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WO2003010512A2 - Separation solide-liquide a grande vitesse - Google Patents

Separation solide-liquide a grande vitesse Download PDF

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Publication number
WO2003010512A2
WO2003010512A2 PCT/US2002/023546 US0223546W WO03010512A2 WO 2003010512 A2 WO2003010512 A2 WO 2003010512A2 US 0223546 W US0223546 W US 0223546W WO 03010512 A2 WO03010512 A2 WO 03010512A2
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WO
WIPO (PCT)
Prior art keywords
affinity
beads
target compound
loop
bead
Prior art date
Application number
PCT/US2002/023546
Other languages
English (en)
Other versions
WO2003010512A3 (fr
Inventor
Bozidar Stipanovic
Edward A. Jakush
Original Assignee
Accurate Polymers, Inc.
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 Accurate Polymers, Inc. filed Critical Accurate Polymers, Inc.
Priority to AU2002355285A priority Critical patent/AU2002355285A1/en
Publication of WO2003010512A2 publication Critical patent/WO2003010512A2/fr
Publication of WO2003010512A3 publication Critical patent/WO2003010512A3/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • the present invention is directed to a method and apparatus for the separation of biological, pharmaceutical and other products.
  • the invention is directed to a method and apparatus for the separation of biological and pharmaceutical products utilizing a centrifuge and reconstituted cellulose beads.
  • Affinity separation is capable of selectively adsorbing a desired protein on a specially designed solid matrix, specific for attracting the protein or a class of proteins.
  • Affinity separation is a powerful method for sequestering a single product from a number of byproducts and impurities in only one step. After washing the product-affinity matrix complex, the product is eluted almost pure.
  • affinity chromatography columns of tightly packed affinity sorbent particles through which a solution containing the target biomolecule is forced to flow, driven by pressure.
  • affinity column chromatography begins to show serious deficiencies.
  • Column affinity chromatography has limitations deliver both high purity and short processing times.
  • Non-porous cellulose beads as for example those described in U.S. Patent No. 5,656,373 to Scarpa, display a high structural integrity and resistance. Furthermore, the non-porous character allows for rapid removal of unbound contaminants, while target biomolecules remain bound selectively on their surface, held by grafted affinity ligands. In addition, non-porous beads do not entrap or retain contaminants inside. Yet, a drawback of non-porous beads is their low surface area.
  • Small, non-porous beads such as Orbicell ® affinity cellulose beads, available from Accurate Polymers in Highland Park, Illinois, are used with great efficiency in a loop system where affinity particles are suspended in a circulating tank and maintained in a homogenous suspension by mechanical means. The mixing motion is mainly affected by pumping action, allowing the particles to repeatedly contact target compounds.
  • the process system is designed as a loop that holds and retains affinity particles, allowing for the clarified liquid component of the suspension to be continuously separated from solids by a solid-liquid separation system such as dynamic filtration and tangential flow filtration systems.
  • the present invention discloses and describes an affinity separation and purification method including (a) incubating reconstituted beads with a fluid containing low concentrations of a target compound, in order to affect the adsorption of said compound to the affinity ligands grafted on the surface of beads in a process coined "loading" the bead; (b) introducing the "loaded" beads, that is, an affinity bead-target compound complex, into an upstream circulating tank, connected with and feeding into a solid-liquid separating system, such as a high speed continuous centrifuge, having two additional ports, one communicating downstream carrying solutes out of the loop by the supernatant liquid (centrate), and the other communicating with the upstream and returning the concentrate back into the circulating tank, thereby effectively keeping the affinity beads within the loop; (c) concentrating the suspension of incubated (loaded) beads to a minimum volume that remains fluid and pumpable; (d) diluting the concentrate in the circulating tank with volumes of fresh buffer and continuing the centrifugation,
  • FIG. 1 is a schematic description of the circulation loop, with supporting vessels, equipment, and material-transfer system for affinity separation comprising Orbicell® affinity beads
  • Figure 2 is one embodiment of a solid-liquid, continuous, high-speed centrifuge applicable to the method, including a Decanter Centrifuge.
  • Figure 2B is another embodiment of a solid-liquid, continuous, high-speed centrifuge, applicable to the method, including a Desludger Centrifuge.
  • Figure 2C is another embodiment of a solid-liquid, continuous, high-speed centrifuge, applicable to the method, including a Nozzle Disc Centrifuge.
  • Figure 2D is another embodiment of a solid-liquid, continuous, high-speed centrifuge, applicable to the method, including a Vortex Nozzle Disc Centrifuge.
  • Figure 3 is a schematic description of a continuous vacuum belt filter for solid-liquid separation including Orbicell® affinity beads.
  • FIG. 4 is a schematic representation of an affinity separation pilot run using a bench top system comprising a high speed centrifuge and Orbicell® affinity beads. DETAILED DESCRIPTION OF THE BEST MODE
  • the very high structural resistance of the beads opens the door for solid-liquid separation equipment like a centrifuge to be incorporated into a circulating loop system for affinity separation and purification.
  • high-speed centrifuges have high capacities and are able to process huge volumes of liquids, automatically and continuously for long periods of time.
  • the resulting solids can be made dryer in centrifuges than can be achieved by a filtration system. Consequently, the volumes of washing solutions are minimized.
  • the present method teaches a new and novel application for high speed centrifuges, namely, affinity separation and purification.
  • a sorbent having non-porous beads has a lot of advantages over porous beads.
  • Non-porous Orbicell® cellulose beads display a high structural integrity and resistance.
  • Porous affinity beads show poor shear and abrasion resistance and a tendency to collapse at high pressures encountered, for instance, during centrifugation steps at high G-s.
