An improved process of purification of protein

Abstract

The invention provides a process of purification of antibody or fusion protein from protein mixture comprising product and process related impurities. The process provides the use of hydroxyapatite chromatography for the separation of low molecular weight impurities and basic variants. In addition, invention further provides a scalable purification process to remove product and process related impurities.

We claim: 
     
         1 . A process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants, the process comprising:
 a. Loading the protein mixture onto Ceramic Hydroxy apatite (CHT) column with suitable buffer;   b. Washing the CHT column with suitable buffer;   c. Eluting the purified protein of interest with suitable gradient of phosphate buffer;   
       wherein the protein of interest is substantially pure and LMW's are below 0.4%, analysed by SE-HPLC Analysis. 
     
     
         2 . A process of purifying a protein of interest from protein mixture comprising protein of interest and one or more low molecular weight (LMW) variants and one or more basic variants (BV), the process comprising:
 a. Loading the protein mixture onto Ceramic Hydroxy apatite (CHT) column with suitable buffer;   b. Washing the CHT column with suitable buffer;   c. Eluting the purified protein of interest with suitable gradient of phosphate buffer;   
       wherein the purified protein of interest is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis. 
     
     
         3 . The process as claimed in  claim 1  or  claim 2 , wherein the purified protein of interest comprises reduced LMWs by at least 97% measured by SE-HPLC analysis. 
     
     
         4 . The process as claimed in  claim 1  or  claim 2 , wherein the purified protein of interest comprises reduced LMWs by at least 95% measured by SE-HPLC analysis. 
     
     
         5 . The process according to  claim 1  or  claim 2 , wherein the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis. 
     
     
         6 . The process according to  claim 1  or  claim 2 , wherein the protein mixture eluted from CHT column comprises LMW below 0.4%, analysed by SE-HPLC Analysis. 
     
     
         7 . The process according to  claim 1  or  claim 2 , wherein the LMW is selected from LC, HC, HH, and 2H1L. 
     
     
         8 . The process according to  claim 7 , wherein the 2H1L amount is selected from about 3% or less, 2.6% or less, 2.5% or less, 2.3% or less, 2% or less, 1.7% or less, 1.5% or less, 1.4% or less measured by CE-SDS. 
     
     
         9 . The process according to  claim 7 , wherein the HH amount is selected from about 0.2% or less, 0.24% or less, 0.21% or less, 0.15% or less, 0.12% or less, 0.09% or less or 0.08% or less, 0.06% or less or 0.04% or less and 0.03% or less measured by CE-SDS. 
     
     
         10 . The process according to  claim 7 , wherein the HC amount is selected from about 0.4% or less, 0.16% or less, 0.15% or less, 0.12% or less and 0.18% or less measured by CE-SDS. 
     
     
         11 . The process according to  claim 7 , wherein the LC amount is selected from about 0.5% or less, 0.4% or less, 0.3% or less and 0.2 or less measured by CE-SDS. 
     
     
         12 . The process according to  claim 7 , wherein the LC, HC, HH and 2H1L present in the amount from about 2.2% to about 2.6% measured by CE-SDS. 
     
     
         13 . A process of purifying a protein of interest from protein mixture comprising protein of interest and one or more basic variants (BV), the process comprising:
 a. Loading the protein mixture onto Ceramic Hydroxy apatite (CHT) column with suitable buffer;   b. Washing the CHT column with suitable buffer;   c. Eluting the purified protein of interest with suitable gradient of phosphate buffer;   
       wherein the purified protein of interest is substantially pure and basic variants are below 15% analysed by CEX-HPLC Analysis. 
     
     
         14 . The process according to  claim 2  or  claim 13 , wherein the wherein the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV. 
     
     
         15 . The process according to  claim 14 , wherein the wherein the protein mixture eluted from CHT column comprises less than 10% BV. 
     
     
         16 . The process according to  claim 14 , wherein the wherein the protein mixture eluted from CHT column comprises 5% BV. 
     
     
         17 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the elution phosphate gradient is a linear gradient wherein the concentration of phosphate is increased gradually from about 24 mM to 104 mM. 
     
     
         18 . The process according to  claim 17 , wherein the concentration of phosphate is increased gradually from about 40 mM to 96 mM. 
     
     
         19 . The process according to  claim 17 , wherein the concentration of phosphate is increased gradually from about 32 mM to 88 mM. 
     
     
         20 . The process according to  claim 17 , wherein the elution phosphate gradient is a linear gradient wherein the concentration of phosphate is increased gradually from about 5% to 25% of buffer B. 
     
     
         21 . The process according to  claim 20 , wherein the elution phosphate gradient is a linear gradient wherein the concentration of phosphate is increased gradually from about 10% to 24% of buffer B. 
     
     
         22 . The process according to  claim 21 , wherein the concentration of phosphate is increased gradually from about 8% to 22% of buffer B. 
     
     
         23 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the gradient elution is performed for 19 CV. 
     
     
         24 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the elution starts at an ascending value of about 1.0 AU/cm and ends at a descending value of about 1.5 AU/cm. 
     
     
         25 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the CHT column is treated with equilibration buffer before the treatment with loading buffer. 
     
     
         26 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the loading and washing buffer has same conductivity and pH. 
     
     
         27 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the loading, washing and elution buffer has pH 7.0±0.2. 
     
