Method for efficient purification of human serum albumin
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Publication Number
US-10981975-B2
Publication Date
2021-04-20
Expiration Date
Abstract
The present invention describes a simple purification process for recombinant human serum albumin. The process results in highly purified protein with limited number of purification steps. The broth containing human albumin is clarified by centrifugation and microfiltration, diafiltered and captured by cation exchange chromatography by a process that allows 140-230 mg of albumin to be captured per ml of resin. Product related impurities are removed by hydrophobic interaction chromatography, optimised to allow 87-97% recovery in flow through mode. The final series of processes are so combined that there is easy transition from one step to the next with minimal interventions and adjustments. The entire process of purification is completed within two days from harvest to final product. Thus a cost-effective process with improved recovery of protein at each step is developed. The purified human serum albumin is analyzed for purity and shows physicochemical characteristics that are similar to standard albumin.
Core Innovation
The invention relates to a process for purification of recombinant human albumin. The process separates plurality of cells from fermentation broth by centrifugation to obtain a cell free supernatant, and then microfilters or diafilters the cell free supernatant to achieve a conductivity of 2 mS/cm or less. The clarified material is concentrated and loaded on a cation exchange column for purification in bind and elute mode.
Monomeric albumin is then separated from aggregates and degradation products by hydrophobic interaction chromatography using a poly propylene glycol resin in a flow through mode, followed by pooling the flow-through and wash and diafiltering to obtain albumin. Hydrophobic interaction chromatography is performed using a buffer comprising 5-30 mM cysteine and 5-30 mM caprylate, and the albumin is then sterile filtered and subjected to pasteurization at 60°C for 1-10 hours.
The application of caprylate and cysteine during hydrophobic interaction chromatography results in increasing recovery of monomeric albumin protein to >87% as compared to hydrophobic interaction chromatography conducted without caprylate and cysteine. Related claimed performance targets include free thiol ratio greater than 0.7 and aggregates less than 2.5%.
Claims Coverage
The document provides two independent claims covering an entire recombinant human albumin purification process, with inventive features centered on multi-step clarification and conductivity-controlled conditioning, chromatography in bind and elute and flow-through modes, and a specific hydrophobic interaction chromatography buffer comprising cysteine and caprylate to increase monomeric albumin recovery while meeting recovery, thiol, and aggregate targets.
Conductivity-restricted microfiltration or diafiltration prior to cation exchange
Separating plurality of cells from fermentation broth or harvesting a plurality of cells by centrifugation to obtain a cell free supernatant, and microfiltering or diafiltering the obtained cell free supernatant to achieve a conductivity of 2 mS/cm or less; concentrating the microfiltrate or diafiltrate against water; and loading the microfiltrate or diafiltrate on a cation exchange column for purification in bind and elute mode.
Hydrophobic interaction chromatography in flow-through mode with polypropylene glycol resin
Separating monomeric albumin from aggregates and degradation products by hydrophobic interaction chromatography using a poly propylene glycol resin with a buffer comprising 5-30 mM cysteine and 5-30 mM caprylate in a flow through mode; pooling the flow-through and wash and diafiltering the same against water to obtain albumin.
Caprylate and cysteine to increase monomeric albumin recovery above 87%
Wherein the application of caprylate and cysteine during hydrophobic interaction chromatography results in increasing the recovery of the monomeric albumin protein to >87% as compared to hydrophobic interaction chromatography conducted without the presence of caprylate and cysteine.
Hydrophobic interaction chromatography performance targets (recovery, thiol ratio, aggregate level)
Separating the monomeric albumin from aggregates and degradation products by hydrophobic interaction chromatography using a poly propylene glycol resin with a buffer comprising 5-30 mM cysteine and 5-30 mM caprylate, wherein recovery of albumin is at least 87%, wherein free thiol ratio is greater than 0.7, and wherein the aggregates are less than 2.5%.
Across the independent claims, the main inventive coverage is the combination of clarification and conductivity-restricted conditioning, cation exchange in bind/elute mode, and hydrophobic interaction chromatography in flow-through mode using poly propylene glycol resin with a buffer containing 5-30 mM cysteine and 5-30 mM caprylate. The claims further anchor the invention to improved monomeric albumin recovery (>87%) and specified product-quality targets including free thiol ratio and aggregate reduction.
Stated Advantages
Increases recovery of the monomeric albumin protein to >87% as compared to hydrophobic interaction chromatography conducted without the presence of caprylate and cysteine.
Documented Applications
No documented applications found
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