  • the non-porous character allows for rapid removal of unbound contaminants, while target biomolecules remain bound selectively on their surface, held by grafted affinity ligands.
  • non-porous beads do not entrap or retain contaminants inside.
  • a drawback of the non-porous beads is their low surface area.
  • the beads used in the instant method are of diameters as small as about 0.2 microns, preferably about 0.1 microns. Such a small size precludes beads used in packed chromatographic columns or in fluidized bed systems.
  • the process and system of the method include: [0034](a) introducing into pump P12 a suspension of affinity beads from the agitated (M - mixer) tank 2 into a slowly agitated incubation tank 1 , holding a liquid that contains low concentrations of the target compound, and incubating the beads with the liquid for a time period sufficient to affect a complete, or nearly complete, adsorption of the target compound onto the surface of the beads;
  • the suspension of affinity bead-target compound complex i.e. loaded beads
  • the suspension of affinity bead-target compound complex is concentrated by effectively removing the supernatant (centrate) out of the loop L and into the waste tank 9, and keeping the beads within the loop L [as in (b), above], maintaining the rate of addition at pump P11 of contents from the incubating tank 1 , equal with the flow rate of supernatant flowing out of the loop L and into the waste tank 9, resulting in a maximum concentration of solids at which the suspension in the circulating tank 5 is still fluid and capable of being pumped and circulated;
  • FIG 1 is only a schematic illustration of a preferred embodiment of the apparatus for practicing the disclosed method, and for someone with ordinary skill in the art it will be possible to practice the teaching of the present method and make modifications and variations in manipulation of pumps, valves, agitators and centrifuges, in order to achieve optimal separating conditions resulting in a product of highest possible purity.
  • the affinity beads used in the present method must be able to survive intact high pressure created by high gravitational forces encountered in high speed solid-liquid centrifuges.
  • the affinity beads must have high structural integrity.
  • the beads must also be substantially non-porous, in order not to entrap impurities and by-products along with the desired target compound.
  • the preferred affinity beads for practicing the method are Orbicell® affinity beads.
  • the beads unlike other non-porous chromatographic sorbents, have, in addition to high structural strength, very high capacity and high chemical resistance, enabling products to be separated in high yields, while still allowing the beads to be regenerated numerous times with very little activity lost in the process.
  • High capacity is, in part, the result of the small size of the beads, with diameters approximately as small as about 0.2 microns, preferably as small as about 0.1 microns. Due to the relatively high density of Orbicell® cellulose (specific gravity of 1.2 to 1.3), the beads sizes of as small as about 0.1 microns can be separated in a high-speed centrifugation process.
  • Chemical and biological stability results from the choice of chemical linkers for grafting and attaching affinity ligands to the bead surfaces: only strong covalent bonds like, but not limited to, -C-0-C-, -C-N-C-, -C-S-C-, -CO-N-C-, and -CO-N-N-CO-, are used for ligand-grafting purposes.
  • an affinity ligand is primarily determined by the nature of the target molecule. A variety of ligands are possible for each target molecule. Possible ligands have been scrutinized and evaluated based on binding constant for the target compound, chemical stability, ease of manufacturing and selectivity toward the target compound. Structurally, a ligand can be, among others or combinations, a large protein, like an antibody, a bacterial protein, like Protein A, a peptide that simulates the activity of above mentioned large proteins, and can also be chosen from groups of small synthetic molecules that behave and mimic large protein interactions with a target compound. Thiophilic and metalo affinity supports are examples of chromatographic supports comprising such synthetic ligands.
  • the present method is not limited in scope to above listed ligands, but may also include a variety of other chromatographic supports, including but not limited to hydrophobic interaction, ion exchange, hydrophilic chromatography and cellulose-binding domain based affinity chromatography.
  • the present novel method of affinity separation and purification relies on two components: (1 ) low porosity reconstituted cellulose affinity beads and (2) a high speed, solid-liquid centrifuge.
  • a decanter centrifuge consists of a rotating drum or bowl containing a scroll or conveyor, with differential speed between bowl and conveyor. The solids are conveyed up the conical end section, leaving the supernatant to drain out the other side.
  • a desludger centrifuge consists of a rotating bowl containing stacked conical discs with small spacing between the discs where the separation takes place. The solids migrate to the bowl periphery where a circular piston periodically drops, shooting the solids out of the bowl.
  • nozzle disc centrifuges have the clarification ability of other disc centrifuges, with the added ability to continuously discharge the thickened solids. Dry cakes are not possible and at higher speeds lower concentrations of solids are obtained.
  • a vortex nozzle disc centrifuge is the preferred model because it can operate at high speed and still deliver a concentrate with a high content of solids. As nozzles are reduced in diameter, the flow that forces the concentrate down the feed tubes to the nozzles is also reduced. Vortex nozzles enable the centrifuge to automatically produce the concentrated phase that has a constant concentration of solids. [0051]
  • the present method is not limited in scope to the described models of centrifuges only. Someone skilled in the art may find the use of slower centrifuges, such as basket type, satisfactory. For example, basket type centrifuges may be used if processing volumes are small and/or the processing times are not of great concern.
  • Figure 3 shows a moving belt continuous filtration system.
  • Different aspects of the moving belt embodiment include the availability of commercial systems in a sanitary design that meet all current stringent regulations for manufacturing of pharmaceuticals, and the availability of durable, chemically resistant and sterilizable filter belt materials with a wide range of porosities.
