Method to produce an immunoglobulin preparation with improved yield

Abstract

The present invention provides improved methods for the manufacturing of IVIG products. These methods offer various advantages such as reduced loss of IgG during purification and improved quality of final products. In other aspects, the present invention provides aqueous and pharmaceutical compositions suitable for intravenous, subcutaneous, and/or intramuscular administration. In yet other embodiments, the present invention provides methods of treating a disease or condition comprising administration of an IgG composition provided herein.

1 - 58 . (canceled) 
     
     
         59 . A method for preparing an enriched IgG composition from plasma, the method comprising the steps of:
 (a) precipitating a cryo-poor plasmid fraction, in a first precipitation step, with from 6% to 10% ethyl alcohol at a pH of from 7.0 to 7.5 to form a first precipitate and a first supernatant;   (b) precipitating IgG from the first supernatant, in a second precipitation step, with from 23% to 27% ethyl alcohol at a pH of from 6.7 to 7.3 to form a second precipitate; and   (c) suspending the second precipitate to form a first suspension, thereby forming an enriched IgG composition,   wherein the ethyl alcohol used in at least one of the first precipitation step and the second precipitation step is added by spray addition.   
     
     
         60 . The method of  claim 59 , wherein the ethyl alcohol used in both the first precipitation step and the second precipitation step is added by spray addition. 
     
     
         61 . The method of  claim 59 , wherein the second precipitation step is performed at a temperature of from −7° C. to −9° C. 
     
     
         62 . The method of  claim 59 , wherein the second precipitate is suspended with an extraction buffer at a ratio of 1 part precipitate to from 12 parts to 18 parts of an extraction buffer. 
     
     
         63 . The method of  claim 62 , wherein the extraction buffer has a pH of from about 4.5 to about 5.0. 
     
     
         64 . The method of  claim 63 , wherein the extraction buffer comprises 5 mM sodium phosphate and 5 mM acetate. 
     
     
         65 . The method of  claim 65 , wherein the extraction buffer comprises from 510 mL to 600 mL of glacial acetic acid per 1000 L of buffer. 
     
     
         66 . The method of  claim 59 , further comprising the step of:
 (d) mixing fumed silica with the first suspension formed in (c) for at least 30 minutes; and   (e) separating a solubilized portion of the first suspension mixed with fumed silica in step (d) from a non-solubilized portion of the first suspension.   
     
     
         67 . The method of  claim 61 , further comprising the step of:
 (d) mixing fumed silica with the first suspension formed in (c) for at least 30 minutes; and   (e) separating a solubilized portion of the first suspension mixed with fumed silica in step (d) from a non-solubilized portion of the first suspension.   
     
     
         68 . The method of  claim 66 , wherein from 0.01 g to 0.07 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         69 . The method of  claim 66 , wherein from 0.02 g to 0.06 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         70 . The method of  claim 66 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         71 . The method of  claim 67 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         72 . The method of  claim 66 , wherein the solubilized portion of the first suspension mixed with fumed silica is separated from the non-solubilized portion of the first suspension by depth filtration. 
     
     
         73 . The method of  claim 72 , wherein the depth filtration further comprises washing a depth filter used in the depth filtration with at least 3 filter void volumes of buffer. 
     
     
         74 . The method of  claim 59 , wherein the first suspension, formed in step (c), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         75 . The method of  claim 59 , wherein the first suspension, formed in step (c), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         76 . The method of  claim 67 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         77 . The method of  claim 67 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         78 . The method of  claim 66 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         79 . The method of  claim 70 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         80 . The method of  claim 71 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         81 . The method of  claim 78 , further comprising treating the solubilized portion of the second suspension separated in step (11) with a solvent and detergent (S/D) treatment step. 
     
     
         82 . The method of  claim 78 , further comprising the steps of:
 (i) binding IgG in the solubilized portion of the second suspension to a cation exchange material;   (j) eluting IgG from the cation exchange material to form a cation exchange eluate.   
     
     
         83 . The method of  claim 82 , further comprising the steps of:
 (k) loading IgG from the cation exchange eluate onto an anion exchange column;   (l) collecting an effluent comprising IgG from the anion exchange column to form an anion exchange flow-through.   
     
     
         84 . The method of  claim 83 , further comprising the step of:
 (m) nanofiltering IgG from the anion exchange flow-through to form a nanofiltrate.   
     
     
         85 . The method of  claim 84 , wherein the nanofiltration is performed with a nanofilter having a mean pore size of about 35 nm. 
     
     
         86 . The method of  claim 84 , further comprising the step of:
 (n) ultrafiltering and diafiltering IgG from the nanofiltrate to form a filtrate having a protein concentration of at least 11% (w/v), thereby obtaining an enriched IgG composition.   
     
