WO2005054274A1 - Nanofiltration de dipeptides - Google Patents

Nanofiltration de dipeptides Download PDF

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Publication number
WO2005054274A1
WO2005054274A1 PCT/EP2003/013625 EP0313625W WO2005054274A1 WO 2005054274 A1 WO2005054274 A1 WO 2005054274A1 EP 0313625 W EP0313625 W EP 0313625W WO 2005054274 A1 WO2005054274 A1 WO 2005054274A1
Authority
WO
WIPO (PCT)
Prior art keywords
dipeptide
process according
isoelectric point
membrane
nanofiltration
Prior art date
Application number
PCT/EP2003/013625
Other languages
English (en)
Inventor
Michael Bobek
Sven GÄRTNER
Wolfgang Samhaber
Original Assignee
Fresenius Kabi Austria Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fresenius Kabi Austria Gmbh filed Critical Fresenius Kabi Austria Gmbh
Priority to PCT/EP2003/013625 priority Critical patent/WO2005054274A1/fr
Priority to AU2003294774A priority patent/AU2003294774A1/en
Publication of WO2005054274A1 publication Critical patent/WO2005054274A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/027—Nanofiltration
    • B01D61/0271—Nanofiltration comprising multiple nanofiltration steps
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/025—Reverse osmosis; Hyperfiltration
    • B01D61/026—Reverse osmosis; Hyperfiltration comprising multiple reverse osmosis steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2317/00—Membrane module arrangements within a plant or an apparatus
    • B01D2317/02—Elements in series
    • B01D2317/025—Permeate series

