WO1995014220A2 - Procede et dispositif pour la separation de melanges de substances dissous et non dissous - Google Patents

Procede et dispositif pour la separation de melanges de substances dissous et non dissous Download PDF

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Publication number
WO1995014220A2
WO1995014220A2 PCT/DE1994/001350 DE9401350W WO9514220A2 WO 1995014220 A2 WO1995014220 A2 WO 1995014220A2 DE 9401350 W DE9401350 W DE 9401350W WO 9514220 A2 WO9514220 A2 WO 9514220A2
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WO
WIPO (PCT)
Prior art keywords
chromatography
gel
separation
gels
desalination
Prior art date
Application number
PCT/DE1994/001350
Other languages
German (de)
English (en)
Other versions
WO1995014220A3 (fr
WO1995014220B1 (fr
Inventor
Wolfgang Pfeiffer
Original Assignee
Brettschneider, Henner
Kaissling-Koschnick, Roswitha
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 Brettschneider, Henner, Kaissling-Koschnick, Roswitha filed Critical Brettschneider, Henner
Priority to AU81392/94A priority Critical patent/AU8139294A/en
Publication of WO1995014220A2 publication Critical patent/WO1995014220A2/fr
Publication of WO1995014220A3 publication Critical patent/WO1995014220A3/fr
Publication of WO1995014220B1 publication Critical patent/WO1995014220B1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • G01N30/6065—Construction of the column body with varying cross section
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • G01N30/6017—Fluid distributors