     
         87 . The method of  claim 86 , wherein the ultrafiltration and diafiltration of step (n) comprises the sub-steps of:
 (n1) concentrating IgG from the nanofiltrate to a protein concentration of 5±2% (w/v) to form a first IgG concentrate;   (n2) diafiltering the IgG concentrate of (n1) against a buffer comprising glycine to form an IgG diafiltrate; and   (n3) concentrating the IgG diafiltrate of (n2) to a protein concentration of at least 11% (w/v).   
     
     
         88 . A method for preparing an enriched IgG composition from plasma, the method comprising the steps of:
 (a) precipitating a cryo-poor plasmid fraction, in a first precipitation step, with from 6% to 10% ethyl alcohol at a pH of from 7.0 to 7.5 to form a first precipitate and a first supernatant;   (b) precipitating IgG from the first supernatant, in a second precipitation step, with from 23% to 27% ethyl alcohol at a pH of from 6.7 to 7.3 to form a second precipitate;   (c) suspending the second precipitate to form a first suspension;   (d) mixing fumed silica with the first suspension formed in (c) for at least 30 minutes; and   (e) separating a solubilized portion of the first suspension mixed with fumed silica in step (d) from a non-solubilized portion of the first suspension, thereby forming an enriched IgG composition.   
     
     
         89 . The method of  claim 88 , wherein the second precipitation step is performed at a temperature of from −7° C. to −9° C. 
     
     
         90 . The method of  claim 88 , wherein the second precipitate is suspended with an extraction buffer at a ratio of 1 part precipitate to from 12 parts to 18 parts of an extraction buffer. 
     
     
         91 . The method of  claim 90 , wherein the extraction buffer has a pH of from about 4.5 to about 5.0. 
     
     
         92 . The method of  claim 91 , wherein the extraction buffer comprises 5 mM sodium phosphate and 5 mM acetate. 
     
     
         93 . The method of  claim 92 , wherein the extraction buffer comprises from 510 mL to 600 mL of glacial acetic acid per 1000 L of buffer. 
     
     
         94 . The method of  claim 88 , wherein from 0.01 g to 0.07 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         95 . The method of  claim 88 , wherein from 0.02 g to 0.06 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         96 . The method of  claim 88 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         97 . The method of  claim 88 , wherein the solubilized portion of the first suspension mixed with fumed silica is separated from the non-solubilized portion of the first suspension by depth filtration. 
     
     
         98 . The method of  claim 97 , wherein the depth filtration further comprises washing a depth filter used in the depth filtration with at least 3 filter void volumes of buffer. 
     
     
         99 . The method of  claim 88 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         100 . The method of  claim 88 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         101 . The method of  claim 88 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         102 . The method of  claim 89 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         103 . The method of  claim 96 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         104 . The method of  claim 101 , further comprising treating the solubilized portion of the second suspension separated in step (h) with a solvent and detergent (S/D) treatment step. 
     
     
         105 . The method of  claim 101 , further comprising the steps of:
 (i) binding IgG in the solubilized portion of the second suspension to a cation exchange material;   (j) eluting IgG from the cation exchange material to form a cation exchange eluate.   
     
     
         106 . The method of  claim 105 , further comprising the steps of:
 (k) loading IgG from the cation exchange eluate onto an anion exchange column;   (l) collecting an effluent comprising IgG from the anion exchange column to form an anion exchange flow-through.   
     
     
         107 . The method of  claim 106 , further comprising the step of:
 (m) nanofiltering IgG from the anion exchange flow-through to form a nanofiltrate.   
     
     
         108 . The method of  claim 107 , wherein the nanofiltration is performed with a nanofilter having a mean pore size of about 35 nm. 
     
     
         109 . The method of  claim 107 , further comprising the step of:
 (n) ultrafiltering and diafiltering IgG from the nanofiltrate to form a filtrate having a protein concentration of at least 11% (w/v), thereby obtaining an enriched IgG composition.   
     
     
         110 . The method of  claim 109 , wherein the ultrafiltration and diafiltration of step (n) comprises the sub-steps of:
 (n1) concentrating IgG from the nanofiltrate to a protein concentration of 5±2% (w/v) to form a first IgG concentrate;   (n2) diafiltering the IgG concentrate of (n1) against a buffer comprising glycine to form an IgG diafiltrate; and   (n3) concentrating the IgG diafiltrate of (n2) to a protein concentration of at least 11% (w/v).   
     
     
         111 . A method for preparing an enriched IgG composition from plasma, the method comprising the steps of:
 (a) precipitating a cryo-poor plasmid fraction, in a first precipitation step, with from 6% to 10% ethyl alcohol at a pH of from 7.0 to 7.5 to form a first precipitate and a first supernatant;   (b) precipitating IgG from the first supernatant, in a second precipitation step, with from 23% to 27% ethyl alcohol at a pH of from 6.7 to 7.3 to form a second precipitate; and   (c) suspending the second precipitate to form a first suspension, thereby forming an enriched IgG composition,   wherein the pH of at least one of the first precipitation step and the second precipitation step is adjusted to the recited value after the concentration of the ethyl alcohol used in the respective step has been achieved.   
     