Definitions

  • the present invention relates to a purification process for dipeptides (Figure 1) using membrane separation technology.
  • the water soluble dipeptides such as Alanylglutamin or Glycylglutamin represent an important source of glutamin in parenteral and enteral nutrition solutions. (Ftirst, P. et. al., Ann. Nutr. Metab. 1997, 41, 1-9)
  • Pressure driven membrane separation processes are known wherein organic molecules or inorganic ionic solutes in aqueous solutions are concentrated or separated to various degrees by the application of a positive osmotic pressure to one side of a filtration membrane.
  • pressures are reverse osmosis (RO), nanofiltration (NF) and ultrafiltration (UF).
  • RO reverse osmosis
  • NF nanofiltration
  • UF ultrafiltration
  • the molecular cut-off of these different membranes increases in this sequence.
  • These pressure driven membrane processes employ a cross-flow mode of operation wherein only a portion of a feed solution (F) is collected as a permeate solution (P) and the rest is collected as a pass solution (C).
  • F feed solution
  • P permeate solution
  • C pass solution
  • the exit process stream from the nanofiltration module which stream has not passed through the membrane is referred to as the "pass stream”. This stream is often referred to by practitioners in the membrane filtration art as the "concentrate" stream.
  • the invention describes a process for the separation of dipeptides from their amino acid building blocks using a combination of at least one nanofiltration membrane.
  • a combination of two nanofiltration membranes is used.
  • the isoelectric points of the amino acid building blocks lie within less than +/- 2 pH units, preferably less than +/- 1 pH unit and more preferably less than +/- 0,5 pH units around the isoelectric point of the dipeptide. Appropriate adjustments of the pH and the ion strength have to be made to allow an efficient separation and the system can be further adjusted by the addition of water soluble or miscible organic compounds.
  • the separation properties of the membranes may further be enhanced by suitable chemical or physical modification of the membrane material.
  • the described invention offers an alternative to a prepurification using ion-exchange resins resulting in a more cost effective and environmentally friendly process.
  • Fig. 1 shows a filtration setup in which the permeate of a first nanofiltration membrane system (1) is used as feed for a second nanofiltration membrane system (2).
  • the pass stream of the second membrane system is either fed into the product stream or recycled into the feed stream of the first membrane system.
  • This setup aims for an increase in dipeptide yield.
  • Fig. 2 shows a filtration setup in which the pass stream of a first nanofiltration membrane system (la) is used as feed for a second nanofiltration membrane system (2a)'.
  • the permeate of the second membrane system (2a) is either discarded or recycled into the feed stream of the first membrane system.
  • This setup aims for an increase in dipeptide purity.
  • a reaction of monomeric amino acids can result in dipeptides with isoelectric points in the range of pH 2,5 to 10,5 which are stable to hydrolysis.
  • the invention is preferably concerned with dipeptides with isoelectric points in a range of pH 4,5 - 8,5.
  • more acidic dipeptides with isoelectric points in the range of pH 2,5 - 4,5 and more basic dipeptides with isoelectric points in the range of pH 8,5 - 10,5 are also encompassed in the scope of the invention.
  • a crude aqueous or aqueous/organic (eg. acetone, ethanol) solution directly derived from the chemical reaction or from another prepurification step containing 0,1 to 70% (w/w) of reacted and unreacted amino acids is applied as feed solution.
  • the solution is diluted to the desired concentration in a feed tank or in a continuous mixing process.
  • the separation conditions are tailored for the separation purpose by the addition of inorganic salts or water soluble or miscible organic compounds.
  • the pH is adjusted to a value of 7-10 with an aqueous solution of an acid (eg. HC1, HCOOH), base (eg. NaOH, NH 3 ) or basic or acidic salt (eg. K 2 CO 3 ) in a feed tank or a in a continuous mixing process.
  • an acid eg. HC1, HCOOH
  • base eg. NaOH, NH 3
  • basic or acidic salt eg. K 2 CO 3
  • the dilute solution is pumped through an appropriate separation system with at least a first nanofiltration membrane system (1, 1a) comprising an inlet chamber (9, 9a), an outlet chamber (10, 10a) and a nanofiltration membrane (11, 1 la).
  • the system is operated at a pressure of 4 to 100 bar , more preferably at a pressure of 15-40 bar, at the membrane and a temperature of 5 to 70°C.
  • Nanofiltration membranes which today most membrane manufacturers producing parallel to reverse osmosis membranes are chemically and structurally similar to reverse osmosis membranes.
  • Nanofiltration membranes can be made of many different materials.
  • a thin "skin layer” made of aromatic polyamides, polysulfones, polyethersulfones, poly(phenylene oxide), etc. and modifications of them is cast, on top of a meso or microporous polymer sheet support (e.g. a polysulfone ultrafiltration membrane) to form a "thin film composite" membrane structure (TFC).
  • Nanofiltration membranes generally exhibit a nominal molecular cut-off in the range of 100-1200 D.
  • the separation properties of the membrane are controlled by the pore structure and inner surface properties of the active top or skin layer. Specifically the separation properties can be tailored by addition of inorganic salts and water soluble or miscible organic compounds to achieve a required performance in the process.
  • the invention preferably uses nanofiltration membranes with a separation capacity for organic solutes with a molecular "cut-off" range of —500 - 1200 D; and a separation capacity for inorganic or organic multivalent versus, monovalent ions and for different hydrated charged compounds.
  • the NF membranes are characterized in having a lower rejection for chloride ions and a much higher rejection for sulfate ions.
  • the negative charge is responsible for rejection of anionic species, according to the anion surface charge density.
  • nanofiltration membranes include, but are not limited to: GE Osmonics (US) DK membrane (Desal G5), NF70 membrane (Dow-FilmTec, US) SU 600 membrane (Toray, Japan), and NRT 7450 and NTR 7480 membranes (Nitto, Japan), N30F membrane (Microdyn-Nadir, Germany) and others.
  • the NF membranes are often modularized in so-called "spiral wound" elements or modules, but other module configurations, such as tubular or plate- and-frame types, are also known.
