WO2008154931A1 - Adsorbent beads suitable for use in separation of biological molecules - Google Patents

Adsorbent beads suitable for use in separation of biological molecules Download PDF

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WO2008154931A1
WO2008154931A1 PCT/DK2008/050153 DK2008050153W WO2008154931A1 WO 2008154931 A1 WO2008154931 A1 WO 2008154931A1 DK 2008050153 W DK2008050153 W DK 2008050153W WO 2008154931 A1 WO2008154931 A1 WO 2008154931A1
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Prior art keywords
beads
surface
plasma
binding
adsorbent
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PCT/DK2008/050153
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French (fr)
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Ayyoob Arpanaei
Timothy John Hobley
Owen Robert Tyrynis Thomas
Bjørn WINTHER-JENSEN
Peter Kingshott
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Danmarks Tekniske Universitet (Technical University Of Denmark)
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Publication of WO2008154931A1 publication Critical patent/WO2008154931A1/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/10Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/26Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265Adsorption chromatography
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268Macromolecular compounds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/34Regenerating or reactivating
    • B01J20/3441Regeneration or reactivation by electric current, ultrasound or irradiation, e.g. electromagnetic radiation such as X-rays, UV, light, microwaves
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/10Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D2215/00Separating processes involving the treatment of liquids with adsorbents
    • B01D2215/02Separating processes involving the treatment of liquids with adsorbents with moving adsorbents
    • B01D2215/021Physically moving or fluidising the adsorbent beads or particles or slurry, excluding the movement of the entire columns

Abstract

The present invention concernsthe construction and use of beads with non- adsorptive surfaces for separation/isolationof biological molecules. The construction was conducted by usingan irradiation method, e.g.plasma treatment of the beads surface. The modified beads are multifunctional and selectively bind molecules of interest (e.g. proteins) without binding tolarger molecules in the liquid, such as DNA. This makes the beads especially adapted for preparative and large/industrial scale separation processes of e.g. proteinsor plasmid DNA.

Description

Adsorbent beads suitable for use in separation of biological molecules

Technical field of the invention

The present invention relates to the field of producing equipment and consumables and use of them for bioprocessing, bio-product capture and purification. In particular the present invention relates to the process of manufacturing and use of adsorbent beads for preparative and large/industrial scale chromatography systems including expanded bed adsorption, and packed bed chromatography, as well as other adsorptive methods, including fluidized bed, batch adsorption, and mixed tank reactors.

Background of the invention

In general, downstream processing accounts for a major part of the total production costs for biological products, especially biopharmaceuticals. Moreover, a significant part of the costs for biopharmaceutical production arises from the chromatography steps which are frequently used in such processes. Chromatographic methods are well established methods for analyzing and purifying biological products. Chromatography may be preparative or analytical. Preparative chromatography seeks to separate and purify or partially purify the components of a mixture and can thus be used in biochemical purification methods. Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC).

Packed bed chromatography is a type of liquid chromatography and is the most conventional type of preparative chromatography used in commercial processes for production of biological products such as therapeutics, foods and fine protein preparations. Packed bed chromatography uses small adsorbent beads of a solid phase packed tightly into a column and held in place with a top and bottom net. The liquid containing the substances to be separated is pumped into the column often from the top in the loading step. Some substances bind to the column (usually those of interest), others do not (usually those not of interest) and pass through. Subsequently the adsorbent beads in the column are washed by percolating a liquid through the column and then the substances bound to the column material are released by percolating a different liquid through the column. Packed bed chromatography is in the wide variety of cases not compatible with crude liquids containing large and undissolved substances in suspension. It is in the majority of cases only compatible with clarified clear liquids in which all undissolved components in suspension have been removed first, so that only dissolved components less than about 0.45 micrometer are present.

Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. Size exclusion chromatography (SEC) is also known as gel permeation chromatography or gel filtration chromatography and separates substances on the basis of size. The major limitation of most types of chromatography is the lack of specificity for only one component. Affinity chromatography (AC) is a method of separating biochemical mixtures, based on a highly specific biological interaction. In the AC method a molecule of interest is reversibly bound on an insoluble matrix, such as agarose beads, functionalized by a so called "affinity ligand", while the remaining molecules flow through the column. The molecule can then be eluted under conditions which separates the molecule from the insoluble matrix. An affinity ligand is a small chemical or biological molecule that is chemically (covalently) fixed to an insoluble matrix to give the matrix (e.g. an adsorbent bead) specificity for binding other substances.

A special form of liquid chromatography is expanded bed adsorption (EBA) chromatography, where the solid phase particles are placed in a column and the liquid phase is pumped in from the bottom and exits at the top, while the bed is fluidized during the loading step. In the loading step some substances bind to the adsorbent beads, whilst others (usually the unwanted ones) do not. The gravity of the particles ensures that the solid phase does not exit the column with the liquid phase. When loading is complete, the adsorbent beads can be washed using a washing buffer. Subsequently the compounds bound to the adsorbent beads can be recovered using an elution solution. EBA is thus a special type of fluidized bed system. The difference between an EBA system and a conventional fluidized bed adsorption chromatography system is that beads are different in size and/or density in EBA. This gives increased chromatographic performance due to lower axial mixing in EBA compared to that in conventional fluidized beds. Conventional fluidised beds typically have adsorbent beads that all have similar size and density. EBA and conventional fluidised beds are compatible with clarified solutions and can potentially be compatible with crude solutions since it can potentially integrate or combine different steps in downstream processing of bioproducts and this can potentially lead to higher yields as well as lower capital and operating costs.

Batch adsorption is compatible with clarified and crude solutions and is a purification and separation technique using adsorbent beads. In batch adsorption, small solid phase adsorbent beads are added to a vessel or tank containing the liquid with the substances to be separated and/or purified and the suspension is mixed to allow adsorption to occur in what is called the loading phase. The liquid is then removed from the solid phase by e.g. draining, decanting, pumping, centrifugation etc. leaving the loaded adsorbents behind. The loaded adsorbents can then be washed and the compounds bound to them released and recovered using suitable elution solutions.

In preparative adsorptive separation methods such as packed bed chromatography, batch adsorption and expanded bed adsorption chromatography, a major problem is binding of unwanted substances to the outer surface of the (usually) porous adsorptive solid phase beads. In particular, the binding of large soluble particulates such as DNA, RNA or viruses or insoluble particulates such as cells, cell debris, protein aggregates, DNA and RNA aggregates to the outer surface is a problem. In packed bed chromatography binding of these substances can lead to blockages in the column or vastly reduced capacity. In batch adsorption, aggregation of the adsorbent solid phase can occur and reduces capacity and causes problems for cleaning and re-use of the solid phase. In EBA, the adsorbent beads can suffer from diminished capacity and aggregation causing the hydrodynamic properties of the column to be significantly impaired.

EBA was originally conceived to be compatible with crude bio-feedstocks containing crude substances or particulates which may be a mixture of insoluble and soluble biological molecules e.g. proteins, peptides, genomic DNA fragments, RNA molecules, viruses, cells and cell debris.

It was hoped that EBA would be a generic solution for combining solid-liquid separation (i.e. clarification) and primary product capture, since in theory, crude substances (insoluble and soluble particulates) can pass through the fluidised bed without causing blockage. Subsequently a number of reports showed that all or part of the clarification, concentration and initial purification steps for a biological product can be combined and integrated into one single unit operation by using this EBA system. Despite the commercialization of the chromatography technology, especially the EBA chromatography (Pharmacia, now GE Healthcare) was found to have a critical problem, namely that for the vast majority of process streams, the hydrodynamic properties of EBA were fatally compromised. This condition arose from cross-linking of the fluidized adsorbent beads by crude substances (particulates) in the feed. Bead cross-linking in EBA is most severe with anion-exchangers and led to the working hypothesis that negatively charged cells, large debris or DNA strands were able to attach to more than one adsorbent bead. The mechanism for cross-linking was hypothesized to be similar to flocculation, and was demonstrated after developing new prototype pellicular EBA adsorbents of smaller than normal size. These new adsorbents were designed for the purification of plasmid DNA. However, results showed that aggregation of the adsorbent beads inhibits the ability of EBA to purify plasmid DNA. In order to relieve the problem of bead cross-linking in EBA systems, a pragmatic approach has been taken where methods were proposed to search for approaches for broth conditioning (e.g. to change pH and/or conductivity) under which interaction of the crude substances (particulates) with the adsorbent beads were minimized. Another proposed approach was simply to break all particulates, debris or DNA into such small pieces that they could not bind to more than one bead.

In EBA the insoluble adsorbent phase, which in this case are the beads are populated with binding sites on the outer surface and within the bead, since the beads are usually at least partially porous. Binding sites on the surface bind both the product of interest and crude material causing cross-linking and thus breakdown of the hydrodynamic properties essential for EBA. Furthermore, applying the above defined broth conditioning approaches usually led to loss of capacity of the adsorbents or of compromised chromatographic properties.

Several attempts have been made to prepare beads with non-adsorptive outer surfaces for chromatography or EBA: i) coating or shielding adsorbent beads by polyelectrolytes; ii) coating or shielding adsorbent beads with a layer of an inert

(non-ionic) polymer; and iii) using adsorbent beads which have been constructed in such a way that no ligands exist in their outer layers (2-3 μm deep) (lid particles). These approaches attempt to combine two functionalities in one: namely adsorption of proteins and size exclusion of large particulates.