     
         28 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the loading, washing and elution buffer does not have any additive selected from sodium chloride, calcium chloride, and glycine. 
     
     
         29 . The process according to  claim 1 ,  claim 2 , or  claim 13 , wherein the CHT column is performed after Protein A chromatography. 
     
     
         30 . The process according to  claim 1 ,  claim 2  or  claim 13 , wherein the CHT column is performed after Anion exchange chromatography. 
     
     
         31 . A process of purifying the protein of interest from protein mixture comprising:
 a. obtaining protein mixture from the mammalian expression system comprising the protein of interest and at least one impurity selected from acidic variant, basic variant, low molecular weight (LMW), High molecular weight (HMW);   b. applying the protein mixture to affinity chromatography column;   c. eluting the protein mixture from affinity chromatography column;   d. performing the viral inactivation of the protein mixture obtained from step;   e. applying the protein mixture obtained from step (d) onto anion exchange chromatography;   f. eluting the protein mixture in flow through mode;   g. applying the protein mixture obtained from step (f) onto Ceramic Hydroxy apatite (CHT) column;   h. Optionally washing the CHT column with a suitable wash buffer;   i. eluting the protein of mixture from CHT column with suitable buffer;   
       wherein the eluted protein mixture is enriched with protein of interest and substantially reduction of impurities selected from acidic variant, basic variant, low molecular weight (LMW), and High molecular weight (HMW). 
     
     
         32 . The process according to  claim 31 , wherein the protein mixture eluted from CHT column comprises LMW selected from about 0.3% or less LMW, 0.2% or less LMW, 0.1% or less LMW analysed by SE-HPLC Analysis. 
     
     
         33 . The process according to  claim 32 , wherein the protein mixture eluted from CHT column comprises LMW below 0.4%, analysed by SE-HPLC Analysis. 
     
     
         34 . The process according to  claim 31 , wherein the wherein the protein mixture eluted from CHT column comprises low BV selected from about 14% or less BV, 13% or less BV, 12% or less BV, 11% or less BV, 10% or less BV, 9% or less BV, 8% or less BV, 7% or less BV, 6% or less BV, 5% or less BV, 4% or less BV, 3% or less BV, 2% or less BV, 1% or less BV. 
     
     
         35 . The process according to  claim 34 , wherein the wherein the protein mixture eluted from CHT column comprises less than 10% BV. 
     
     
         36 . The process according to  claim 35 , wherein the wherein the protein mixture eluted from CHT column comprises 5% BV. 
     
     
         37 . The process according to  claim 31 , wherein anion exchange chromatography removes substantial acidic variants. 
     
     
         38 . The process according to  claim 37 , wherein the acidic variant is less than about 14% or less AV, 13% or less AV, 12% or less AV, 11% or less AV, 10% or less AV, 9% or less AV, 8% or less AV, 7% or less AV, 6% or less AV, 5% or less AV, 4.5% or less AV, 4% or less AV, 3% or less AV, 2% or less AV, 1% or less AV. 
     
     
         39 . The process according to  claim 31 , wherein anion exchange chromatography removes substantial high molecular weight impurity (HMW). 
     
     
         40 . The process according to  claim 39 , wherein High molecular weight impurity is selected from about 0.5% or less, about 0.4% or less or 0.3% or less or 0.2% or less or 0.1% or less. 
     
     
         41 . The process according to any of the preceding claims, protein of interest is selected from IgG1 antibody or fragment thereof and fusion protein. 
     
     
         42 . The process according to  claim 41 , wherein the IgG1 antibody or fusion protein has isoelectric point from 6 to 9. 
     
     
         43 . The process according to  claim 41 , wherein the IgG1 antibody or fusion protein are selected from Etanercept, Rituximab, Palivizumab, Infliximab, Trastuzumab, Alemtuzumab, Adalimumab, Ibritumomab, Omalizumab, Cetuximab, Bevacizumab, Natalizumab, Eculizumab, Certolizumab pegol, Ustekinumab, Canakinumab, Golimumab, Ofatumumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Brentuximab vedotin, Pertuzumab, Trastuzumab emtansine, Raxibacumab, Obinutuzumab, Siltuximab, Ramucirumab, Vedolizumab, Nivolumab, Pembrolizumab, Darucizumab, Necitumumab, Dinutuximab, Secukinumab, Mepolizumab, Alirocumab, Evolocumab, Daratumumab, Elotuzumab, Ixekizumab, Reslizumab, Olaratumab, Bezlotoxumab, Atezolizumab, Obiltoxaximab, Sarilumab, Ocrelizumab, Tildrakizumab, Romosozumab, Brolucizumab, Crizanlizumab. 
     
     
         44 . The process according to  claim 41 , wherein the IgG1 antibody binds to IgE. 
     
     
         45 . The process according to  claim 44 , wherein the IgG1 antibody binds is Omalizumab. 
     
     
         46 . A pharmaceutical composition of antibody binds to IgE comprising substantially purified monomer of said antibody having purity at least 90% and LMW less than about 0.2% measure by SE-HPLC. 
     
     
         47 . A pharmaceutical composition of omalizumab comprising substantially purified monomer of said Omalizumab having purity at least 90% and LMW less than about 5% measure by SE-HPLC. 
     
     
         48 . The pharmaceutical composition as claimed in  claim 46  or  claim 47 , wherein the monomer purity is selected from more than 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99%.