  • the systems can be cleaned-in-place, sterilized and validated with fewer difficulties than is inherent with most of tangential flow filter cartridge systems. Additional benefits can be realized because belt continuous filtration systems allow non-porous affinity particles with no resistance to pressure to be used for affinity separations.
  • Orbicell ® affinity beads are capable of being successfully used in a moving belt system for affinity separations of target biomolecules that may be sensitive to shear forces, which exist in both tangential flow filtration and in a solid-liquid centrifuge system.
  • FIG. 3 is gives a schematic illustration of an affinity separation moving belt system, using non-porous affinity particles.
  • a slurry of affinity particles is fed onto the moving belt 26 while vacuum suction boxes 34, 35, 36, and 37 remove the filtrate with contaminants that did not bind with the affinity particles.
  • a fresh washing buffer solution 22 is fed over the cake 27 and filtrate is collected in receiving vessels 30, 31 , 32, and 33. Washing nozzles 23 used for counter-current wash can be also be used with fresh washing buffer instead.
  • Back- washing nozzle 25 is used to help complete the removal of cake 27, comprising loaded affinity particles that are subsequently collected form loaded outlet 28, while waste, containing unbound byproducts and contaminants is collected from byproduct outlet 38.
  • loaded affinity particles from loaded outlet 28 are contacted with an elution buffer and the slurry is again processed on the moving belt system in the same manner as above, but now the stream 38 contains the target compound in the filtrate.
  • two systems in tandem would be used for these two distinct, adsorption and elution, stages of an affinity separation process.
  • the suspension using pump P10, was fed through the inlet feed port F into the centrifuge C, run at 7,000 rpm and controlled by a speed controller 50, the centrifuge having two functioning outlet ports 45 and 46. Port 47 is used only as an outlet for lighter-than-water fractions and was kept closed in the experiment.
  • the addition pump P10 was stopped and solids ejection mechanism was actuated hydraulically by means of a tap-water inlet, controlled with valves 51 (flow control) and 48 (actuation control). This ejected concentrate through port 46 (containing 350 ml per each ejection) was collected in a circulating container 42, while supernatant was collected in container 44.
  • Example 2 Purification of human IgG using a continuous centrifuge and Orbicell® Simul TM M affinity beads.
  • An affinity separation of an IgG antibody was conducted using the Orbicell® thiophilic support (Orbicell® Simul MTM) and a bead circulating loop system depicted in Figure 4.
  • Human immunonoglobulin (IgG) was obtained by purifying it from commercial sources of plasma paste I, II and III. Before use, the IgG was subjected to ultra-filtration through a 100 K molecular weight cut off membrane, to eliminate fragments like F c or F at> - The Simul MTM beads were routinely recycled by washing in 0.2 M NaCI and 1 M acetic acid, then 6 M urea, then water until a pH of near neutrality was obtained.
  • the beads were incubated in the same buffer as used for washing, namely: 0.45 M Na 2 S0 4 and 5 mM phosphate, pH 8.0 (binding-washing-buffer, BWB). About 320 g of wet beads (approximately 120 g of dry weight) were mixed with 600 ml of IgG solution, containing 1 mg/ml of protein. The thoroughly dispersed suspension was stirred (magnetically) for 1.5 hours at room temperature. The suspension was fed into the centrifuge C (run at 5000 rpm and at a feed rate of about 420 ml/min ) and operated in the same manner as shown in Fig.
  • Example 2 the supernatant discharge was collected in fractions, that were monitored by OD 280 and washing was stopped when ODs approached background values.
  • the elution of the IgG was affected with a buffer comprising 50 mM sodium acetate, at pH 5.0, and fraction 7 to 14 were collected; (the elution speed depends on the binding constant between antibody and affinity ligands, grafted on the surface of the beads, and not on the centrifuge).
  • the total OD 28 o of the wash (BWB) material was 234, and of elute was 452 OD units. Since the total of the applied OD units was 706, and the total washed + eluted (234+ 452) OD units amounted to 686, the recovery of proteins is calculated to be a high, 93%.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Separation Of Solids By Using Liquids Or Pneumatic Power (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé et un dispositif pour la séparation et la purification d'affinité. Les étapes sont les suivantes: (a) incubation de perles reconstituées avec un fluide à faibles concentrations de composé cible (charge); (b) introduction des perles 'chargées' dans un réservoir de circulation en amont, relié à un système de séparation solide-liquide aux fins d'alimentation (du type centrifugeuse continue à grande vitesse); le système en question présente deux points d'accès supplémentaires, l'un communiquant vers l'aval pour le transport de solutés hors de la boucle par le liquide surnageant (centrifugat), et l'autre communiquant vers l'amont pour le retour de concentré dans le réservoir de circulation, ce qui permet de maintenir efficacement les perles d'affinité dans la boucle; (c) concentration de la suspension des perles incubées en un volume minimum restant fluide et pompable; (d) dilution du concentré dans le réservoir de circulation avec des volumes de solution tampon fraîche et poursuite de la centrifugation, ce qui permet d'éliminer les sous-produits et les impuretés solubles non liés, dans le liquide surnageant (centrifugat); (e) modification des conditions dans le réservoir de circulation pour libérer efficacement à partir du complexe le composé cible avec les perles d'affinité, puis centrifugation pour séparer le composé cible des perles non chargées, et transport de ces perles hors de la boucle vers l'aval par le liquide surnageant (centrifugat); et (f) lavage des perles d'affinité restant dans la boucle, puis transfert de ces perles vers un réservoir de rétention pour des opérations supplémentaires de nettoyage, de stérilisation et de validation, avant le retour dans la boucle.
PCT/US2002/023546 2001-07-23 2002-07-23 Separation solide-liquide a grande vitesse WO2003010512A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002355285A AU2002355285A1 (en) 2001-07-23 2002-07-23 High-speed, solid-liquid separation