By using such an approach the selectivity of the adsorptive method is increased. In the case of EBA, this is supposed to allow the EBA system to exploit its potential and to act as a multifunctional system. Nevertheless, the above approaches to design of new EBA beads are problematic. Shielding adsorbent beads with an electrolyte polymer could lead to unspecific or non-specific adsorption, because electrolytes are charged. Furthermore, the reusability of such shielded beads is questionable. Coating adsorbents with a layer of agarose or other gel-forming polymers (i.e. the second approach above) is problematic since any inert layer must be ultra thin in order not to decrease mass transfer (molecular diffusion) of protein into the particle interior where the ligands for binding are situated. Intra-particle mass transfer has been shown as the main parameter affecting dynamic capacity of an EBA support. The coating layers obtained by using the second strategy have been too thick (with a diameter of a few microns) and it is expected they can not meet a condition of efficient mass transfer. This has limited the application of such coated adsorbent beads only to the processes in which very small molecules (e.g. organic acids) are target molecules. The same problem of mass transfer limitations can be predicted for the 'lid' beads since controlling the chemical methods used, which involve either selective ligand elimination from the surface, or removing functional groups prior to ligand coupling is extremely difficult. Over-reaction is likely to be difficult to avoid, leading to a deep layer around the adsorbent devoid of ligand and thus a long diffusion path length before the product reaches the ligands for binding. Hence further improvements of the adsorbents are needed. Plasma treatment/irradiation such as e.g. plasma, corona discharge, photo activation (UV), laser, ion beam, electron beam and gamma radiation is a method which can be used for obtaining surface modifications. "Plasma" is defined as a partially or wholly ionized medium consisting of radicals, electrons, ions and possibly neutrals and photons, which also meet additional criteria. To conduct a surface modification by plasma, a glow discharge is created in an evacuated vessel refilled by preferably a low pressure gas, since increasing the pressure leads to increased temperature. Then radio frequency (RF), microwave (MW), alternating current (AC), or direct current (DC) is used to energize the gas. Surfaces in contact with plasma will be bombarded by energetic species (e.g. ions, electrons, radicals), which transfer energy to the surface causing chemical and physical reactions. For example, during an oxygen plasma treatment, the exposed surface is oxidized and atoms and chemical groups existing on the surface will be replaced by hydroxyl and carbonyl groups. Removing molecular or atomic layers on the surface can also be conducted by using plasma etching.

The main advantages of plasma treatment of surfaces are: i) low temperature reactions, ii) changes occur to the chemical structure of the surface can be in a shallow nano-scale layer with no change to the bulk properties depending on the plasma type and power, iii) a very wide range of surface modifications are possible, and iv) low amounts of toxic by-products are formed during the treatment.

Plasma technology has also been used in the fabrication of restricted access media and materials for analytical chromatography supports and packings. These packing materials consist of two parts; (i) an internal active region for binding of metabolites which is not accessible for macromolecules like proteins and (ii) an inert outer layer or barrier. By using columns packed by restricted access media, there is no need to use an off-line pretreatment of biological sample to remove proteins and macromolecules that adhere to the packing materials causing fouling. For construction of such packings, plasma is generally used to activate the surface, which is then coated by grafting a polymer which has negligible nonspecific adsorption of macromolecules. For example, octadecylsilylated silica gel beads were treated by oxygen plasma to produce silanol groups on the beads surface. Then, the silanol groups were chemically silylated to give restricted access media.

In JP 57079448 a method has been described for the surface inactivation of packing materials for a gas chromatography column by using plasma treatment. In this method, a gaseous alkane like methane, ethane, propane, butane, pentane, hexane or an inert gas such as argon, helium, xenon was introduced to form the plasma atmosphere. Argon or oxygen plasma treatment of packing materials used for gas chromatography, in particular plasma treatment of diatomaceous earth coated with different compounds, was shown to improve the chromatographic properties of such systems by reducing adsorption of the support and was attributed to the formation of an inert coating on the support surface. In these reports, it was also shown that plasma treatment of packing material leads to reducing tailing of aromatic hydrocarbon peaks. In another report, the stationary polymer phase of the packing material of a packed column or interior capillary column surface used for gas chromatography was cross-linked by using low-temperature plasma (US 4,966,785). That led to a non-extractable polymer layer surrounding the stationary packing material. Plasma treatment has also been used to introduce oxygen or nitrogen atoms directly bonded to the surface of porous membranes with ion exchange groups (WO 98/32790). Different moieties and functionalities were bonded to the membrane through oxygen and nitrogen atoms. Plasma treatment was used in this method for activation of an inactive surface to make it more susceptible for chemical functionality (WO 98/32790).

Beads used in gas chromatography are much smaller than adsorbent beads used in preparative chromatography and EBA. Gas chromatography is an analytical technique using tiny amounts of sample which are volatilised into the gas phase and cannot be used for the preparative purification or recovery of biological compounds and substances. In analytical liquid chromatography methods such as high performance liquid chromatography (HPLC), adsorbent beads with a size range of 1-20 micron are used, but in preparative and process scale chromatography, EBA and batch adsorption, beads are usually bigger than 90 microns, although some adsorbents down to 40 microns in size have been reported. Surface modification by irradiation methods such as plasma can be used for activating a surface for further chemical reaction or modification. None of these methods have been used for surface modification of chromatography beads used for EBA, packed bed chromatography for purification, fluidized bed chromatography, batch adsorption or other adsorbent bead based unit operations for the separation and full or partial purification of proteins, DNA etc.

Summary of the invention

An object of the present invention is to provide adsorptive beads which do not aggregate with substances in the liquid, e.g. DNA, and wherein such beads are suitable for use for purifying/isolating biological molecules.

The present invention relates to a method for manufacturing chromatographic beads or other adsorptive beads, suitable for use in separation of biological molecules, said method comprising modifying beads using irradiation methods such as e.g. plasma technology. The present invention furthermore relates to beads obtained/obtainable using such methods as well as use of such beads in various separation methods.

In the present invention irradiation, such as plasma, is used to modify the beads. The modified beads do not have the above mentioned drawbacks of cross-linking when the applied liquid contains cell debris, and large molecules such as DNA. Thus the compatibility of plasma treated beads for adsorption of biological products such as proteins and exclusion of bigger materials such as DNA, cells or cell debris makes the beads especially adapted for bioprocesses, such as EBA. Beads used for preparative chromatography such as EBA are larger than beads for analytic chromatography, which are in the size range of 1-20 micron.

Brief description of the figures

Figure 1 shows Strategies applied for plasma surface modification of adsorbent beads. Here '+' represents a quaternary amine ligand distributed on the surface and throughout an adsorbent. Figure 2 shows Schematic diagram of the experimental apparatus used for plasma treatment of Q HyperZ beads.

Figure 3 shows Chemical structures of the monomers used for graft polymerization with plasma.

Figure 4 shows Light microscopy images of unmodified (a), surface-etched (b) and surface-coated (c) Q HyperZ beads.

Figure 5 shows scanning electron microscopy (SEM) images of unmodified (a), surface-etched (b) and surface-coated (c) Q HyperZ beads, with magnification of 1000 (top) and 10000 (bottom). The bar represents a size of 10 micron (top) or 1 micron (bottom).

Figure 6 shows reduction in binding capacity in batch binding tests for surface- coated and surface-etched beads expressed as a percentage of the capacity of untreated Q Hyper Z adsorbents. Batch biding tests with the protein bovine serum albumin (BSA) for 2 (black bar) and 30 (dark grey bar) minutes, and homogenized calf thymus DNA for 2 (white bar) or 30 (light grey bar) minutes.

Figure 7 shows comparison of the kinetics of DNA and protein binding to modified and unmodified Q Hyper Z adsorbents in batch reactions with homogenized calf thymus DNA (a and b), plasmid pUG6 (c and d) and BSA (e and f). Legend : unmodified adsorbents (squares, black bar), surface-coated (VA170-3) (triangles, grey bar) and surface-etched (Et220-3) (diamonds, white bar). Here, C/Co represents the ratio of unbound molecule in the supernatant after binding at a given time point (C) versus the amount of the molecule originally present before binding began (Co).

Figure 8 shows comparison of the reduction in binding capacity of calf thymus DNA (circles), plasmid DNA (squares) and protein BSA (triangles) to (A) VA170-3 or (B) Et220-3 adsorbents compared to the unmodified adsorbents. The data in Figures 7 B, D and F was used for generation of the results. At each time point for the particular plasma modified adsorbent, the reduction in binding capacity for DNA or protein was calculated as a percentage of the DNA or protein capacity measured at that time point for the unmodified adsorbents.

Figure 9 shows breakthrough curves during EBA for a) protein BSA, b) homogenized calf thymus DNA and c) plasmid DNA. DNA and BSA concentrations in the feeds were 0.06 mg.mL"1 and 1 mg.mL"1, respectively, in 50 mM Tris-HCI pH 8 buffer containing 100 mM NaCI. Unmodified Q HyperZ (squares), ET220-3 beads (diamonds), VA170-3 beads (triangles).

Figure 10 shows bed contraction profiles in EBA for a) protein BSA, b) homogenized calf thymus DNA and c) plasmid DNA. DNA and BSA concentrations in the feeds were 0.06 mg.mL"1 and 1 mg.mL"1, respectively, in 50 mM Tris-HCI pH 8 buffer containing 100 mM NaCI. Unmodified Q HyperZ (squares), Et220-3 (diamonds), VA170-3 (triangles).

The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Purification is herein intended to encompass any full or partial purification or isolation of biological molecules from a mixture comprising different compounds. Purification is obtained by separating the molecules of interest from other components. What is thus obtained is an enrichment/purification of the molecules of interest. Examples of biological molecules that may be isolated/purified include various proteins such as e.g. antibodies, hormones, enzymes, etc. as well as peptides, oligosaccharides, viruses, virus-like particles, protein complexes, oligonucleotide molecules, small molecules, antibiotics, etc Irradiation is defined herein as the process by which an item is exposed to radiation. In common usage the term refers specifically to ionizing radiation. Plasma treatment is a kind of irradiation technique.

