WO2013190453A2

WO2013190453A2 - Compositions for separation methods

Info

Publication number: WO2013190453A2
Application number: PCT/IB2013/054968
Authority: WO
Inventors: Tracy Thompson; Bernd Helmut Adam Rehm; Andrew Brian HERBERT; Edward George Saravolac; Paul B. MCDERMOTT; Jenny Lyn DRAPER
Original assignee: Tracy Thompson; Bernd Helmut Adam Rehm; Herbert Andrew Brian; Edward George Saravolac; Mcdermott Paul B; Draper Jenny Lyn
Priority date: 2012-06-18
Filing date: 2013-06-18
Publication date: 2013-12-27
Also published as: WO2013190453A3

Abstract

This invention relates generally to the fields of separation and conversion technologies, and more particularly to materials for use in chromatography techniques. The chromatography materials are useful in a wide range of separation and conversion processes, including those reliant on reverse osmosis, microfiltration, ultrafiltration, nanofiltration, including those using permeable or semipermeable filtration, and provide efficient methods for purifying or producing various target substances.

Description

COMPOSITIONS FOR SEPARATION METHODS

TECHNICAL FIELD

[0001] This invention relates generally to the fields of separation and conversion technologies, and more particularly to materials for use in chromatography methods, for example column chromatography techniques such as affinity chromatography. The chromatography materials and methods of the present invention are useful in a wide range of separation and conversion processes, and provide efficient methods for purifying or producing various target substances.

BACKGROUND OF THE INVENTION

[0002] The separation of desirable target substances from undesirable substances, for example from a complex composition, is a fundamental step in the production of many important commodities, including foods, chemicals, pharmaceuticals, and biologies such as cells, viruses, polypeptides, polynucleotides, and metabolites. Similarly, the conversion of one or more precursor substances into a target substance, for example by enzymatic conversion, optionally coupled with enrichment or separation of the target substance, for example from the precursor substance(s), is a fundamental step in many methods of manufacture.

[0003] As a consequence, there has been a great deal of expenditure to develop methods and technologies to enable the efficient separation and production of desirable target substances.

Techniques for the separation of one or more desirable substances, typically one or more liquids or one or more dissolved or suspended solids from a second component, such as another liquid or other dissolved or suspended solids, have been developed over many years.

[0004] Most commonly used is packed bed chromatography where stationary phase particles are packed into a bed and a solution containing the target molecule or molecules is passed through the column and the target or targets bind to the stationary phases. A particular challenge of this method is the formation of irregular flow channels. These irregular flow channels prevent the efficient purification of the target as well as preventing efficient cleaning of the stationary phase, thereby creating a potential for contamination. A further challenge is the total binding capacity of a packed bed column, where low binding density of, for example, affinity chromatography stationary phases can negatively impact purification efficiency.

[0005] In the production of monoclonal antibodies, for example, it has been suggested that packing the bed with ten- fold excess stationary phase could address the issue of irregular flow path. Clearly, there are cost and efficiency consequences in adopting such a suggestion. [0006] One solution to reducing the risk of irregular flow path is to reduce the operating pressure of the column. While this has the desired effect of reducing the risk of channelling, the negative consequence of an increase in processing times. Maximising stationary phase packing density is a possible solution to problems with the total binding capacity of a column, but will typically require increased operating pressures (and thus exposure to the concordant risks of channelling), or increased processing times.

[0007] A related and serious challenge, particularly to the use of non-rigid chromatography stationary phases, is lowered packed bed stability, or stationary phase deformation or aggregation, such that flow through the packed bed is restricted or even stopped. Low volumetric productivities are associated with, for example, the rapid increase in back pressure that results from particle compression and the concomitant increase in pack pressure, even at low flow rates. Taken together, these problems frequently render non-rigid chromatography stationary phases unsuitable or less suitable for use in many column chromatography applications, particularly large scale preparative methods. [0008] It is an object of the present invention to overcome or at least ameliorate some of the above disadvantages, to provide improved compositions and methods for the preparation and purification of target substances, in particular by column chromatographic methods, for example by affinity column chromatography, or at least to provide the public with a useful choice.

[0009] Other objects of the invention may become apparent from the following description which is given by way of example only.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a method for preparing one or more target substances from a source material, the method generally comprising providing a chromatography stationary phase comprising a population of support particles and a population of polymer particles, contacting the source material with the stationary phase for a time sufficient to allow the polymer particles to bind one or more target substances or one or more precursors of a target substance or one or more contaminants, separating by chromatography the one or more contaminants from the particle-bound target substance or precursor thereof or the one or more target substances or precursor thereof from a particle-bound contaminant, and recovering the target substance. [0011] In one embodiment, the population of polymer particles comprises, consists essentially of, or consists of amorphous polymer particles. [0012] In one embodiment, the population of support particles is a homogeneous population.

In another embodiment, the population of support particles is a heterogeneous population.

[0013] In one embodiment, the population of support particles comprises, consists essentially of, or consists of substantially non-deformable particles. In one embodiment, the substantially non- deformable particles are rigid particles.

[0014] In one embodiment, the population of support particles comprises, consists essentially of, or consists of substantially inert particles. In one embodiment, the substantially inert particles are non-derivatized particles.

[0015] In one embodiment, the population of polymer particles, such as the population of amorphous polymer particles, is a homogeneous population. In another embodiment, the population of polymer particles, for example the population of amorphous polymer particles, is a heterogeneous population.

[0016] In one embodiment, one or more of the polymer particles, for example the population of amorphous polymer particles, comprises one or more biopolymers selected from a polyester, polyester, polythioester or a polyhydroxyalkanoate.

[0017] In one embodiment, one or more of the polymer particles, for example one or more of the amorphous polymer particles, is or is capable of being synthesised by a particle-forming protein. In one embodiment, substantially all of the population of polymer particles, for example the population of amorphous polymer particles, is or is capable of being synthesised by a particle - forming protein.

[0018] In one embodiment, one or more of the polymer particles, for example one or more of the amorphous polymer particles, comprises a polymer particle-forming protein, such as a polymer synthase or a polymer synthase fusion.

[0019] In one embodiment, one or more of the polymer particles, for example one or more of the amorphous polymer particles, comprises a ligand or binding domain capable of binding one or more of the population of support particles.

[0020] In one embodiment, the recovery of the target substance is by elution from the polymer particle. In other embodiments, for example in embodiments where one or more contaminants binds to the one or more polymer particles, for example one or more of the amorphous polymer particles, the recovery of the target substance is by collection of the

chromatography wash or eluate prior to elution of the one or more contaminants. [0021] Accordingly, in one aspect, the present invention provides a method for separating or purifying one or more target substances from a source material, the method comprising providing a chromatography stationary phase comprising a population of support particles and a population of polymer particles, for example one or more of the amorphous polymer particles, contacting the source material with the stationary phase for a time sufficient to allow one or more of the polymer particles, for example one or more of the amorphous polymer particles, to bind one or more target substances, separating one or more contaminants from the particle-bound target substance by chromatography, and recovering the target substance, wherein one or more of the polymer particles, for example one or more of the amorphous polymer particles, comprises: " a biopolymer selected from a polyester, a polythioester or a polyhydroxyalkanoate; or

^■ a polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion; or

^■ both of the above.

[0022] Accordingly, in another aspect, the present invention provides a method for separating or purifying one or more target substances from a source material, the method comprising providing a chromatography stationary phase comprising a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, contacting the source material with the stationary phase for a time sufficient to allow one or more of the polymer particles to bind one or more contaminants, separating one or more target substances from the particle - bound contaminants by chromatography, and recovering the target substance, wherein one or more of the polymer particles, for example one or more of the amorphous polymer particles, comprises:

^■ a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or

^■ both of the above.

[0023] In another aspect, the present invention provides a method for preparing one or more reaction products, the method comprising contacting in or prior to introduction into a

chromatography system, a source material comprising one or more reaction substrates with a stationary phase comprising a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, for a sufficient time to allow the one or more polymer particles to bind a desired fraction of the one or more reaction substrates, optionally separating one or more contaminants from the polymer particles by chromatography, and recovering the reaction product, wherein the one or more polymer particles comprise a catalyst of the reaction, and wherein one or more of the polymer particles comprises:

^■ a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or

^■ both of the above.

[0024] Accordingly, in one exemplary embodiment the invention provides a purification method for purifying one or more antibodies, which comprises providing a source material comprising one or more antibodies, contacting the source material with a chromatography stationary phase comprising a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, wherein the one or more polymer particles, for example one or more of the amorphous polymer particles, comprise a ligand or binding domain capable of binding an antibody, and recovering the antibody.

[0025] In a further aspect, the present invention provides a chromatography stationary phase, wherein the chromatography stationary phase comprises a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, wherein one or more of the polymer particles comprises: " a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or

^■ both of the above. [0026] In another aspect, the present invention provides a chromatography stationary phase, wherein the chromatography stationary phase comprises a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, wherein one or more of the polymer particles comprises:

^■ a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or ^■ a polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion; or

^■ both of the above, and

^■ a ligand or binding domain capable of binding one or more of the support particles. [0027] In another aspect, the present invention provides a method for preparing a

chromatography stationary phase, the method comprising providing a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, admixing as a suspension the population of support particles and the population of polymer particles at a sufficient concentration such that substantially no excess liquid is present in the suspension prior to or during introduction into a chromatography system, to provide a chromatography stationary phase, wherein one or more of the polymer particles comprises:

^■ a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or

^■ both of the above.

[0028] In another aspect, the present invention provides a population of polymer particles, for example a population of amorphous polymer particles, for use in chromatography, wherein one or more of the polymer particles comprises: " a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or

^■ both of the above, and " a ligand or binding domain capable of binding one or more of the support particles.

[0029] In a further aspect, the invention provides a method for making a filter for use in chromatography, the method comprising providing a permeable or semipermeable support, and providing a chromatography stationary phase, wherein the chromatography stationary phase comprises a population of support particles and a population of polymer particles, for example a population of amorphous polymer particles, associating one or more support particles with the permeable or semipermeable support, associating one or more of the polymer particles with one or more of the support particles to provide a semipermeable filter, wherein one or more of the polymer particles comprises: " a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate; or

^■ both of the above, and

^■ a ligand or binding domain capable of binding one or more of the support particles. [0030] In a further aspect, the invention provides a method for making a filter for use in chromatography, the method comprising providing a permeable or semipermeable support, and providing a chromatography stationary phase, wherein the chromatography stationary phase comprises a population of polymer particles, for example a population of amorphous polymer particles, associating one or more polymer particles with the permeable or semipermeable support, to provide a semipermeable filter, wherein one or more of the polymer particles comprises:

^■ a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate; or

^■ both of the above, and " a ligand or binding domain capable of binding the support.

[0031] In a further aspect, the invention provides a method for preparing polymer particles, for example a population of amorphous polymer particles, wherein one or more of the polymer particles comprises:

^■ a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate; or " a polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion; or both of the above, and a ligand or binding domain capable of binding one or more support particles; wherein the method comprises separating one or more contaminants from the polymer particles, and recovering the polymer particles.

[0032] In one embodiment, the separation is by contacting the one or more polymer particles with one or more support particles to which the ligand or binding domain binds. [0033] Another aspect of the invention relates to an isolated, purified or recombinant nucleic acid comprising at least one nucleotide sequence encodinga polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion; and at least one nucleotide sequence encoding a ligand or binding domain capable of binding one or more support particles.

[0034] In one embodiment the at least one nucleic acid sequence encoding the a polymer particle-forming polypeptide and the at least one nucleic acid sequence encoding the ligand or binding domain capable of binding one or more support particles are present as a single open reading frame.

[0035] In one embodiment, the nucleic acid comprises 12 or more contiguous nucleotides from a sequence selected from one of SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 5 herein. [0036] Another aspect of the invention relates to an expression construct, the expression construct comprising nucleic acid sequence encoding

^■ a polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion; and

^■ a ligand or binding domain capable of binding one or more support particles. [0037] In one embodiment the at least one nucleic acid sequence encoding the a polymer particle-forming polypeptide and the at least one nucleic acid sequence encoding the ligand or binding domain capable of binding one or more support particles are present as a single open reading frame.

[0038] Another aspect of the present invention relates to a vector comprising an expression construct of the invention, or a host cell comprising an expression construct or a vector of the invention.

[0039] Another aspect of the invention relates to a method of producing fusion polypeptides, fusion polypeptide particles, or polymer particles, for example amorphous polymer particles, comprising a fusion polypeptide, the method comprising: ^■ providing a host cell comprising an expression construct or vector of the invention, the expression construct or vector comprising nucleic acid sequence encoding a polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion, and a ligand or binding domain capable of binding one or more support particles; and

^■ maintaining the host cell under conditions suitable for expression of the expression construct; and

^■ separating the fusion polypeptides, the fusion polypeptide particles, or the polymer particles comprising a fusion polypeptide, from the host cells.

[0040] In one embodiment, the expression construct comprises a strong promoter.

[0041] Another aspect of the invention relates to a fusion polypeptide, fusion polypeptide particle, or polymer particle comprising a fusion polypeptide of the invention, for example an isolated or purified fusion polypeptide, fusion polypeptide particle, or polymer particle comprising a fusion polypeptide of the invention, the fusion polypeptide a polymer particle-forming polypeptide, such as a polymer synthase or a polymer synthase fusion, and a ligand or binding domain capable of binding one or more support particles.

[0042] In one embodiment, the fusion polypeptide comprises 12 or more contiguous amino acids from a sequence selected from SEQ ID NO. 1 or SEQ ID NO. 3 herein.

[0043] In one embodiment, the fusion polypeptide is encoded by nucleic acid comprising 12 or more contiguous nucleotides from a sequence selected from one of SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 5 herein.

[0044] Another aspect of the present invention relates to a host cell, culture, or culture supernatant comprising a fusion polypeptide, a fusion polypeptide particle or a polymer particle comprising a fusion polypeptide of the invention.

[0045] The invention further provides compositions, membranes, filters, filter apparatuses

(such as filter cartridges), columns (including pre-packed columns) comprising one or more chromatography stationary phases, or one or more polymer particles, for example one or more amorphous polymer particles, as described herein.

[0046] In one embodiment, the invention provides a chromatography device comprising a column having a cylindrical interior for accepting a stationary phase; and a particulate stationary phase packed within the column; wherein the stationary phase comprises one or more populations of support particles as described herein and one or more populations of polymer particles, for example a population of amorphous polymer particles, as described herein.

[0047] The invention further relates to a chromatography device prepared by the steps of providing a column having a cylindrical interior for accepting a stationary phase, and forming a particulate stationary phase within said column, wherein the particulate stationary phase comprises one or more populations of support particles as described herein and one or more populations of polymer particles, for example a population of amorphous polymer particles, as described herein.

[0048] In another embodiment, the invention provides a method of making a

chromatography device comprising the steps of providing a column having a cylindrical interior for accepting a stationary phase, providing one or more populations of support particles, and providing one or more populations of polymer particles, for example a population of amorphous polymer particles, and forming a particulate stationary phase within said column, wherein the forming step comprises the steps of admixing the one or more populations of support particles and the one or more populations of polymer particles, and placing the particulate stationary phase into said column to thereby produce the chromatography device.

[0049] The following embodiments may relate to any of the aspects of the invention herein described.

[0050] In various embodiments, the population of support particles comprises, consists essentially of, or consists of one or more of the following and not limited to: silica, diatomaceous earth, zeolite, silica gel, fused silica, glass beads, sintered glass, perlite, dowex, aluminum oxides, alumina, and polymer(divinlypolystyrene) dextrans, crosslinked dextrans, cellulose and

polymethacrylate.

[0051] In various embodiments, one or more of the polymer particles, for example one or more amorphous polymer particles, comprises one or more of the following:

^■ a polymer particle-binding polypeptide;

^■ a polypeptide fusion partner;

^■ an affinity ligand or binding domain;

^■ an enzyme; a fusion polypeptide comprising two or more of the above; or any combination of any two or more of the above. [0052] In various embodiments, substantially all of the polymer particles comprise:

^■ a biopolymer, such as a biopolymer selected from poly-beta-hydroxy acids,

biopolylactates, biopolythioesters, and biopolyesters; or

^■ both of the above.

[0053] In one embodiment, the polymer particle-forming polypeptide is covalendy bound to the surface of the polymer particle.

[0054] In various embodiments, one or more of the one or more amorphous polymer particles has a degree of crystallinity below about 20%, for example, below about 15%, below about 10%, below about 5%, including for example below 2%, or has a degree of crystallinity of 0%. In an exemplary embodiment, substantially all of the amorphous polymer particles have a degree of crystallinity below about 20%, for example, below about 15%, below about 10%, below about 5%, including for example below 2%, or has a degree of crystallinity of 0%. [0055] In various embodiments, one or more of the one or more amorphous polymer particles has a glass transition temperature T _Gin the range from 0° to 60° C, for example in the range of from 0° to 50° C, of from 0° to 40° C, of from 0° to 35° C, and in particular of from 0° to 30° C. In an exemplary embodiment, substantially all of the amorphous polymer particles have a glass transition temperature T _Gin the range from 0° to 60° C, for example in the range of from 0° to 50° C, of from 0° to 40° C, of from 0° to 35° C, and in particular of from 0° to 30° C.

[0056] In one embodiment, one or more of the polymer particles, for example one or more amorphous polymer particles, comprises one or more ligands or binding domains displayed on the surface thereof. In one example, the one or more polymer particles, for example one or more amorphous polymer particles, comprises at least one ligand or binding domain capable of binding a target substance, reaction substrate, or contaminant, and at least one ligand capable of binding at least one support particle.

[0057] In one example, one or more of the polymer particles, for example one or more amorphous polymer particles, comprises a silica binding ligand, a silica-binding domain, a zeolite- binding domain or a cellulose-binding domain. [0058] In one embodiment, the polymer particles, for example the amorphous polymer particles, are bound to, associated with or comprise a permeable or semipermeable support, such as a semipermeable membrane, stationary phase, filter, filter cartridge, or the like.

[0059] In one embodiment, the source material is or is derived from a cell lysate. In one embodiment, the source material is or is derived from a protein expression system, including an in vitro protein expression system.

[0060] In one embodiment, the source material is or is derived from a food, including a dairy product or dairy processing stream, a fermentate including a wine or beer fermentate, and the like.

[0061] In one embodiment, the source material is a solution, including a reaction solution, a chemical synthesis solution, a chemical synthesis intermediate, and the like.

[0062] In one embodiment, the target substance is a polypeptide, including for example, a recombinant polypeptide, an antibody, an enzyme, a hormone, and the like.

[0063] In one embodiment, the target substance is a polynucleotide, including for example, a recombinant polynucleotide, a vector, an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, an miRNA, an siRNA, or a tRNA, or a DNA molecule such as a cDNA.

[0064] In one embodiment, the target substance is a cellular metabolite, including a secreted metabolite.

[0065] In various embodiments, the polymer particle comprises a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate (PHA). Most preferably the polymer comprises polyhydroxyalkanoate, preferably poly(3-hydroxybutyrate) (PHB).

[0066] In various embodiments, the polymer constituting the particle consists essentially of, or consists a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate (PHA). In one embodiment, the polymer comprises polyhydroxyalkanoate, preferably poly(3- hydroxybutyrate) (PHB).

[0067] In various embodiments the polymer particle comprises a polymer particle encapsulated by a phospholipid monolayer.

[0068] In various embodiments the polymer synthase is bound to the polymer particle or to the phospholipid monolayer or is bound to both.