     
         112 . The method of  claim 111 , wherein the pH of both the first precipitation step and the second precipitation step is adjusted to the recited value after the concentration of the ethyl alcohol used in the respective step has been achieved. 
     
     
         113 . The method of  claim 111 , wherein the pH of the first precipitation step (a) is maintained at from 7.0 to 7.5 for the entire first precipitation step. 
     
     
         114 . The method of  claim 111 , wherein the pH of the second precipitation step (b) is maintained at from 6.7 to 7.3 for the entire second precipitation step. 
     
     
         115 . The method of  claim 111 , wherein the second precipitation step is performed at a temperature of from −7° C. to −9° C. 
     
     
         116 . The method of  claim 111 , wherein the second precipitate is suspended with an extraction buffer at a ratio of 1 part precipitate to from 12 parts to 18 parts of an extraction buffer. 
     
     
         117 . The method of  claim 116 , wherein the extraction buffer has a pH of from about 4.5 to about 5.0. 
     
     
         118 . The method of  claim 117 , wherein the extraction buffer comprises 5 mM sodium phosphate and 5 mM acetate. 
     
     
         119 . The method of  claim 118 , wherein the extraction buffer comprises from 510 mL to 600 mL, of glacial acetic acid per 1000 L of buffer. 
     
     
         120 . The method of  claim 111 , further comprising the step of:
 (d) mixing fumed silica with the first suspension formed in (c) for at least 30 minutes; and   (e) separating a solubilized portion of the first suspension mixed with fumed silica in step (d) from a non-solubilized portion of the first suspension.   
     
     
         121 . The method of  claim 115 , further comprising the step of:
 (d) mixing fumed silica with the first suspension formed in (c) for at least 30 minutes; and   (e) separating a solubilized portion of the first suspension mixed with fumed silica in step (d) from a non-solubilized portion of the first suspension.   
     
     
         122 . The method of  claim 120 , wherein from 0.01 g to 0.07 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         123 . The method of  claim 120 , wherein from 0.02 g to 0.06 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         124 . The method of  claim 120 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         125 . The method of  claim 121 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         126 . The method of  claim 120 , wherein the solubilized portion of the first suspension mixed with fumed silica is separated from the non-solubilized portion of the first suspension by depth filtration. 
     
     
         127 . The method of  claim 126 , wherein the depth filtration further comprises washing a depth filter used in the depth filtration with at least 3 filter void volumes of buffer. 
     
     
         128 . The method of  claim 111 , wherein the first suspension, formed in step (c), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         129 . The method of  claim 111 , wherein the first suspension, formed in step (c), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         130 . The method of  claim 120 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         131 . The method of  claim 120 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         132 . The method of  claim 120 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         133 . The method of  claim 124 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         134 . The method of  claim 125 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         135 . The method of  claim 132 , further comprising treating the solubilized portion of the second suspension separated in step (h) with a solvent and detergent (S/D) treatment step. 
     
     
         136 . The method of  claim 132 , further comprising the steps of:
 (i) binding IgG in the solubilized portion of the second suspension to a cation exchange material;   (j) eluting IgG from the cation exchange material to form a cation exchange eluate.   
     
     
         137 . The method of  claim 136 , further comprising the steps of:
 (k) loading IgG from the cation exchange eluate onto an anion exchange column;   (l) collecting an effluent comprising IgG from the anion exchange column to form an anion exchange flow-through.   
     
     
         138 . The method of  claim 137 , further comprising the step of:
 (m) nanofiltering IgG from the anion exchange flow-through to form a nanofiltrate.   
     
     
         139 . The method of  claim 138 , wherein the nanofiltration is performed with a nanofilter having a mean pore size of about 35 nm. 
     
     
         140 . The method of  claim 138 , further comprising the step of:
 (n) ultrafiltering and diafiltering IgG from the nanofiltrate to form a filtrate having a protein concentration of at least 11% (w/v), thereby obtaining an enriched IgG composition.   
     
     
         141 . The method of  claim 140 , wherein the ultrafiltration and diafiltration of step (n) comprises the sub-steps of:
 (n1) concentrating IgG from the nanofiltrate to a protein concentration of 5±2% (w/v) to form a first IgG concentrate;   (n2) diafiltering the IgG concentrate of (n1) against a buffer comprising glycine to form an IgG diafiltrate; and   (n3) concentrating the IgG diafiltrate of (n2) to a protein concentration of at least 11% (w/v).   
     