Plasma as used herein is defined as a partially or wholly ionized medium consisting of electrons, ions and possibly neutrals and photons. Gas plasma can for example be generated by glow discharge in a gas atmosphere at reduced pressure ("vacuum"). It creates a stable, partially ionized gas that may be utilized for effecting reactions on the surface of the substrate because the gas plasma environment activates even chemical compounds that are un-reactive under normal conditions. To conduct surface modification by plasma, an electric field is created in an evacuated vessel refilled by low pressure gas. Radio frequency, microwave, altering current or direct current is used to energize the gas. The plasma system generates plasma through the ionization of a particular gas. Such gas may be for example, selected from the group of gaseous alkanes like methane, ethane, propane, butane, pentane, hexane. The gas could further be oxygen, water, air, nitrogen, ammonia. Under this condition, the gaseous element reacts with the surface and forms a reacted layer on that. For example, if oxygen is used oxygen atoms will react with the atoms on the surface and form oxidized forms of those atoms. For example for Carbon, plasma treatment of the surface with oxygen or air forms hydroxyl, carbonyl and carboxylic groups on the surface. The reactability of atoms and elements is different from each other. Furthermore, plasma treatment can be carried out to coat a surface by polymerization of an introduced monomer or oligomer on the surface or coupling polymer chains to the surface. To introduce monomer, oligomer or polymers into the chamber, inert gases including helium, neon, argon, krypton and xenon are usually used as carrier. Besides plasma, different irradiation treatments including corona discharge, photo activation (UV), laser, ion beam, electron beam and gamma radiation also can be used for surface treatment.

A typical plasma generator consists of a reaction chamber, a high frequency generator and matching network, high vacuum system, gas delivery system and temperature controllers. The main advantages of plasma treatment of surfaces are: i) low temperature reactions, ii) changes can occur to the chemical structure of the surface in a shallow nanoscale layer with no change to the bulk properties, iii) a very wide range of surface modifications are possible, and iv) low amounts of toxic by- products are formed during the treatment.

The plasma technology is, according to the invention, used in two different strategies to either: (i) shave off only those ligands/binding sites located on the surface of adsorbent beads (surface etching and oxidation), or (ii) to cover surface ligands by coating the adsorbent beads with a nanoscale polymer layer (surface polymerization).

The method can also be applied during preparation of adsorptive media. In preparation of adsorptive media sometimes a two-step method is used. In the first step (so-called activation), some reactive sites are created and in the second step (so-called functionalization) these reactive sites are functionalized by coupling particular ligands to the reactive sites. One can use plasma treatment after the first step to deactivate reactive sites located on the bead exterior by either i) shaving off reactive groups or ii) adding a polymer on the surface. Therefore, in the next step (functionalization), ligands can be coupled to the reactive sites which only exist within the bead and not on the surface.

According to strategy i) the liqands/bindinq sites are shaved off from the surface of the beads. By shaving off is meant that the binding sites are removed from the surface and the beads do not bind substances on the surface. This is achieved by etching and oxidation of the surface of the beads using plasma together with an oxidizing gas such as oxygen or air which contains oxygen. With oxygen or air plasma treatment the exposed surface is oxidized and atoms and chemical groups existing on the surface will be replaced by mainly hydroxyl and carbonyl groups. By plasma etching the molecular or atomic layers on the surface is removed. Vacuum inside the reaction chamber is implemented before the plasma is created, vaporizes the water within the beads, and the pressure is kept low. The oxidizing gas is added and the gas is energized by applying an electric field. According to strategy ii) the binding sites on the surface of the beads are covered by a thin layer of polymer. This is termed surface polymerization which is to cover the ligands by coating the adsorbent beads with a nano-scale polymer layer. Although the layer is on the nano-scale, thicker layers can also be applied. Nano- scale is defined below. The polymers on the surface are obtained by adding monomers, oligomer or a polymer solution together with a carrier gas to the chamber with the beads and vacuum. The monomers are added as long as the beads are subjected to the plasma and activated by oxygen or other activating gaseous materials. The plasma polymerization process is initiated and perpetuated by means of an electric field. The exact procedure is outlined in example 1

Nano-scale is defined here as a modification with a size or thickness or length that can be measure in the nano-meter scale. This may range from e.g. 0.001 nano- meters to 10000 nano-meters.

Carrier gas is a gas which carries monomers, oligomers or polymers into the plasma chamber. Often, argon is used as the inert diluent or carrier gas which aids in the polymerization of the precursor. Other gases which can be used include, but are not limited to, helium, neon, xenon.

To conduct surface modification by plasma, a glow discharge is created in an evacuated vessel refilled by a gas at low pressure e.g. 10-20 Pa, since increasing the pressure leads to increased temperature. Glow discharge refers to the illuminating environment created in a plasma reactor due to a decay in the energy level of excited species (radicals, ions etc.) in the plasma chamber.

The method concerns steps of treating beads with irradiation e.g. plasma in a reactor, and adding gases such as argon, helium, xenon etc and/or monomers, oligomers or polymers, such as e.g. vinyl alcohol, vinyl actetae, ethylene oxide, etc. and oligomers and polymers of the aforementioned monomers. After a certain time depending on the plasma method (e.g. from 2 minutes to 2-3 hours or even up to 24 hours, for microwave plasma in a rotating reactor or 2 minutes up to 24 hours for other types of reactors and other types of irradiation methods), the plasma is stopped, and the beads are collected, washed and used. The result of the plasma treatment is a change in the surface but not in the bulk properties of the beads.

Monomers are small molecules that may become chemically bonded to other monomers to form a polymer. Examples of monomers are hydrocarbons such as the alkene and arene homologous series. Hydrocarbon monomers such as phenylethene and ethene may form polymers such as plastics like polyphenylethene, e.g. polystyrene and polyethene e.g. polyethylene or polythene. Other monomers include acrylic monomers such as acrylic acid, methyl methacrylate, and acrylamide. Amino acids are natural monomers, and polymerize to form proteins. Glucose monomers can also polymerize to form starches, amylopectins and glycogen polymers. The lower molecular weight compounds built from monomers are also referred to as dimers, trimers, tetramers, quadramers, pentamers, octamers, 20-mers, etc. if they have 2, 3, 4, 5, 8, or 20 monomer units, respectively. Any number of these monomer units may be indicated by the appropriate prefix, e.g., c/ecamer, being a 10-unit monomer chain or polymer. The monomers can e.g. be such as vinyl acetate, vinylpyrrolidinone, safrole, vinyl alcohol. Suitable monomer sources are those that provide a poly (ethylene oxide) or essentially similar poly(ethylene glycol) type structure when vaporized and plasma deposited on a substrate. Examples of compounds which may be utilized in the plasma coating process of the invention include ethers such as 12-crown-4, 15-crown-5, tri(ethylene glycol) dimethyl ether, tetra(ethylene glycol)dimethyl ether, tri(ethylene glycol) divinyl ether; triethylene glycol; poly(ethylene glycol) of various molecular weights; ethylene oxide; propylene oxide; dioxane; polysaccharides such as dextrin; acrylonitrile; 2-hydroxyethyl methacrylate; and quaternary ammonium compounds. Any monomers able of polymerization can be used in the present invention.

Oligomers are short polymers with a relatively low numbers of units.

Polymers the term polymer molecule is defined as a molecule formed by covalent linkage of two or more monomers to form a long linear or branched chain. The term "polymer" may be used interchangeably with the term "polymer molecule".

Polymer coating In preparing a multifunctional bead, for process scale of e.g. EBA chromatography, batch adsorption or packed bed chromatography, a bead with different functionalities on the surface and within the interior is required, for which the outer zone or neutral layer must be as thin as possible to maximize the availability of binding groups in the interior active zone. Irradiation methods have the ability to give nano-scale modification of surfaces. By appropriate selection of monomer and plasma parameters, uniform and very thin layers of polymer may be deposited on the surface. The polymer layer is in the range of nano-scale. The polymer layer is formed by plasma polymerization of the corresponding monomer. Suitable monomers are, for example, vinyl acetate, vinyl alcohol, ethylene oxide, vinylpyrrolidone etc., or substituted derivatives thereof wherein the substituent may be a halogen atom such as a fluoro or chloro group, or any other group, such as a nitro group which does not substantially interfere with the polymerization. The method proposed here has the potential to be used for modifying adsorptive or reactive surfaces with new generations of 'smart' polymers with multiple and different functionalities. For example, beads could be coated by thermo/pH/photo sensitive polymers through a graft polymerization treatment. These beads could be used for separation of biological products or other applications in bioprocesses.

Polymerization is a process of bonding monomers, or "single units" together through a variety of reaction mechanisms to form polymers. Polymer coating monomers are covalently attached to the surface of the beads building a polymer layer on the surface and creating a non-adsorptive surface, that is inactive/neutralized/non-adsorptive and to which molecules can not adhere. An inactive surface can be one which contains chemical groups which do not interact with other species of concern in the solution being processed. In other words other species of concern are not attracted to the groups on the surface, cannot stick to them appreciably through e.g. electrostatic, hydrophobic, van der Waals, or affinity interactions, and do not react chemically with them e.g. do not form covalent bonds.