[0069] In various embodiments the polymer particle comprises two or more different fusion polypeptides. [0070] In various embodiments the polymer particle comprises two or more different fusion polypeptides on the polymer particle surface.

[0071] In various embodiments the polymer particle comprises three or more different fusion polypeptides, such as three or more different fusion polypeptides on the polymer particle surface. [0072] In various embodiments the polymer particle further comprises at least one substance bound to or incorporated into the polymer particle, or a combination thereof.

[0073] In various embodiments the substance is bound to the polymer particle by cross- linking.

[0074] In various embodiments the polymer synthase is bound to the polymer particle or to the phospholipid monolayer or is bound to both.

[0075] In various embodiments the polymer synthase is covalently or non-covalently bound to the polymer particle it forms.

[0076] In various embodiments the polymer synthase is a PHA synthase from the class 1 genera Adnetobacter, Vibrio, Aeromonas, Chromobacterium, Pseudomonas, 7.oogloea, Alcaligenes, Delfiia, Burkholderia, Ralstonia, Rhodococcus, Gordonia, Rhodobacter, Paracoccus, Rickettsia, Caulobacter,

Methylobacterium, A^orhi^obium, Agrobacterium, Rhi^obium, Sinorhi^obium, Rickettsia, Crenarchaeota, Synechoystis, Ectothiorhodospira, Thiocapsa, Thyocystis and Allochromatium, the class 2 genera Burkho/deria and Pseudomonas, or the class 4 genera Bacillus, more preferably from the group comprising class 1 Adnetobacter ¹ sp. RA3849, Vibrio cholerae, Vibrio parahaemoyl ticus, Aeromonas punctata FA440, Aeromonas hydrophila, Chromobacterium molaceum, Pseudomonas sp. 61-3, 7.oogloea ramigera, Alcaligenes latus, Alcaligenes sp. SH-69, Delfiia addovorans, Burkholderia sp. DSMZ9242, Ralstonia eutrophia H16, Burkholderia cepacia, Rhodococcus rubber PP2, Gordonia rubripertinctus, Rickettsia prowa^ekii, Synechoystis sp. PCC6803, Ectothiorhodospira shaposhnikovii N 1 , Thiocapsa pfennigii 9111, Allochromatium vinosum D, Thyocystis molacea 2311, Rhodobacter sphaeroides, Paracoccus denitrificans, Rhodobacter capsulatus, Caulobacter crescentus,

Methylobacterium extorquens, A^orhi^obium caulinodans, Agrobacterium tumefaciens, Sinorhi^obium meliloti 41, Rhodospirillum rubrum HA, and Rhodopirillum rubrum ATCC25903, class 2 Burkholderia caryophylli, Pseudomonas chloraphis, Pseudomonas sp. 61-3, Pseudomonas putida U, Pseudomonas okovorans, Pseudomonas aeruginosa, Pseudomonas stationary phaseovorans, Pseudomonas

Pseudomonas mendocina, Pseudomonas pseudolcaligenes, Pseudomonas putida BM01, Pseudomonas nitroreducins, Pseudomonas chloraphis, and class 4 Bacillus megaterium and Bacillus sp. INT005.

[0077] In other embodiments the polymer synthase is a PHA polymer synthase from Gram- negative and Gram -positive eubacteria, or from archaea. [0078] In various examples, the polymer synthase may comprise a PHA polymer synthase from C. necator, P. aeruginosa, A. vinosum, B. megaterium, H. mansmortui, P. aureofaciens, or P. putida, which have Accession No.s AY836680, AE004091, AB205104, AF109909, YP137339, AB049413 and AF 150670, respectively. [0079] Other polymer synthases amenable to use in the present invention include polymer synthases, each identified by it accession number, from the following organisms: R. eutropha

(A34341), T. pfennigii (X93599), A punctata (032472), Pseudomonas sp. 61 -3 (AB014757 and

AB014758), R. sphaeroides (AAA72004), C. violaceum (AAC69615), A. borkumensis SK2 (CAL17662), A. borkumensis SK2 (CAL16866), R, sphaeroides KD131 (ACM01571 and YP002526072), R opac s A (BAH51880 and YP002780825), B. multivorans ATCC 17616 (YP001946215 and BAG43679), A. borkumensis SK2(YP693934 and YP693138), R, rubrum (AAD53179), gamma proteobacterium HTCC5015 (ZP05061661 and EDY86606),

sp. BH72 (YP932525), C. violaceum ATCC 12472 (NP902459), Limnobacter sp. MED105 (ZP01915838 and EDM82867), . algicola OG893 (ZP01895922 and EDM46004), R. phaeroides (CAA65833), C. violaceum ATCC 12472 (AAQ60457), A. latus (AAD10274, AAD01209 and AAC83658), S. maltophilia K279a (CAQ46418 and

YP001972712), R. solanaceamm IPO1609 (CAQ59975 and YP002258080), B. multivorans ATCC 17616 (YP001941448 and BAG47458), Pseudomonas sp. gll3 (ACJ02400), Pseudomonas sp. gl06 (ACJ02399), Pseudomonas sp. glOl (ACJ02398), R. sp. gl32 (ACJ02397), R. leguminosarum bv. viciae 3841

(CAK10329 and YP770390), A_Zoarcus sp. BH72 (CAL93638), Pseudomonas sp. LDC-5 (AAV36510), L. nitroferrum 2002 (ZP03698179), Thauera sp. MZ1T (YP002890098 and ACR01721), M. radiotolerans JCM 2831 (YP001755078 and ACB24395), Methylobacterium sp. 4-46 (YP001767769 and ACA15335), L nitroferrum 2002 (EEG08921), P. denitrificans (BAA77257), M. gryphiswaldense (ABG23018),

Pseudomonas sp. USM4-55 (ABX64435 and ABX64434), A. hydrophila (AAT77261 and AAT77258), Bacillus sp. INT005 (BAC45232 and BAC45230), P. putida (AAM63409 and AAM63407), G.

rubnpertinctus (AAB94058), B. megatenum (AAD05260), D. acidovorans (BAA33155), P. seriniphilus

(ACM68662), Pseudomonas sp. 14-3 (CAK18904), Pseudomonas sp. LDC-5 (AAX18690), Pseudomonas sp. PCI 7 (ABV25706), Pseudomonas sp. 3Y2 (AAV35431 , AAV35429 and AAV35426), P. mendodna (AAM10546 and AAM10544), P. nitroreducens (AAK19608), P. pseudoalcaligenes (AAK19605), P.

stationary phaseovorans (AAD26367 and AAD26365), Pseudomonas sp. USM7-7 (ACM90523 and ACM90522), P. fluorescens (AAP58480) and other uncultured bacterium (BAE02881, BAE02880, BAE02879, BAE02878, BAE02877, BAE02876, BAE02875, BAE02874, BAE02873, BAE02872, BAE02871, BAE02870, BAE02869, BAE02868, BAE02867, BAE0286, BAE02865, BAE02864, BAE02863, BAE02862, BAE02861 , BAE02860, BAE02859, BAE02858, BAE02857, BAE07146, BAE07145, BAE07144, BAE07143, BAE07142, BAE07141, BAE07140, BAE07139, BAE07138, BAE07137, BAE07136, BAE07135, BAE07134, BAE07133, BAE07132, BAE07131 , BAE07130, BAE07129, BAE07128, BAE07127, BAE07126, BAE07125, BAE07124, BAE07123, BAE07122, BAE07121, BAE07120, BAE07119, BAE07118, BAE07117, BAE07116, BAE07115, BAE07114,

BAE07113, BAE07112, BAE07111, BAE07110, BAE07109, BAE07108, BAE07107, BAE07106,

BAE07105, BAE07104, BAE07103, BAE07102, BAE07101, BAE07100, BAE07099, BAE07098,

BAE07097, BAE07096, BAE07095, BAE07094, BAE07093, BAE07092, BAE07091, BAE07090,

BAE07089, BAE07088, BAE07053, BAE07052, BAE07051, BAE07050, BAE07049, BAE07048,

BAE07047, BAE07046, BAE07045, BAE07044, BAE07043, BAE07042, BAE07041, BAE07040,

BAE07039, BAE07038, BAE07037, BAE07036, BAE07035, BAE07034, BAE07033, BAE07032,

BAE07031, BAE07030, BAE07029, BAE07028, BAE07027, BAE07026, BAE07025, BAE07024,

BAE07023, BAE07022, BAE07021, BAE07020, BAE07019, BAE07018, BAE07017, BAE07016,

BAE07015, BAE07014, BAE07013, BAE07012, BAE07011, BAE07010, BAE07009, BAE07008,

BAE07007, BAE07006, BAE07005, BAE07004, BAE07003, BAE07002, BAE07001, BAE07000,

BAE06999, BAE06998, BAE06997, BAE06996, BAE06995, BAE06994, BAE06993, BAE06992,

BAE06991, BAE06990, BAE06989, BAE06988, BAE06987, BAE06986, BAE06985, BAE06984,

BAE06983, BAE06982, BAE06981, BAE06980, BAE06979, BAE06978, BAE06977, BAE06976,

BAE06975, BAE06974, BAE06973, BAE06972, BAE06971, BAE06970, BAE06969, BAE06968,

BAE06967, BAE06966, BAE06965, BAE06964, BAE06963, BAE06962, BAE06961, BAE06960,

BAE06959, BAE06958, BAE06957, BAE06956, BAE06955, BAE06954, BAE06953, BAE06952,

BAE06951, BAE06950, BAE06949, BAE06948, BAE06947, BAE06946, BAE06945, BAE06944,

BAE06943, BAE06942, BAE06941, BAE06940, BAE06939, BAE06938, BAE06937, BAE06936,

BAE06935, BAE06934, BAE06933, BAE06932, BAE06931, BAE06930, BAE06929, BAE06928,

BAE06927, BAE06926, BAE06925, BAE06924, BAE06923, BAE06922, BAE06921, BAE06920,

BAE06919, BAE06918, BAE06917, BAE06916, BAE06915, BAE06914, BAE06913, BAE06912,

BAE06911, BAE06910, BAE06909, BAE06908, BAE06907, BAE06906, BAE06905, BAE06904,

BAE06903, BAE06902, BAE06901, BAE06900, BAE06899, BAE06898, BAE06897, BAE06896,

BAE06895, BAE06894, BAE06893, BAE06892, BAE06891, BAE06890, BAE06889, BAE06888,

BAE06887, BAE06886, BAE06885, BAE06884, BAE06883, BAE06882, BAE06881, BAE06880,

BAE06879, BAE06878, BAE06877, BAE06876, BAE06875, BAE06874, BAE06873, BAE06872,

BAE06871, BAE06870, BAE06869, BAE06868, BAE06867, BAE06866, BAE06865, BAE06864,

BAE06863, BAE06862, BAE06861, BAE06860, BAE06859, BAE06858, BAE06857, BAE06856,

BAE06855, BAE06854, BAE06853 and BAE06852).

[0080] In various embodiments the polymer synthase can be used for the in vitro production of polymer particles by polymerising or facilitating the polymerisation of the substrates (R)- Hydroxyacyl-CoA or other CoA thioester or derivatives thereof. [0081] In various embodiments the substrate or the substrate mixture comprises at least one optionally substituted amino acid, lactate, ester or saturated or unsaturated fatty acid, preferably acetyl-CoA.

[0082] In various embodiments, the catalyst is an enzyme. In a representative example, the catalyst is an enzyme, the precursor substance is a substrate of the enzyme, and the target substance is a product of the reaction catalysed by the enzyme. In further embodiments, the population of polymer particles, for example a population of amorphous polymer particles, may comprise more than one or more enzyme. Particularly contemplated are embodiments wherein a population of polymer particles, for example a population of amorphous polymer particles, comprises two or more enzymes wherein the product of a reaction catalysed by one enzyme is the substrate of the or an other enzyme, such as, for example, two or more enzymes comprising part or all of a synthetic or catalytic pathway. Alternative embodiments, wherein one population of polymer particles, for example a population of amorphous polymer particles, comprises one or more enzymes wherein the product of one or more reactions catalysed by said enzyme(s) is a substrate for one or more enzymes present on another population of polymer particles, for example another population of amorphous polymer particles, are also contemplated.

[0083] In one embodiment, the one or more polymer particles, for example the one or more amorphous polymer particles, or the one or more support particles, or both, are permanendy associated with the permeable or semipermeable support. In another embodiment, the one or more polymer particles, for example the one or more amorphous polymer particles, or the one or more support particles, or both, are reversibly associated with the permeable or semipermeable support.

[0084] In various embodiments the one or more polymer particles, for example the one or more amorphous polymer particles, are covalently or non-covalently bound to the semipermeable filter. For example, the one or more polymer particles, for example the one or more amorphous polymer particles, are adsorbed onto a semipermeable support or membrane. In another example, the one or more polymer particles, for example the one or more amorphous polymer particles, comprise a ligand or binding domain capable of binding to the semipermeable support or membrane.

[0085] In various embodiments the one or more support particles are covalently or non- covalendy bound to the permeable or semipermeable filter. For example, the one or more support particles are adsorbed onto a permeable or semipermeable support or membrane. In another example, the one or more support particles comprise a ligand or binding domain capable of binding to the permeable or semipermeable support or membrane. [0086] In various embodiments, the permeable or semipermeable support comprises one or more of the following: polyethersulfone, PVDF, PP, PEES HDPE (high density polyethylene), PP (polypropylene), PEEK (polyetheretherketone), PET and FEP (fluorinated ethylene propylene). In another embodiment, the permeable or semipermeable support comprises a polysaccharide including, for example, cellulose, derivatised cellulose, or stabilised cellulose. In yet another embodiment, the permeable or semipermeable support comprises one or more ceramics.

[0087] In various embodiments, the permeable or semipermeable filter is in one of the following configurations: spirally-wound, plate & frame, flat sheet, hollow fibre, spin-disc, or tubular. Examples thereof may conveniently be provided as a cassette or cartridge.

[0088] In various embodiments, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, are prepared, separated, or purified by tangential- flow filtration, for example, in the presence of one or more of the following: a detergent, a pH modifier, one or more solvents, one or more chaotropes, one or more enzymes, and one or more thiols. In certain embodiments the method includes a chemical treatment such as acid or base treatments.

[0089] In various embodiments, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, has improved stability.

[0090] In various embodiments, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, is reusable. In one example, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, has improved reusability. In one exemplary embodiment, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, retains activity after 5 or more, 10 or more, 15 or more, or 20 or more uses. In another exemplary embodiment, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, retains activity after 30 or more, 40 or more, 50 or more, 100 or more , 200 or more, or 300 or more uses.

[0091] In various embodiments, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, is capable of retaining activity for two weeks, three weeks, or four weeks or more of storage, for example at 4°C. [0092] In various embodiments, the method of preparing, separating, or purifying one or more substances or one or more polymer particles, for example a population of amorphous polymer particles, using tangential-flow filtration comprises or is preceded or followed by homogenisation, microfluidization, sonication, centrifugation or any combination thereof. [0093] In various embodiments, the ligand or binding domain capable of binding an antibody is selected from the group comprising protein A, protein G, protein A/ G , protein L, a recombinant variant thereof, a functional fragment thereof including recombinant functional fragments thereof, such as the Z domain of protein A, and any combination thereof, such as a ZZ domain comprising a contiguous repeat of the Z domain of protein A. [0094] In one embodiment, one or more of the polymer particles, for example a population of amorphous polymer particles, comprises a fusion polypeptide comprising a polymer particle- forming polypeptide and one or more GB1 domain of protein G from Streptococcus spp.

[0095] In one embodiment, the fusion polypeptide is or comprises a GB1 domain encoded by a polynucleotide sequence comprising 12 or more contiguous nucleotides of SEQ ID NO. 5 or a complement thereof. In another embodiment, the fusion polypeptide is or comprises a polypeptide encoded by a polynucleotide sequence comprising 12 or more contiguous nucleotides of SEQ ID NO. 5 or a complement thereof.

[0096] An exemplary ligand or binding domain capable of binding an antibody is encoded by a DNA sequence (SEQ ID NO. 5 in the attached Sequence ID Listing) or a complement thereof encoding an N-terminal linker (LEVLAVIDKRGGGGGSGGGSGGGSGGGG, [SEQ ID NO. 6]) and three GB1 binding domains from protein G (Streptococcus sp.), each separated by a linker region (SGGGSGGGSGGGGS, [SEQ ID NO. 7]).

[0097] In one embodiment, said polymer particle has a immunoglobulin binding capacity of greater than 30mg immunoglobulin/ g wet polymer particle. [0098] In one embodiment, the binding capacity is at least about 35mg immunoglobulin/ g wet polymer particle, about 40mg immunoglobulin/ g wet polymer particle, about 45mg

immunoglobulin/ g wet polymer particle, about 50mg immunoglobulin/ g wet polymer particle, about 55mg immunoglobulin/ g wet polymer particle, or about 60mg immunoglobulin/ g wet polymer particle. [0099] In one embodiment, the immunoglobulin is IgG.

[00100] In one embodiment, one or more of the polymer particles, for example one or more amorphous polymer particles, comprises a fusion polypeptide comprising a polymer particle- forming polypeptide, one or more antibody-binding domains, and one or more support particle- binding domains.

[00101] For example, one or more of the polymer particles, for example the one or more amorphous polymer particles, comprises a fusion polypeptide comprising a polymer particle - forming polypeptide, one or more antibody-binding domains, and one or more support particle- binding domains, wherein the antibody-binding domain is selected from the group comprising protein A, protein G, protein A/ G , protein L, a recombinant variant thereof, a functional fragment thereof including recombinant functional fragments thereof, such as the Z domain of protein A, and any combination thereof, such as a ZZ domain comprising a contiguous repeat of the Z domain of protein A.

[00102] For example, one or more of the polymer particles, for example the one or more amorphous polymer particles, comprises a fusion polypeptide comprising a polymer particle- forming polypeptide, one or more antibody-binding domains, and one or more support particle- binding domains, wherein the antibody-binding domain is selected from the group comprising protein A, protein G, protein A/ G , protein L, a recombinant variant thereof, a functional fragment thereof including recombinant functional fragments thereof, such as the Z domain of protein A, and any combination thereof, such as a ZZ domain comprising a contiguous repeat of the Z domain of protein A, and wherein the support particle-binding domain is selected from the group comprising a silica-binding domain, a cellulose-binding domain, or a zeolite-binding domain. [00103] In one embodiment, one or more of the polymer particles, for example the one or more amorphous polymer particles, comprises a fusion polypeptide comprising a polymer particle- forming polypeptide, one or more GB1 domains of protein G from Streptococcus spp., and one or more silaffin domains.

[00104] In various embodiments, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, comprises a population of support particles comprising or consisting of an inert media of a particle size range selected to allow adequate process flows.

[00105] In various embodiments, the chromatography stationary phase or the one or more populations of polymer particles, for example a population of amorphous polymer particles, comprises or consists of one or more polymer particles, for example one or more amorphous polymer particles, covalently linked to the support particles.

[00106] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[00107] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[00108] Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DETAILED DESCRIPTION OF THE INVENTION [00109] The present invention relates to methods and compositions for use in

chromatography, for example column chromatography techniques. Chromatography is a separation technology in which the feedstock is run over or around a stationary phase. Various forms of chromatography are well known in the art, and exemplary methods suited to the application of the present invention include various forms of affinity chromatography, where the selective binding or retention of desired or undesired substances is the basis for separation of target substances from contaminating material.