Definitions

  • the invention relates to extraction, filtration and chromatography processes and their optimization and combination for the preparative separation of dissolved and precipitated substance mixtures, in particular in precipitant gradients stabilized by molecular sieves during fractional crystallization.
  • the methods and the devices for their effective implementation are described.
  • the first process step often consists in working up suspensions, for example cell homogenates or protein precipitations. These process steps usually involve labor-intensive and lossy centrifugation, filtration and extraction to separate solids and liquids.
  • the process described here allows the solid and the liquid part of the suspension to be obtained loss-free in one work step by directly applying a precipitated stock suspension.
  • Protein precipitations were carried out directly on gel chromatography columns by applying proteins in dissolved form and allowing them to migrate into a precipitant gradient preformed on the column. Serum proteins were fractionated in this way, but not, as we intended, to desalinate and recover the serum at the same time:
  • the high-performance chromatography to be developed should have a large flat application area in order to reduce the risk of
  • the tube extension at the sample application point provides a much larger application area compared to a cylindrical tube. This reduces the risk of blocking. Overall, a larger amount of substance can be worked up than previously with comparatively the same filling quantities. This applies both to the first filtration step and in particular to the subsequent chromatography. In this, the dividing tray volume adapts to the falling capacity requirements as the separation progresses.
  • the separation matrix is therefore used more effectively overall. This manifests itself in comparatively lower filling quantities, shorter cycle times and less separation-related dilution.
  • the effective use of the separation matrix requires that the distorted elution front is compensated by a corresponding curvature of the flow adapter. As shown in Fig. 3, it is irrelevant where this curvature lies and in which direction it is flowed through.
  • the arrangement A is particularly suitable for the combination of filtration, extraction and chromatography because of the flat application surface, whereas the arrangement B is more favorable for the chromatography of solutions because the amount of separation matrix on the application is additionally increased by the curvature.
  • a precipitated protein suspension is applied to a desalination gel under high salt conditions, the protein suspension remains on the gel and can be washed with the precipitation buffer free of soluble "impurities" (here e.g. serum).
  • impurities here e.g. serum
  • the salt concentration in the elution buffer is subsequently reduced, the precipitated substances are extracted from the precipitate in the application zone in the order of their solubility and, because they cannot penetrate the gel, migrate in the exclusion volume of the gel bed to the position at which they again meet the limit achieve their solubility. Here they crystallize and fail. Different bands of precipitation form one after the other in the order of their solubilities.
  • the extraction in combination with the accelerated migration in the exclusion volume leads to one
  • Protein crystals on a hydrophobic matrix.
  • the hydrophobicity of the matrix is of central importance because it can be adjusted in a targeted manner, thereby defining the range in which the separation process can be controlled by means of precipitant gradients.
  • the hydrophobic adsorption on the gel matrix can be specifically changed.
  • this requires a fine adjustment of the hydrophobicity between the surface of the separation matrix and the protein to be cleaned.
  • Various examples are shown in the examples for this fine-tuning.
  • Fig. 1 To determine the optimal shape for the separating device, three different funnel-shaped tubes were filled with the same volume (73 ml) of desalination gel (Fig. 1) and the relative elution profile of a low molecular weight dye (bromophenol blue) was determined (Fig. 2).
  • the elution profile of tube A showed the steepest elution profile. This means that the elution front deviates the least from a flat surface in this example.
  • the bottom of an arbitrarily shaped chromatography tube can be adapted to the shape of the elution front using the same method.
  • a liquid flow through a conical chromatography tube on the wall has a longer path than in the center. If you sketch different streamlined separating pipes and if you draw equal distances on the streamlines and connect them, you get an idea of how the interference front advances depending on the time (Fig. 3A). It is clear from this that, in the case of a flat application surface in an arbitrarily shaped conical chromatography tube, an external curvature of the base can compensate for the different lano separation distances during the chromatography.
  • the entire separation tube forms: one expediently streamlined.
  • the maximum dimension should not exceed 120 cm. This is the upper limit up to which accessories can still be obtained relatively easily.
  • the upper and lower tube of at least 10 cm height are brought to the desired distance. Their outer edges are never connected with a dashed auxiliary line. Their center point later becomes the turning point of the curvature in the streamlined separation tube. The tangent at the turning point Fig. 4. serves as a construction aid line.
  • the conical separating tube is constructed using the auxiliary lines designated under 1, as described in Fig. 4.
  • a separation tube as shown in Fig. 1A (upper inner diameter 10cm, lower inner diameter 5cm) was filled with approx. 700ml Eiogel F6 fine and equilibrated with physiological buffer.
  • the elution profiles are shown in Fig. 6 with the corresponding scale curves. Only the strongest curvature shows a sharper elution profile. This method can be used to determine the most suitable bottom curvature for an arbitrarily shaped separating pipe.
  • the curved elution front is equalized by shaping the bottom of the chromatography tube so that it has the same curvature as the distorted elution front.
  • the bracket is designed to e.g. via a plug or Luer-lock connection (5th) can easily be exchanged for other filter housings with different filter curvature so that parts of "flow adapters" (10th) are already available on the market.
  • a conical chromatography tube according to Fig. 1A with a large diameter of 5 cm and a small diameter of 2.5 cm is closed at the bottom with a river adapter as the bottom, the curvature of which has been changed as described.
  • the tube is filled with desalination gel based on acrylamide (73ml: Biorad, PG6, fine) because this gel does not allow proteins to penetrate the gel particles due to its low exclusion limit.
  • the gel was swollen in physiological salt buffer and the packed chromatography tube was covered with a filter and equilibrated with the same buffer. The excess buffer, which was formed by setting the gel bed, is carefully drained off with the tube open. The filter is carefully covered with the smallest possible volume of a 12% by weight Na 2 SO 4 solution. It is important that the filter lies very evenly on the gel.
  • the salt solution is carefully soaked up from the gel bed - but make sure that no air penetrates between the gel and the filter.
  • the serum is eluted with citrate buffer, the concentration of which corresponds to the precipitation buffer. After this pretreatment, the serum elutes free of antibodies, pyrogens and viruses. As soon as the protein concentration drops at the buffer outlet and the citrate concentration rises, the serum elution is ended.
  • the elution is interrupted and the separation tube is backwashed with PBS buffer for equilibration and anti-blocking solution.
  • the antibodies are known because they are collected in the. In contrast to the inactivated viruses, do not bind to the quaternary anine under low salt conditions.
  • the desalination gel derivatized with quaternary amine is lifted off at the filter separation layer and can be used again after regeneration and drying.
  • the separator which is open at the top, is now ready for the next serum separation 8.
  • Example 6 the same device as in Example 6 is used.
  • the chromatography gel is swollen in saturated Na 2 SO 4 , filled into the chromatography tube, equilibrated with the same buffer and covered with a filter. Under these high salt conditions, the gel shows a lower degree of swelling than at a physiological salt concentration. Accordingly, one must provide an equalizing volume in the upper cylindrical tube to expand the gel.
  • the suspension of the precipitated serum proteins is applied to the filter of the device and smoothed out.
  • the chromatography tube filled with gel is washed with saturated sodium sulfate solution until sodium chloride is no longer detectable in the eluate.
  • a gradient consisting of saturated sodium sulfate solution in the mixing vessel and physiological buffer in the feed is then connected.
  • the gradient can be adapted to the particular separation problem. This applies both to the gradient shape and to its composition and, moreover, also to the shape of the separating tube.
  • hydrophobic groups are additionally coupled to the desalination gel.
  • Hydroxypropylated Sephadex LH20
  • LH20 Hydroxypropylated Sephadex
  • the amides of acrylamide e.g. BioRad: Biogel P6 and IBF: Trisacryl GF 05 as desalination gels
  • alkylhydrazines or alkylamines can be converted into the corresponding amides and hydrazides by exchanging the amidic nitrogen.
  • hydrophobic desalting gels based on acrylamide are used for fractional crystallization with improved separation.
  • a known method is used to prepare the alkylhydrazides and alkylamides:
  • the improved separation manifests itself in an increase in band sharpness and a larger elution volume between two precipitation bands in the desired area of the elution profile.
  • the modification reactions mentioned in Example 10 in particular when separating hydrophobic peptides, only insufficiently increased the hydrophilicity of small-pore gels.
  • the binding via strong ion exchange groups can be neglected for fractional crystallization, it is advisable to introduce such charged ionic groups into highly crosslinked gels for this special application.
  • modification of the acrylamide gels for example, aminomethane and aminoethanesulfonic acid are available, which can be reacted via the amino group by an exchange reaction with acrylamide, which results in a strong cation exchanger.
  • the desalination gels based on polysaccharide can in turn be reacted in alkaline with an epoxy such as (2,3-epoxypropyltrimethylammonium chloride), which creates a strong anion exchanger.
  • an epoxy such as (2,3-epoxypropyltrimethylammonium chloride

