Method for efficient purification of human serum albumin
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Publication Number
US-10377812-B2
Publication Date
2019-08-13
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, optimized 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 includes separating plurality of cells from fermentation broth or harvest by centrifugation, microfiltering the obtained cell free supernatant, and concentrating the microfiltered and diafiltering the broth against water. It further includes loading the diafiltered sample on a cation exchange column for purification in bind and elute mode.
A key feature is separating the monomeric albumin from aggregates and degradation products by hydrophobic interaction chromatography. The hydrophobic interaction chromatography utilizes polypropylene glycol (PPG) resin in a flow through mode, with 10 mM of cysteine and 10 mM of caprylate, achieving recovery of albumin at least 87%, with free thiol ratio greater than 0.7 and aggregates less than 2.5%.
After hydrophobic interaction chromatography, the albumin is loaded on anion exchange resin for chromatography in bind and elute mode. The eluted protein is concentrated and diafiltered against water, sterile filtered, and subjected to pasteurization at 60°C for 1-10 hours. The overall workflow is directed to obtain purified recombinant human albumin with controlled aggregate and degradation profiles and a specified free thiol ratio.
Claims Coverage
The independent claim defines a multi-step purification process for recombinant human albumin with sequential cation exchange and hydrophobic interaction chromatography in flow-through mode using PPG resin with cysteine and caprylate, followed by anion exchange, sterile filtration, and pasteurization. The claim set also includes dependent claims that refine product quality thresholds, define the fermentation broth sources, and specify resin loading and HIC additives.
Cell separation, microfiltration, and concentration/diafiltration against water
Separating plurality of cells from fermentation broth or harvest by centrifugation; microfiltering the obtained cell free supernatant; and concentrating the microfiltered and diafiltering the broth against water.
Cation exchange bind and elute purification
Loading the diafiltered sample on a cation exchange column for purification in bind and elute mode.
Flow-through hydrophobic interaction chromatography with PPG resin, cysteine and caprylate
Separating the monomeric albumin from aggregates and degradation products by hydrophobic interaction chromatography which utilizes polypropylene glycol (PPG) resin in a flow through mode, with 10 mM of cysteine and 10 mM of caprylate.
Pooling, diafiltration, and anion exchange bind and elute purification
Pooling the flow-through and wash and diafiltering the same against water; loading the albumin on anion exchange resin for chromatography in bind and elute mode; concentrating the eluted protein and diafiltering against water.
Sterile filtration and pasteurization at 60°C
Sterile filtering the protein and subjecting to pasteurization at 60°C for 1-10 hours.
Recombinant albumin quality thresholds and process time window
Producing recombinant human albumin with purity >97%, free thiol ratio >0.75, aggregates <2.5%, and degradation <2% within 60 hours from harvesting to bottling.
Defined fermentation broth sources
Selecting fermentation broth from bacteria, fungi, mammalian cells, or homogenate of transgenic plant producing recombinant human albumin.
Increased cation exchange resin loading via resident time
Loading cation exchange resin by gradual increase in resident time to increase loading capacity by 75-150% above the resin’s labelled binding capacity.
Use of fatty acids in HIC flow-through mode
Employing hydrophobic interaction chromatography in flow-through mode and further adding one or more fatty acids.
Overall claim coverage centers on a purification process for recombinant human albumin that uses sequential cation exchange and anion exchange purification steps, with monomeric albumin separation from aggregates and degradation products via hydrophobic interaction chromatography in flow-through mode using PPG resin with cysteine and caprylate. Dependent claims further specify albumin quality thresholds and time window, restrict fermentation broth sources, and refine cation exchange resin loading and HIC flow-through composition through resident time and fatty acids.
Stated Advantages
At least 87% recovery of albumin in the hydrophobic interaction chromatography flow-through step.
Free thiol ratio greater than 0.7.
Aggregates less than 2.5%.
Purity greater than 97%.
Degradation less than 2%.
Improved cation exchange resin loading capacity by 75-150% above labelled binding capacity.
Separation of monomeric albumin from aggregates and degradation products.
Documented Applications
Purification of recombinant human albumin from fermentation broth or harvest, including production within a defined time window from harvesting to bottling and generation of a purified albumin suitable for further processing after sterile filtration and pasteurization.
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