Definitions

[00110] As used herein, the term "amorphous polymer" is one substantially free of crystallinity, and is to be understood as those polymers which are solids at room temperature in spite of an irregular arrangement of the molecule chains. Particularly contemplated amorphous polymers are those polymers that were or are essentially non-crystalline having a degree of crystallinity at neutral pH below 20%, preferably below 5%, below 10%, below 5%, preferably below 2%, or is 0%, for example as assessed using the ATR-FTIR methodology as described in Porter MM, Yu J,

Crystrallization Kinetics of Poly(3-Hydroxybutyrate) Granules in Different Environmental

Conditions, Journal of Biomaterials and Nanobiotechnology 2 pp301-310, 2011. Additionally, those amorphous polymers are particularly suitable whose glass transition temperature T _Gis in the range from 0° to 60° C, preferably 0° to 50° C, preferably 0° to 40° C, preferably 0° to 35° C, and in particular 0° to 30° C, at neutral pH. [00111] As used herein, the term "amorphous polymer particle" is to be understood as those polymer particles comprising an amorphous polymer. Consequently, an amorphous polymer particle comprises, consists essentially of, or consists of polymer which is substantially free of crystallinity, for example those polymers which are solids at room temperature in spite of an irregular arrangement of the polymer molecule chains. Particularly contemplated amorphous polymers particles are those comprising, consisting essentially of, or consisting of polymers that were or are essentially non-crystalline having a degree of crystallinity at neutral pH below 20%, preferably below 5%, below 0%, below 5%, preferably below 2%, or is 0%, for example as assessed using the ATR- FTIR methodology as described in Porter MM, Yu J, CrystraUization Kinetics of Poly(3- Hydroxybutyrate) Granules in Different Environmental Conditions, Journal of Biomaterials and Nanobiotechnology 2 pp301-310, 2011. Those amorphous polymers are particularly suitable for forming amorphous polymer particles of the invention whose glass transition temperature T _Gis in the range from 0° to 60° C, preferably 0° to 50° C, preferably 0° to 40° C, preferably 0° to 35° C, and in particular 0° to 30° C, at neutral pH. It will be appreciated by those skilled in the art that, depending on the storage or operative conditions, some increase in degree of crystallinity of the amorphous polymer particles of the invention may occur. It will also be appreciated that in certain circumstances a reduction in crystallinity can be achieved, by for example, exposing the polymer particles to different (typically non-acidic) pH, aqueous conditions, or different temperature or pressure. Accordingly, the term amorphous polymer particle as used herein contemplates those polymer particles having at some point after their preparation a degree of crystallinity as outlined above, for example, substantially free of crystallinity when at neutral pH in an aqueous environment.

[00112] As used herein, the term "biopolymer" is to be understood as those polymers which are able to be synthesised by a biological system or entity, such as but not limited to an organism, a cell, or a protein. Accordingly, the terms "biopolyester" and "biopolythioester" is to be understood as those polyesters, and polythioesters, respectively, which are able to be synthesised by a biological system or entity. Examples include polyesters and polyhydroxycarboxylates produced by various bacteria and archea, typically as a means to store carbon or energy, such as but not limited to polythioesters and polyhydroxyalkanoates.

[00113] The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/ or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences. [00114] The term "comprising" as used in this specification means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. [00115] The term "contaminant" refers to a substance or substances in the source material that differ from the target substance, and are desirably excluded from the final target substance preparation. Typical contaminants of biological source materials include nucleic acids, proteins, peptides, endotoxins, viruses, etc. Contaminants that can be removed by the practice of the inventive method have one or more properties that differ from those of the desired product, e.g., molecular weight, charge, specific affinity for various ligands or binding domains, and so on.

[00116] The term "filter" and grammatical equivalents refers herein to a type of filter module or filter cassette that comprises a porous, permeable or semipermeable filter element through which the source medium to be filtered is flowed, typically in a substantially perpendicular fashion, for example for permeation through the filter element of selected component(s) or contaminants of the source medium.

[00117] The term "coupling reagent" as used herein refers to an inorganic or organic compound that is suitable for binding at least one substance or a further coupling reagent that is suitable for binding a coupling reagent on one side and at least one substance on the other side. Examples of suitable coupling reagents, as well as exemplary methods for their use including methods suitable for the chemical modification of particles or fusion proteins of the present invention, are presented in PCT/DE2003/002799, published as WO 2004/020623 (Bernd Rehm), herein incorporated by reference in its entirety.

[00118] The term "expression construct" refers to a genetic construct that includes elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5' to 3' direction:

(1) a promoter, functional in the host cell into which the construct will be introduced,

(2) the polynucleotide to be expressed, and

(3) a terminator functional in the host cell into which the construct will be introduced.

[00119] Expression constructs of the invention are inserted into a replicable vector for cloning or for expression, or are incorporated into the host genome. [00120] Examples of expression constructs amenable for adaptation for use in the present invention are provided in PCT/DE2003/002799 published as WO 2004/020623 (Bernd Rehm) and PCT/NZ2006/000251 published as WO 2007/037706 (Bernd Rehm) which are each herein incorporated by reference in their entirety. [00121] The terms "form a polymer particle" and "formation of polymer particles", as used herein in relation to particle-forming proteins refer to the activity of a particle-forming protein as discussed herein.

[00122] A "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the enzymatic or binding activity and/ or provides three dimensional structure of the polypeptide.

[00123] The term "fusion polypeptide", as used herein, refers to a polypeptide comprising two or more amino acid sequences, for example two or more polypeptide domains, fused through respective amino and carboxyl residues by a peptide linkage to form a single continuous polypeptide. It should be understood that the two or more amino acid sequences can either be directly fused or indirectly fused through their respective amino and carboxyl termini through a linker or spacer or an additional polypeptide.

[00124] In one embodiment, one of the amino acid sequences comprising the fusion polypeptide comprises a particle-forming protein. In one embodiment, one of the amino acid sequences comprising the fusion polypeptide comprises a polymer synthase. [00125] In one embodiment, one of the amino acid sequences comprising the fusion polypeptide comprises a fusion partner.

[00126] The term "fusion partner" as used herein refers to a polypeptide such as a protein, a protein fragment, a binding domain, a target-binding domain, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody (a VHH for example), a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')2 antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain (a ZZ domain for example), an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, biotin, a biotin derivative, an avidin, a streptavidin, a substrate, an enzyme, an abzyme, a co-factor, a receptor, a receptor fragment, a receptor subunit, a receptor subunit fragment, a ligand, an inhibitor, a hormone, a lectin, a polyhistidine, a coupling domain, a DNA binding domain, a FLAG epitope, a cysteine residue, a library peptide, a reporter peptide, an affinity purification peptide, or any combination of any two or more thereof. [00127] It should be understood that two or more polypeptides listed above can form the fusion partner.

[00128] In one embodiment the amino acid sequences of the fusion polypeptide are indirectly fused through a linker or spacer, the amino acid sequences of said fusion polypeptide arranged in the order of polymer synthase-linker- fusion partner, or fusion partner -linker-polymer synthase. In other embodiments the amino acid sequences of the fusion polypeptide are indirectly fused through or comprise an additional polypeptide arranged in the order of polymer synthase-additional polypeptide- fusion partner, or polymer synthase-linker- fusion partner -additional polypeptide. Again, N-terminal extensions of the polymer synthase are expressly contemplated herein. [00129] In one exemplary embodiment the amino acid sequences of the fusion polypeptide are indirectly fused through a linker or spacer, the amino acid sequences of said fusion polypeptide arranged in the order of polymer synthase-linker- antibody binding polypeptide or antibody binding polypeptide-linker-polymer synthase, or polymer synthase-linker-enzyme or enzyme-linker-polymer synthase, for example. In other exemplary embodiments the amino acid sequences of the fusion polypeptide are indirectly fused through or comprise an additional polypeptide arranged in the order of polymer synthase-additional polypeptide- antibody binding polypeptide or polymer synthase- additional polypeptide-enzyme, or polymer synthase-linker- antibody binding polypeptide -additional polypeptide or polymer synthase-linker-enzyme-additional polypeptide. Again, N-terminal extensions of the polymer synthase are expressly contemplated herein. [00130] A fusion polypeptide according to the invention may also comprise one or more polypeptide sequences inserted within the sequence of another polypeptide. For example, a polypeptide sequence such as a protease recognition sequence is inserted into a variable region of a protein comprising a particle binding domain.

[00131] Conveniently, a fusion polypeptide of the invention is encoded by a single nucleic acid sequence, wherein the nucleic acid sequence comprises at least two subsequences each encoding a polypeptide or a polypeptide domain. In certain embodiments, the at least two subsequences will be present "in frame" so as comprise a single open reading frame and thus will encode a fusion polypeptide as contemplated herein. In other embodiments, the at least two subsequences are present "out of frame", and are separated by a ribosomal frame-shifting site or other sequence that promotes a shift in reading frame such that, on translation, a fusion polypeptide is formed. In certain embodiments, the at least two subsequences are contiguous. In other embodiments, such as those discussed above where the at least two polypeptides or polypeptide domains are indirectly fused through an additional polypeptide, the at least two subsequences are not contiguous. [00132] Reference to a "binding domain" or a "domain capable of binding" is intended to mean one half of a complementary binding pair and may include binding pairs from the list above. For example, antibody-antigen, antibody-antibody binding domain, biotin-streptavidin, receptor- ligand, enzyme-inhibitor pairs. A target-binding domain will bind a target molecule in a sample, and are an antibody or antibody fragment, for example. A polypeptide-binding domain will bind a polypeptide, and are an antibody or antibody fragment, or a binding domain from a receptor or signalling protein, for example.

[00133] Examples of substances that are bound by a binding domain include a protein, a protein fragment, a peptide, a polypeptide, a polypeptide fragment, an antibody, an antibody fragment, an antibody binding domain, an antigen, an antigen fragment, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, a pharmaceutically active agent, a biologically active agent, an adjuvant or any combination of any two or more thereof. Such substances are "target components" in a sample that is analysed according to a method of the invention. [00134] Accordingly, a "domain capable of binding a target substance" and grammatical equivalents will be understood to refer to one component in a complementary binding pair, wherein the other component is the target substance.

[00135] The term "genetic construct" refers to a polynucleotide molecule, usually double- stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. In various embodiments, the insert polynucleotide molecule is derived from the host cell, or is derived from a different cell or organism and/ or is a recombinant polynucleotide. In one embodiment, once inside the host cell the genetic construct becomes integrated in the host genome, such as the host chromosomal DNA. In one example the genetic construct is linked to a vector.

[00136] The term "host cell" refers to a bacterial cell, a fungi cell, yeast cell, a plant cell, an insect cell or an animal cell such as a mammalian host cell that is either 1) a natural PHA particle producing host cell, or 2) a host cell carrying an expression construct comprising nucleic acid sequences encoding at least a thiolase and a reductase and optionally a phasin. Which genes are required to augment what the host cell lacks for polymer particle formation will be dependent on the genetic makeup of the host cell and which substrates are provided in the culture medium. [00137] The term "linker or spacer" as used herein relates to an amino acid or nucleotide sequence that indirectly fuses two or more polypeptides or two or more nucleic acid sequences encoding two or more polypeptides. In some embodiments the linker or spacer is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100 amino acids or nucleotides in length. In other embodiments the linker or spacer is about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or about 1000 amino acids or nucleotides in length. In still other embodiments the linker or spacer is from about 1 to about 1000 amino acids or nucleotides in length, from about 10 to about 1000, from about 50 to about 1000, from about 100 to about 1000, from about 200 to about 1000, from about 300 to about 1000, from about 400 to about 1000, from about 500 to about 1000, from about 600 to about 1000, from about 700 to about 1000, from about 800 to about 1000, or from about 900 to about 1000 amino acids or nucleotides in length.

[00138] In one embodiment the linker or spacer may comprise a restriction enzyme recognition site. In another embodiment the linker or spacer may comprise a protease cleavage recognition sequence such as enterokinase, thrombin or Factor Xa recognition sequence, or a self-splicing element such as an intein. In another embodiment the linker or spacer facilitates independent folding of the fusion polypeptides.

[00139] The terms "mixed population" or "heterogeneous population", as used herein, refers to two or more populations of entities, each population of entities within the mixed population differing in some respect from another population of entities within the mixed population. For example, when used in reference to a mixed population of expression constructs, this refers to two or more populations of expression constructs where each population of expression construct differs in respect of the fusion polypeptide encoded by the members of that population, or in respect of some other aspect of the construct, such as for example the identity of the promoter present in the construct. Alternatively, when used in reference to a mixed population of fusion polypeptides, this refers to two or more populations of fusion polypeptides where each population of fusion polypeptides differs in respect of the polypepetides, such as polymer synthase, the fusion partner such as an antibody binding domain or an enzyme, the members that population contains. For example, in the context of use in the preparation of a purified antibody, a mixed population of fusion polypeptides refers to two or more populations of fusion polypeptides where each population of fusion polypeptides differs in respect of the polypepetides, such as polymer synthase, the antibody binding domain, the members that population contains. Similarly, in the context of use in the preparation of a target substance a mixed population of fusion polypeptides refers to two or more populations of fusion polypeptides where each population of fusion polypeptides differs in respect of the polypepetides, such as polymer synthase, the enzyme, the precursor binding domain, or the enzyme-substrate binding domain the members that population contains. Still further, when used in reference to a mixed population of polymer particles, for example a population of amorphous polymer particles, this refers to two or more populations of polymer particles where each population of polymer particles differs in respect of the fusion polypeptide or fusion polypeptides the members of that population carry. Heterogeneous populations of polymer particles comprising two or more subpopulation of polymer particles, where each subpopulation may comprise one or more of the fusion polypeptides described herein (such as those above) are specifically contemplated. Similarly, heterogeneous populations of support particles comprising two or more subpopulation of support particles, for example where each subpopulation differs in a physical, chemical, or compositional characteristic to another, are again specifically contemplated.

[00140] The term "monolithic", for example when used with reference to a filter element matrix, contemplates a porous, three-dimensional material having a continuous interconnected pore structure in a single piece.

[00141] As used herein the term "non-deformable" when used in reference to for example support particles means a particle having a reduced degree of deformation or compressibility, for example when compared to one or more of the amorphous polymer particles comprising a chromatography stationary phases of the present invention.

[00142] As used herein the term "non-derivatized" when used in reference to for example support particles means a particle that has not been modified, including physically, chemically or biologically modified, for example so as to comprise (for example by cross-linking, adsorption, or absorption) a functional moiety.

[00143] The term "nucleic acid" as used herein refers to a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof. The term includes reference to a specified sequence as well as to a sequence complimentary thereto, unless otherwise indicated. The terms "nucleic acid" and "polynucleotide" are used herein interchangeably.

[00144] "Operably-linked" means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators. [00145] The term "over-expression" generally refers to the production of a gene product in a host cell that exceeds levels of production in normal or non-transformed host cells. The term "overexpression" when used in relation to levels of messenger RNA preferably indicates a level of expression at least about 3-fold higher than that typically observed in a host cell in a control or non- transformed cell. More preferably the level of expression is at least about 5-fold higher, about 0- fold higher, about 5-fold higher, about 20-fold higher, about 25-fold higher, about 30-fold higher, about 35-fold higher, about 40-fold higher, about 45-fold higher, about 50-fold higher, about 55- fold higher, about 60-fold higher, about 65-fold higher, about 70-fold higher, about 75-fold higher, about 80-fold higher, about 85-fold higher, about 90-fold higher, about 95-fold higher, or about 100-fold higher or above, than typically observed in a control host cell or non-transformed cell.

[00146] Levels of mRNA are measured using any of a number of techniques known to those skilled in the art including, but not limited to, Northern blot analysis and RT-PCR, including quantitative RT-PCR. [00147] The term "particle-binding protein", as used herein refers to proteins and protein domains capable of binding to the particle. Such binding may be mediated directly through interaction with the polymer, or via interaction with a moiety bound to the polymer, such as via a polymer synthase covalendy bound to the polymer. Particle-binding proteins suitable for use herein include one or more particle binding domains from proteins capable of binding to the polymer particle core, such as the C-terminal fragment of PHA synthase protein or the particle binding domain of polymer depolymerise.

[00148] The term "particle-forming protein", as used herein, refers to proteins involved in the formation of the particle. It may, for example, be selected from the group of proteins which comprises a polymer depolymerase, a polymer regulator, a polymer synthase and a particle size- determining protein. Preferably the particle-forming protein is selected from the group comprising a thiolase, a reductase, a polymer synthase and a phasin. A particle-forming protein such as a synthase may catalyse the formation of a polymer particle by polymerising a substrate or a derivative of a substrate to form a polymer particle. Alternatively, a particle-forming protein such as a thiolase, a reductase or a phasin may facilitate the formation of a polymer particle by facilitating

polymerisation. For example, a thiolase or reductase may catalyse production of suitable substrates for a polymerase. A phasin may control the size of the polymer particle formed. Preferably the particle-forming protein comprises a particle binding domain and a particle forming domain.

[00149] As used herein, the term "particle-forming reaction mixture" refers to at least a polymer synthase substrate if the host cell or expression construct comprises a synthase catalytic domain or a polymer synthase and its substrate if the host cell or expression construct comprises another particle-forming protein or a particle binding domain that is not a polymer synthase catalytic domain. [00150] A "particle size-determining protein" refers to a protein that controls the size of the polymer particles. It may for example be derived from the family of phasin-like proteins, preferably selected from the those from the genera Ralstonia, Alcaligenes and Pseudomonas, more preferably the phasin gene phaP from Ralstonia eutropha and the phasin gene phaF from Pseudomonas oleovorans. Phasins are amphiphilic proteins with a molecular weight of 14 to 28 kDa which bind tightly to the hydrophobic surface of the polymer particles. It may also comprise other host cell proteins that bind particles and influence particle size.

[00151] A polymer synthase comprises at least the synthase catalytic domain at the C-terminus of the synthase protein that mediates polymerisation of the polymer and attachment of the synthase protein to the particle core. Polymer synthases for use in the present invention are described in detail in Rehm, 2003, which is herein incorporated by reference in its entirety. For example, the polymer synthase is a PHA synthase from the class 1 genera Adnetobacter, Vibrio, Aeromonas, Chromobacterium, Pseudomonas, T_^oogloea, Alcaligenes, Delflia, Burkholderia, Ralstonia, Rhodococcus, Gordonia, Rhodobacter, Paracoccus, Rickettsia, Caulobacter, Methylobacterium, A^orhi^obium, Agrobacterium, Rhi^obium, Sinorhi^obium, Rickettsia, Crenarchaeota, Synechocystis, Ectothiorhodopira, Thiocapsa, Thyocystis and

Allochromatium, the class 2 genera Burkholderia and Pseudomonas, or the class 4 genera Bacillus, more preferably from the group comprising class 1 Adnetobacter sp. RA3849, Vibrio cholerae, Vibrio parahaemoyl ticus, Aeromonas punctata FA440, Aeromonas hydrophila, Chromobacterium molaceum, Pseudomonas sp. 61-3, Zoogloea ramigera, Alcaligenes latus, Alcaligenes sp. SH-69, Delftia acidovorans, Burkholderia sp. DSMZ9242, Ralstonia eutrophia HI 6, Burkholderia cepacia, Rhodococcus rubber PP2, Gordonia rubripertinctus, Rickettsia prowa^ekii, Synechocystis sp. PCC6803, Ectothiorhodospira shaposhnikovii Nl, Thiocapsa pfennigii 9111, Allochromatium vinosum I ⁾. Thyoystis violacea 2311, Rhodobacter phaeroides, Paracoccus denitrificans, Rhodobacter capsulatus, Caulobacter crescentus, Methylo bacterium extorquens, A^orhi^obium caulinodans, Agrobacterium tumefaciens, Sinorhi^obium meliloti 41, Rhodospirillum rubrum HA, and Rhodopirillum rubrum ATCC25903, class 2 Burkholderia caryophylli, Pseudomonas chloraphis, Pseudomonas sp. 61-3, Pseudomonas putida U, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas stationary phaseovorans, Pseudomonas Pseudomonas mendocina, Pseudomonas pseudolcaligenes, Pseudomonas putida BM01, Pseudomonas nitroreducins, Pseudomonas chloraphis, and class 4 Bacillus megaterium and Bacillus sp. INT005.

