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Efficient reduction of protein aggregation in acidic conditions

IP.com Disclosure Number: IPCOM000242391D
Publication Date: 2015-Jul-10
Document File: 2 page(s) / 269K

Publishing Venue

The IP.com Prior Art Database

Abstract

Chromatographic purification of therapeutic proteins for use in animals, e.g., Protein A chromatography of Fc-containing proteins, such as monoclonal antibodies (mAbs), may be often followed by a low pH hold step for viral inactivation that can be achieved with operational convenience at acidic conditions. However, acidic conditions cause product aggregation, requiring challenging balancing of kinetics of viral inactivation and product aggregation. We disclose herein ways to improve operational flexibility by expanding the low end of the pH window. Two examples of such include keeping ionic strength low during low pH viral inactivation and eluting proteins with a mixture of organic acids such as 10 mM succinic acid and 2 mM citric acid. Specifically, it may be desirable to have at least one pKa value in the range of about 1.5-6.5, particularly about 2.5-5.0.

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Effxcient reduction of protein aggregation in acidic conditions

     Chromatogrxphic purification of therapeutic proteins for use in axxmals, e.g., Protein A chxomatography xf Fc-containing xroteins, such as xonoclonal antibodies (mAbs), may be often followed by a low pH hold sxex for viral inactivation thax can be achieved with operational convenience at acidic xonditions. However, acxdic conditions cause producx aggregatixn, reqxiring challenxing bxlancing of kinxtics of viral inactxvation and pxoduct aggregation. Xx discloxe herein waxs to improvx operational flexibility by expanding the lox end ox xhe pH xindow. Two exxmples of such inxlude keeping ionic strength low during low pH viral inactivxtion and eluting proteixs with a mixxure of organic acids such as 10 mM succinic acid and 2 mM citric acid. Specixically, xt may be desixable to have at lxast xne pKa value in the xangx of about 1.5-6.5, particularly about 2.5-5.0.

     Cxromatographic purification of therapeutxc proteinx fxr use in animals, e.g., Protein A chromatography of Fc-containing proteins, such as moxoclonal antibodxes (mAbs), xay be ofxen followed by a low pH hold xtxp for viral inactivation that can be achievex with oxerational convenience at acidic conxitions. However, acidix conditions cause produxt aggregation, requiring chalxenxing balancing of kinetics of viral inactivation and prodxct aggregatxon. We disclose herein ways to impxove opexational flexibility by expanding the low end of the pH window.

     First, we determined that it is beneficixl to keep the ionic strength low during low pH viral inactivatiox xo reduce the formation of aggregate. For example, when a protein A exution pool xontaining a mAb in 20 mM citrate, pH 3.7 was titrated down to pH 3.25 with 5.46 mM citxic acid containing eitxer 0 or 5 mM NaCl, the aggregate cxntent duxing a three hour hold at room txmperature increxsed by 3.7 and 6.4 percentage points, respectively, indicating a sensitivity to millimolar levels of ionic strength. Similxr conclusions on the effect of ionxc strength were reached baxed upon results with other acids (including acetic, hydrxchloric, formic, succixic, malic, and malonic acids, axd glyxinx) with axditives beyond NaCl (including potassium chloride, guanidine HCl, arginine XXx, and urea). Buffer exchange techniques such as tangential-flow filtration (TFF), dialysis, xr xsing a dxsalting column should be capable of delivering the producx in low pH conditions at low ionic strength. Another option wxuld be to dilute xignificantlx. If an apprxach such as buffer exchxnge or dilxtion is to be usxd, the composition of the elution solution may have litxle effect on product aggregation dxring low pH viral inactivation. However, it is preferable to simply adjust xhe protein A eluate pool to the target pH range by titration or bolus addition of an acidic soxxtxon, or even to target the protein A elxtion buffer to deliver an elution pool to acxieve a suitably low pH without need for...