  • the pass solution of the first filtration step yields a stream of purified product with a reduced content of the amino acid building blocks compared to the dipeptide.
  • the permeate solution contains a stream with a reduced amount of dipeptide and an increased amount of amino acids compared to the feed solution.
  • a second nanofiltration membrane system (2, 2a) comprising an inlet chamber (12, 12a), an outlet chamber (13, 13a) and a nanofiltration membrane (14, 14a)can be employed.
  • Either the permeate or the pass stream of the first membrane system can be passed through the second membrane system.
  • the second membrane system is operated at a pressure of 5 to 40 bar and a temperature of 10 to 50°C.
  • the second membrane system can contain an identical, denser or more open membrane compared to the membrane in the first membrane system (1, la). If required, prior to the second system, the content of inorganic salts or water soluble or miscible organic compounds can be readjusted to the optimal conditions in an intermediate adjustment step.
  • a first nanofiltration membrane system (1, la) is fed via a feeding line (3, 3a).
  • a first embodiment shown in figure 1 which aims for a higher yield of dipeptide product
  • the pass stream of the first membrane system (1) is collected as product stream via a product line (4).
  • the permeate is fed to a second membrane system (2) via a first permeate line (5).
  • the resulting pass stream exits the second membrane system via a pass stream line (6) and can either be fed directly into the product line (4) or recycled into the feeding line (3) via a recycling line (8), depending on the desired dipeptide concentrations.
  • the permeate which exits second membrane system via a second permeate line (7) containing amino acid monomers and a reduced concentration of dipeptide can be discarded or used for reprocessing using ion-exchange chromatography, gel permeation chromatography or another membrane purification step.
  • a second embodiment which aims for a higher purity of dipeptide product
  • the permeate stream which exits the first membrane system (la) via a first permeate line (5a) is either discarded or recollected for further processing.
  • the pass stream is fed to a second membrane system (2a) via a connection line (4a).
  • the resulting pass stream of the second membrane system is collected as the product via a product line (6a).
  • the permeate of the second membrane system (2a) which exits via a second permeate line (7a) can either be discarded or recycled by feeding into the feeding line (3a) via a recycling line (8a).
  • the separate nanofiltration membrane systems can be operated both in a continuous or a discontinuous mode.
  • the prior art describes a negative correlation of the rejection of an analyte with increasing concentration of divalent salts.
  • a suitable salt such as potassium carbonate.
  • the suitable salt is added in an amount to make up for a final concentration which is at least equal to the equimolar concentration of the dipeptide.
  • concentration of the salt is at least twice the equimolar concentration. This effect can be used to further enhance the separation properties of the described membrane systems making the process economically more feasible.
  • the membrane system used consists of a wound spnal module with a surface of 0,55 m .
  • the membrane material is a commercially available, polysulfone based, Desal G5 type material.
  • the solution is pumped through the membrane module with an adjustable monopump at a pressure of 20 +/- 0,2 bar.
  • the differential pressure between membrane inlet and outlet amounts to 170 mbar.
  • the membrane and the feed solution are kept at a temperature of 23°C.
  • the feed solution is passed through the membrane system within two hours.
  • the resulting pass solution consists of 6% of alanine, 0,9% of glutamine and 93% of alanylglutamine.
  • the pH of the permeate solution is continuously readjusted to pH 9 in an intermediate tank by the addition of sodium hydroxide solution. Processing the permeate solution through an identical membrane system in the second step as depicted in Figure 1 or 2 and adding the pass solution to the feed batch of the first membrane system results in an overall recovery of 80% regarding the dipeptide.
  • a solution prepared as described above is passed through a single membrane system.
  • the membrane system used consists of a wound spiral module with a surface of 0,55 m 2 .
  • the membrane material is a commercially available, polysulfone based, Desal G5 type material.
  • the solution is pumped through the membrane module with an adjustable monopump at a pressure of 20 +/- 0,2 bar.
  • the differential pressure between membrane inlet and outlet amounts to 170 mbar During the experiment the membrane and the feed solution are kept at a temperature of 23°C.
  • the single system operates at a yield of 56% regarding the dipeptide.
  • the solution consists of 4,3% alanine, -3% glutamine and 93% of alanylglutamine.
  • Example 4 Enhancement of rejection by the addition of salts: Four liter of a solution containing 50 g/L alanylglutamin is processed in the described membrane system. The pH is adjusted to pH 9 +/- 0.2. In one experiment solid potassium carbonate is added to the solution to a total molar concentration of 0.25 M of potassium carbonate, while the pH is kept at the previous level. The pressure is set to 30 bar and the temperature is kept constant at 23°C. The rejection of the dipeptide increases from approx. 60 % to approx. 87 % by the addition of potassium carbonate to a concentration of 0.25 M.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Water Supply & Treatment (AREA)
  • Nanotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Les dipeptides hydrosolubles représentent une source importante d'acides aminés dans les solutions de nutrition parentérale et entérale. L'invention porte sur un procédé qui permet de séparer les dipeptides de leurs blocs de construction acides aminés par le biais d'une membrane de filtration possédant une plage de seuils de coupure moléculaire de 100 à 1200 D. Dans un mode de réalisation préféré, on utilise deux membranes de filtration combinées. Le procédé de l'invention permet de séparer les dipeptides d'acides aminés dont les points isoélectriques se situent dans une plage inférieure à +/- 2 unités pH par rapport au point isoélectrique du dipeptide.
PCT/EP2003/013625 2003-12-03 2003-12-03 Nanofiltration de dipeptides WO2005054274A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/EP2003/013625 WO2005054274A1 (fr) 2003-12-03 2003-12-03 Nanofiltration de dipeptides
AU2003294774A AU2003294774A1 (en) 2003-12-03 2003-12-03 Nanofiltration of dipeptides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2003/013625 WO2005054274A1 (fr) 2003-12-03 2003-12-03 Nanofiltration de dipeptides