Beads as used herein, refers to beads/particles that are porous either in part or completely porous, or non-porous, are spherical in shape or ovoid, and can have other regular shapes such as cylinders, cubes, diamonds and pyramids, but can also be irregularly shaped and may be modified for example, such polymeric beads may include but be not limited to polystyrene, polyolefins such as polyethylene and polypropylene, polyurethanes, polyvinyl acetates, polyvinyl alcohols, polyesters such as polyethylene terephthalate, nylon such as nylon 6 and nylon 6,6, poly (vinylpyrrolidone), poly (vinylfluoride), poly (vinylchloride), poly (methylmethacrylate), poly (methacrylate), and copolymers such as poly (styrene butadiene) and composite materials; and natural polymers and modified natural polymers such as agarose, cellulose, chitosan, chitin and other hydrogel based adsorbent beads (i.e. composed of hydrated polymers) and inorganic beads like silicones, silica and also composite materials. Such composite beads can contain inorganic core materials imbedded in them or forming a skeleton structure such as zirconia, silica, glass, tungsten carbide, stainless steel, steel, or other metals, quartz and other natural or man made materials. Such inorganic particles may include, but be not limited to silicon dioxide (silica available in various grades such as Fumed Silica Aerosil 2) from Degussa, South Plainfield, N. J. ), titanium dioxide, (available from Whittaker, Clark Daniels, Inc, also of South Plainfield, N. J.), alumina, alumina coated silica, carbon nanotubes, ceramics, glass beads (available from Superior Micropowders, Alberquerque, N. M.), iron oxide, zinc oxide, magnesium oxide, zeolites, alumina silicates, boron oxide, silicon nitride, PZT piezoelectric ceramics, silicon oxynitride, and tantalum pentoxide, hydroxyl apatite. Beads used for separation bioprocesses are preferably in the size range of 40 micron to 1000 micron, more preferably in the range of 50-100 micron for packed bed adsorbents, and again more preferably 50-300 micron EBA adsorbents and 50 - 400 micron for batch adsorption materials.

Beads include adsorptive beads which are defined herein as beads that can bind substances on the surface and within the bead, including the inner surface of porous beads, through adsorption e.g. have binding sites/liqands on the surface. The term "binding site/ligand" is defined herein as a chemical group that is coupled or fixed to the bead matrix and is able to adsorb or bind to molecules in solution due to special properties like positive or negative charge (i.e. ion exchange), hydrophobicity, affinity, Van der Waals, hydrophobic charge induction, ion pairing etc. It is these binding sites/ligands on the surface of the adsorbent which unspecifically or non-specifically bind materials such as DNA, RNA, cells, cell debris, viruses and insoluble agglomerates and can cause unwanted aggregation of the adsorbents or blockage of the pores of the adsorbents.

Non-adsorptive/neutralized/inactive surface beads as used herein refer to any beads which do not appreciably bind substances on the surface, and which do not have binding sites/ligands on their surface e.g. no charges on the outside. The terms non-adsorptive, inactive and neutralized are used interchangeably in the present application. The two types of irradiation treatment i) and ii) as described above on the adsorbent beads reduced or eliminated binding sites on the surface. Surface modified beads are defined as beads, which are treated in such a way that the surface is modified e.g. changed in any way in relation to the surface of not treated beads as defined above (beads). Surface modification includes any kind of addition or removing of substances on the surface of the adsorbent beads e.g. surface shaving as well as e.g. polymer coating. Surface-shaved beads are adsorbent beads with no ligands e.g. no binding sites are left on the outside of the beads, but the binding sites inside are intact. Surface-coated beads are when polymer or monomer is added and the binding ligands are covered by a thin layer, but the binding sites inside are intact and the pores of the beads are not significantly blocked. The result is that the beads have no binding or no accessible binding sites on the surface, but still have accessible binding sites inside. In contrast to commonly used beads, such as EBA adsorbents, which have binding sites on the surface and therefore potentially cross-react with substances in the liquid and with each other. The plasma treated beads do not comprise binding ligands on the surface, only in the inside of the beads and thereby prevent binding of larger molecules such as DNA, cell debris or cell walls or intact cells. Thus the beads are multifunctional with the surface inactive and their interior zone active. Further the obtained beads do not cross-link to each other or binding substances in the applied liquid, such as DNA. The term cross-linking herein describes the situation that arises from cross-linking of the fluidized adsorbent beads or bridging of the fluidized adsorbent beads by crude substances (particulates) in the liquid. This may be due to beads linking to each other or to the crude substances in the feed. Bead cross-linking in EBA is most severe with anion-exchangers and led to the working hypothesis that negatively charged cells, large debris or DNA strands were able to attach to more than one adsorbent bead. Due to the modifying of beads using plasma as described above the beads according to the invention have no or limited adsorption sites on the surface and testing those beads demonstrated that the problems of aggregation of adsorbents due to e.g. DNA was removed. The terms aggregation and cross-linking are used interchangeably in the present application.

The plasma modified beads are defined as beads subjected to plasma in such a way that the surface is modified. By modified is meant any changes on molecular or atom in the surface and any changes which alters or enhances the functionality of the beads. By functionality is meant the characteristics or properties of the beads both chemically and physically, and in particular the ability or lack of ability to bind or interact with other substances. Plasma modified beads with a nano- scale polymer coating or a shaved surface have high mass transfer rate of e.g. protein into the interior of the beads and to its binding sites in the interior of the beads, to permit binding rates and capacities for substances of interest such as proteins to remain high. Mass transfer as defined herein is the efficiency whereby the particular binding molecule e.g. the protein of interest is reaching the binding sites in the interior of the beads. Mass transfer is affected by the distance that the binding molecule must pass and the sizes of the pores it must pass through on its way to the interior of the bead to bind with the binding sites. Fast mass transfer allowing high efficiency of reaching the binding sites in the interior is promoted by short distances and large pores. Surface modification of the beads should not significantly affect the distance the molecule of interest must traverse before encountering the binding sites/ligands inside or significantly narrow the pores that it must pass through.

Beads subjected to the plasma treatment and polymer coated beads according to the invention are especially suitable for use in methods for separation of biological molecules including all adsorptive methods, which could be use for separating biological molecules, such as, but not limited to; chromatography processes and batch adsorption processes that rely on non-specific binding, affinity binding, ion exchange binding (e.g., anionic and cationic exchange), hydrophobic charge induction binding, hydrophobic binding, and any system such as packed bed chromatography, fluidized bed chromatography, expanded bed chromatography (EBA) and batch adsorption; and chromatography processes that rely on affinity/specificity such as affinity chromatography used in packed bed chromatography, fluidized bed chromatography, expanded bed chromatography (EBA) or batch adsorption. In the present context the term packed bed relates to embodiments wherein the adsorbent particles are employed in columns operating with the particles in a settled or packed state wherein all particles are fixed on top of each other. Often packed bed columns are equipped with top and bottom adaptors defining and fixing the whole adsorbent bed to avoid any movement of the beads during operation. In the present context the term "expanded bed" relates to embodiments wherein the adsorbent particles are employed in columns allowing the adsorbent to expand with an upward liquid flow through the column. The column will be designed to avoid excessive liquid mixing and turbulence in the column by using adsorbent particles with different sizes and/or densities while the individual adsorbent particles are kept in a non-fixed, dynamic state moving only in a narrow local zone in the column. While preferred expanded beds have a small mixing zone in the bottom part of the column where incoming liquid is distributed throughout the cross-section of the column, expanded beds generally operate under plug flow conditions in similarity with packed beds. In the case where the adsorbent is not held within a column it may be used as a solid phase such as in a batch adsorption process where the adsorbents are mixed with the bio-solution in a mixing vessel such as a tank and then the adsorbent is separated from the liquid by draining, settling, decantation, centrifugation, filtration.

Non-specific binding, the term non-specific binding as used herein, refers to the interactions between a protein of interest and a ligand or other compound, which is fixed to a solid phase matrix, through non-specific interactions, e.g., through electrostatic forces or hydrophobic forces at an interaction site, but lacking the structural complementarity that enhances the effects of the non-structural forces such as in affinity (specific) binding.

Unspecific binding as used herein refers to the unwanted attachment of substances to a surface, which does not occur due to interactions with a particular ligand, such as a specific affinity ligand or a non-specific binding ligand, but rather due to an ill-defined association with one or more substances that compose the particular surface. Such unspecific binding is usually weak and can be reduced or eliminated by populating the surface with desired ligands or with a particular type of covering, such as a polymer. Fouling refers to the binding of unwanted substances to the surface of adsorbent beads which thus hinders the binding of other desired products by blocking access to the ligands inside the beads and thus reducing the capacity of the adsorbents for the substances of interest. Fouling can also lead to the release of unwanted compounds that have stuck to the surface of the beads, during recovery of the wanted substances during e.g. elution steps. Fouling also refers to difficulty in cleaning the adsorbents due to the presence of unwanted substances bound to the surface of the beads that are difficult or impossible to remove by cleaning agents and solutions, which thus restricts or prohibits re-use of the adsorbents.

Chromatographic methods normally refers to the process by which a solute of interest, e.g., a protein of interest, in a mixture is separated from other solutes in the mixture by passing the mixture through a column containing adsorbent beads, which adsorbs or retains a solute more or less strongly due to properties of the solute, such as isoelectric point, hydrophobicity, size and structure, under particular buffering conditions of the process. Use of the term "chromatography" includes column and membrane types as well as monolith types. Also, according to the present invention chromatographic methods may include batch purification where a solute of interest is separated from other solutes in a mixture by subjecting the mixture to an stationary phase adsorbent in e.g. a tank, without the use of a column, and then separating the liquid from the absorbed solute by e.g. draining, decanting, pumping, centrifugation etc. leaving the loaded adsorbents behind. The loaded adsorbents can then be washed and the compounds bound to them released and recovered using suitable elution solutions. In preparative chromatography, after passing of the mixture through the adsorbents, the adsorbents are washed by passing a washing solution through the adsorbents, which is then followed by passing an elution solution through the adsorbents in order to release the solutes retained on the adsorbents so that they can be collected in a purified or semipurified state. Chromatographic methods are sometimes used to separate contaminants (any unwanted molecule or compound existing in the liquid/fluid phase together with the compound of interest) from the liquid/fluid phase rather than the molecule of interest. This invention also includes such an application.