[00152] Other polymer synthases amenable to use in the present invention include polymer synthases, each identified by it accession number, from the following organisms: C. necator

(AY836680), P. aeruginosa (AE004091), A vinosum (AB205104), B. megaterium (AF109909), H. marismortui (YP137339), P. aureofaciens (AB049413), P. putida (AF150670), R. eutropha (A34341), T. pfennigii (X93599), A punctata (032472), Pseudomonas p. 61-3 (AB014757 and AB014758), R.

phaeroides (AAA72004, C. molaceum (AAC69615), A. horkumensis SK2 (CAL17662), A. horkumensis SK2 (CAL16866), R. phaeroides KD131 (ACM01571 AND YP002526072), R. opacus B4 (BAH51880 and YP002780825), B. multivorans ATCC 17616 (YP001946215 and BAG43679), borkumensis SK2(YP693934 and YP693138), R. rubrum (AAD53179), gamma proteobacterium HTCC5015

(ZP05061661 and EDY86606), Azoarcus sp. BH72 (YP932525), C. violaceum ATCC 12472

(NP902459), Umnobacter p. MED105 (ZP01915838 and EDM82867), M. algicola DG893

(ZPOl 895922 and EDM46004), R. phaeroides (CAA65833), C. violaceum ATCC 12472 (AAQ60457), A. lotus (AAD10274, AAD01209 and AAC83658), S. maltophilia K279a (CAQ46418 and

YP001972712), R. solanaceamm IPO1609 (CAQ59975 and YP002258080), B. multivorans ATCC 17616 (YP001941448 and BAG47458), Pseudomonas sp. gll3 (ACJ02400), Pseudomonas sp. gl06 (ACJ02399), Pseudomonas sp. glOl (ACJ02398), R. p. gl32 (ACJ02397), R. leguminosarum bv. ήάα6 3841 (CAK10329 and YP770390), Azoarcus p. BH72 (CAL93638), Pseudomonas p. LDC-5 (AAV36510), L. nitroferrum 2002 (ZP03698179), Thauera p. MZ1T (YP002890098 and ACR01721), M. radiotolerans JCM 2831 (YP001755078 and ACB24395), Methylobacterium p. 4-46 (YP001767769 and ACA15335), L.

nitroferrum 2002 (EEG08921), P. denitrificans (BAA77257), M. gryphiswaldense (ABG23018), Pseudomonas sp. USM4-55 (ABX64435 and ABX64434), A. hydrophila (AAT77261 and AAT77258), Bacillus sp. INT005 (BAC45232 and BAC45230), P. putida (AAM63409 and AAM63407), G. rubripertinctus (AAB94058), B. megaterium (AAD05260), D. acidovorans (BAA33155), P. seriniphilus (ACM68662), Pseudomonas sp. 14-3 (CAK18904), Pseudomonas p. LDC-5 (AAX18690), Pseudomonas p. PCI 7 (ABV25706), Pseudomonas sp. 3Y2 (AAV35431, AAV35429 and AAV35426), P. mendocina

(AAM10546 and AAM10544), P. nitroreducens (AAK19608), P. pseudoalcaligenes (AAK19605), P.

stationary phaseovorans (AAD26367 and AAD26365), Pseudomonas p. USM7-7 (ACM90523 and

ACM90522), P. fluorescens (AAP58480) and other uncultured bacterium (BAE02881, BAE02880, BAE02879, BAE02878, BAE02877, BAE02876, BAE02875, BAE02874, BAE02873, BAE02872, BAE02871, BAE02870, BAE02869, BAE02868, BAE02867, BAE0286, BAE02865, BAE02864, BAE02863, BAE02862, BAE02861, BAE02860, BAE02859, BAE02858, BAE02857, BAE07146, BAE07145, BAE07144, BAE07143, BAE07142, BAE07141, BAE07140, BAE07139, BAE07138, BAE07137, BAE07136, BAE07135, BAE07134, BAE07133, BAE07132, BAE07131, BAE07130, BAE07129, BAE07128, BAE07127, BAE07126, BAE07125, BAE07124, BAE07123, BAE07122, BAE07121, BAE07120, BAE07119, BAE07118, BAE07117, BAE07116, BAE07115, BAE07114, BAE07113, BAE07112, BAE07111, BAE07110, BAE07109, BAE07108, BAE07107, BAE07106, BAE07105, BAE07104, BAE07103, BAE07102, BAE07101, BAE07100, BAE07099, BAE07098, BAE07097, BAE07096, BAE07095, BAE07094, BAE07093, BAE07092, BAE07091, BAE07090, BAE07089, BAE07088, BAE07053, BAE07052, BAE07051, BAE07050, BAE07049, BAE07048, BAE07047, BAE07046, BAE07045, BAE07044, BAE07043, BAE07042, BAE07041, BAE07040, BAE07039, BAE07038, BAE07037, BAE07036, BAE07035, BAE07034, BAE07033, BAE07032, BAE07031, BAE07030, BAE07029, BAE07028, BAE07027, BAE07026, BAE07025, BAE07024, BAE07023, BAE07022, BAE07021, BAE07020, BAE07019, BAE07018, BAE07017, BAE07016, BAE07015, BAE07014, BAE07013, BAE07012, BAE07011, BAE07010, BAE07009, BAE07008, BAE07007 BAE07006, BAE07005, BAE07004, BAE07003 BAE07002, BAE07001 BAE07000, BAE06999 BAE06998, BAE06997, BAE06996, BAE06995 BAE06994, BAE06993 BAE06992, BAE06991 BAE06990, BAE06989, BAE06988, BAE06987 BAE06986, BAE06985 BAE06984, BAE06983 BAE06982, BAE06981, BAE06980, BAE06979 BAE06978, BAE06977 BAE06976, BAE06975 BAE06974, BAE06973, BAE06972, BAE06971 BAE06970, BAE06969 BAE06968, BAE06967 BAE06966, BAE06965, BAE06964, BAE06963 BAE06962, BAE06961 BAE06960, BAE06959 BAE06958, BAE06957, BAE06956, BAE06955 BAE06954, BAE06953 BAE06952, BAE06951 BAE06950, BAE06949, BAE06948, BAE06947 BAE06946, BAE06945 BAE06944, BAE06943 BAE06942, BAE06941, BAE06940, BAE06939 BAE06938, BAE06937 BAE06936, BAE06935 BAE06934, BAE06933, BAE06932, BAE06931 BAE06930, BAE06929 BAE06928, BAE06927 BAE06926, BAE06925, BAE06924, BAE06923 BAE06922, BAE06921 BAE06920, BAE069 9 BAE069 8, BAE06917, BAE06916, BAE06915 BAE06914, BAE06913 BAE06912, BAE06911 BAE069 0, BAE06909, BAE06908, BAE06907 BAE06906, BAE06905 BAE06904, BAE06903 BAE06902, BAE06901, BAE06900, BAE06899 BAE06898, BAE06897 BAE06896, BAE06895 BAE06894, BAE06893, BAE06892, BAE06891 BAE06890, BAE06889 BAE06888, BAE06887 BAE06886, BAE06885, BAE06884, BAE06883 BAE06882, BAE06881 BAE06880, BAE06879 BAE06878, BAE06877, BAE06876, BAE06875 BAE06874, BAE06873 BAE06872, BAE0687 BAE06870, BAE06869, BAE06868, BAE06867 BAE06866, BAE06865 BAE06864, BAE06863 BAE06862, BAE06861, BAE06860, BAE06859 BAE06858, BAE06857 BAE06856, BAE06855 BAE06854, BAE06853 and BAE06852).

[00153] The N-terminal fragment of PHA synthase protein (about amino acids 1 to 200, or 1 to 150, or 1 to 100) is highly variable and in some examples is deleted or replaced by an enzyme, an antibody binding domain, or another fusion partner without inactivating the synthase or preventing covalent attachment of the synthase via the polymer particle binding domain (i.e. the C-terminal fragment) to the polymer core. The polymer particle binding domain of the synthase comprises at least the catalytic domain of the synthase protein that mediates polymerisation of the polymer core and formation of the polymer particles.

[00154] In some embodiments the C-terminal fragment of PHA synthase protein is modified, partially deleted or partially replaced by an enzyme, an antibody binding domain, or another fusion partner without inactivating the synthase or preventing covalent attachment of the synthase to the polymer particle.

[00155] In certain cases, the enzyme, the antibody binding domain, or another fusion partner are fused to the N-terminus and/ or to the C-terminus of PHA synthase protein without inactivating the synthase or preventing covalent attachment of the synthase to the polymer particle. Similarly, in other cases the enzyme, the antibody binding domain, or another fusion partner, are inserted within the PHA synthase protein, or indeed within the particle-forming protein. Examples of PhaC fusions are known in the art and presented herein.

[00156] In one specific example, the N-terminal fragment of PHA synthase protein (about amino acids 1 to 200, or 1 to 50, or 1 to 100) is deleted or replaced by an antibody binding domain such as the Z domain of protein A or a tandem repeat of same without inactivating the synthase or preventing covalent attachment of the synthase to the polymer particle.

[00157] A "polymer depolymerase" as used herein refers to a protein which is capable of hydrolysing existing polymer, such as that found in a polymer particle, into water soluble monomers and oligomers. Examples of polymer depolymerases occur in a wide variety of PHA-degrading bacteria and fungi, and include the PhaZ — PhaZ7 extracellular depolymerases from Paucimonas lemoignei, the PhaZ depolymerases from Addovomx sp., A.faecalis (strains AE 22 and T ), Delfiia (Comamonas) acidovorans strain YM 069, Comamonas testosteroni, Comamonas sp., ain HS, Pseudomonas sp. strain GM101 (acession no. AF293347), P. fluorescens strain G

R.

pickettii (strains A and K , acession no. J04223, D253 5), S. exfoliatus K 0 and Streptomyces hygroscopicus (see Jendrossek D., and Handrick, R., Microbial Degredation of Poyl hydroxyalkanoates, Annual Review of Microbiology, 2002, 56:403-32).

[00158] The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention are purified natural products, or are produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide variant, or derivative thereof.

[00159] The term "promoter" refers to non transcribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.

[00160] When used in respect of a polymer particle or stationary phase of the invention, the phrase "retaining activity" and grammatical equivalents and derivatives thereof is intended to mean that the polymer particle or stationary phase still has useful binding activity, for example, useful support particle binding activity, or target substance binding activity, or both useful support particle binding activity and target substance binding activity. Preferably, the retained activity is at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the original activity, and useful ranges may be selected between any of these values (for example, from about 35 to about 100%, from about 50 to about 00%, from about 60 to about 100%, from about 70 to about 100%, from about 80 to about 100%, and from about 90 to about 100%). For example, preferred polymer particles or stationary phases of the invention retain activity for a given storage period, for example retain at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the original activity of the polymer particle or stationary phase after about 1 month or more at 4 °C.

Similarly, preferred stationary phases of the invention are capable of supporting the maintenance of useful activity of the polymer particles they comprise, and can be said to retain activity, ideally until applied using the methods contemplated herein.

[00161] As used herein, the term "improved stability" when used in relation to a stationary phase of the invention means a stationary phase capable of retaining binding activity, supporting activity, usability or resuability for a given period, or under particular conditions, or both, for example, after 1 month at 4°C.

[00162] As used herein, the term "reusable" or "reusability" and grammatical equivalents and derivatives thereof, is intended to mean that the polymer particle or stationary phase has retained useful binding activity, for example, useful support particle binding activity, or target substance binding activity, or both useful support particle binding activity and target substance binding activity after multiple cycles of reuse, including reuse after a period of storage of, for example, one month or more at 4 °C. Preferably, the polymer particle or stationary phase is reusable up to 5, 10, 15, or 20 or more times. [00163] The term "substance" when referred to in relation to being bound to or absorbed into or incorporated within a polymer particle is intended to mean a substance that is bound by a fusion partner or a substance that is able to be absorbed into or incorporated within a polymer particle.

[00164] As used herein, the term "support particle" contemplates a particle comprising a stationary phase for chromatography of the present invention, wherein an interaction of the particle with one or more target substances is incidental. In other words, the primary purpose of the support particle (including, for example, substantially all of a population of support particles) is not to interact directly with one or more target substances or precursors thereof, but to support the one or more amorphous polymer particles.

[00165] The term "terminator" refers to sequences that terminate transcription, which are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions. [00166] The term "variant" as used herein refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants are naturally-occurring allelic variants, or non- naturally occurring variants. Variants are from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polynucleotides and polypeptides possess biological activities that are the same or similar to those of the wild type polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

Polynucleotide and polypeptide variants [00167] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 5 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. A number of nucleic acid analogues are well known in the art and are also contemplated.

[00168] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is preferably at least 5 nucleotides in length. The fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods. [00169] The term "fragment" in relation to promoter polynucleotide sequences is intended to include sequences comprising cis-elements and regions of the promoter polynucleotide sequence capable of regulating expression of a polynucleotide sequence to which the fragment is operably linked.

[00170] Preferably fragments of promoter polynucleotide sequences of the invention comprise at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 00, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800, more preferably at least 900 and most preferably at least 000 contiguous nucleotides of a promoter polynucleotide of the invention.

[00171] The term "primer" refers to a short polynucleotide, usually having a free 3ΌΗ group, that is hybridized to a template and used for priming polymerization of a polynucleotide

complementary to the template. Such a primer is preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.

[00172] The term "probe" refers to a short polynucleotide that is used to detect a

polynucleotide sequence that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.

[00173] The term "variant" as used herein refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants are naturally-occurring allelic variants, or non- naturally occurring variants. Variants are from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polynucleotides and polypeptides possess biological activities that are the same or similar to those of the wild type polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein. Polynucleotide variants

[00174] Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, at least 100 nucleotide positions, or over the entire length of the specified polynucleotide sequence.

[00175] Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI

(ftp:/ / ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

[00176] The identity of polynucleotide sequences can be examined using the following unix command line parameters: bl2seq -i nucleotideseql— j nucleotideseq2 -F F -p blastn

[00177] The parameter— F F turns off filtering of low complexity sections. The parameter— p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities = ".

[00178] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/ emboss/ align/.

[00179] Alternatively the GAP program can be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235. [00180] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).

[00181] The similarity of polynucleotide sequences can be examined using the following unix command line parameters: bl2seq -i nucleotideseql— j nucleotideseq2 -F F -p tblastx

[00182] The parameter— F F turns off filtering of low complexity sections. The parameter— p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

[00183] Variant polynucleotide sequences preferably exhibit an E value of less than x 0⁴⁰, more preferably less than 1 x 10^~20, less than 1 x 10^~30, less than 1 x 10^~40, less than 1 x 10^~50, less than 1 x 10^~60, less than 1 x 10™, less than 1 x lO^"80, less than 1 x 10^~90, less than 1 x 10⁴⁰⁰, less than 1 x 10⁴¹⁰, less than 1 x 10⁴²⁰ or less than 1 x 10⁴²³ when compared with any one of the specifically identified sequences.

[00184] Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.

[00185] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

[00186] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30°C (for example, 10 °C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 962, PNAS 84: 390). Typical stringent conditions for polynucleotide of greater than 00 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in X SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.

[00187] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 0°C below Tm. On average, the Tm of a polynucleotide molecule of length less than 00 bp is reduced by approximately

(500/oligonucleotide length)°C.

[00188] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA- PNA hybrid having a length less than 100 bases are 5 to 0°C below the Tm.

[00189] Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a

polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG

(tryptophan), in some examples other codons for the same amino acid are changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

[00190] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[00191] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence can be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2. 0 [Oct 2004]) from NCBI

(ftp:/ / ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.

Polypeptide Variants [00192] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, at least 100 amino acid positions, or over the entire length of a polypeptide of the invention.

[00193] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2. 0 [Oct 2004]) in bl2seq, which is publicly available from NCBI (ftp:/ / ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[00194] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[00195] Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides can be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences can be examined using the following unix command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp

[00196] Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 ¹⁰, more preferably less than 1 x 10^~20, less than 1 x 10^~30, less than 1 x 10^~40, less than 1 x 10^~50, less than 1 x 10^~60, less than 1 x 10™, less than 1 x 10^~80, less than 1 x 10^~90, less than 1 x O ⁰⁰, less than 1 x 10 ¹⁰, less than 1 x 0⁴²⁰ or less than 1 x 0⁴²³ when compared with any one of the specifically identified sequences.

[00197] The parameter— F F turns off filtering of low complexity sections. The parameter— p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. [00198] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[00199] A polypeptide variant of the present invention also encompasses that which is produced from the nucleic acid encoding a polypeptide, but differs from the wild type polypeptide in that it is processed differendy such that it has an altered amino acid sequence. For example, a variant is produced by an alternative splicing pattern of the primary RNA transcript to that which produces a wild type polypeptide.

[00200] The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. In certain examples the vector is capable of replication in at least one additional host system, such as E. colt.

Chromatography

[00201] Generally, the invention finds application in chromatography, for example column chromatography technologies. For example, in one embodiment the invention relates to a process for preparing one or more target substances from a source liquid, the process comprising: contacting the source liquid with a population of polymer particles, for example a population of amorphous polymer particles, in or prior to addition to a chromatography system, for example a

chromatography column, wherein one or more of the following steps are performed: concentrating the population of polymer particles, separating one or more contaminants from the one or more polymer particle-bound target substances or a polymer particle-bound precursor thereof, eluting the target substance from the polymer particles; and recovering the target substance.

[00202] In embodiments relating to a particle-bound precursor of the target substance, one or more of the polymer particles comprises one or more enzymes capable of catalysing the conversion of the precursor to the target substance, or to a further precursor to the target substance. For example, in one embodiment the precursor of the target substance is a substrate of an enzyme capable of catalysing the conversion of the substrate to the target substance, and one or more of the polymer particle comprises the enzyme. In another example, the precursor of the target substance is a substrate of an enzyme capable of catalysing the conversion of the substrate to a further precursor to the target substance, which itself is the substrate of a second enzyme capable of catalysing the conversion of the further precursor to the target substance, and one or more of the polymer particles comprises the first enzyme, the second enzyme, or both the first and the second enzyme. It will be appreciated that by providing one or more polymer particles comprising appropriately chosen enzymes, a series of catalytic steps in the conversion of a precursor to the target substance can be employed.

[00203] In another embodiment the invention relates to a process for preparing one or more target substances from a source liquid, the process comprising: contacting the source liquid with a population of polymer particles, for example a population of amorphous polymer particles, comprising a chromatography stationary phase of the invention, wherein one or more of the following steps are performed: concentrating the population of polymer particles, separating one or more target substances or a precursor thereof from one or more polymer particle-bound contaminants, and recovering the target substance. [00204] In one embodiment, the contacting the source liquid with a population of polymer particles, for example a population of amorphous polymer particles, occurs prior to introduction into a chromatography system.

[00205] The compositions, methods, and polymer particles of the invention have application in conjunction with existing chromatography systems and technologies. A great variety of such systems exist. [00206] The compositions, methods, and polymer particles of the invention have application in conjunction with existing packed bed chromatography systems, for example expanded bed chromatography.