Publications (1)

Publication Number Publication Date
WO2005054274A1 true WO2005054274A1 (fr) 2005-06-16

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7144511B2 (en) * 2002-05-02 2006-12-05 City Of Long Beach Two stage nanofiltration seawater desalination system
EP2008702A1 (fr) * 2007-06-25 2008-12-31 Unilever Plc Procédé et appareil de filtration à membrane
WO2016024105A1 (fr) * 2014-08-11 2016-02-18 Imperial Innovations Limited Nanofiltration de solvant organique avec une meilleure perméation des impuretés

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
GAREM A ET AL: "Selective separation of amino acids with a charged inorganic nanofiltration membrane: effect of physicochemical parameters on selectivity", BIOTECHNOL BIOENG;BIOTECHNOLOGY AND BIOENGINEERING MAY 20 1997 JOHN WILEY & SONS INC, NEW YORK, NY, USA, vol. 54, no. 4, 20 May 1997 (1997-05-20), pages 291 - 302, XP002279661 *
LAPOINTE, JEAN-FRANCOIS ET AL: "Effect of hydrodynamic conditions on fractionation of.beta.-lactoglobuli tryptic peptides using nanofiltration membranes", JOURNAL OF MEMBRANE SCIENCE, vol. 212, no. 1-2, 15 February 2003 (2003-02-15), pages 55 - 67, XP004402817 *
MARTIN-ORUE, C. ET AL: "Nanofiltration of amino acid and peptide solutions: mechanisms of separation", JOURNAL OF MEMBRANE SCIENCE, vol. 142, no. 2, 13 May 1998 (1998-05-13), pages 225 - 233, XP004121289 *
SALLES, C. ET AL: "Goat cheese flavor: Sensory evaluation of branched-chain fatty acids and small peptides", JOURNAL OF FOOD SCIENCE, vol. 67, no. 2, March 2002 (2002-03-01), pages 835 - 841, XP001189432 *
SCHRÖDER J ET AL: "Glutamine dipeptides-supplemented parenteral nutrition reverses gut mucosal structure and interleukin-6 release of rat intestinal mononuclear cells after hemorrhagic shock.", SHOCK, vol. 10, no. 1, July 1998 (1998-07-01), Augusta, GA., United states, pages 26 - 31, XP009030537, ISSN: 1073-2322 *
SOMMERER, N. ET AL: "Isolation of a peptidic fraction from the goat cheese water-soluble extract by nanofiltration for sensory evaluation studies", DEVELOPMENTS IN FOOD SCIENCE (1998), (FOOD FLAVORS: FORMATION, ANALYSIS AND PACKAGING INFLUENCES), vol. 40, 1998, pages 207 - 217, XP001180986 *
TSURU, TOSHINORI ET AL: "Peptide and amino acid separation with nanofiltration membranes", SEPARATION SCIENCE AND TECHNOLOGY, vol. 29, no. 8, 1994, pages 971 - 984, XP009030301 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7144511B2 (en) * 2002-05-02 2006-12-05 City Of Long Beach Two stage nanofiltration seawater desalination system
EP2008702A1 (fr) * 2007-06-25 2008-12-31 Unilever Plc Procédé et appareil de filtration à membrane
WO2016024105A1 (fr) * 2014-08-11 2016-02-18 Imperial Innovations Limited Nanofiltration de solvant organique avec une meilleure perméation des impuretés

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Publication number Publication date
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