Chromatography process for purification of insulin analogues
Inventors
Nagarajan, Ramprabu • HASHIM, MINAL • SHAIKH, AZIMODDIN MINYASAB • HEDGE, SANDHYA • Hazra, Partha
Assignees
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Publication Number
US-11440937-B2
Publication Date
2022-09-13
Expiration Date
Abstract
The present invention relates to a process for purification of non-glycosylated insulin analogues 5 from a mixture comprising glycosylated and non-glycosylated proteins by removal of glycosylated proteins. The removal is achieved by combination of two RP-HPLC steps.
Core Innovation
The invention relates to a two step process for purification of non-glycosylated insulin analogue(s) from a complex mixture comprising both non-glycosylated, and glycosylated insulin analogue(s). The process uses RP-HPLC performed with acetonitrile as an organic modifier and an ion pairing agent. In the first step, the complex mixture is treated at acidic pH ranging from about 3.5 to about 3.9 to yield a first mixture comprising partially purified non-glycosylated insulin analogue.
In the second step, the first mixture is further purified by performing RP-HPLC at alkaline pH ranging from about 7.3 to about 8.7 using an ion pairing agent different from the first step, while still using acetonitrile as the organic modifier. The process is formulated to obtain at least 99.95% purified non-glycosylated insulin analogue. The ion pairing agent is selected from octane sulphonic acid, sodium perchlorate and tetrabutyl ammonium bisulphate (TBAB).
An alternative sequence is also described in which the complex mixture is first purified at alkaline pH in the presence of an ion pairing agent, yielding a partially purified non-glycosylated insulin analogue, and then purified at acidic pH using an ion pairing agent different from the first step. The purification is directed to remove glycosylated variants and to reach very low glycosylated levels in the purified fractions. The document reports glycoform identification by mass spectrometry-based glycoform identification.
Claims Coverage
The independent claim covers a two step RP-HPLC purification of non-glycosylated insulin analogue(s) from a complex mixture that includes glycosylated insulin analogue(s), using acetonitrile and an ion pairing agent in acidic and alkaline pH windows with different ion pairing agents between steps; it requires at least 99.95% purified non-glycosylated insulin analogue, and it is drafted to allow either acidic-then-alkaline or alkaline-then-acidic operation.
Two step RP-HPLC purification from mixed glycosylated and non-glycosylated insulin analogues
Purifying non-glycosylated insulin analogue(s) from a complex mixture comprising both non-glycosylated, and glycosylated insulin analogue(s) using two RP-HPLC steps.
Acidic first RP-HPLC step with acetonitrile and ion pairing agent
Performing RP-HPLC with the complex mixture in the presence of an ion pairing agent in combination with acetonitrile as the organic modifier at acidic pH ranging from about 3.5 to about 3.9 to yield a first mixture comprising partially purified non-glycosylated insulin analogue (step a1 or a2 order variant).
Alkaline second RP-HPLC step at different ion pairing agent
Performing RP-HPLC on the a1 (or a2) first mixture in the presence of an ion pairing agent different from the ion pairing agent used in step a1 (or a2), in combination with acetonitrile as the organic modifier at alkaline pH ranging from about 7.3 to about 8.7 to obtain at least 99.95% purified non-glycosylated insulin analogue (step b1 or b2).
Ion pairing agent selection across octane sulphonic acid, sodium perchlorate, and TBAB
Defining the ion pairing agent as selected from the group consisting of octane sulphonic acid, sodium perchlorate and tetrabutyl ammonium bisulphate (TBAB), used so that the ion pairing agent in the second step differs from that in the first step.
Overall, the claim requires a two step RP-HPLC separation using acetonitrile, with a first RP-HPLC step at acidic pH and a second RP-HPLC step at alkaline pH (or the reverse), where different ion pairing agents are applied across the steps, and the outcome is at least 99.95% purified non-glycosylated insulin analogue.
Stated Advantages
Achieves at least 99.95% purified non-glycosylated insulin analogue.
Provides purification directed to removal of glycosylated variants, with purified fractions reported to have glycosylated levels reduced to <0.05% / BLOQ.
Documented Applications
Purification of non-glycosylated insulin analogues from complex mixtures containing glycosylated and non-glycosylated insulin analogue(s), including insulin glargine and insulin lispro.
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