[00207] Filters suitable for use in the present invention include microfiltration, ultrafiltration, nanofiltration and reverse osmosis filter systems. In one exemplary embodiment, the filter comprises a multiplicity of filter sheets (filtration membranes) in a stacked arrangement. In another embodiment, the filter comprises a monolithic element, such as a monolithic matrix.

[00208] As will be appreciated, many chromatography systems are currently available and are suitable for use in conjunction with the present invention. Commercially available systems include, for example, the Profina System (BioRad), the AKTA system (GE), the K-Prime system (Millipore) and PKP chromatography system (Pall) employing suitable affinity chromatography media such as (MabSelect GE), ProSep (Millipore) and HyperD Ceramic (Pall) and Affiprep protein A(BioRad).

[00209] A general outline of exemplary chromatography processes applicable to the present invention is as follows: an exemplary, simple system utilizes a feed pump to allow circulation of various liquids, including feedstock and buffers in the system from reservoir (s). Unbound compounds and solutions pass through the column. The source liquid may optionally be preprocessed, for example, to remove particulate or solid matter (for example by centrifugation or filtration techniques well known in the art), concentrated, or diluted, as required for subsequent purification. [00210] The source liquid is then contacted with a population of polymer particles, for example a population of amorphous polymer particles, for a time sufficient to allow the formation of particle:target complexes. Generally, the source liquid is contacted with the polymer particles for a time sufficient to lead to binding of a desirable proportion of the target(s) to the polymer particles. Processing of the source liquid and polymer particles in the chromatography system will typically be so as to ensure optimal contact and binding of the target(s).

[00211] The particle:target complexes are thus concentrated, then the complexes are washed (typically selected to dissociate non-specifically bound contaminants from the particle:target complexes), whereupon the target substance is eluted from the particles, (d) the target substance is then separated from the polymer particles, thereby to recover purified target substance. The target substance may optionally be (e) further processed, for example by concentration.

[00212] In a further exemplary embodiment, the source liquid comprises a precursor of the target substance, for example, a substrate of one more enzymes, the product of which is a desired target substance. In this embodiment, the source liquid comprising the precursor substance is contacted with one or more polymer particles, for example one or more of the amorphous polymer particles, comprising one or more enzymes capable of catalysing the conversion of the precursor to the target substance. As described above, the source liquid and polymer particles are contacted for a time sufficient to form complexes, albeit in this case a particle:enzyme-substrate complex. The source liquid and biopolymer particle mixture is maintained for a time sufficient to enable both a desirable proportion of precursor to be bound by the biopolymer particle, and to enable the conversion of the precursor molecule to the target substance.

[00213] A further general scheme for the purification of a target substance using the methods of the invention wherein the polymer particles, for example one or more amorphous polymer particles, are used to enrich the target substance by removal of one or more contaminants. In this case, the source liquid is contacted with one or more populations of polymer particles capable of binding one or more contaminants present in the source liquid. Here, the formation of

particlexontaminant complexes allows the elution and recovery of target substance(s) which may then be further processed (including for example, via one or more chromatography methods of the present invention as described herein). Subsequent washing (typically with a second wash buffer) allows dissociation with the particlexontaminant complexes, wherein the biopolymer particle comprising the chromatography system may be reused.

[00214] Accordingly, those skilled in the art will recognise that the various embodiments of the invention (including those representative examples outlined above) contemplate the elution of desired target substances from the chromatography system either before, or after, elution of various contaminants, depending on the particular configuration or the particular population of polymer particles used.

Support particles

[00215] The support particles of the invention may comprise a wide variety of stationary phase materials commonly used in chromatographic methods.

[00216] In certain embodiments, in particular for application in HPLC systems, the support particles have an average size/ diameter of about 0.5 um to about 10.0 um, or more particularly about 3 um to about 5.0 um. In other embodiments, including for example larger scale preparative methods, the support particles have an average size/ diameter of about 5μηι to 500μηι, for example from about 5μηι to 500μπι, from about 50μηι to 400μπι, or from about 50μηι to about 300μηι. In certain circumstances, particle size distribution is controlled, for example such that it remains within approximately 10% of the mean. The stationary phase material is porous in some examples, but will frequently be non-porous, and will in certain embodiments be chemically modified or otherwise derivatised.

[00217] In certain embodiments spherically shaped particles, rather than irregularly shaped particles, are used. It is generally considered in the art that irregularly-shaped materials are more difficult to pack than spherical materials, and that spherical materials exhibit greater packed bed stability than columns packed with irregularly-shaped materials of the same size. However, irregularly-shaped particles are in certain embodiments desired, for example to provide increased surface area or interstitial volume for interaction with the polymer particles, for example the one or more amorphous polymer particles, of the invention. [00218] In general, any particulate stationary phase material known in the art for use in column chromatography, such as those known for use in FPLC columns including prepacked FPLC columns, are suitable for use as support particles in the chromatography stationary phases, devices, and methods of the present intention. Examples of suitable materials for use include alumina, silica, titanium oxide, zirconium oxide, ceramic materials, organic polymers, zeolites, diatomaceous earths, or any mixtures of two or more thereof. Such materials that have been bonded with a surface modifier are also contemplated. Such surface modifiers may be an alkyl group, alkenyl group, alkynyl group, aryl group, cyano group, amino group, diol group, nitro group, ester group, or an alkyl or aryl group containing an embedded polar functionality. For example, an alkyl group surface modifier group may be a methyl, ethyl, propyl isopropyl, butyl, tert-butyl sec-butyl pentyl, isopentyl, hexyl, cyclohexyl, octyl or octadecyl group. Further examples of suitable materials for use as support particle materials include alkyl-bonded, phenyl-bonded, cyano-bonded, diol-bonded, and amino-bonded silica, and mixtures thereof. Suitable materials are readily available from a wide variety of commercial sources, including Waters Corporation (Milford, Mass., USA), Alltech Associates, Inc. (Deerfield, 111., USA), Beckman Instruments, Inc. (Fullerton, Calif, USA), Gilson, Inc. (Middleton, Wis., USA), EM Science (Gibbstown, N.J., USA), Supelco, Inc. (Bellefonte, Pa., USA).

Source materials

[00219] The present invention relates to the preparation of a target substance from a source material. A "source material" as used herein refers to a material, typically a liquid, containing at least one and frequently more than one substance, usually a biological substance, or product of value which are sought to be extracted or purified from other substances present in the source material. Generally, source materials may for example be aqueous solutions, organic solvent systems, or aqueous/ organic solvent mixtures or solutions. The source materials are often complex mixtures or solutions containing many biological molecules such as proteins, antibodies, hormones, and viruses as well as small molecules such as salts, sugars, lipids, and the like. While a typical source material of biological origin may begin as an aqueous solution or suspension, it may also contain organic solvents used in earlier separation steps such as solvent precipitations, extractions, and the like. Examples of source liquids that may contain valuable biological substances amenable to the purification method of the invention include, but are not limited to, a culture supernatant from a bioreactor, a homogenized cell suspension, plasma, plasma fractions, and dairy processing streams such as milk, colostrum and whey such as cheese whey.

[00220] In various embodiments, the source material comprises one or more liquids selected from the group consisting of serum, plasma, plasma fractions, whole blood, milk, colostrum, whey, cell fluids, tissue culture fluids, plant cells fluids, plant cell homogenates, and tissue homogenates. For example, the source material is a plant extract, such as a fruit juice or a vegetable juice.

Fermentates are particularly contemplated, as are cultures or culture supernatants, particularly those of cultures expressing one or more recombinant proteins, such as one or more monoclonal antibodies. Target substances

[00221] As above, the present invention relates to the preparation of a target substances from source materials, including source materials comprising a precursor of the target substance. The term "target substance" as used herein refers to the one or more desired product or products to be prepared or purified from the source liquid. Target substances are typically biological products of value, for example, immunoglobulins, clotting factors, vaccines, antigens, antibodies, selected proteins or glycoproteins, peptides, enzymes, metabolites, and the like.

[00222] In various embodiments, the target substance is selected from the group consisting of vaccines, clotting factors, immunoglobulins, antigens, antibodies, proteins, glycoproteins, peptides, sugars, carbohydrates, and enzymes. [00223] In various embodiments, the one or more affinity ligands or binding domains bind at least one of the target species selected from the group consisting of proteins, nucleic acids, viruses, sugars, carbohydrates, immunoglobulins, clotting factors, glycoproteins, peptides, antibodies, antigens, hormones, or polynucleotides.

[00224] However, the invention finds application in the preparation of a wide variety of target substances other than those typically considered to be 'biological', as will be appreciated on recognition of the multiplicity of functional moieties which may be associated with the polymer particles, for example the one or more amorphous polymer particles, described herein. For example, the polymer particles of the invention may be conveniendy functionalised with metal or metal-ion binding moieties, such as metal or metal-ion co-ordinating polypeptides, for example by expression of a polymer synthase:metal-binding polypeptide fusion polypeptide. Indeed, the ability to fuse one or more protein functionalities to the polymer- forming protein or polymer-binding protein comprising the polymer particles allows for application in the preparation of an extremely varied range of target substances.

[00225] The fusion polypeptides comprising the polymer particles of the invention may conveniently be produced using biotechnological techniques well known in the art, including the use of one or more expression constructs.

Expression Constructs [00226] Processes for producing and using expression constructs for expression of fusion polypeptides in microorganisms, plant cells or animal cells (cellular expression systems) or in cell free expression systems, and host cells comprising expression constructs useful for forming polymer particles for use in the invention are well known in the art (e.g. Sambrook et al., 1987; Ausubel et al., 1987). [00227] Expression constructs for use in methods of the invention are in one embodiment inserted into a replicable vector for cloning or for expression, or in another embodiment are incorporated into the host genome. Various vectors are publicly available. The vector is, for example, in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more selectable marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known in the art. [00228] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.

[00229] In one embodiment the expression construct is present on a high copy number vector.

[00230] In one embodiment the high copy number vector is selected from those that are present at 20 to 3000 copies per host cell. [00231] In one embodiment the high copy number vector contain a high copy number origin of replication (ori), such as ColEl or a ColEl-derived origin of replication. For example, the ColE- 1 derived origin of replication may comprise the pUC19 origin of replication.

[00232] Numerous high copy number origins of replication suitable for use in the vectors of the present invention are known to those skilled in the art. These include the ColEl -derived origin of replication from pBR322 and its derivatives as well as other high copy number origins of replication, such as Ml 3 FR ori or pl5A ori. The 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. [00233] Preferably, the high copy number origin of replication comprises the ColEl -derived pUC19 origin of replication.

[00234] The restriction site is positioned in the origin of replication such that cloning of an insert into the restriction site will inactivate the origin, rendering it incapable of directing replication of the vector. Alternatively, the at least one restriction site is positioned within the origin such that cloning of an insert into the restriction site will render it capable of supporting only low or single copy number replication of the vector.

[00235] Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker to detect the presence of the vector in the transformed host cell. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[00236] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance.

[00237] Examples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up expression constructs, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al.,1980. A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

[00238] An expression construct useful for forming polymer particles preferably includes a promoter which controls expression of at least one nucleic acid encoding a polymer synthase, particle-forming protein or fusion polypeptide.

[00239] Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., 1978; Goeddel et al., 1979), alkaline phosphatase, a tryptophan (trp) promoter system

(Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al., 983). Promoters for use in bacterial systems also will contain a Shine-

Dalgarno (S.D.) sequence operably linked to the nucleic acid encoding a polymer synthase, particle- forming protein or fusion polypeptide.

[00240] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[00241] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol

dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

[00242] Examples of suitable promoters for use in plant host cells, including tissue or organ of a monocot or dicot plant include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters are those from the host cell, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating expression constructs using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/ 00894, which is herein incorporated by reference. [00243] Examples of suitable promoters for use in mammalian host cells comprise those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis- B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[00244] Transcription of an expression construct by higher eukaryotes is in some examples increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, oc- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 00-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Typically, the enhancer is spliced into the vector at a position 5' or 3' to the polymer synthase, particle-forming protein or fusion polypeptide coding sequence, but is preferably located at a site 5' from the promoter.

[00245] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polymer synthase, particle-forming protein or fusion polypeptide.

[00246] In one embodiment the expression construct comprises an upstream inducible promoter, such as a BAD promoter, which is induced by arabinose. [00247] In one embodiment the expression construct comprises a constitutive or regulatable promoter system.

[00248] In one embodiment the regulatable promoter system is an inducible or repressible promoter system. [00249] While it is desirable to use strong promoters in the production of recombinant proteins, regulation of these promoters is essential since constitutive overproduction of

heterologous proteins leads to decreases in growth rate, plasmid stability and culture viability.

[00250] A number of promoters are regulated by the interaction of a repressor protein with the operator (a region downstream from the promoter). The most well known operators are those from the lac operon and from bacteriophage A. An overview of regulated promoters in E. coli is provided in Table 1 of Friehs & Reardon, 99 .

[00251] A major difference between standard bacterial cultivations and those involving recombinant E. coli is the separation of the growth and production or induction phases.

Recombinant protein production often takes advantage of regulated promoters to achieve high cell densities in the growth phase (when the promoter is "off and the metabolic burden on the host cell is slight) and then high rates of heterologous protein production in the induction phase (following induction to turn the promoter "on").

[00252] In one embodiment the regulatable promoter system is selected from Lad, Trp, phage γ and phage RNA polymerase.

[00253] In one embodiment the promoter system is selected from the lac or Ptac promoter and the lad repressor, or the trp promoter and the TrpR repressor.

[00254] In one embodiment the Lacl repressor is inactivated by addition of isopropyl-B-D- thiogalactopyranoside (IPTG) which binds to the active repressor causes dissociation from the operator, allowing expression.

[00255] In one embodiment the trp promoter system uses a synthetic media with a defined tryptophan concentration, such that when the concentration falls below a threshold level the system becomes self-inducible. In one embodiment 3-B-indole-acrylic acid is added to inactivate the TrpR repressor. [00256] In one embodiment the promoter system may make use of the bacteriophage γ repressor cl. This repressor makes use of the γ prophage and prevent expression of all the lytic genes by interacting with two operators termed OL and OR. These operators overlap with two strong promoters PL and PR respectively. In the presence of the cl repressor, binding of RNA polymerase is prevented. The cl repressor can be inactivated by UV-irradiation or treatment of the cells with mitomycin C. A more convenient way to allow expression of the recombinant polypeptide is the application of a temperature-sensitive version of the cl repressor cI857. Host cells carrying a γ-based expression system can be grown to mid-exponential phase at low temperature and then transferred to high temperature to induce expression of the recombinant polypeptide.

[00257] A widely used expression system makes use of the phage T7 RNA polymerase which recognises only promoters found on the T7 DNA, and not promoters present on the host cell chromosome. Therefore, the expression construct may contain one of the T7 promoters (normally the promoter present in front of gene 0) to which the recombinant gene will be fused. The gene coding for the T7 RNA polymerase is either present on the expression construct, on a second compatible expression construct or integrated into the host cell chromosome. In all three cases, the gene is fused to an inducible promoter allowing its transcription and translation during the expression phase.

[00258] The E. coli strains BL21 (DE3) and BL21 (DE3) pLysS (Invitrogen, CA) are examples of host cells carrying the T7 RNA polymerase gene (there are a few more very suitable and commercially available E. coli strains harbouring the T7RNA polymerase gene such as e.g. KRX and XJ (autolysing)). Other cell strains carrying the T7 RNA polymerase gene are known in the art, such as Pseudomonas aeruginosa ADD1976 harboring the T7 RNA polymerase gene integrated into the genome (Brunschwig & Darzins, 1992) and Cupriavidus necator (formerly Ralstonia eutropha) harboring the T7 RNA polymerase gene integrated into the genome under phaP promoter control (Barnard et al., 2004).

[00259] The T7 RNA polymerase offers three advantages over the host cell enzymes: First, it consists of only one subunit, second it exerts a higher processivity, and third it is insensitive towards rifampicin. The latter characteristic can be used especially to enhance the amount of fusion polypeptide by adding this antibiotic about 10 min after induction of the gene coding for the T7 RNA polymerase. During that time, enough polymerase has been synthesised to allow high-level expression of the fusion polypeptide, and inhibition of the host cell enzymes prevents further expression of all the other genes present on both the plasmid and the chromosome. Other antibiotics which inhibit the bacterial RNA polymerase but not the T7 RNA polymerase are known in the art, such as streptolydigin and strep to varicin.

[00260] Since all promoter systems are leaky, low-level expression of the gene coding for T7 RNA polymerase may be deleterious to the cell in those cases where the recombinant polypeptide encodes a toxic protein. These polymerase molecules present during the growth phase can be inhibited by expressing the T7-encoded gene for lysozyme. This enzyme is a bifunctional protein that cuts a bond in the cell wall of the host cell and selectively inhibits the T7 RNA polymerase by binding to it, a feed-back mechanism that ensures a controlled burst of transcription during T7 infection. The E. coli strain BL21 (DE3) pLysS is an example of a host cell that carries the plasmid pLysS that constitutively expresses T7 lysozyme.

[00261] In one embodiment the promoter system makes use of promoters such as API or APR which are induced or "switched on" to initiate the induction cycle by a temperature shift, such as by elevating the temperature from about 30-37°C to 42°C to initiate the induction cycle.

[00262] A strong promoter may enhance fusion polypeptide density at the surface of the particle during in-vivo production.

[00263] Preferred fusion polypeptides for use in one embodiment of the present invention comprise a (i) a polymer synthase and (ii) a fusion partner comprising at least one antibody binding domain.

[00264] A nucleic acid sequence encoding both (i) and (ii) for use herein comprises a nucleic acid encoding a polymer synthase and a nucleic acid encoding a fusion partner comprising at least one antibody binding domain. Once expressed, the fusion polypeptide is able to form or facilitate formation of a polymer particle. [00265] In one embodiment the nucleic acid sequence encoding at least polymer synthase is indirectly fused with the nucleic acid sequence encoding a particle-forming protein or the nucleic acid encoding a fusion partner through a polynucleotide linker or spacer sequence of a desired length.

[00266] In one embodiment the amino acid sequence of the fusion polypeptide encoding at least one fusion partner is contiguous with the C-terminus of the amino acid sequence comprising a polymer synthase.

[00267] In one embodiment the amino acid sequence of the fusion protein comprising at least one fusion partner is indirectly fused with the N-terminus of the amino acid sequence comprising a polymer synthase fragment through a peptide linker or spacer of a desired length that facilitates independent folding of the fusion polypeptides.

[00268] In one embodiment the amino acid sequence of the fusion polypeptide encoding at least one fusion partner is contiguous with the N-terminus of the amino acid sequence comprising a particle-forming protein or a C-terminal synthase fragment.

[00269] In one embodiment the amino acid sequence of the fusion protein encoding at least one fusion partner is indirectly fused with the C-terminus of the amino acid sequence comprising a particle-forming protein or a N-terminal polymer synthase fragment through a peptide linker or spacer of a desired length to facilitate independent folding of the fusion polypeptides.

[00270] In one embodiment the amino acid sequence of the fusion polypeptide encoding at least one fusion partner is contiguous with the N-terminus of the amino acid sequence encoding a depolymerase, or a C-terminal depolymerase fragment.

[00271] One advantage of the fusion polypeptides according to the present invention is that the modification of the proteins binding to the surface of the polymer particles does not affect the functionality of the proteins involved in the formation of the polymer particles. For example, the functionality of the polymer synthase is retained if a recombinant polypeptide is fused with the N- terminal end thereof, resulting in the production of recombinant polypeptide on the surface of the particle. Should the functionality of a protein nevertheless be impaired by the fusion, this shortcoming is offset by the presence of an additional particle-forming protein which performs the same function and is present in an active state.