An adsorbent is any solid substance capable of adsorbing another substance to its surface by adhesion by either direct interaction with the molecule of interest or interaction with compounds that are attached to the adsorbent. Adsorbents that are useful in various types of chromatography are well known in the art and are readily available through commercial sources.

Adsorption is operative in most natural physical, biological, and chemical systems, and is widely used in industrial applications such as activated charcoal, synthetic resins and water purification. Adsorptive chromatography is sorption processes in which certain adsorbates (molecules of interest) are selectively transferred from the fluid phase to the surface of and within insoluble, particles suspended in a vessel or packed in a column. The terms molecule of interest and adsorbate are used interchangeably.

Affinity chromatography and "protein affinity chromatography" are used interchangeably to refer to a protein separation technique in which a protein of interest is reversibly and specifically bound to a biospecific ligand, usually as a combination of spatial complementarity and/or one or more types of chemical interactions, e.g., electrostatic forces, hydrogen bonding, hydrophobic forces, and/or van der Waals forces at the binding site. These interactions are not just due to the general properties of the molecule such as isoelectric point, hydrophobicity or size but are a result of specific interactions from the molecule of interest and the ligand such as the hydrophobic and precise protein domain fit for protein A and antibody interactions, for example. Protein A is an example of an affinity liqand, which can be fixed to a solid support, e.g., Sepharose, for binding molecules that contain an Fc region. See Ostrove (1990) in Guide to Protein.

Chromatographic beads are thus beads which can be used in any of the above mentioned methods and include but are not limited to ion exchange beads, cation exchange beads, anion exchange beads, affinity beads, adsorptive beads, non adsorptive beads, expanded bed chromatography beads, hydrophobic beads, hydrophobic charge induction beads, etc.

The present invention solves the problem of unwanted substances sticking to the outer surface of adsorptive beads, which in itself is unwanted due to a number of problems such as the risk of fouling and to risk of reduction in binding capacity and risk of release of the unwanted substances into the final solution, but which can also cause unwanted side effects such as adsorbent aggregation, by providing adsorptive beads, which are modified in such a way that they selectively bind molecules of interest (e.g. proteins) without binding to bigger molecules in the liquid, they have a high mass transfer rate (of e.g. protein into its binding site in the beads). Due to the very thin nano-scale surface modification there is no unnecessary destruction of ligands inside the adsorbent and they can be reused since they are easy to clean since the surfaces do not have unwanted substances stuck to them. The plasma treated beads do not comprise liqand on the surface, only in the inside of the beads, and thereby prevent binding of larger molecules such as DNA, RNA or other big particulates like intact cells and cell debris, viruses, virus like particles, plasmids.

Until now no commercially viable method for producing beads without surface ligands are known and the method disclosed herein therefore provides a huge advance in the field of preparative chromatography as well as other adsorptive methods in preparative and large/industrial scales.

The present invention thus concerns a method for modifying beads by using irradiation, such as plasma, in particular glow discharge plasma, to shave molecules off the surface of the adsorptive beads or add polymers to the surface. The objective is to stop binding on the surface and at the same time not change the binding properties inside the beads.

As mentioned above, the present invention relates to a method using irradiation for the preparation of multifunctional beads with non-adsorptive surfaces for adsorptive separations of use in bioprocessing, said method comprising An irradiation method, in particular plasma treatment, was used for nano-scale modification of the surface of beads used for EBA bioprocessing, batch adsorption, packed bed chromatography and fluidised bed adsorption, to achieve a multifunctional bead with one functionality on the surface and another one in the interior of the treated beads. The surface modification was based on the inherent characteristics of an irradiation method like plasma, to make a nano-scale thin hydrophilic neutral layer surrounding the bead. In particular, beads which had two functionalities were obtained by plasma treatment: (a) insignificant adsorption of big particulates like DNA molecules and (b) adsorption of smaller molecules like proteins. Plasma treatment was shown to affect only a shallow layer of the surface of the beads and not to change bulk properties or the structure of the treated beads. The applied procedure for modifying the adsorbent surface is depicted in Figure 1. Here, plasma technology was used in two different strategies to either: (i) shave off only those ligands located on the surface of adsorbent beads (surface etching and oxidation), or (ii) to cover surface ligands by coating the adsorbent beads with a nano-scale polymer layer (surface graft polymerization).

Adsorbent beads for industrial and large scale bioprocesses/isolation of biological molecules are manufactured using "wet chemistry" methods such as e.g. polymerization reactions for obtaining porous hydrogels (typically agarose), as well as for activation and functionalisation of the beads during the attachment of ligands or for other modifications.

This is also reflected in the reported attempts to make EBA beads without ligands as discussed above. Even new generations of EBA adsorbents such as those based on porous zirconia are filled with a hydrogel obtained using wet chemistry methods. It thus follows that it has not previously been suggested to use any surface modification process conducted under dry vacuum conditions (i.e. plasma technology).

The other feature of the present invention relates to the application of such an adsorbent in a bioprocess, an adsorbent with a non-adsorptive surface where the thickness of the non-adsorptive layer is in nano-scale, can be used for separation of a molecule of interest e.g. proteins away from excluded bigger molecules or moieties in the liquid, such as DNA molecules, intact cells and cell debris. Also the applications include separation processes in which proteins and other small molecules like RNA molecules or fragments are considered as contaminants, and bigger moieties like plasmid DNA molecules and viruses are considered as the molecule of interest. In such an application the adsorbents with non binding surfaces described here, can be used to separate contaminants (e.g. proteins, RNA molecules, etc.) from a solution containing plasmid DNA, viruses etc.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. The present invention thus relates to a method for the manufacture of beads suitable for use in purification of biological molecules, said method comprising modifying beads using an irradiation method. According to a preferred embodiment of the present invention, the irradiation method is plasma treatment.

According to another preferred embodiment of the present invention, the surface of the beads are etched and oxidized.

According to yet another preferred embodiment of the present invention, compounds selected from the list consisting of: monomers, oligomers, polymers, and any mixture thereof are covalently attached to the surface of the bead.

In yet another embodiment of the present invention, the oxidized layer and/or the layer of covalently attached compounds, respectively are 1 nanometer to 10 microns. The layer may alternatively be 0.001 nm to 10 microns, or from 1 to 1000 nanometer, or from 1 to 100 nanometer, or from 1 to 10 nanometer.

In another preferred embodiment of the present invention, the beads have a diameter of 40 to 1000 microns.

The present invention thus furthermore relate to beads suitable for use in purification of biological molecules, wherein said beads are obtainable and/or obtained by a method according to the present invention.

Another aspect relates to use of beads according to the present invention in expanded bed adsorption chromatography.

Yet another aspect relates to use of beads according to the present invnetion in batch adsorption processes.

Yet another aspect relates to use of beads according to the present invention in fluidized bed processes. A final aspect relates to use of beads according to the present invention in packed bed chromatography processes.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further detail in the following non-limiting examples.

Examples Plasma reactor

To implement a surface modification of beads used for bioprocesses, a plasma atmosphere can be created in a horizontal chamber. To do that, both electrodes (anode and cathode) can be attached on the surface of the chamber. An electrical field is created within the chamber by using an electric energy generator which is equipped with a frequency converter and generates a certain frequency. The chamber could be made from glass (Pyrex) or quartz or any other electrically resistant material. In another configuration, one electrode can be a metal bar mounted along the center of the horizontal chamber and another electrode attached on the surface. To increase the volume of the plasma atmosphere and homogeneity of it, the second electrode can be a metal sheet covering the outer surface of the plasma chamber wall. To be able to better expose the whole surface of the beads to the plasma, the said chamber can be rotated or tumbled.

Another configuration for the reactor can be a vertical reactor which has been equipped with 2 electrodes on the wall or one inside in the center and another one on the wall.

To expose the whole surface to the plasma atmosphere, an active gas like oxygen, air, water vapor (for surface etching and oxidation) or a carrier gas like argon carrying a monomer or oligomer or polymer can be applied from one side of the chamber for the first configuration, or in the case of the second configuration from the bottom of the chamber/column through a net which keeps the beads inside the chamber but allows them to be fluidised. A circulation tube can be connected to the chamber. In both configurations, the chamber is connected to a vacuum pump to achieve the desired pressure inside the column, and also to the active gas (e.g. oxygen, air, and monomer/oligomer/polymer) and carrier gas (e.g. argon) reservoirs. h

Plasma treatment

Surface etching and oxidation in a rotating (tumbling) plasma reactor with the set up shown schematically in Figure 2 was used for the plasma treatment of adsorbent beads. A certain amount of beads were placed within the plasma chamber. The air inside the chamber was evacuated by using a vacuum pump. This leads to a low pressure within the chamber, which is needed for a low temperature/low pressure glow discharge plasma treatment. The vacuum also vaporizes the water within the beads and is necessary for achieving the low pressure required. After achieving a certain value of vacuum, the pressure inside the plasma chamber was increased by opening the oxidation gas (e.g. air or oxygen or water) valve to adjust the pressure to the desired value (e.g. 10 Pa). Tumbling was started with a low rate (e.g. 10-30 rpm). Electrodes were connected and power was turned on. Visible radiation showed that plasma was being generated. After treatment, the tumbling was stopped and power was turned off. Electrodes were then disconnected and evacuation was ceased. The air valve was opened to allow pressure to increase to atmospheric pressure. Then, the chamber was opened and treated beads were taken out. Equipment in contact with reactive materials was cleaned before the next batch of treatment. The beads were then washed by different solvents (e.g. acetone, ethanol and water).