     
         142 . A method for preparing an enriched IgG composition from plasma, the method comprising the steps of:
 (a) precipitating a cryo-poor plasmid fraction, in a first precipitation step, with from 6% to 10% ethyl alcohol at a pH of from 7.0 to 7.5 to form a first precipitate and a first supernatant;   (b) precipitating IgG from the first supernatant, in a second precipitation step, with from 23% to 27% ethyl alcohol at a pH of from 6.7 to 7.3 and temperature of from 7° C. to 9° C. to form a second precipitate; and   (c) suspending the second precipitate to form a first suspension, thereby forming an enriched IgG composition.   
     
     
         143 . The method of  claim 142 , wherein the second precipitate is suspended with an extraction buffer at a ratio of 1 part precipitate to from 12 parts to 18 parts of an extraction buffer. 
     
     
         144 . The method of  claim 143 , wherein the extraction buffer has a pH of from about 4.5 to about 5.0. 
     
     
         145 . The method of  claim 144 , wherein the extraction buffer comprises 5 mM sodium phosphate and 5 mM acetate. 
     
     
         146 . The method of  claim 145 , wherein the extraction buffer comprises from 510 mL to 600 mL of glacial acetic acid per 1000 L of buffer. 
     
     
         147 . The method of  claim 142 , further comprising the step of:
 (d) mixing from 0.01 g to 0.07 g fumed silica per g of the second precipitate formed in step (b) with the first suspension formed in (c) for at least 30 minutes; and   (e) separating a solubilized portion of the first suspension mixed with fumed silica in step (d) from a non-solubilized portion of the first suspension.   
     
     
         148 . The method of  claim 147 , wherein from 0.02 g to 0.06 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         149 . The method of  claim 147 , wherein from 0.03 g to 0.05 g fumed silica per g of the second precipitate formed in step (b) is mixed with the first suspension in step (d). 
     
     
         150 . The method of  claim 147 , wherein the solubilized portion of the first suspension mixed with fumed silica is separated from the non-solubilized portion of the first suspension by depth filtration. 
     
     
         151 . The method of  claim 150 , wherein the depth filtration further comprises washing a depth filter used in the depth filtration with at least 3 filter void volumes of buffer. 
     
     
         152 . The method of  claim 142 , wherein the first suspension, formed in step (c), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         153 . The method of  claim 142 , wherein the first suspension, formed in step (c), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         154 . The method of  claim 147 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 85% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         155 . The method of  claim 147 , wherein the soluble portion of the first suspension, separated in step (e), contains at least 90% of the IgG content of the cryo-poor plasma fraction used in step (a). 
     
     
         156 . The method of  claim 147 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         157 . The method of  claim 149 , further comprising the steps of:
 (f) precipitating IgG from the solubilized portion of the suspension, in a third precipitation step, with from 22% to 28% ethyl alcohol at a pH of from 6.7 to 7.3 to form a third precipitate;   (g) suspending the third precipitate to form a second suspension;   (h) separating a solubilized portion of the second suspension formed in step (g) from a non-solubilized portion of the second suspension.   
     
     
         158 . The method of  claim 147 , further comprising treating the solubilized portion of the second suspension separated in step (h) with a solvent and detergent (S/D) treatment step. 
     
     
         159 . The method of  claim 147 , further comprising the steps of:
 (i) binding IgG in the solubilized portion of the second suspension to a cation exchange material;   (j) eluting IgG from the cation exchange material to form a cation exchange eluate.   
     
     
         160 . The method of  claim 159 , further comprising the steps of:
 (k) loading IgG from the cation exchange eluate onto an anion exchange column;   (l) collecting an effluent comprising IgG from the anion exchange column to form an anion exchange flow-through.   
     
     
         161 . The method of  claim 160 , further comprising the step of:
 (m) nanofiltering IgG from the anion exchange flow-through to form a nanofiltrate.   
     
     
         162 . The method of  claim 161 , wherein the nanofiltration is performed with a nanofilter having a mean pore size of about 35 nm. 
     
     
         163 . The method of  claim 161 , further comprising the step of:
 (n) ultrafiltering and diafiltering IgG from the nanofiltrate to form a filtrate having a protein concentration of at least 11% (w/v), thereby obtaining an enriched IgG composition.   
     
     
         164 . The method of  claim 163 , wherein the ultrafiltration and diafiltration of step (n) comprises the sub-steps of:
 (n1) concentrating IgG from the nanofiltrate to a protein concentration of 5±2% (w/v) to form a first IgG concentrate;   (n2) diafiltering the IgG concentrate of (n1) against a buffer comprising glycine to form an IgG diafiltrate; and   (n3) concentrating the IgG diafiltrate of (n2) to a protein concentration of at least 11% (w/v).