[00272] In this manner, it is possible to ensure a stable bond of the recombinant polypeptide bound to the polymer particles via the particle-forming proteins.

[00273] It should be appreciated that the arrangement of the proteins in the fusion polypeptide is dependent on the order of gene sequences in the nucleic acid contained in the plasmid.

[00274] For example, it may be desired to produce a fusion polypeptide wherein the fusion partner is indirectly fused to the polymer synthase. The term "indirectly fused" refers to a fusion polypeptide comprising a particle-forming protein, preferably a polymer synthase, and at least one fusion partner that are separated by an additional protein which may be any protein that is desired to be expressed in the fusion polypeptide.

[00275] In one embodiment the additional protein is selected from a particle-forming protein or a fusion polypeptide, or a linker or spacer to facilitate independent folding of the fusion polypeptides, as discussed above. In this embodiment it would be necessary to order the sequence of genes in the plasmid to reflect the desired arrangement of the fusion polypeptide.

[00276] In one embodiment the fusion partner is direcdy fused to the polymer synthase. The term "direcdy fused" is used herein to indicate where two or more peptides are linked via peptide bonds. [00277] It may also be possible to form a particle wherein the particle comprises at least two distinct fusion polypeptides that are bound to the polymer particle. For example, a first fusion polypeptide comprising a binding domain capable of binding at least one enzyme product fused to a polymer synthase could be bound to the polymer particle, and a second fusion polypeptide comprising the enzyme could be bound to the polymer particle.

[00278] In one embodiment the expression construct is expressed in vivo. Preferably the expression construct is a plasmid which is expressed in a microorganism, preferably Escherichia colt. [00279] In one embodiment the expression construct is expressed in vitro. Preferably the expression construct is expressed in vitro using a cell-free expression system.

[00280] In one embodiment one or more genes can be inserted into a single expression construct, or one or more genes can be integrated into the host cell genome. In all cases expression can be controlled through promoters as described above. [00281] In one embodiment the expression construct further encodes at least one additional fusion polypeptide comprising an antigen capable of eliciting a cell-mediated immune response or a binding domain capable of binding at least one antigen capable of eliciting a cell-mediated immune response and a particle-forming protein, preferably a polymer synthase, as discussed above.

[00282] Plasmids useful herein are shown in the examples and are described in detail in

PCT/DE2003/002799 published as WO 2004/020623 (Bernd Rehm) and PCT/NZ2006/000251 published as WO 2007/037706 (Bernd Rehm) which are each herein incorporated by reference in their entirety.

Hosts for Particle Production

[00283] The particles of the present invention are conveniently produced in a host cell, using one or more expression constructs as herein described. Polymer particles of the invention can be produced by enabling the host cell to express the expression construct. This can be achieved by first introducing the expression construct into the host cell or a progenitor of the host cell, for example by transforming or transfecting a host cell or a progenitor of the host cell with the expression construct, or by otherwise ensuring the expression construct is present in the host cell. [00284] Following transformation, the transformed host cell is maintained under conditions suitable for expression of the fusion polypeptides from the expression constructs and for formation of polymer particles. Such conditions comprise those suitable for expression of the chosen expression construct, such as a plasmid in a suitable organism, as are known in the art. For example, and particularly when high yield or overexpression is desired, provision of a suitable substrate in the culture media allows the particle-forming protein component of a fusion polypeptide to form a polymer particle. [00285] Preferably the host cell is, for example, a bacterial cell, a fungi cell, yeast cell, a plant cell, an insect cell or an animal cell, preferably an isolated or non-human host cell. Host cells useful in methods well known in the art (e.g. Sambrook et al., 987; Ausubel et al., 1987) for the production of recombinant polymer particles are frequently suitable for use in the methods of the present invention, bearing in mind the considerations discussed herein.

[00286] Suitable prokaryote host cells comprise, for example, eubacteria, such as Gram- negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. colt. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. i»£ X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include other Enterobacteriaceae such as Escherichia spp., Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella tphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as 13. subtilis, B. megaterium and 13. licheniformis, Pseudomonas such as P. aeruginosa, and Actinomycetes such as Streptomyces, Rhodococcus, Corynebacterium and Mycobaterium.

[00287] In some embodiments, for example, E. coli strain W3 0 may be used because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3 0 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3 0 strain A2, which has the complete genotype tonA ; E. coli W3 0 strain 9E4, which has the complete genotype tonA ptr3; E. coli Wii iO strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E 5 (argF-lac) 69 degP ompT kanr; E. coli W3 0 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanr; E. coli W3 0 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation.

[00288] In some preferred embodiments, for example, Eactococcus lactis strains that do not produce lipopolysaccharide endotoxins may be used. Examples of Eactococcus lactis strains include MG1363 and Eactococcus lactis subspecies cremoris NZ9000.

[00289] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for use in the methods of the invention, for example. Examples include Saccharomyces cerevisiae, a commonly used lower eukaryotic host microorganism. Other examples include Schi^osaccharomyces pombe (Beach and Nurse, 1981; EP 139,383), Kluyperomyces hosts (U.S. Patent No. 4,943,529; Fleer et al., 1991) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., 1983), Kfragilis (ATCC 12,424), K bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilamm (ATCC 36,906; Van den Berg et al, 1990), K.

thermotolerans, and A. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., 1988); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., 1979); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolpocladium (WO 91 /00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., 1983; Tilburn et al., 1983; Yelton et al., 1984) and A. niger (Kelly and Hynes, 1985). Methylo tropic yeasts are suitable herein and comprise yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in Anthony, 1982.

[00290] Examples of invertebrate host cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[00291] Examples of useful mammalian host cell lines are monkey kidney CV1 line

transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al.,

1980); mouse Sertoli cells (TM4, Mather, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[00292] Eukaryotic cell lines, for example mammalian cell lines, will be preferred when, for example, the fusion partner, such as an enzyme or an antibody binding domain requires one or more post-translational modifications, such as, for example, glycation. For example, one or more enzymes may require post-translational modification to be optimally active, and may thus be usefully expressed in an expression host capable of such post-translational modifications.

[00293] In one embodiment the host cell is a cell with an oxidising cytosol, for example the E. coli Origami strain (Novagen). [00294] In another embodiment the host cell is a cell with a reducing cytosol, preferably E. coli.

[00295] The host cell, for example, may be selected from the genera comprising Ralstonia, Acaligenes, Pseudomonas and Halobiforma. Preferably the microorganism used is selected from the group comprising, for example, Ralstonia eutropha, Alcaligenes lotus, Escherichia coli, Pseudomonas fragi, Pseudomonas putida, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Halobiforma haloterrestris. This group comprises both microorganisms which are naturally capable of producing biocompatible, biodegradable particles and microorganisms, such as for example E. coli, which, due to their genetic makeup, are not capable of so doing. The genes required to enable the latter-stated microorganisms to produce the particles are introduced as described above. [00296] Extremely halophilic archaea produce polymer particles with lower levels of unspecific binding of protein, allowing easier isolation and purification of the particles from the cells.

[00297] In principle, any culturable host cell may be used for the production of polymer particles by means of the above-described process, even if the host cell cannot produce the substrates required to form the polymer particles due to a different metabolism. In such cases, the necessary substrates are added to the culture medium and are then converted into polymer particle by the proteins which have been expressed by the genes which have been introduced into the cell.

[00298] Genes utilized to enable the latter-stated host cells to produce the polymer particles include, for example, a thiolase, a reductase or a polymer synthase, such as phaA thiolase, phaB ketoacyl reductase or phaC synthase from Ralstonia eutropha. Which genes are used to augment what the host cell lacks for polymer particle formation will be dependent on the genetic makeup of the host cell and which substrates are provided in the culture medium.

[00299] The genes and proteins involved in the formation of polyhydroxyalkanoate (PHA) particles, and general considerations for particle formation are reported in Madison, et al, 1999; pubHshed PCT International AppHcation WO 2004/020623 (Bernd Rehm); and Rehm, 2003;

Brockelbank JA. et al., 2006; Peters and Rehm, 2006; Backstrom et al, (2006) and Rehm, (2006), all of which are herein incorporated by reference.

[00300] A polymer synthase alone can be used in any host cell with (R)-Hydroxyacyl-CoA or other CoA thioester or derivatives thereof as a substrate.

[00301] The polymer particle can also be formed in vitro. Preferably, for example, a ceU free expression system is used. In such systems a polymer synthase is provided. Purified polymer synthase, such as that obtainable from recombinant production, or in ceU free systems capable of protein translation, that obtainable in the cell free system itself by way of introduction of an expression construct encoding a polymer synthase, will be preferred. In order to produce an environment to allow particle formation in vitro the necessary substrates for polymer particle formation should be included in the media.

[00302] The polymer synthase can be used for the in vitro production of functionalised polymer particles using (R)-Hydroxyacyl-CoA or other CoA thioester as a substrate, for example.

[00303] The fusion polypeptides can be purified from lysed cells using a cell sorter, centrifugation, filtration or affinity chromatography prior to use in in vitro polymer particle production.

[00304] In vitro polymer particle formation enables optimum control of surface composition, including the level of fusion polypeptide coverage, phospholipid composition and so forth.

[00305] It will be appreciated that the characteristics of the polymer particle may be influenced or controlled by controlling the conditions in which the polymer particle is produced. This may include, for example, the genetic make-up of the host cell, eg cell division mutants that produce large granules, as discussed in Peters and Rehm, 2005. The conditions in which a host cell is maintained, for example temperature, the presence of substrate, the presence of one or more particle-forming proteins such as a particle size-determining protein, the presence of a polymer regulator, and the like.

[00306] In one embodiment, a desirable characteristic of the polymer particle is that it is persistent. The term "persistent" refers to the ability of the polymer particle to resist degradation in a selected environment. An additional desirable characteristic of the polymer particle is that it is formed from the polymer synthase or particle-forming protein and binds to the C- or N-terminal of the polymer synthase or particle-forming protein during particle assembly.

[00307] In some embodiments of the invention it is desirable to achieve overexpression of the expression constructs in the host cell. Mechanisms for overexpression a particular expression construct are well known in the art, and will depend on the construct itself, the host in which it is to be expressed, and other factors including the degree of overexpression desired or required. For example, overexpression can be achieved by i) use of a strong promoter system, for example the T7 RNA polymerase promoter system in prokaryotic hosts; ii) use of a high copy number plasmid, for example a plasmid containing the colEl origin of replication or iii) stabilisation of the messenger RNA, for example through use of fusion sequences, or iv) optimization of translation through, for example, optimization of codon usage, of ribosomal binding sites, or termination sites, and the like. The benefits of overexpression may allow the production of smaller particles where desired and the production of a higher number of polymer particles. [00308] The composition of the polymers forming the polymer particles may affect the mechanical or physiochemical properties of the polymer particles. For example, polymer particles differing in their polymer composition may differ in half-life or may release biologically active substances, in particular pharmaceutical active ingredients, at different rates. For example, polymer particles composed of C₆-C₁₄ 3-hydroxy fatty acids exhibit a higher rate of polymer degradation due to the low crystallinity of the polymer. An increase in the molar ratio of polymer constituents with relatively large side chains on the polymer backbone usually reduces crystallinity and results in more pronounced elastomeric properties. By controlling polymer composition in accordance with the process described herein and known in the art, it is accordingly possible to influence the

biodegradability of the polymer particles and thus affect the duration the polymer particles (and when present the one or more fusion partners are maintained in, for example, a chromatography system, or to affect the binding, catalysis, or release of one or more target substances or precursors thereof to, on, or from the polymer particles.

[00309] At least one fatty acid with functional side groups is preferably introduced into the culture medium as a substrate for the formation of the polymer particles, with at least one hydroxy fatty acid and/ or at least one mercapto fatty acid and/ or at least one β-amino fatty acid particularly preferably being introduced. "Fatty acids with functional side groups" should be taken to mean saturated or unsaturated fatty acids. These also include fatty acids containing functional side groups which are selected from the group comprising methyl groups, alkyl groups, hydroxyl groups, phenyl groups, sulfhydryl groups, primary, secondary and tertiary amino groups, aldehyde groups, keto groups, ether groups, carboxyl groups, O-ester groups, thioester groups, carboxylic acid amide groups, hemiacetal groups, acetal groups, phosphate monoester groups and phosphate diester groups. Use of the substrates is determined by the desired composition and the desired properties of the polymer particle. [00310] The substrate or the substrate mixture may comprise at least one optionally substituted amino acid, lactate, ester or saturated or unsaturated fatty acid, preferably acetyl-CoA.

[00311] In one embodiment one or more substances is provided in the substrate mixture and is incorporated into the polymer particle during polymer particle formation, or is allowed to diffuse into the polymer particle. [00312] The polymer particle may comprise a polymer selected from poly-beta-hydroxy acids, polylactates, polythioesters and polyesters, for example. Most preferably the polymer comprises polyhydroxyalkanoate (PHA), preferably poly(3-hydroxybutyrate) (PHB). [00313] The polymer synthase or polymer particle preferably comprises a phospholipid monolayer that encapsulates the polymer particle. Preferably said particle-forming protein spans said lipid monolayer.

[00314] The polymer synthase or particle-forming protein is preferably bound to the polymer particle or to the phospholipid monolayer or is bound to both.

[00315] The particle-forming protein is preferably covalendy or non-covalently bound to the polymer particle it forms.

[00316] Preferably at least about 1%, 2%, 3%, 4%, 5%, 0%, 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the surface area of the polymer particle is covered by fusion polypeptides.

[00317] In certain circumstances it may be desirable to control the size of the particles produced in the methods of the invention, for example to produce particles particularly suited to a given application. For example, it may be desirable to produce polymer particles comprising one or more fusion partners at a relatively large size, for example to support robust durability. For example, in the context of particles for use in the preparation of one or more antibodies, it may be desirable to produce polymer particles comprising one or more antibody binding domains of a relatively large size to ensure durability and functionality in chromatography systems. In other examples, such as in the catalysis of an enzyme substrate to a target substance, it may be desirable to produce polymer particles comprising one or more enzymes of a relatively small size, for example to enable a high relative concentration of enzyme in the chromatography system. Methods to control the size of polymer particles are described in PCT/DE2003/002799 published as WO

2004/020623, and PCT/NZ2006/000251 published as WO 2007/037706.

[00318] In some embodiments, particle size is controlled by controlling the expression of the particle-forming protein, or by controlling the expression of a particle size-determining protein if present, for example.

[00319] In other embodiments of the present invention, for example, particle size control may be achieved by controlling the availability of a substrate, for example the availability of a substrate in the culture medium. In certain examples, the substrate may be added to the culture medium in a quantity such that it is sufficient to ensure control of the size of the polymer particle. [00320] It will be appreciated that a combination of such methods may be used, allowing the possibility for exerting still more effective control over particle size. [00321] In various embodiments, for example, particle size may be controlled to produce particles having a diameter of from about 10 nm to 3 μηι, preferably from about 10 nm to about 900 nm, from about 10 nm to about 800 nm, from about 10 nm to about 700 nm, from about 10 nm to about 600 nm, from about 10 nm to about 500 nm, from about 10 nm to about 400 nm, from about 10 nm to about 300 nm, from about 10 nm to about 200 nm, and particularly preferably of from about 10 nm to about 100 nm.

[00322] In other embodiments, for example, particle size may be controlled to produce particles having a diameter of from about 10 nm to about 90 nm, from about 10 nm to about 80 nm, from about 10 nm to about 70 nm, from about 10 nm to about 60 nm, from about 10 nm to about 50 nm, from about 10 nm to about 40 nm, from about 10 nm to about 30 nm, or from about 10 nm to about 20 nm.

[00323] Other ranges of average polymer size, for example, including ranges within the above recited ranges, are specifically contemplated, for example polymer particles having a diameter of from about 50 to about 500 nm, from about 150 to about 250 nm, or from about 100 to about 500 nm, etc.

[00324] In various embodiments, for example, 90% of the particles produced have a diameter of about 200 nm or below, 80 % have a diameter about 150 nm or below, 60 % have a diameter about 100 nm or below, 45 % have a diameter about 80 nm or below, 40 % have a diameter about 60 nm or below, 25 % have a diameter about 50 nm or below, and 5 % have a diameter about 35 nm or below

[00325] In various embodiments, for example, the method produces polymer particles with an average diameter less than about 200 nm, less than about 150 nm, or less than about HOnm.

[00326] The invention consists in the foregoing and also envisages constructions of which the following gives examples only. DESCRIPTION OF THE DRAWINGS

[00327] Figure 1 demonstrates binding and elution of IgG on a PolyBind-Z™-based affinity media column as described herein in Example 2. A PolyBind-Z™- Celite^® column was equilibrated with PBS and then loaded with IgG (2 and 4 ml of a 2 mg/ ml solution, indicated in solid lines). The column was washed with 15 ml PBS and then 20 ml glycine-saline buffer pH 2.75 was applied at a flow rate of approximately 2 ml/ min. Fractions were collected and the absorbance at 280 nm was measured. The dotted line indicates elution of 8 mg IgG on a matching control (Celite^® only) column. [00328] Figure 2 presents a photograph of SDS-PAGE analysis of IgG bind-elute fractions from a PolyBind-Z™- Celite^® affinity media column as described herein in Example 2. Selected fractions from the bind-elute study of Figure 1 were analysed by SDS-PAGE on an 8-16 % gradient gel. [00329] Figure 3 demonstrates the recovery of IgG over multiple bind-elute-wash cycles on a PolyBind-Z™- Celite^® column as described herein in Example 3. The column was loaded with 3 ml affinity media (1 g Celite^® with 0.4 g PolyBind-Z™) and equilibrated with PBS. During each bind cycle the PBS equilibrated column was loaded with 2.5 ml of 2 mg/ml human polyclonal IgG, washed with PBS and eluted with 50 mM citrate-saline buffer pH 3.0. After three cycles, the column was cleaned in place (CIP) with 0.1N NaOH and re-equilibrated with PBS. Fractions (1.0 ml) were collected and assessed for 280 nm absorbance. Recovery (%) was determined by comparing the total amount of IgG eluted with the amount (5 mg) loaded on the column.

[00330] Figure 4 demonstrates the binding and elution of IgG on a PolyBind-Z™-Celpure^® affinity media FPLC column as described herein in Example 3. An FPLC column was packed with PolyBind-Z™-Celpure^® affinity media to a bed volume of 3 ml as described in Example 1 and equilibrated with PBS. The dynamic binding capacity of the column was assessed by loading the column with 15 ml of 2 mg/ ml human polyclonal IgG. The column was washed with 5 ml PBS (from fraction 15) and the IgG eluted with 10 mL 50 mM citrate-saline buffer pH 2.75 (from fraction 20). Fractions (1 ml) were collected and the absorbance at 280 nm was measured to quantify the recovery of IgG from the column.

[00331] Figure 5 demonstrates the separation and purification of IgG from a contaminating protein using a PolyBind-Z™-Celpure^® affinity FPLC column as described herein in Example 4. The PBZ-Celpure^® affinity column was run at 1 ml/ minute, equilibrated with PBS and loaded with a mixture of 5 mg human polyclonal IgG and 2.5 mg BSA in 5 ml PBS. After loading the BSA + IgG mixture, the column was washed with 15 ml PBS and then 50 mM citrate-saline pH 3.0 elution buffer was applied to the column. The absorbance at 280 nm over time is presented.