Coating beads by surface polymerization

The first part of this treatment was performed as described above for the surface etching and oxidation treatment up to the point where vacuum was formed inside the plasma chamber. Next, a monomer or oligomer or polymer solution as well as a carrier gas reservoir was connected to the plasma chamber. The vacuum was started once all valves were closed. After reaching a pressure slightly less than the desired value (e.g. 10 Pa), valves 4 and 1 (Figure 2) were opened to evacuate gas within all the connecting lines. Once the pressure started decreasing, valve 3 was opened completely. Once pressure again started decreasing, valve 3 was closed to avoid losing the active compound (e.g. monomer or oligomer or polymer). Then, the carrier gas was started to flow into the active compound reservoir. Valve 3 was opened very slowly to achieve a desired pressure (e.g. 15- 20 Pa). Tumbling was started and then power was connected. Visible radiation showed that plasma was being generated. Once treatment was finished, first the carrier gas flow was stopped and then valve 4 and 3 were closed. For a description of the equipment configuration and in particular the position of the valves, see Figure 2.

In the following, a number of examples are provided to illustrate how the present invention can be exploited. We give systematic examples of how low temperature plasma discharge can be used to rapidly create a variety of modified adsorbent beads. For the examples we have chosen to modify one type of bead which is used for packed bed chromatography and EBA and have elected to demonstrate them in batch binding and EBA tests. Using two examples we show how the plasma modified beads can be screened in batch binding tests to find the best candidates for adsorptive bioprocesses. In the final example we have chosen to use expanded bed adsorption chromatography experiments to demonstrate the superiour performance of the adsorption beads produced using the invention.

Example 1: Modification of adsorbent beads with plasma treatment

Below, an example of how the invention can be used is given. Firstly we describe one possible process set-up exploiting low temperature discharge plasma to modify the adsorbent Q Hyper Z, then we demonstrate how the invention is used in practice for the modification of the adsorbents using two different strategies: (i) shave off only those ligands located on the surface of the adsorbent beads (surface etching and oxidation), or (ii) to cover the ligands by coating adsorbent beads with a nano-scale polymer layer (surface polymerization) (see Figure 1).

Materials The anion exchange adsorbent Q HyperZ was obtained from BioSepra (now PALL). All chemicals including calf thymus DNA, bovine serum albumin, safrole, vinyl pyrrolidinone and vinyl acetate were purchased from Sigma-Aldrich and were of analytical grade. A home-made reactor was used and the experimental set up is shown in Figure 2. The plasma was created in a horizontal chamber made from Pyrex (40 mm x 250 mm) and a copper/nylon paper was coated on the outside to act as one electrode. A stainless steel bar was used as the second electrode and was centered in the middle of the plasma chamber (Figure 2). The plasma chamber was rotated at a rate of 10-30 rpm during treatment to ensure complete exposure of the surface of the beads to the plasma. An electrical field was created within the chamber by using a 20 KHz AC generator powered by a standard 0-240V vario transformer.

Plasma treatment

Surface etching and oxidation

In this procedure ligands are removed from the surface when oxygen atoms become excited and lead to formation of mainly hydroxyl and then carbonyl groups on the surface of the beads. The rotating plasma reactor shown in Figure 2 was used and 5-8 grams of Q HyperZ beads were added within the plasma chamber. The air inside the chamber was removed using a vacuum pump to reduce the pressure to below 10 Pa. Reduced pressure within the chamber, is needed for a low temperature, low pressure glow discharge plasma treatment and necessitates that the water within the beads vapourises. The pressure inside the plasma chamber was then increased by opening the oxidation gas (e.g. in this case air or oxygen or water) valve (valve 2) and the pressure adjusted to the desired value (e.g. 10 Pa). Reactor rotation was started with a low rate (e.g. 10- 30 rpm), the electrodes were connected and plasma was sustained by applying an electric potential of 220 volts (corresponding to 35.8 W. L"1 power applied per volume) and a frequency of 20 KHz. A visible radiation showed that plasma was being generated. After treatment for 2-3 h, the tumbling was stopped, power turned off, electrodes disconnected and gas evacuation was ceased. Valve 2 was opened to allow the pressure to rise to atmospheric pressure. Then, the chamber was opened and the treated beads were taken out. Equipment in contact with reactive materials was cleaned before the next batch was treated. Beads were washed with water, 0.1 M NaOH, 0.1 M HCI, 20% v/v ethanol solutions and again with water. Then, they were suspended in 50 mM Tris-HCI buffer pH 8 overnight before use.

Coating beads by surface polymerization The procedure was initially the same as described above, namely that the rotating plasma reactor shown in Figure 2 was used and 5-8 grams of Q HyperZ beads were added within the plasma chamber. The air inside the chamber was removed using a vacuum pump to reduce the pressure to below 10 Pa. However, in this treatment, the solution containing monomer, as well as carrier gas reservoirs were connected to the plasma chamber. After lowering the pressure to a value slightly less than that desired (e.g. 10 Pa), valves 4 and 1 (Figure 2) were opened to evacuate gas within all connecting lines. Once the pressure started decreasing, valve 3 was opened completely. When the pressure once again started decreasing, valve 3 was closed to avoid losing the active compound (i.e. the monomer to be used during coating). Then, the carrier gas (argon) was started to flow into the active compound reservoir. In the next step, valve 3 was opened very slowly to achieve the desired pressure (e.g. 15-20 Pa) and flow rate of carrier gas (5 mL.min"1) in the reactor. Reactor rotation was started and then power was connected at the voltages mentioned in the text below. Once treatment was finished, the carrier gas flow was first stopped and then valve 4 and 3 were closed. The beads were collected and washed as described above. The monomers, vinyl pyrrolidinone, vinyl acetate and safrole (Figure 3) were used and the treatment time varied from 0.5, 1, 2 or 3 hours depending on the monomer type.

Testing plasma-treated beads

Study of the structure and surface of the beads

Light microscopy of the beads was performed with a Nikon Optiphot microscope (Nikon, Melville, NY, USA) fitted with a Kappa CF-8/1 FMC monochrome video camera (Kappa Opto-electronics GmbH, Gleichen, Germany) and digitized images and average size measurements were produced with the aid of Image-Pro® Plus software (version 4.1 for Windows™; Media Cybernetics, Silver Spring, MD, USA).

The surface topography of the beads was monitored with scanning electron microscopy (SEM; DSM-960, Zeiss). Before the SEM imaging, the samples were coated with a thin layer of palladium/gold to minimize the charging effect and increase the image contrast.

The elemental composition of the surface was studied by x-ray photo electron spectroscopy (XPS). XPS was performed using a SPECS Sage 100 instrument (Specs, Berlin, Germany) using a non-monochromatic MgK3 X-ray source at a power of 275 Watts (11 keV and 25 mA). The pressure in the chamber was always below 1 x 10"7 mbar. The elements were identified and quantified from survey spectra, acquired at 100 eV pass energy in the range from 0 to 1100 eV. The samples were mounted on the holder by use of double-side sticky tape. XPS data was used for the evaluation of the surface changes after a plasma treatment and in particular to determine if plasma treatment was able to coat or eliminate active groups (ligands), e.g. quaternary amine groups from the surface.

Strategies applied for plasma treatment

The commercial adsorbent Q HyperZ was chosen for this work. It has a rigid ceramic skeleton that can be expected to maintain the shape and protect the ligand containing gel inside from potential abrasion in the plasma reactor, and the effects of dehydration in the low pressure atmosphere used. However other types of adsorbent typically used for packed bed, EBA, or batch adsorption or adsorption resins not composed of ceramic can also be used. Two strategies were employed to provide a multifunctional adsorbent devoid of ligands on the surface (Figure 1). The plasma method was used to either: (i) shave off only those ligands located on the surface of the adsorbent beads (surface etching and oxidation), or (ii) to cover the ligands by coating adsorbent beads with a nano-scale polymer layer (surface graft polymerization). Although the layer is on the nano-scale, thicker layers can also be applied. The plasma treatments used and the beads obtained in each case are summarized in Table 1.

Table 1 : Conditions used in the plasma treatment of Q HyperZ adsorbents.

Figure imgf000033_0001

Etching and

Et220-2 oxidation by na 2 220 35.8 10 air plasma

Etching and

Et220-3 oxidation by na 3 220 35.8 10 air plasma

Coating by Vinyl

VPlOO plasma pyrrolidin 1 100 7.4 15-20 polymerization -one

Coating by Vinyl

VP140 plasma pyrrolidin 1 140 9 15-20 polymerization -one

Coating by

Vinyl

VA170-2 plasma 2 polymerization acetate 170 16.5 15-20

Coating by

Vinyl

VA170-3 plasma 170 16.5 15-20 polymerization acetate 3

Coating by

SA130 plasma Safrole 0.5 130 8.2 15-20 polymerization

Table legend : na indicates no monomer was used.

Characterization of plasma treated beads

Physical structure

Light microscopy images showed no difference between untreated beads (Figure

4a), surface-etched beads (Et220-3) (Figure 4b) and surface-coated beads (VA170-3) (Figure 4c). The diameters of at least 800 beads of each type were measured using light microscopy and no significant difference was seen, the average diameters were 73.3, 72.2 and 73.2 micron for the untreated, surface- etched (Et220-3) and surface-coated (VA170-3) beads, respectively. Furthermore, there was no evidence of fines or broken beads seen. The results imply that plasma treatment is a mild process and does not adversely affect the size or shape of the beads. SEM images of untreated, Et220-3 and VA170-3 beads are shown in Figure 5 and no differences between the adsorbents can be discerned. This implies that the pores were not filled up or blocked or otherwise damaged by the treatment and that the treatment was therefore truly at the nano scale or even smaller and that no unwanted mass transfer limitations are to be expected for the treated beads.