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Le procédé permet une filtration, une extraction et une chromatographie de mélanges de substances dissous et non dissous, et il permet notamment d'obtenir du sérum déminéralisé et également de réaliser une cristallisation fractionnante, appelée également chromatograpie par précipitation ou précipitation par zones. Pour une exécution combinée efficace de ses procédés, on a mis au point un dispositif de séparation constitué d'une colonne de chromatographie qui est maintenue en place par des surfaces d'extrémité poreuses au moins partiellement convexes et qui est remplie d'un tamis moléculaire. Dans sa forme de réalisation particulière, la colonne présente un évasement en entonnoir vers le haut. Les dispositifs de séparation décrits peuvent être exploités non seulement avec des gels de déminéralisation mais aussi avec des matériaux chromatographiques quelconques, ce qui permet l'exécution des procédés classiques de filtration, d'extraction et de chromatographique de manière plus avantageuse. Du fait de ses possibilités universelles d'application, ce procédé permet tout particulièrement d'effectuer, de façon plus efficace, les nettoyages biochimiques, en particulier les étapes initiales de tels nettoyages.
PCT/DE1994/001350 1993-11-16 1994-11-15 Procede et dispositif pour la separation de melanges de substances dissous et non dissous WO1995014220A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU81392/94A AU8139294A (en) 1993-11-16 1994-11-15 Method and device for the separation of dissolved and undissolved mixtures

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4339166.4 1993-11-16
DE4339166 1993-11-16

Publications (3)

Publication Number Publication Date
WO1995014220A2 true WO1995014220A2 (fr) 1995-05-26
WO1995014220A3 WO1995014220A3 (fr) 1995-08-10
WO1995014220B1 WO1995014220B1 (fr) 1995-09-28

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PCT/DE1994/001350 WO1995014220A2 (fr) 1993-11-16 1994-11-15 Procede et dispositif pour la separation de melanges de substances dissous et non dissous

