution.
The invention therefore provides a process for fractionating a mixture of PEG-protein adducts comprising partitioning the PEG-protein adducts in a PEG-containing aqueous biphasic system. Preferably the process further comprises the step of recovering a PEG-protein adduct of pre-determined degree of PEG substitution from one phase of the biphasic system. Whilst any PEG protein adduct mixture may be fractionated in accordance with the invention, it is preferred to use adducts of monomethoxyPEG preferably those formed by reaction of the protein with tresyl monomethoxyPEG, (TMPEG). In a particular aspect of the invention, unreacted TMPEG is destroyed or the adduct-forming reaction is quenched, by addition of lysine or albumin. Partitioning may be performed batchwise or continuously, for instance by counter currents of the two phases and may be repeated to obtain additional fractionation.
Claim 1 of 3 Claims
What is claimed is:
1. An adduct of polyethylene glycol (PEG) and granulocyte-macrophage
colony stimulating factor, said granulocyte-macrophage colony stimulating
factor having 6 lysine residues.
____________________________________________
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full
patent.
| [
Outsourcing Guide
] [
Cont. Education
] [
Software/Reports
] [
Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ] [ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ] [ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ] [ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ] [ Site Map ] |