Elemental analysis of the adsorbent surface by XPS

The Q HyperZ bead is a composite material consisting of a porous core made from yttrium-stabilized zirconium oxide filled and coated with a gel material synthesized through polymerization of methacryloylaminopropyl- trimethylammonium, as functionalized monomer and N, N' methyl bis methacrylamide (a bifunctional acrylic monomer), as cross-linker (Voute and Boschetti, 1999). A successful plasma treatment can be expected to lead to a change in the elemental composition of the surface, which was analyzed by using x-ray photo electron spectroscopy (XPS) and the results are shown in Table 2. After ligand shaving by air/oxygen plasma etching (beads Et220-2 and Et220-3) it was found that the amount of nitrogen on the surface decreased to an undetectable level (Table 2). This implies that the quaternary amine ligands originally present on the surface were efficiently removed by the treatment. The percentage of oxygen increased for these beads (Et220-2 and Et220-3) due to oxidation of the surface, a result that is in agreement with what was observed by Kim et al. (1998) through their investigation on the effect of the oxygen plasma treatment on SiO2 aerogel film. Changing the treatment time from 2 hours to 3 hours did not affect the surface composition significantly (Table 2).

Table 2: Data obtained from XPS analysis of the surface of untreated and plasma- treated beads.

Bead Zirconium

Carbon% Oxygen% Nitrogen% Yttrium% name %

Untreated 45.9 33.3 5.1 14.1 1.5

Et220-2 20.2 58.3 ND* 17.9 3.6

Et220-3 30.3 50.1 ND* 17.0 2.5

VPlOO 63.6 20.9 10.0 4.9 0.6 VP140 70.3 17.0 11.6 1.0 ND*

VA170-2 58.6 30.4 5.7 4.6 0.7

VA170-3 64.3 28.8 3.2 2.4 0.3

SA130 74.4 22.2 1.3 1.7 0.4

*ND: Not detected

Treatment of the beads with plasma together with all of the different monomers applied led to a large increase in the percentage of carbon and a decrease in zirconium on the beads (Table T) indicating that coating of the surface had occurred. The SA130 beads had less surface nitrogen than the VA170 types suggesting more efficient coating with safrole occurred than vinyl acetate. The VPlOO and VP140 beads had an increased percentage of nitrogen on the surface compared to the untreated types, however this result does not provide direct evidence that the quaternary amine ligands have been covered, due to the fact that vinyl pyrrolidinone contains nitrogen, but does suggest that a modification to the surface has occurred. Increasing the voltage/power during treatment led to better coating as shown by a decrease in the percentages of zirconium and yttrium on the surface of VP140 beads compared to VPlOO. Moreover, the XPS data for zirconium and yttrium suggest that the VP140 beads have been coated more efficiently than beads coated by vinyl acetate (VA170) and safrole (SA130). Increasing the treatment time from 2 to 3 hours also led to a more efficient coating treatment as shown by the change in the percentages of carbon, nitrogen, zirconium and yttrium for the VA170-2 compared to VA170-3 beads.

Example 2.

Selection of low temperature plasma discharge modification method for producing beads with selective adsorption properties.

Here an example is given of how different plasma modification methods can be employed and screened to predict which conditions are likely to produce the best beads for reduced DNA binding with negligible impact on protein binding. First, the invention depicted in example 1 is used to modify the adsorbents used in example 1 in the same way as described in example 1. Second, a screening study is conducted in which the binding capacity of the adsorbents for the test protein bovine serum albumin (BSA) and test DNA molecules (calf thymus DNA) in simple batch binding experiments is used to demonstrate how to predict which of the plasma modification methods shows the most promise. 5

The plasma reactor was set up as described in example 1

Q HyperZ adsorbents were treated with low discharge plasma as described in example 1 and given the same names as in example 1. 10

Preparation of DNA feedstocks for batch binding tests

Calf thymus DNA was dissolved slowly overnight in 50 mM Tris-HCI, pH 8, to give a 2 mg.mL"1 solution and then sonicated on ice with an MSE soniprep 150 (MSE

Scientific Instruments Ltd., Sussex, L)K). Then, the solution was centrifuged at 15 20000xg in the SS-34 rotor of a SORVAL RC5C laboratory centrifuge for 30 minutes at 4 0C. The sonicated DNA (0.5-10 kbp) in the supernatant was portioned into sterile tubes and stored at -20 0C.

Protein and DNA batch binding tests

20 The capacity of the plasma treated Q HyperZ adsorbent beads was tested in batch binding studies using bovine serum albumin (BSA) and homogenized calf thymus DNA. Untreated or plasma-treated Q HyperZ beads (0.1 mL) were equilibrated in 50 mM Tris-HCI pH8 buffer then incubated in 4 mL BSA solution (4 mg.mL"1) or 1.5 mL DNA solution (1.5 mg.mL"1) prepared in equilibration buffer. Either 2 or 30

25 minutes was used for binding, with shaking on an orbital shaker (Infors, Basel, Switzerland). The beads were then settled by a short (30 s) centrifugation at 2000xg in a microfuge (Biofuge pico, HAEREUS Instruments, Osterode, Germany), the supernatant collected and analysed at 280 or 260 nm in a Lambda 20 UV-Vis spectrophotometer (Perkin-Elmer Analytical Instrument, Shelton, CT,

30 USA) for BSA and DNA, respectively.

Observations of bead behaviour

In all cases the beads were equilibrated in binding buffer (50 mM Tris-HCI pH 8) over night. It was observed that the adsorbents treated by safrole agglomerated

35 in the buffer solution suggesting a hydrophobic interaction between the beads. This is consistent with the hydrophobic nature of the polymer layer formed using the safrole monomer (Figure 3), confirming coating had occurred, however agglomeration is not a suitable trait for an adsorbent. In contrast, none of the other beads (see Table 1 in example 1) were observed to self-agglomerate in solution.

Batch binding characteristics of plasma modified beads

To examine the effects of the different treatments on protein and DNA binding, the different modified beads were reacted with BSA or calf thymus DNA and the supernatants collected and analysed. The results in Figure 6 show that the Et220- 3 beads had the most promising performance, with the largest reduction in DNA binding after 2 or 30 minutes as compared to the other bead types. Furthermore the reduction in BSA binding for the Et220-3 was less than that observed for the VA170-3 and the SA-130 types. Excluding the SA130 beads, which had an unwanted propensity for agglomeration (as mentioned above), the VA170 adsorbents showed the next best reduction in capacity for DNA binding. Thus one can choose the Et220-3 and the VA170 treatments as the most promising.

Example 3 Selection of the best plasma discharge modification method for producing adsorbents.

This is an example of how the two most promising plasma discharge modification methods found in example two are employed and the results used to select the single best adsorbent bead candidate.

The plasma reactor was set up as described in example 1.

Q HyperZ adsorbents ET220-3 and VA170 were produced with low discharge plasma as described in example 1.

Calf thymus DNA and protein solutions were prepared as described in example 2.

Preparation of plasmid DNA In order to prepare stock solutions of pUG6 (4.009 kbp plasmid), Escherichia coli DH5alpha cells containing pUG6 (Guldener et al., 1996) were grown in Luria Bertani (LB) medium (tryptone 10 mg.mL"1, yeast extract mg.mL"1 and NaCI 10 mg.mL"1) containing 100 μg.mL"1 ampicillin in a 5-L batch fermentor. The biomass was harvested after ~20 hours when the dry weight had reached ~10 mg.mL"1 by centrifugation for 30 min at 4 0C at 10000xg in the SLA 3000 rotor of a SORVAL RC5C centrifuge. The cell paste was then washed by resuspension in 50 mM Tris- HCI pH 8 buffer before recentrifuging at 10000xg. The cell paste was stored at - 20 0C. A QIAGEN kit (QIAGEN Sciences, Maryland, USA) was used for the purification of the plasmid using the protocol described by the manufacturers.

The kinetics of adsorption of the plasmid pUG6, sonicated calf thymus DNA and BSA to the plasma-treated and untreated adsorbent beads were studied batch wise in a similar way as the protein and DNA batch binding studies in example 2 were described, with the following exceptions. For each time point in all tests, 0.1 mL of adsorbent beads was used and the initial concentrations of BSA (5 mL), plasmid DNA (2.5 mL) and homogenized calf thymus DNA (2.5 mL) solutions were 3.2, 0.7 and 1 mg.mL"1, respectively.

