Stabilized compositions of proteins having a free thiol moiety

Abstract

Compositions of proteins having free thiols, and methods of making and using such compositions, are described.

1 . A composition comprising a protein having a free thiol and a carbohydrate, wherein the carbohydrate is present in an amount sufficient to maintain the stability of the protein and wherein the pH of the composition is less than 7.0. 
     
     
         2 . The composition of  claim 1 , further comprising an antioxidant, wherein the antioxidant and the carbohydrate are present in amounts sufficient to maintain the stability of the protein and wherein the pH of the composition is less than 7.0. 
     
     
         3 . The composition of  claim 1 , further comprising a surfactant. 
     
     
         4 . The composition of  claim 1 , wherein the pH of the composition is between about 4.5 and about 6.5. 
     
     
         5 . The composition of  claim 1 , wherein the stability is at least 5-80% greater, under preselected conditions, than the stability of a composition which differs by lacking the carbohydrate. 
     
     
         6 . The composition of  claim 1 , wherein the carbohydrate is present in an amount sufficient to increase the stability of the protein. 
     
     
         7 . The composition of  claim 1 , wherein the carbohydrate is present in an amount sufficient to inhibit the reaction of a free thiol on a first molecule of the protein with a free thiol on a second molecule of the protein to form an aggregate. 
     
     
         8 . The composition of  claim 1 , wherein the carbohydrate is present in an amount sufficient to inhibit the formation of an aggregate formed by the reaction of a free thiol on a first molecule of the protein with a free thiol on a second molecule of the protein by at least 5-80%, under pre-selected conditions, as compared to the same composition lacking the carbohydrate. 
     
     
         9 . The composition of  claim 1 , wherein the carbohydrate is present in an amount sufficient that upon storage, in a gas tight container, at a temperature of 2-8° C., for a period of 6 months, the composition will retain at least 85% of the stability the composition had prior to storage. 
     
     
         10 . The composition of  claim 9 , wherein the storage occurs in darkness. 
     
     
         11 . The composition of  claim 1 , wherein the carbohydrate is present in an amount sufficient to have stability comparable to that of a lyophilized composition comprising sucrose, 0.01% polysorbate-20, pH 6.0, 50 mM Citrate. 
     
     
         12 . The composition of  claim 1 , comprising about 1-40% carbohydrate. 
     
     
         13 . The composition of  claim 1 , wherein the carbohydrate is sucrose or trehalose. 
     
     
         14 . The composition of  claim 1 , wherein the composition is a liquid. 
     
     
         15 . The composition of  claim 1 , wherein the composition contains less than about 10% O 2 . 
     
     
         16 . The composition of  claim 1 , wherein the composition is made by a method comprising physical removal of O 2  from the composition. 
     
     
         17 . The composition of  claim 1 , wherein the protein containing a free thiol has two, three, or more free thiol groups and has zero, two, four, or more thiol groups which form sulfhydryl bridges, per active unit of protein. 
     
     
         18 . The composition of  claim 1 , wherein the protein having a free thiol is selected from the group consisting of glucocerebrosidase (GCB), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIBl), beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies, antibody fragments, antibodies and antibody fragments engineered to introduce cysteine residues, core 2 beta 1,6-N-acetylglucosaminyltransferase-M (C2GnTM), core 2 beta 1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), platelet-derived growth factor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT), p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase, desfuroylceftiofur (DFC), apolipoprotein B 100 (apoB) and other low density lipoprotein domains, apolipoprotein A-I variants, hypoxia-inducible factor-1 alpha (HIF-1 alpha), von Willebrand factor (VWF), proteins and peptide mimetics that contain the CAAX motif, mucolytics, carboxypeptidase Y, cathepsin B, cathepsin C, skeletal muscle Ca 2+  release channel/ryanodine receptor (RyRl), nuclear factor kappa B (NF-KB), AP-1, proteindisulfide isomerase (PDI), glycoprotein lb alpha (GPlb alpha), calcineurin (CaN), fibrillin-1, CD4, S100A3, ionotropic glutamate receptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin (alpha2AP), thrombospondin, gelsolin, mucins, creatine kinase, Factor VIII, phospholipase D (PLD), insulin receptor beta subunit, acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha 2M), glutathione reductase (GR), complement component C2, complement component C3, complement component 4, complement Factor B, alpha-lactalbumin, beta-D-galactosidase, endoplasmic reticulum Ca 2+ -ATPase, RNase inhibitor, lipocortin 1, proliferating cell nuclear antigen (PCNA), actin, coenzyme A (CoA), acyl-CoA synthetase, 3-2trans-enoyl-CoA-isomerase precursor, atrial natriuretic factor (ANF) sensitive guanylate cyclase, Pz-peptidase, aldehyde dehydrogenase, P-450, NADPH-P-450 reductase, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyl tetrahydropterin synthetase, lutropin receptor, low moleculat weight acid phosphatase, serum cholinesterase (BChE), adrenodoxin, hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2), phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome c1 heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I, muscle AMP deaminase, lactate dehydrogenase, and somatostatin-binding protein. 
     
     
         19 - 46 . (canceled) 
     
     
         47 . A method of treating a patient, the method comprising administering the composition of  claim 1  to the patient. 
     
     
         48 . The method of  claim 47 , wherein the administration is by IV infusion or subcutaneous administration. 
     
     
         49 - 50 . (canceled)