Method of purifying a long-acting human growth hormone
Inventors
Kang, Gil Bu • Lee, Chung Min • Ahn, Jee Won • Lee, Dong Eok • Jun, Chang Bong • Lee, Won Jeong • Cho, Sung Yoo • Park, Chi Hye • Kim, Ki Wan • Lee, Yoon Jung • Moon, Ji Hyun • Oh, Hoo Keun • Park, Young Joon • Lee, Sang Hyun • Lim, Hyoung Taek • Park, Soon Jae • Chung, Hye Shin • Lee, Sang Mee
Assignees
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Publication Number
US-9902753-B2
Publication Date
2018-02-27
Expiration Date
Abstract
The present invention relates to a long-acting human growth hormone NexP-hGH protein and its production method. More specifically, it relates to a specific isoform of long-acting human growth hormone NexP-hGH protein in which human growth hormone is fused with a highly glycosylated alpha-1 antitrypsin mutant whereby long-acting properties in vivo are increased. The present invention also relates to a high-purity purification method for NexP-hGH, which includes the steps of: (a) carrying out anion-exchange resin chromatography on a biological emulsion comprising NexP-hGH in which human growth hormone is fused with an alpha-1 antitrypsin mutant; (b) carrying out hydrophobic resin chromatography on the biological emulsion comprising NexP-hGH, or on the eluate produced in step (a); and (c) carrying out affinity chromatography, entailing packing with a resin to which anti-alpha-1 antitrypsin antibody fragments are attached, on the biological emulsion comprising NexP-hGH and on the eluate produced in step (a) or step (b).
Core Innovation
The invention relates to a long-acting human growth hormone that is a fusion protein of human growth hormone (hGH) and an alpha-1 antitrypsin mutant lacking protease inhibitor activity. The fusion protein is highly glycosylated and has an isoelectric point (pI) of 5.2 or less, and the document links long-acting behavior to glycosylation and pI.
The fusion protein preparation comprises structural isoforms that differ by glycan patterns and isoelectric points. The document further relates isoform composition to pharmacodynamic or biological effects, and states that structural isoforms with different glycan patterns and pI are separated during purification.
A purification strategy uses sequential chromatography for high purity. The method includes applying a biological fluid comprising the long-acting human growth hormone to anion-exchange resin chromatography, followed by hydrophobic resin chromatography, and then affinity chromatography using a column filled with resin to which anti-alpha-1 antitrypsin antibody binding fragments are attached. During the affinity step, MgCl2 is used to separate and elute structural isoforms.
Claims Coverage
The document provides one independent claim that covers a multi-step purification method for a specific long-acting hGH fusion protein, including affinity-based separation of structural isoforms using MgCl2. The coverage focuses on sequential chromatography and explicit isoform separation behavior during the affinity step, with additional narrowing in dependent claims for resin selections and chromatographic buffer constraints.
Sequential chromatography purification of long-acting hGH fusion
A method of purifying a long-acting human growth hormone wherein the long-acting human growth hormone is a fusion protein of human growth hormone (hGH) and an alpha-1 antitrypsin mutant without protease inhibitor activity, comprising applying the biological fluid to anion-exchange resin chromatography, applying the fluid or an eluate to hydrophobic resin chromatography, and applying the fluid or an eluate to an affinity chromatography column with resin to which anti-alpha-1 antitrypsin antibody binding fragments are attached.
MgCl2-driven structural isoform separation in affinity chromatography
Separating and eluting structural isoforms of the long-acting human growth hormone with different glycan patterns and isoelectric points with 50 to 200 mM MgCl2 during the purification process using the affinity chromatography column filled with resin to which anti-alpha-1 antitrypsin antibody binding fragments are attached, wherein isoform 1 is eluted and collected at about 100 mM MgCl2 and isoform 2 is eluted and collected at about 200 mM MgCl2.
Specific anion-exchange resin functional group selection
The anion-exchange resin functional group is selected from quaternary amine (Q), diethylaminoethyl (DEAE), and quaternary amino ethyl (QAE).
Specific hydrophobic resin functional group selection
The hydrophobic resin functional group is selected from phenyl, octyl, isopropyl, butyl, and ethyl.
Affinity step wash and elution buffer constraints with NaCl and MgCl2
In the affinity chromatography step, the method adsorbs the long-acting human growth hormone (or an eluate from the preceding steps) onto equilibrated resin bearing anti-alpha-1 antitrypsin antibody binding fragments, washes with pH 6.5 to 8.5 buffer containing 0 to 200 mM NaCl, and elutes with pH 6.5 to 8.5 buffer containing 50 to 200 mM MgCl2.
Hydrophobic chromatography wash and elution buffer constraints
In the hydrophobic resin chromatography step, the method equilibrates and washes with pH 6 to 8 buffer containing 1 to 3 M NaCl and elutes with pH 6 to 8 buffer containing 0 to 1 M NaCl.
Anion-exchange chromatography adsorption and elution buffer constraints
In the anion-exchange resin chromatography step, the method adsorbs (diafiltered culture broth) onto an equilibrated anion-exchange resin at pH 6 to 9 with 0 to 100 mM NaCl, washes the resin under those conditions, and elutes the long-acting human growth hormone using pH 6 to 9 buffer with 100 to 1,000 mM NaCl.
Overall claim coverage centers on purifying a long-acting hGH/alpha-1 antitrypsin mutant fusion protein using a three-stage chromatography sequence (anion-exchange, hydrophobic, then antibody-fragment affinity). The principal inventive focus is that structural isoforms differing in glycan patterns and isoelectric points are separated and eluted in the affinity step by using MgCl2 at specified concentrations, with additional narrowing by selected resin chemistries and explicit wash/elution constraints.
Stated Advantages
Improves purity of the long-acting human growth hormone after sequential chromatography stages, with reported high purification purity after each step.
Enables separation of structural isoforms with different glycan patterns and isoelectric points during affinity chromatography using MgCl2.
Provides a relationship between isoform composition and pharmacodynamic/biological effects in rats.
Documented Applications
Use in purification and collection of structural isoforms of a long-acting human growth hormone fusion protein for evaluation of pharmacodynamic/biological effects in rats (body weight and body length).
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