Applications Claiming Priority (2)

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US30742601P 2001-07-23 2001-07-23
US60/307,426 2001-07-23

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WO2003010512A2 true WO2003010512A2 (fr) 2003-02-06
WO2003010512A3 WO2003010512A3 (fr) 2003-10-02

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1942939B1 (fr) 2005-09-30 2018-05-30 Medimmune Limited Composition comprenant un anticorps anti-interleukine 13
CN113069790A (zh) * 2021-03-30 2021-07-06 临沂大学 用于光系统ii内周天线cp43和cp47的提取制备装置及方法
US11155575B2 (en) 2018-03-21 2021-10-26 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8600582D0 (en) * 1986-01-10 1986-02-19 Ca Minister Nat Defence Purifying biological materials
CA2127605A1 (fr) * 1993-12-23 1995-06-24 Peter J. Degen Methode de separation par affinite
US6315900B1 (en) * 1998-06-03 2001-11-13 Accurate Polymers Static separation method using non-porous cellulose beads

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1942939B1 (fr) 2005-09-30 2018-05-30 Medimmune Limited Composition comprenant un anticorps anti-interleukine 13
EP1942939B2 (fr) † 2005-09-30 2021-07-07 Medimmune Limited Composition comprenant un anticorps anti-interleukine 13
US11155575B2 (en) 2018-03-21 2021-10-26 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods
US12091433B2 (en) 2018-03-21 2024-09-17 Waters Technologies Corporation Non-antibody high-affinity-based sample preparation, sorbent, devices and methods
CN113069790A (zh) * 2021-03-30 2021-07-06 临沂大学 用于光系统ii内周天线cp43和cp47的提取制备装置及方法

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Publication number Publication date
WO2003010512A3 (fr) 2003-10-02
AU2002355285A1 (en) 2003-02-17

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