LNnT Purification Using SMB Chromatography and Crystallization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing purification processes for lacto-N-neotetraose from microbial fermentation are complex, uneconomical, and inefficient, particularly in removing by-products and contaminants such as lacto-N-triose II, para-lacto-N-neohexaose, and para-lacto-N-neooctaose, while also dealing with recombinant DNA and proteins from genetically modified organisms.
Innovation Solution
A process involving membrane filtration, simulated moving bed chromatography, and crystallization steps to purify lacto-N-neotetraose, including nanofiltration to reduce higher saccharides, SMB chromatography to enhance purity, and crystallization with alcohol washing to achieve high purity, followed by homogenization to obtain a solid powder product.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional purification processes are used for lacto-N-neotetraose from microbial fermentation, then the product can be obtained, but the process is complex, uneconomical, and inefficient with poor separation of by-products and contaminants
Solution Approach 1:
The purification process is divided into distinct sequential stages: membrane filtration (nanofiltration/ultrafiltration) to remove larger contaminants, ion exchange chromatography to separate charged impurities, and crystallization to achieve final high purity. Each stage targets specific types of contaminants, breaking down the complex purification task into manageable segments that collectively achieve ≥90% purity while controlling overall process complexity
Solution Approach 2:
The process utilizes parameter changes in different purification stages: membrane filtration exploits molecular size differences, ion exchange chromatography exploits charge differences at controlled pH and ionic strength, and crystallization exploits solubility changes with temperature and concentration. These parameter-based separations enable efficient removal of different contaminant types without requiring overly complex equipment
2Manufacturing precision
If multiple purification steps are implemented to remove by-products like lacto-N-triose II and para-lacto-N-neohexaose, then product purity increases, but production time and cost increase
Solution Approach 1:
Membrane filtration (nanofiltration or ultrafiltration) is performed as a preliminary step before chromatography and crystallization. This preliminary action removes larger molecular weight contaminants and concentrates the lacto-N-neotetraose solution, reducing the load on subsequent purification steps and enabling faster, more efficient processing in later stages
Solution Approach 2:
The ion exchange chromatography step operates continuously to remove charged contaminants including by-products like lacto-N-triose II and para-lacto-N-neohexaose. The continuous flow through the ion exchange resin bed maintains constant separation efficiency without interruption, reducing total purification time while achieving the necessary purity levels
3Manufacturing precision
If conventional purification methods are used, then some contaminants are removed, but efficient separation of specific by-products and contaminants is not achieved
Solution Approach 1:
Ion exchange resin serves as an intermediary substance that selectively binds charged contaminant molecules (such as lacto-N-triose II and other charged by-products) while allowing neutral lacto-N-neotetraose to pass through or be selectively eluted. This intermediary mechanism enables highly efficient separation based on charge differences without requiring complex equipment or difficult-to-control conditions
Solution Approach 2:
The crystallization step exploits changes in solubility parameters with temperature and concentration to achieve final purification. By controlling cooling rate and final concentration, lacto-N-neotetraose crystallizes in high purity form while remaining impurities stay in the mother liquor. This parameter-based separation is simple to implement and highly effective for achieving ≥90% purity
4Ease of manufacture
If purification process is simplified to reduce complexity and cost, then production becomes more economical, but product purity and contaminant removal efficiency decrease
Solution Approach 1:
The purification train is segmented into three relatively simple, well-established unit operations: membrane filtration, ion exchange chromatography, and crystallization. Each segment uses standard, commercially available technology rather than complex custom equipment, keeping individual step costs and complexities low while the combination achieves the required ≥90% purity
Solution Approach 2:
Crystallization is used as the final purification step because it is a simple, low-cost operation that exploits fundamental solubility parameter changes. By controlling temperature and concentration, high purity crystals are obtained without requiring sophisticated equipment or complex process control, achieving both cost-effectiveness and high purity simultaneously
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The process achieves lacto-N-neotetraose purity of ≥90% with efficient separation of contaminants, suitable for nutritional, medical, and cosmetic applications, while being cost-effective and scalable.
Implementation Method 1
the solution resulting from step 1 membrane filtration is subjected to nanofiltration by applying 1-50 bar, more preferably 2-30 bar, more preferably 3-10 bar and more preferably 4-5 bar of pressure
Implementation Method 2
The at least one simulated moving bed chromatography step has: i) at least 4 columns, more preferably at least 8 columns, more preferably at least 12 columns
Implementation Method 3
c) at least one crystallization step from water, obtaining a crystal mass
Implementation Method 4
d) at least one washing step with alcohol, alcohol/water mixture or solvent or solvent/water mixture in order to wash off remaining smaller saccharides
Implementation Method 5
e) at least one homogenization step of the washed, drained crystal mass, obtaining a homogenized, dried lacto-N-neotetraose product
Data Source
AI summary
Disclosed is a process for the purification of LNnT (lacto-N-neotetraose) from a fermentation broth, the process comprises subjecting a fermentation broth to a first step of membrane filtration, thereby providing a filtrated solution, such filtrated solution is subjecting to a second step of simulated moving bed chromatography, obtaining a purified solution thereof, then subjecting this purified solution to a third step of crystallization, obtaining crystals containing the LNnT of interest, and subjecting the crystals to a fourth and final step of drying, thereby providing a highly purified powder of LNnT.