[00332] Figure 6 presents a photograph of SDS-PAGE analysis of A280 peak fractions collected during the purification and separation of IgG from BSA on a PBZ-Celpure^® affinity column as described herein in Example 4. Fractions were collected from the column eluate and analysed by SDS-PAGE on a 10-16% gradient gel. Early fractions (1-4) were collected during column washing with PBS, and show removal of BSA from the column. The citrate elution buffer was initiated at fraction 5, and the elution of IgG is shown in fractions 6 - 8 (lanes 6-8). Fraction 14 is an aliquot of a 0.1 N NaOH column wash. [00333] Figure 7 demonstrates the separation and purification of IgG from contaminating proteins using a PBZ-Celpure^® affinity FPLC column as described herein in Example 6. The PBZ- Celpure^® affinity column was run at 1 ml/ minute, equilibrated in PBS and loaded with a solution of 5 mg human polyclonal IgG in 5 ml spent DMEM tissue culture fluid (TCF). After loading the TCF + IgG solution, the column was washed with approximately 7 ml PBS and then 50 mM citrate-saline pH 3.0 elution buffer was applied to the column at approximately 20 minutes. The absorbance at 280 nm over time is presented.

[00334] Figure 8 presents a photograph of SDS-PAGE analysis of selected peak fractions collected during the purification and separation of human polyclonal IgG from spent tissue culture fluid as described herein in Example 5. Fractions were collected from the PolyBind-Z™-Celpure^® affinity column eluate and analysed by SDS-PAGE on a 10-16% gradient gel. Early fractions (1-7) were collected during column washing with PBS. The citrate elution buffer was initiated at fraction 8. Fraction 14 is an aliquot of a 0.1N NaOH column wash.

[00335] Figure 9 depicts a) SN-Pl and, b) SN-P3 PBZ plasmid constructs and the fusion polypeptides encoded thereby as described herein in Example 6.

[00336] Figure 10 demonstrates binding of silaffin-tagged PBZ (SN-PBZ) polymer particles to glass beads as described herein in Example 7. Left: glass beads (white top layer) with Nile-Red stained SN-PBZ polymer particles (bottom layer). Right: SN-PBZ-glass bead stationary phase samples after mixing, incubation, and centrifugation, displaying the SN-PBZ-bound glass (bottom layer) and unbound SN-PBZ (top layer).

[00337] Figure 11 presents a photograph of purified samples of SN-PBZ-bound glass beads as described herein in Example 7. The pink color indicates the presence of the Nile-Red stained PBZ polymer particles. From the left, the samples are: SN-Pl, SN-Pl, P3, and PhaC.

[00338] Figure 12 presents a photograph of columns comprising 200μ1 Celite^® mixed with: 1) P4, 2) P3, 3) nothing, 4) SN-Pl polymer particles as described herein in Example 7. Pink colour indicates the presence of Nile-Red stained PHB polymer particles.

[00339] Figure 13 presents a photograph of columns comprising 200μ1 Celite^® mixed with: 1) P4, 2) P3, 3) nothing, 4) SN-Pl polymer particles after 1 month storage at 4°C as described herein in Example 7. Pink colour indicates the presence of Nile-Red stained PHB polymer particles. [00340] Figure 14 demonstrates the binding and elution of human polyclonal IgG on an affinity stationary phase comprising glass beads and PHB polymer particles bearing a silica affinity tag as herein described in Example 8. Columns were prepared and tested as described in Example 6. "Unbound": unbound IgG which flowed through the column; "Elution": IgG released upon column elution. Samples are: glass alone - no PHB added control; PhaC - plain PHB bead control (no binding domains); P3 - normal PHB polymer particles; SN-P bioreactor - SN-P1 polymer particles grown by fermentation; SN-P1 flask - SN-P1 polymer particles grown in a flask culture; SN-P1 flask recycle: same column from previous bar (SN-P1 flask), washed and re-used one week later.

[00341] Figure 5 demonstrates the binding and elution of human polyclonal IgG on an affinity stationary phase comprising Celite^® and PHB polymer particles bearing a silica affinity tag as herein described in Example 9. Columns were prepared and tested as described in Example 8. Unbound - unbound IgG that flowed through the column; Elution - IgG released upon column elution. Samples are: Celite^® plain - no PHB; P3 - normal PHB; P4 - PHB with extra ZZ domains; SN-P1 - SN-P1 polymer particles; IgG - IgG sample initially added to the columns.

[00342] Figure 16. Recovery of IgG Polybind-Z™-Celpure affinity FPLC column. The Polybind-Z™ -Celpure affinity column was run at 1 ml/ minute, equilibrated in PBS and loaded with 5 mg IgG in PBS (2.5 ml of 2 mg/ ml). After loading the IgG mixture the column was washed with PBS and then eluted with 0.1 M citric acid-pH 3.0. After every 10 bind-elute cycles a CIP was performed with 5 ml 0.1 M NaOH. Each block of 10 bind-elute cycle data (between CIPs) was averaged and plotted. [Note: A total of 290 cycles were recorded by the machine, some due to air bubble amalgamation of peaks, others due to machine error. An air trap was used after the first 100 cycles to remove bubbles interfering with the readings.] EXAMPLES

Example 1: Stationary phase assessments.

Introduction

[00343] This example describes experiments to assess various chromatography stationary phases for use in the present invention. Here, the Polybind polyhydroxybutyrate bead expressing the Z domain (Polybind-Z™) of Protein A was used to create an affinity media packed into liquid chromatography columns for the affinity separation and purification of proteins, in this case IgG. This example describes the process for passively mixing the Polybind-Z™ with various inert support particles and assessing flow characteristics of liquid chromatographic columns suited for affinity purification of the target protein from a mixture of proteins. Method

[00344] Inert support particles suitable for use in the present invention were assessed by determining the pressure produced at a given rate of flow and column height. In the current example a small manually operated liquid chromatography column was packed with a range of inert granular support particles (see Table 1 below) and a solution of phosphate buffered saline was pumped through the column. Using an in line pressure gauge, the pressure resulting from various applied flow rates was recorded (Table 1). The best flow conditions were determined for each stationary phase, being those that allowed the highest flow rate with the lowest back pressure. In the current example, Celite was established as having preferred flow characteristics.

Table 1: Pressure Flow Studies withPerlite A60, A40 and Celite

Perlite AP60 - 2.5 cm column height by 1 cm i.d.

Pump Speed Pressure Flow Net Flow

5 rpm 2.5 psi 10 ml/8 min 1.25 ml/min

10 rpm 4 psi 13 ml/ 5 min 2.6 ml / min

15 rpm 6 psi 35 ml/ min 3.8 ml/min

Perlite AP 40 - 2.5 cm column height by 1 cm i.d.

Pump Speed Pressure (psi) Flow Net Flow

5 rpm 1.5 7 ml/5 min 1.4 ml/min

10 rpm 2.5 13 ml / 5 min 2.6 ml/min

15 rpm 3.75 19 ml / 5 min 3.8 ml/min

20 rpm 4.75 26 ml/ 5 min 5.2 ml/ min

Ceilite 3 cm column height by 1 cm i.d.

Pump Speed Pressure (psi) Flow Net Flow

15 rpm 0 psi 20 ml/5 min 4 ml/ min

25 rpm 1.5 psi 33 ml/ min 6.6 ml/min

Example 2: Preparation of an affinity media column and its use in a method for the binding and elution of IgG.

[00345] This example describes the preparation of an affinity media column comprising PolyBind polyhydroxybutyrate polymer particles expressing the Z domain of Protein A (PolyBind- Z™) and a population of inert support particles. The use of the affinity media column in a chromatography method for the binding and elution of IgG is also described.

Materials and Methods

Affinity media preparation [00346] Celite^®, a filtration material comprising diatomaceous earth, was employed as an inert support material in the preparation of an affinity stationary phase for use in column chromatography. A PolyBind-Z™ (PBZ) Celite^® column was prepared by mixing 2 ml of a 20% PBZ suspension (0.4 g) and 1 g of dry Celite^® into a homogeneous paste, which was transferred directly to a laboratory chromatography column (10 x 1 cm). Once the paste had settled in the column to form a stationary phase, a layer of phosphate buffered saline (PBS) was allowed to cover the bed surface of the stationary phase and gradually the PBS was pumped through the column.

Binding and elution of IgG

[00347] After equilibrating a PBZ: Celite^® column or a Celite^®-only column (each with a 3 ml bed of stationary phase) with PBS, the column was loaded with a 2 mg/ml solution of human polyclonal IgG in PBS to a total of 4 or 8 mg of IgG. The column was washed with 15 ml PBS and the IgG was eluted with 50 mM glycine-saline pH 2.75 at a rate of approximately 2 ml/min. Fractions were collected during the wash and elution steps.

Results [00348] The absorbance at 280 nm of each fraction was measured (Figure 1). The elution profile demonstrates that the IgG bound to the PBZ: Celite^® column, and was eluted with a low pH wash. IgG did not bind the column comprising Celite^® only. This is consistent with the conditions typically required to release IgG from Protein A.

[00349] SDS-PAGE analysis of the fractions confirms that IgG was eluted from the affinity column during the glycine-buffer elution (Figure 2). IgG was absent from the bind-wash fractions (lanes 2 - 8) and eluted in the glycine-saline buffer elution fractions (lanes 9-11).

Discussion

[00350] This example clearly demonstrates that the methods of the present invention employing an affinity stationary phase comprising a mixed population of PBZ polymer particles with an inert support material to form an affinity stationary phase of the present invention are well-suited to the binding and elution of a target molecule.

Example 3: Demonstration of the consistent and reproducible recovery of IgG over multiple bind-elute cycles and cleaned in place cycles on an affinity media column. [00351] This example describes the binding and elution of IgG on a PBZ- Celite^® affinity column after multiple bind-elute and cleaned in place (CIP) cycles. Materials and Methods

[00352] Multiple IgG bind-elute cycles as described in Example 2 were run in series on a PBZ- Celite^® affinity column, interspersed with cleaned in place (CIP) cycles of 5 ml 0.1N NaOH.

Results [00353] Recycling of a PBZ-Celite^® affinity column over multiple IgG bind-elute-wash cycles resulted in a consistent and reproducible recovery of IgG as determined by measuring the absorbance at 280 nm (Figure 3). There was no loss of IgG binding activity over the course of ten cycles of reuse interspersed with three CIP cycles. The average recovery of IgG on the PBZ- Celite^® affinity column was 63.6% +/- 9.8. Discussion

[00354] This example clearly demonstrates that the affinity stationary phase of the present invention comprising a mixed population of PBZ polymer particles with an inert support material is robust and stable and is suitable for reuse. This example further illustrates the robustness and stability of a passively loaded column loaded with the affinity stationary phase of the present invention.

Example 4: Determination of the dynamic loading capacity of an affinity media fast protein liquid chromatography (FPLC) column.

[00355] This example describes the preparation of an FPLC column packed with PBZ- Celpure^® affinity medium. A dynamic binding study to determine the capacity of the column to bind IgG is also described.

Materials and Methods

[00356] An affinity chromatography stationary phase was prepared using a refined, pharmaceutical grade diatomaceous earth (Celpure^® 1000). An affinity stationary phase comprising 1.3 g of purified Celpure^® 1000 and 0.4 g PBZ was prepared as described in Example 1. A fast- protein-liquid-chromatography (FPLC) column was packed with the PBZ- Celpure^® affinity stationary phase to a bed volume of 3 ml and a bed height of 3.8 cm by preparing of a column with 1.3 g purified Celpure 1000 mixed with 0.4 g Polybind-Z™ and then run at 10 rpm pump speed, where a flow of 2.45 gave a pressure of 70 kPa (11 psi). The dynamic binding capacity of the column was assessed by loading the column with 15 ml of 2 mg/ ml human polyclonal IgG. The column was washed with PBS and the IgG eluted with 50 mM citrate-saline buffer pH 2.75. Results

[00357] Fractions (1 ml) were collected and the absorbance at 280 nm was measured to quantify the recovery of IgG from the column. IgG was loaded to saturation and a dynamic binding capacity of 12 mg IgG per gram of PBZ was determined column upon elution with citrate (Figure 4). Assuming 60% recovery, an estimated 20 mg human polyclonal IgG binding to the FPLC column was calculated.

Discussion

[00358] This example clearly demonstrates that the affinity stationary phase of the present invention comprising a mixed population of PBZ polymer particles with an inert support material is suitable for use with an FPLC system. The affinity stationary phase of the present invention has sufficient capacity to bind and elute a target molecule.

Example 5: Demonstration of the separation and purification of IgG from a contaminating protein using an affinity media FPLC column. [00359] This example describes the separation and purification of human IgG from bovine serum albumin (BSA) using a PBZ-Celpure^® affinity stationary phase packed in an FPLC column.

Materials and Methods

[00360] An affinity stationary phase was prepared and packed in a column using the method described in Example 1. The affinity stationary phase comprised 0.73 g of Celpure^® 1000 and 0.4 g PBZ yielding an initial bed height of 3.2cm when packed in an FPLC column. Backpressure was low-moderate at 0.18-0.34Mpa. The PBZ-Celpure^® affinity stationary phase column was run at 1 ml/minute, equilibrated in PBS and loaded with a mixture of 5 mg human polyclonal IgG and 2.5 mg BSA in 5 ml PBS. After loading the BSA + IgG mixture, the column was washed with PBS and then 50 mM citrate-saline pH 3.0 was applied. Fractions (1 ml) were collected and analysed using SDS-PAGE.

Results

[00361] Measurement of the absorbance at 280 nm over time (Figure 5) demonstrates that BSA was washed from the PBZ-Celpure^® affinity column in the PBS wash volume (2-10 minutes). The IgG was eluted from the column in the citrate-saline elution buffer (12-14 minutes). [00362] SDS-PAGE analysis of fractions collected during the wash and elution steps (Figure 6) confirms that BSA was removed from the column during the column wash with PBS. IgG was eluted when the column was loaded with citrate-saline elution buffer (lanes 6-8, Figure 6).

Discussion

[00363] This example clearly demonstrates that the affinity stationary phase of the present invention comprising a mixed population of PBZ polymer particles with an inert support material is suitable for use in a method to separate a target molecule from contaminating proteins.

Example 6: Demonstration of the separation and purification of IgG from a complex mixture using an affinity media FPLC column.

[00364] This example describes the separation and purification of human IgG from the proteins in spent tissue culture fluid using a PBZ-Celpure^® affinity stationary phase packed in an FPLC column.

Materials and Methods [00365] 5 mg of human polyclonal IgG was added to 5 ml of spent Dulbecco's Modified Eagle Media (DMEM) tissue culture fluid that had been incubated with cells for 3-4 days. The mixture was loaded on to a PBZ-Celpure^® affinity FPLC column at a rate of 1 ml/ min. The column was washed with PBS and then 50 mM citrate-saline pH 3.0 elution buffer was applied. Fractions (2 ml) were collected and analysed using SDS-PAGE. Results

[00366] Measurement of the absorbance at 280 nm (Figure 7) demonstrates that contaminating proteins derived from the tissue culture fluid were removed from the PBZ-Celpure^® affinity column in the wash volume. The IgG was eluted from the column in the citrate-saline elution buffer at 23 minutes. [00367] SDS-PAGE analysis of fractions collected during the wash and elution steps (Figure 8) confirms that the proteins of the tissue culture fluid were removed from the column during the column wash with PBS (fractions 1-6). IgG was eluted from the column when it was loaded with the citrate-saline buffer (fractions 7-12). Discussion

[00368] This example clearly demonstrates that the affinity stationary phase of the present invention comprising a mixed population of PBZ polymer particles with an inert support material is suitable for use in a method to separate a target molecule from contaminating proteins in a complex mixture.

Example 7: Preparation of an affinity stationary phase comprising affinity-tagged PHB polymer particles bound to glass beads.

[00369] This example describes the preparation of an affinity stationary phase comprising PHB polymer particles expressed with a silica affinity tag bound to an inert silica support material.

Materials and Methods

Bacterial strains

[00370] Escherichia coli (E. coli) BL21 (DE3) cells were transformed with the plasmid pMCS69 (Hoffmann, N., Amara, A.A., Br. Beermann, Qi, Q., B., Hinz, H.-J., Rehm, B.H.A. (2002) J. Biol. Chem, 277:42926-42936) a pBBRlMCS derivative containing genes phaA and phaB from C. necator colinear to the /^-promoter that mediates provision of the activated precursors of

polyhydroxybutyrate in E. coli. plus one of the following plasmids (depicted in Figure 9):

[00371] SN-Pl: pET14b_SN-G5-ZZ-PhaC (Jahns AC, Haverkamp RG, Rehm BH. (2008) Bioconjug Chem. Oct; 19(10): 2072-80). This construct expresses a PBZ bead with a single ZZ domain and a silaffin affinity tag, both at the N-terminus. The amino acid sequence of the expressed fusion protein is presented herein as SEQ ID NO. 1, and the nucleotide sequence encoding same is presented herein as SEQ ID NO. 2.

[00372] SN-P3: _PET14b_SN-ZZlZZ-PhaC-L-ZZ. This construct expresses a PBZ bead with a C-terminal and two N-terminal ZZ domains as depicted in Figure 9. A silaffin tag is expressed on the N-terminus. The amino acid sequence of the expressed fusion protein is presented herein as

SEQ ID NO. 3, and the nucleotide sequence encoding same is presented herein as SEQ ID NO. 4.

[00373] PhaC: pET14b_PhaC . This construct consists of the polymerase protein PhaC, with no binding domains.

[00374] P3: pET14b_ZZlZZ-PhaC-L-ZZ (PHB with three ZZ domains [00375] P4: pET14b_ZZlZZ-PhaC-L-ZZlZZ (PHB with four ZZ domains) Bacteria were grown in LB media comprising 00 μg/ ml ampicillin, 50 μg/ ml chloramphenicol, 1 % glucose. 1 mM IPTG was added to the LB media to induce protein expression.

Buffers and Chemicals

[00376] The following buffer solutions were employed in the method: [00377] Storage buffer: PBS with 20% EtOH, pH 7.4

[00378] Nile Red: 25 mg/ml Nile Red dye dissolved in DMSO

PBZ bead expression and purification

[00379] PBZ bead expression and purification was performed as described in ***. Briefly, transformed E. coli expressing the above plasmids were grown in flasks or bioreactor. The cells were lysed, and the polymer particles separated from the cell lysate by gradient centrifugation or detergent washing. Isolated PBZ polymer particles were stored in storage buffer at 4°C before use.

Column materials

[00380] Glass beads were employed as a population of inert silica support particles. The beads were borosilicate spheres (MO-SCI Specialty Products LLC) either large (74 μπι ) or small (20-40 μηι).

Preparation ofiPBZ-glass bead affinity stationary phase

[00381] PBZ polymer particles were used on columns (Pierce (ThermoFisher) spin columns, product #69705) both with and without Nile Red staining for visualisation. For the stained samples, PBZ polymer particles in storage buffer were stained by addition of Nile Red dye at an approximate ratio of 2:1 (μΐ Nile Red solution: mg wet bead mass). Stained polymer particles were washed three times in PBS.

[00382] PBZ polymer particles were mixed with glass beads at varying ratios to a maximum of approximately 1 mg glass : 1 mg PBZ. The mixtures were resuspended in an appropriate volume of PBS, and rotated end-over-end at room temperature for 1-2 hours to allow PHB to attach to the glass surface (Figure 0).

Results

[00383] The PBZ-bound glass particles were harvested by centrifugation, and excess free PHA was removed by gradient centrifugation or pipetting, resulting in a homogenous stationary phase of PBZ-bound glass beads (Figure ). Only the PBZ polymer particles bearing the silica affinity tag (SN-P1 and SN-P2) and not PBZ polymer particles without the silica affinity tag (P3 and PhaC) remained attached to the glass beads (Figure 11).