Kinetics of protein, homogenized calf thymus DNA and plasmid DNA adsorption When the calf thymus DNA binding kinetics were examined for unmodified beads as well as the ET220-3 and VA170 types, the results in Figure 7 showed that equilibrium was essentially reached after ca. 15 minutes (Figure 7a) for all three bead types, but that the capacity (Figure 7b) and the kinetics were lower for the Et220-3 than the VA170-3 and both were lower than the unmodified beads. In particular Figure 7b shows that there was a large amount of DNA (2 mg.mL"1 beads) bound to the unmodified adsorbents within 30 s which was not seen for the Et220-3 type. When binding of plasmid DNA to the three bead types was examined (Figures 7c and 7d), a similar pattern was observed to that for calf thymus DNA (Figure 7a and 7b). However in this case although a very large amount of DNA (~2.5 mg.mL"1 beads) was bound to the unmodified adsorbents within 30 s, only a small amount of further adsorption occurred (Figure 7a and 7b) compared to that seen with calf thymus DNA. Much less plasmid binding occurred to the VA170-3 and Et220-3 adsorbents within 30 s than to the unmodified type, however binding continued until 30-45 minutes had elapsed when equilibrium was reached. Nevertheless, the plasmid binding capacities at equilibrium were less than half of those for calf thymus DNA. The results show that binding of sonicated calf thymus DNA to the Et220-3 (surface-etched) adsorbents is much slower than that of plasmid DNA (Figures 7a and 7b). This could be due to the non-uniformity 5 of size of the pieces of calf thymus DNA formed during the sonication procedure. Sonicated calf thymus DNA contains a smear of fragments with a molecular size range of ~0.5-10 kbp when analysed by agarose gel electrophoresis (results not shown). In contrast the plasmid DNA is a discrete size of ~4 kbp. The results show that large molecules of plasmid or calf thymus DNA have no accessibility to

10 the ligands located inside the adsorbent gel and thus only interact with the outer surface. However very small DNA molecules produced during sonication might diffuse slowly to a small extent into the pores, accounting for the extended time to reach equilibration as seen in Figure 7b as compared to Figure 7d. When the time course of binding of the protein BSA was examined the results for the

15 modified adsorbents were the same as for the unmodified types after the first 30 s, at which time ~27 mg BSA. mL"1 beads had bound (Figure 7e and 7f). For the unmodified types, equilibrium (70 mg.mL"1) was reached after 10 minutes, whereas for both the modified adsorbents binding continued for at least 60 minutes, at which time the capacity for the VA170-3 and Et220-3 was ~80 and

20 90%, respectively, of that for the unmodified beads. The data at each time point in Figures 7b, 7d and 7f was used to calculate the reduction in binding capacity of calf thymus DNA, plasmid DNA and BSA for each of the modified beads as a percentage of the capacity of the unmodified ones at the corresponding time point. The results in Figures 8a and 8b for VA170-3 and Et220-3, respectively,

25 show that in the beginning of the binding reaction (e.g. after 30 s) there is almost no difference in BSA binding capacity for the modified beads compared to the unmodified types (see open and closed triangles), however at the same time point there is an extremely large reduction in binding capacity for calf thymus and plasmid DNA for both modified adsorbents, but with the best performance seen

30 for the Et220-3 (Figure 8b). As the binding reactions progressed, the differences between the modified and unmodified adsorbents were reduced. At all time points the Et220-3 had a greater reduction in binding capacity for calf thymus or plasmid DNA than for BSA protein binding (Figure 8b), however for the VA170-3 the reduction in BSA binding capacity after 5 minutes was greater than for the calf

35 thymus DNA (Figure 8a). The results show that the Et220-3 has better performance than the VA170-3 with respect to minimizing DNA binding whilst having the least impact on BSA binding. The results from both modified adsorbent types show that the plasma modification has successfully generated adsorbents with a greater reduction in binding of large molecules (i.e. DNA molecules) to the quaternary amine ligands in the adsorbent than that for protein (i.e. BSA). The results show that removing the surface ligands by etching is more successful than covering them via graft polymerization. In addition the results for BSA binding show that accessibility to the ligands located in the gel material of the etched adsorbent bead (Et220-3) is better than that of the surface-coated one (VA170- 3). Thus it is concluded that the Et220-3 is the most promising candidate adsorbent for batch adsorption, packed bed chromatography or EBA produced by the plasma method.

Example 4. Plasma modification of beads and testing in expanded bed adsorption trials.

This is an example of how the best beads produced by low temperature discharge plasma can be used in expanded bed adsorption. The binding capacity and bed aggregation characteristics of the best adsorbents produced in example 3 are demonstrated and described.

The plasma reactor was set up as described in example 1.

Q HyperZ adsorbents were modified by surface ligand removal (ET220-3) or surface coating (VA170-3) with low discharge plasma as described in example 1.

Calf thymus DNA and protein solutions were prepared as described in example 2.

Plasmid DNA was prepared as described in example 3.

EBA column setup and use

A 1-cm diameter EBA column (UpFront Chromatography A/S, Copenhagen, Denmark) with ~5 cm settled bed height, i.e. ~4 ml. adsorbent was used. The expanded bed column was connected to a FPLC system equipped with a peristaltic pump, flow through L)V detector (260 or 280 nm) and fraction collector (Pharmacia Biotech, Uppsala, Sweden). The column was equilibrated with > 50 column volumes (CV) of 50 mM Tris-HCI pH 8 buffer containing 100 mM NaCI. The feedstock was loaded at a superficial flow velocity of 350±20 cm.h"1 or 308±10 cm.h"1 which gave two fold expansion of the bed for untreated and plasma-treated beads, respectively. During loading the bed height was recorded and fractions were collected, which were analysed at 280 or 260 nm in a Lambda 20 UV-Vis spectrophotometer (Perkin-Elmer Analytical Instrument, Shelton, CT, USA) for BSA and DNA, respectively. Washing and elution of the bed was not examined and the beads were not reused. The feedstock was 1 mg.mL"1 BSA or 0.06 mg.mL"1 DNA (either homogenized calf thymus or plasmid) solution in 50 mM Tris-HCI, pH 8 buffer containing 100 mM NaCI (conductivity ~11 mS.cm"1).

The voidage (epsion; ε) corresponding to the expanded bed height (H) at each time point was calculated by using the following equation (Anspach et al., 1999):

A = !zi! (1)

H0 l -ε Here, H0 and (epsilono) ε0 are the height and voidage of the settled bed, respectively. A value of 0.4 was assumed for (epsilono) εo. The ratio between voidage at each time to the initial voidage (before starting loading of the feed) ε/ε, (epsilon/epsilon,) was used to study the bed contraction profile.

Evaluation of the plasma-treated beads performance in an EBA system

The dynamic binding characteristics of the plasma modified beads were examined using EBA. The results in Figure 9a show that there was no significant difference in the protein BSA binding capacity of the Et220-3 and PV170-3 adsorbents as compared to the unmodified beads. The dynamic BSA binding capacity averaged for the three bead types examined was 33.7±1.01 mg.mL"1 of gel at 10% breakthrough. In contrast, a very large reduction in the dynamic binding capacity of calf thymus DNA to the modified adsorbents was seen (Figure 9b). The lowest capacity at 10% breakthrough (1.29 mg applied DNA. mL"1 gel) was observed for the surface-etched support (Et220-3) as compared to 6.92 mg applied DNA. mL"1 gel for the unmodified support. A similar but less dramatic pattern was observed when the capacity for plasmid DNA was studied (Figure 9c). The Et220-3 beads had the lowest dynamic binding capacity for plasmid of 0.76 mg.mL"1 gel compared to that for the unmodified beads (1.5 mg.mL"1 gel). The results are consistent with what was observed in the batch binding studies in which the Et220-3 adsorbents were found to have the greatest reductions in DNA binding and least effects on BSA capacity (Figures 8a and 8b). The results suggest that the Et220-3 plasma modified beads perform the best and are much better than the unmodified beads, since DNA binding is drastically reduced without significantly affecting protein binding.

The expanded bed contraction profiles measured during the EBA experiments in Figures 9a, 9b and 9c are shown in Figures 10a to 10c. It is observed that there is no significant difference between bed contraction for unmodified and plasma- treated adsorbent beads (Et220-3 and VA170-3) during loading of the protein BSA (Figure 10a). However, bed contraction is more severe for the unmodified beads compared to that for both the plasma treated beads (Et220-3 and VA-170) when a feedstock containing homogenized calf thymus DNA is loaded (10b). The results are consistent with the dynamic binding capacities shown above in which calf thymus DNA binding was dramatically reduced for the plasma modified beads. However, there was little difference in bed contraction during the loading of plasmid DNA (Figure 10c), which may be due to the much lower binding capacity for plasmid DNA of the unmodified adsorbents compared to that for calf thymus DNA (compare Figures 9b and 9c). The results therefore show that the Et220-3 beads are much better than the unmodified beads and are the best of the types produced here for use in an adsorption process, such as an EBA process, packed bed or batch binding, in which protein binding capacity should be high, and where DNA binding capacity should be low and in which contraction of the EBA bed should be as insignificant as possible. It is clear that the invention has produced a multifunctional bead which is suitable for bioprocessing.

Claims

Claims
1. A method for the manufacture of beads suitable for use in purification of biological molecules, said method comprising modifying beads using an irradiation method.
2. The method according to claim 1, wherein the irradiation method is plasma treatment.
3. The method according to claim 1, wherein the surface of the beads are etched and oxidized.
4. The method according to claim 1, wherein compounds selected from the list consisting of: monomers, oligomers, polymers, and any mixture thereof are covalently attached to the surface of the bead.
5. The method according to claims 3 and 4, wherein the oxidized layer and/or the layer of covalently attached compounds, respectively are 1 nanometer to 10 microns.
6. The method according to claim 1, wherein the beads have a diameter of 40 to 1000 microns.
7. Beads suitable for use in purification of biological molecules, wherein said beads are obtainable by a method according to any of the claims 1-6.
9. Use of beads according to claim 7 in expanded bed adsorption chromatography.
10. Use of beads according to claim 7 in batch adsorption processes.
11. Use of beads according to claim 7 in fluidized bed processes.
12. Use of beads according to claim 7 in packed bed chromatography processes.
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US9297032B2 (en) 2012-10-10 2016-03-29 Apdn (B.V.I.) Inc. Use of perturbants to facilitate incorporation and recovery of taggants from polymerized coatings
US9919512B2 (en) 2012-10-10 2018-03-20 Apdn (B.V.I.) Inc. DNA marking of previously undistinguished items for traceability
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US9790538B2 (en) 2013-03-07 2017-10-17 Apdn (B.V.I.) Inc. Alkaline activation for immobilization of DNA taggants
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