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AU (1) AU8139294A (fr)
DE (1) DE4440805C2 (fr)
WO (1) WO1995014220A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040734A1 (fr) * 1997-03-12 1998-09-17 Merck Patent Gmbh Piece terminale pour colonne chromatographique
DE19940131B4 (de) * 1998-08-24 2004-12-02 Pfefferle, Christoph, Dr. Verfahren und Vorrichtung zur Gewinnung von biologisch aktiven Substanzen
CN109107223A (zh) * 2018-10-15 2019-01-01 潘仲巍 一种从当归中富集阿魏酸的设备及方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19718652C2 (de) * 1996-05-03 2000-07-20 Pfeiffer Wolfdietrich Vorrichtung für die Flüssigkeitschromatographie
DE19900684A1 (de) * 1999-01-04 2000-07-06 Mark Lisso Verfahren zur chromatographischen Trennung von Stoffgemischen in reine Stoffe mittels Flüssig-Chromatographie
DE102016105997A1 (de) * 2016-04-01 2017-10-05 Flavologic Gmbh Adsorptionssystem und Verfahren zum Betreiben eines Adsorptionssystems

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1340295A (en) * 1971-07-01 1973-12-12 Merck Patent Gmbh Assembled column for chromatography
EP0021817A1 (fr) * 1979-06-22 1981-01-07 Sekisui Kagaku Kogyo Kabushiki Kaisha Remplissage pour chromatographie en phase liquide, procédé pour séparer des substances solubles dans l'eau à l'aide de ce remplissage et utilisation de ce remplissage dans la séparation de substances biochimiques solubles dans l'eau
EP0023322A1 (fr) * 1979-07-20 1981-02-04 Kureha Kagaku Kogyo Kabushiki Kaisha Emploi d'un gel de polyvinylpyrrolidone réticulé comme gel chromatographique
EP0248379A2 (fr) * 1986-06-06 1987-12-09 Magyar Tudományos Akadémia Kutatási Eszközöket Kivitelezö Vállalata Colonne réapprovisionnable pour la chromatographie préparative à basse pression
DE3821619A1 (de) * 1987-08-11 1989-02-23 Petrolchemisches Kombinat Vesikulaere fuellkoerper fuer die chromatographie aus denaturierten mikroorganismen und verfahren zur herstellung
US4832916A (en) * 1986-03-17 1989-05-23 Armin Gilak Chromatographic column for immunological determining methods

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1055531A1 (ru) * 1982-08-05 1983-11-23 Ленинградский Ордена Ленина И Ордена Трудового Красного Знамени Государственный Университет Им.А.А.Жданова Колонка дл препаративной жидкостной хроматографии

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1340295A (en) * 1971-07-01 1973-12-12 Merck Patent Gmbh Assembled column for chromatography
EP0021817A1 (fr) * 1979-06-22 1981-01-07 Sekisui Kagaku Kogyo Kabushiki Kaisha Remplissage pour chromatographie en phase liquide, procédé pour séparer des substances solubles dans l'eau à l'aide de ce remplissage et utilisation de ce remplissage dans la séparation de substances biochimiques solubles dans l'eau
EP0023322A1 (fr) * 1979-07-20 1981-02-04 Kureha Kagaku Kogyo Kabushiki Kaisha Emploi d'un gel de polyvinylpyrrolidone réticulé comme gel chromatographique
US4832916A (en) * 1986-03-17 1989-05-23 Armin Gilak Chromatographic column for immunological determining methods
EP0248379A2 (fr) * 1986-06-06 1987-12-09 Magyar Tudományos Akadémia Kutatási Eszközöket Kivitelezö Vállalata Colonne réapprovisionnable pour la chromatographie préparative à basse pression
DE3821619A1 (de) * 1987-08-11 1989-02-23 Petrolchemisches Kombinat Vesikulaere fuellkoerper fuer die chromatographie aus denaturierten mikroorganismen und verfahren zur herstellung

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040734A1 (fr) * 1997-03-12 1998-09-17 Merck Patent Gmbh Piece terminale pour colonne chromatographique
US6171502B1 (en) 1997-03-12 2001-01-09 Merck Patentgesellschaft End piece for a chromatographic column
DE19940131B4 (de) * 1998-08-24 2004-12-02 Pfefferle, Christoph, Dr. Verfahren und Vorrichtung zur Gewinnung von biologisch aktiven Substanzen
CN109107223A (zh) * 2018-10-15 2019-01-01 潘仲巍 一种从当归中富集阿魏酸的设备及方法

Also Published As

Publication number Publication date
WO1995014220A3 (fr) 1995-08-10
AU8139294A (en) 1995-06-06
DE4440805A1 (de) 1995-07-20
DE4440805C2 (de) 1998-11-26

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