Stable formulations of polypeptides and uses thereof

Abstract

Formulations are provided that contain single variable domains with a good solubility and good stability under different storage, transportation and stress conditions. The formulations are useful as pharmaceutical formulation. The formulation comprises an aqueous carrier with a pH of 5.5 to 8.0, a buffer selected from the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0; an excipient; and/or a surfactant selected from polysorbate 80, polysorbate 20 and poloxamers. The formulation is further characterized that it has an inorganic salt concentration of 150 mM or lower. The invention further relates to containers and pharmaceutical units comprising such formulations and to methods for preparing and prophylactic and therapeutic uses of the formulations and pharmaceutical units of the invention.

1 . A formulation comprising an aqueous carrier having a pH of 5.5 to 8.0 and a polypeptide comprising one or more single variable domains at a concentration of 1 mg/mL to 200 mg/mL, said formulation being formulated for administration to a human subject and said formulation further comprising:
 a histidine pH 6.0-6.5 buffer at a concentration of 10 mM to 100 mM,   wherein said formulation has an inorganic salt concentration of 150 mM or lower and wherein the polypeptide comprises at least one single variable domain that specifically binds human serum albumin (HSA).   
     
     
         2 . The formulation of  claim 1 , that does not contain any inorganic salt. 
     
     
         3 . The formulation of  claim 1 , wherein the concentration of polypeptide is about 1 to 200 mg/ml or more, about 5 to 100 mg/mL or more, about 5 to 50 mg/mL or more, about 5 to 30 mg/mL or more, around 5 mg/mL, around 10 mg/mL, around 20 mg/mL, around 30 mg/mL, around 40 mg/mL, around 50 mg/mL, around 60 mg/mL, around 70 mg/mL, around 80 mg/mL, around 90 mg/mL, around 100 mg/mL, or around 150 mg/mL. 
     
     
         4 . The formulation of  claim 1 , wherein the polypeptide comprises two or more single variable domains, or two or three single variable domains. 
     
     
         5 . The formulation of  claim 1 , wherein the at least one single variable domain that specifically binds HSA has following sequence: 
       
         
           
                 
               
                   (SEQ ID NO: 21) 
                 
                   
                     EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSS 
                   
                 
                   
                     ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGG 
                   
                 
                     SLSRSSQGTLVTVSS . 
                 
             
                
                
                
                
               
            
           
         
       
     
     
         6 . The formulation of  claim 1 , wherein
 the polypeptide has a solubility of at least 20 mg/mL, 50 mg/mL or more, 90 mg/mL or more, 120 mg/mL or more, 150 mg/mL or more, or even 200 mg/mL or more, as determined by the polyethylene glycol (PEG) exclusion method or by a concentration experiment;   the polypeptide has a melting temperature of at least 59° C. or more, at least 60° C. or more, at least 61° C. or more, at least 62° C. or more, or at least 63° C. or more as measured by the thermal shift assay (TSA) and/or differential scanning calorimetry (DSC);   no particulates are present as measured by OD320/OD280 and/or elastic light scattering;   less than 10% of the polypeptide forms pyroglutamate at the N-terminal glutamic acid during storage at a temperature of 37±5° C. up to at least 2 weeks, at least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at least 6 months, at least 1 year, at least 1.5 years or at least 2 years, the % of pyroglutamate as measured by Reversed Phase High Performance Liquid Chromatography (RP-HPLC);   less than 10% of the polypeptide forms dimers during storage at a temperature of 37±5° C. up to at least 2 weeks, at least 3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at least 6 months, at least 1 year, at least 1.5 year or at least 2 years, the % of dimers as measured by Size Exclusion High Performance Liquid Chromatography (SE-HPLC);   at least 80% of the polypeptide retains its binding activity to at least one of its targets after storage at 37±5° C. up to at least 2 week, at least 3 weeks, at least 5 weeks, at least 2 months, at least 6 months, at least 1 year, 1.5 year or even 2 years or more compared to the binding activity prior to storage, said binding activity as measured by enzyme-linked immunosorbent assay (ELISA) and/or Surface Plasmon Resonance; and/or   the polypeptide is stable during mechanical stress.   
     
     
         7 . (canceled) 
     
     
         8 . The formulation of  claim 1 , further comprising an excipient at a concentration of 1% to 20% (w:v). 
     
     
         9 . (canceled) 
     
     
         10 . The formulation of  claim 1 , wherein the histidine buffer has a concentration of 10 to 50 mM, 10 to 20 mM, 10 mM or 15 mM. 
     
     
         11 . The formulation of  claim 8 , wherein the excipient is a saccharide, a non-reducing sugar and/or polyol. 
     
     
         12 . The formulation of  claim 11 , wherein the excipient is selected from the group consisting of mannitol and sucrose. 
     
     
         13 . The formulation of  claim 8 , wherein the excipient has a concentration of 2.5% to 15% (w:v), 5% to 10% (w:v), 5% (w:v), 7.5% (w:v), 8% (w:v) or 10% (w:v). 
     
     
         14 . The formulation of  claim 1 , further comprising a surfactant at a concentration of 0.001% to 1% (v:v) selected from polysorbate 80, polysorbate 20 or a poloxamer. 
     
     
         15 . The formulation of  claim 14 , wherein the surfactant has a concentration of 0.01% to 0.1% (v:v), 0.01% to 0.05% (v:v), 0.01% (v:v) or 0.005% (v:v). 
     
     
         16 . The formulation of  claim 1 , comprising:
 a) a histidine pH 6.5 or pH 6.0 buffer at a concentration of 10 mM to 100 mM;   b) an excipient at a concentration of 1% to 20% (w:v); and   c) a surfactant at a concentration of 0.001% to 1% (v:v) selected from polysorbate 80, polysorbate 20 or a poloxamer.   
     
     
         17 .- 20 . (canceled) 
     
     
         21 . A method for the preparation of a formulation of  claim 1 , at least comprising the step of concentrating the polypeptide and exchanging it with the selected buffer and/or excipient. 
     
     
         22 . A sealed container containing a formulation according to  claim 1 . 
     
     
         23 . A pharmaceutical unit dosage form suitable for parenteral administration to a human, comprising a formulation according to  claim 1  in a suitable container. 
     
     
         24 . A kit comprising one or more of the sealed containers according to  claim 22  and instructions for use of the formulation. 
     
     
         25 . (canceled) 
     
     
         26 . Method for prevention and/or treatment of one or more diseases and/or disorders, comprising administering to a subject in need thereof a formulation according to  claim 1 . 
     
     
         27 . (canceled) 
     
     
         28 . A kit comprising one or more of the pharmaceutical unit dosage forms according to  claim 23 , and instructions for use of the formulation.