Discussion [00384] This example clearly demonstrates that the construct of the present invention, whereby a PHB bead is expressed with a silica affinity tag, may be employed to produce a stable affinity stationary phase whereby the silica-affinity tagged PBZ bead is bound to a silica inert support material.

Example 8: Preparation of an affinity stationary phase comprising affinity-tagged PBZ polymer particles bound to a porous silicate material.

[00385] This example describes the preparation of an affinity stationary phase comprising PBZ polymer particles expressed with a silica affinity tag bound to an inert silica support material.

Materials and Methods [00386] PBZ polymer particles prepared as described in Example 6 were combined with Celite^® (a filtration medium comprising diatomaceous earth as described in Example 1).

Results

Due to the porosity of Celite^®, all types of PBZ polymer particles, those with and without the silica affinity tag, were trapped in the stationary phase (Figure 12), compared to the glass spheres (Example 6), which only retain PBZ polymer particles containing the SN- peptide. However, after 1 month of storage in storage buffer (as described in Example 6), PBZ polymer particles which do «o bear the silica affinity tag float to the surface of Celite^® bed, while PBZ polymer particles with the tag remain attached in an homogenous stationary phase with the Celite^® (Figure 13).

Discussion [00387] This example demonstrates that the PBZ bead of the present invention, bearing a silica affinity tag, may be employed using the methods of the present invention to prepare an affinity stationary phase in combination with a porous silicate material that is stable over long term storage. Example 9: Demonstration of binding and elution of a target molecule on an affinity media column comprising silica- affinity tagged PBZ polymer particles and glass beads.

[00388] This example describes the use of an affinity media column comprising SN-tagged PBZ polymer particles and an inert silica support material in a chromatography method for the binding and elution of IgG.

Materials and Methods

[00389] Spin columns comprising PBZ polymer particles and glass beads were prepared as described in Examples 7.

[00390] The columns were rinsed twice with PBS, pH 7.4, followed by centrifugation. 200 μΐ of 5 mg/ ml human polyclonal IgG in PBS was added to the column and incubated for 5 minutes, then removed by centrifugation at 100 x g. The column was rinsed three times with PBS to remove unbound IgG. To elute the bound IgG, 200 μΐ of 50 mM glycine, pH 2.7 (elution buffer) was then applied to the columns and incubated for 5 minutes. The columns were then centrifuged at 100 x g for 1 minute. Columns were rinsed three times in PBS, and stored in 500 μΐ storage buffer at 4°C between uses (as described in Example 9).

Results

Detection of IgG concentration in samples was performed by Bradford Assay. Approximately 0.3 mg/ml IgG was recovered from spin columns prepared with 50 μΐ of the glass + SN-P1 bead mixture, representing a binding capacity of approximately 1 μg IgG per μΐ stationary phase (Figure 14). In contrast, columns with an affinity stationary phase comprising PBZ polymer particles without the silica affinity tag (PhaC, P3) did not bind IgG. The binding capacity of the glass columns diminished only slighdy when re-used after a week in storage (Figure 14).

Discussion

[00391] This example demonstrates that an affinity stationary phase of the present invention, comprising a mixed population of PBZ polymer particles bearing a silica-affinity tag of the present invention and glass beads is well-suited to the binding and elution of IgG. Example 10: Demonstration of binding and elution of a target molecule on an affinity media column comprising silica- affinity tagged PBZ polymer particles and Celite®.

[00392] This example describes the use of an affinity media column comprising SN-tagged PBZ polymer particles and an inert silica support material in a chromatography method for the binding and elution of IgG.

Materials and Methods

[00393] Spin columns comprising PBZ polymer particles and Celite^® were prepared as described in Example 7.

[00394] Columns were loaded with human polyclonal IgG and then washed with PBS and the IgG eluted according to the method described in Example 9.

Results

[00395] IgG was bound and eluted from all affinity stationary phases comprising PBZ polymer particles, regardless of whether or not they expressed the silica affinity tag (Figure 15).

Discussion [00396] This example demonstrates that an affinity stationary phase of the present invention, comprising a mixed population of PBZ polymer particles of the present invention and an inert support of Celite^® particles is well-suited to the binding and elution of IgG.

Example 11: Demonstrating 300 cycles of binding and elution of IgG and cleaning of a column packed with Polybind-Z™- + Celpure using an FPLC.

[00397] The robustness of the Polybind-Z™ -Celpure affinity FPCL column for IgG binding activity was demonstrated by running 300 bind-elute-CIP cycles on an FPLC. A Polybind-Z™- Celpure 1000 column was made by mixing lg (2 ml of a 50% slurry) of PBZ with 4 ml PBS pH 9.0 and 1.7 g of Celpure 1000 into a paste and rolling for >2 hours in a 50 ml Falcon tube. This was poured into a CIO/10 GE column, connected to an AKTA FPLC and washed through with 0.1% tween followed by PBS pH 7.4. The method involved three programmed and automated FPLC experiments of 100 cycles each. Cycles were separated into blocks of 10 IgG bind-elute cycles followed by a CIP cycle of 0.1N NaOH. The average recovery of IgG in the first block was 78% (StDev 17.8) compared to the average recovery in the final block of 67% (StDev 6.0). Recycling the IgG bind-wash-elute cycle on a PBZ affinity column resulted in a consistent and reproducible recovery of IgG as determined by absorbance at 280 nm with litde loss of recovery over the course of 300 cycles (Figure 16). This example illustrates the robustness and stability of the passively loaded column.

Example 12: Support particle assessments [00398] This example describes the assessment of additional support particle populations to identify those suitable for use in particular embodiments of the present invention.

In an addition to the stationary phases listed in Example 2, further inert support particles were examined for their potential as a support for PBZ polymer particles, by the same method. A support material was mixed with 0.4 g wet weight of PBZ and a small manually operated liquid

chromatography column was packed. A solution of phosphate buffered saline was pumped through the column and pressure monitored using an in-line pressure gauge. The pressure resulting from various applied flow rates was recorded (Table 2).

Results

[00399] The data shown in Table 2 below establish that a number of support materials can be copacked with polymer particles of the present invention. Celpure 000 remains a preferred support particle population for particular applications of the present invention.

Table 2: Pressure Flow Studies with Dextran cross-linked G-25, Sigmacell Cellulose, Silica gel and Amporphous silica. All columns made with 0.4 g wet-weight PBZ and varying support mass.

rpm . . .

Example 13: Demonstration of an enzyme application in a column format using -amylase polymer particles + Celpure 1000 support, using an FPLC.

[00400] The Celpure 000 support particles can also be used to support other types of polymer particles, including enzymes. Here 0.5 g wet weight (2.5 ml of a 20% slurry) of polymer particles, containing immobilized a-amylase as the ligand, were mixed with 0.8 g of Celpure 1000 and poured into a GE C 0/ 0 mini-column. Using an AKTA Prime FPLC a flow was established of 1 ml/ min with a back pressure of 0.02 Mpa and a bed height of 2.7 cm (volume 2.1 cm³).

For the experiment, a solution of 80 ml 1% soluble starch in 0.5 mM sodium phosphate, 8 mM sodium chloride as minimal buffering agent, at neutral pH was run in a recycling format through the column at 0.5 ml/min for 18 hours (160 minutes for one pass). Samples were taken at 0 minutes, 160 minutes (2.67 hr) and 080 minutes ( 8 hr) and measured for conversion of starch to maltose vs a maltose control. Maltose production was measured at 540 nm by the addition of an equal measure of colour reagent (from the Sigma a-amylase assay) and heating in an oil bath at OOC for 5 minutes and diluting in water 50-fold.

Results

[00401] As shown in Table 3 below, at time = 0 no maltose was present, but after one pass 71% of starch had been converted to maltose and 92% was converted after 18 hours. The enzyme remained active on the column and was immobilized by the Celpure 000 support particles. Table 3: Production of maltose from a column of a-amylase polymer particles in a population of Celpure support particles.

Sample time (minutes) A540 Conversion (%)

0 (starch only, 1%) 0.01 0

160 0.17 71

1080 0.22 92

Maltose control (1%) 0.24 -

[00402] Thus, the majority of starch was converted to maltose after one pass of the substrate, with virtually all (or all, depending on margin of error) after 8 hours.

This example clearly demonstrates that the particle populations and chromatography solutions of the present invention are able to convert a precursor substance to a desired product during chromatography. INDUSTRIAL APPLICATION

[00403] The polymer particles and methods of the invention have application in a wide range of purification and preparation technologies, including the separation of target substances from complex compositions and the preparation of reaction products from compositions comprising one or more reaction substrates.

Claims

CLAIMS . A chromatography stationary phase, wherein the chromatography stationary phase

comprises a population of support particles and a population of polymer particles, wherein one or more of the amorphous polymer particles comprises: i. a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. both (i) and (ii) above.

2. The chromatography stationary phase of claim 1 , wherein one or more of the polymer

particles is one or more amorphous polymer particles having a degree of crystallinity below about 20%.

3. The chromatography stationary phase of claim 2, wherein substantially all of the amorphous polymer particles have a degree of crystallinity below about 20%.

4. The chromatography stationary phase of claim 3, wherein substantially all of the amorphous polymer particles have a degree of crystallinity below about 2%.

5. The chromatography stationary phase of any one of claims 1 to 4, wherein one or more of the polymer particles is one or more amorphous polymer particles having a glass transition temperature T _Gin the range from 0° to 60° C.

6. The chromatography stationary phase of claim 5, wherein substantially all of the amorphous polymer particles have a glass transition temperature T _Gin the range from 0° to 60° C.

7. The chromatography stationary phase of claim 6, wherein substantially all of the amorphous polymer particles have a glass transition temperature T _Gin the range from 0° to 30° C.

8. The chromatography stationary phase of any one of claims 1 to 7, wherein one or more of the polymer particles comprises a ligand or binding domain capable of binding one or more of the support particles.

9. The chromatography stationary phase of any one of claims 1 to 8, wherein the population of support particles comprises, consists essentially of, or consists of one or more of the following: silica, diatomaceous earth, zeolite, silica gel, fused silica, glass beads, sintered glass, perlite, dowex, aluminum oxides, alumina, polymer (divinlypolystyrene), dextran, crosslinked dextran, cellulose, or polymethacrylate.

10. The chromatography stationary phase of any one of claims 1 to 9 wherein the population of support particles comprises one or more of silica, diatomaceous earth, or zeolite, or any combination thereof. . The chromatography stationary phase of any one of claims 1 to 0 wherein one or more of the polymer particles comprises one or more of the following: i. a polymer particle-binding polypeptide; ii. a polypeptide fusion partner; iii. an affinity ligand or binding domain; iv. an enzyme; v. a fusion polypeptide comprising two or more of the above; or vi. any combination of any two or more of (i) to (v) above.

12. The chromatography stationary phase of any one of claims 1 to 11 wherein one or more of the polymer particles comprises one or more ligands or binding domains displayed on the surface thereof.

13. The chromatography stationary phase of any one of claims 1 to 12 wherein the one or more polymer particles comprises at least one ligand or binding domain capable of binding a target substance, reaction substrate, or contaminant, and at least one ligand capable of binding at least one support particle.

14. The chromatography stationary phase of any one of claims 1 to 13 wherein one or more of the polymer particles comprises an immunoglobulin binding ligand or a immunoglobulin- binding domain. 5. The chromatography stationary phase of any one of claims 1 to 14 wherein one or more of the polymer particles comprises a silica binding ligand, a silica-binding domain, a zeolite- binding domain or a cellulose-binding domain. 6. A population of polymer particles for use in chromatography, wherein one or more of the amorphous polymer particles comprises: i. a biopolymer selected from a polyester, polyester, polythioester or a polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. both (i) and (ii) above; and iv. a ligand or binding domain capable of binding one or more of the support particles.

17. The population of polymer particles of claim 6, wherein one or more of the polymer

particles is one or more amorphous polymer particles having a degree of crystallinity below about 20%. 8. A method for preparing one or more target substances from a source material, the method comprising providing a stationary phase for chromatography comprising a population of support particles and a population of polymer particles, contacting the source material with the stationary phase for a time sufficient to allow the polymer particles to bind one or more target substances or one or more precursors of a target substance or one or more contaminants, separating by chromatography the one or more contaminants from the particle-bound target substance or precursor thereof or the one or more target substances or precursor thereof from a particle-bound contaminant, and recovering the target substance, wherein one or more of the polymer particles comprises: i. a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. both (i) and (ii) above. 9. The method of claim 8, wherein one or more of the polymer particles is one or more amorphous polymer particles having a degree of crystallinity below about 20%.

20. The method of claim 8 or claim 19, wherein the method comprises providing a stationary phase for chromatography comprising a population of support particles and a population of amorphous polymer particles, contacting the source material with the stationary phase for a time sufficient to allow one or more of the amorphous polymer particles to bind one or more contaminants, separating one or more target substances from the particle-bound contaminants by chromatography, and recovering the target substance.

21. The method of claim 18 or 19, wherein the method comprises contacting in or prior to introduction into a chromatography system a source material comprising one or more reaction substrates with a stationary phase for chromatography comprising a population of support particles and a population of polymer particles for a sufficient time to allow the one or more polymer particles to bind a desired fraction of the one or more reaction substrates, optionally separating one or more contaminants from the polymer particles by

chromatography, and recovering the reaction product, wherein the one or more polymer particles comprise a catalyst of the reaction.

22. A method for making a filter for use in chromatography, the method comprising providing a permeable or semipermeable support, and providing a stationary phase for chromatography, wherein the stationary phase for chromatography comprises a population of polymer particles, associating one or more of the amorphous polymer particles with the permeable or semipermeable support to provide a semipermeable filter, wherein one or more of the polymer particles comprises:

i. a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. both (i) and (ii) above; and iv. a ligand or binding domain capable of binding the support.

23. A method for making a filter for use in chromatography, the method comprising providing a permeable or semipermeable support, and providing a stationary phase for chromatography, wherein the stationary phase for chromatography comprises a population of support particles and a population of polymer particles, associating one or more support particles with the permeable or semipermeable support, associating one or more of the polymer particles with one or more of the support particles to provide a semipermeable filter, wherein one or more of the polymer particles comprises:

i. a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. both (i) and (ii) above; and iv. a ligand or binding domain capable of binding one or more of the support particles.

24. A method for preparing amorphous polymer particles, wherein one or more of the amorphous polymer particles comprises:

i. a biopolymer selected from a polyester, polythioester or a polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. both (i) and (ii) above; and iv. a ligand or binding domain capable of binding one or more of the support particles; wherein the method comprises separating one or more contaminants from the amorphous polymer particles, and recovering the amorphous polymer particles.

25. The method of claim 24 wherein the separation is by contacting the one or more amorphous polymer particles with one or more support particles to which the ligand or binding domain binds.

26. A method of making a chromatography device, the method comprising the steps of

providing a column having a cylindrical interior for accepting a stationary phase, providing one or more populations of support particles, and providing one or more populations of polymer particles, and forming a particulate stationary phase within said column, wherein the forming step comprises the steps of admixing the one or more populations of support particles and the one or more populations of polymer particles, and placing the particulate stationary phase into said column to thereby produce the chromatography device, wherein one or more of the polymer particles comprises: i. a biopolymer selected from a polyester, polyester, polythioester or a

polyhydroxyalkanoate; or ii. a polymer particle-forming polypeptide; or iii. a polymer particle-binding polypeptide; iv. a polypeptide fusion partner; v. an affinity ligand or binding domain; vi. an enzyme; vii. a fusion polypeptide comprising two or more of the above; or

Vlll. any combination of any two or more of (i) to (vii) above.

27. A chromatography device prepared by the steps of providing a column having a cylindrical interior for accepting a stationary phase, and forming a particulate stationary phase within said column, wherein the particulate stationary phase comprises a stationary phase as claimed in any one of claims 1 to 5. 28. An isolated, purified or recombinant nucleic acid comprising at least one nucleotide

sequence encodinga polymer particle-forming polypeptide and at least one nucleotide sequence encoding a ligand or binding domain capable of binding one or more support particles.

29. The nucleic acid of claim 28 additionally comprising at least one nucleotide sequence

encoding a ligand or binding domain capable of binding one or more target substances.

30. The nucleic acid of claim 29 wherein the ligand or binding domain capable of binding one or more target substances is an immunoglobuHn-binding ligand or immunoglobulin-binding domain, or is capable of binding an antibody.

31. An expression construct comprising a nucleic acid sequence as claimed in any one of claims 28 to 30.

32. A vector comprising a nucleic acid of any one of claims 28 to 30 or an expression construct of claim 31.

33. A host cell comprising a nucleic acid of any one of claims 28 to 30 or an expression

construct of claim 3 or a vector of claim 32. 34. A fusion polypeptide, fusion polypeptide particle, or polymer particle comprising a fusion polypeptide, wherein the fusion polypeptide comprises a polymer particle-forming polypeptide and a ligand or binding domain capable of binding one or more support particles.

35. The fusion polypeptide, fusion polypeptide particle, or polymer particle of claim 34

comprising a fusion polypeptide, wherein the fusion polypeptide comprises a ligand or binding domain capable of binding one or more target substances.

36. The fusion polypeptide, fusion polypeptide particle, or polymer particle of claim 34 or 35 wherein the ligand or binding domain capable of binding one or more target substances is an immunoglobulin-binding ligand or immunoglobulin-binding domain, or is capable of binding an antibody.

37. A host cell, culture, or culture supernatant comprising a fusion polypeptide, a fusion polypeptide particle or a polymer particle of any one of claims 34 to 36.

38. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of any one of claims 1 to 37 wherein one or more of the polymer particles is one or more amorphous polymer particles having a degree of crystallinity below about 20%.

39. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of any one of claims 1 to 38 wherein, when present, the ligand or binding domain capable of binding an antibody is selected from the group comprising protein A, protein G, protein A/ G , protein L, a recombinant variant thereof, a functional fragment thereof including recombinant functional fragments thereof, the Z domain of protein A, and any combination thereof, including a ZZ domain comprising a contiguous repeat of the Z domain of protein A.

40. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of claim 39 wherein one or more of the polymer particles comprises a fusion polypeptide comprising a polymer particle-forming polypeptide and one or more GB domain of protein G from Streptococcus.

41. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of claim 40 wherein the GB1 domain is encoded by a polynucleotide sequence comprising 12 or more contiguous nucleotides of SEQ ID NO. 5.

42. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of any one of claims 1 to 41 wherein the polymer particles have an immunoglobulin binding capacity of greater than 30mg immunoglobulin/g wet polymer particle.

43. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of any one of claims 1 to 42 wherein one or more of the polymer particles comprises a fusion polypeptide comprising a polymer particle-forming polypeptide, one or more antibody-binding domains, and one or more support particle-binding domains.

44. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of any one of claims 1 to 43 wherein one or more of the polymer particles comprises a fusion polypeptide comprising a polymer particle-forming polypeptide, one or more antibody-binding domains, and one or more support particle-binding domains, wherein the antibody-binding domain is selected from the group comprising protein A, protein G, protein A/ G , protein L, a recombinant variant thereof, a functional fragment thereof including recombinant functional fragments thereof, the Z domain of protein A, and any combination thereof, including a ZZ domain comprising a contiguous repeat of the Z domain of protein A.

45. The chromatography stationary phase, population of particles, method, device, nucleic acid, polypeptide, or particle of any one of claims 1 to 44 wherein the support particle-binding domain is selected from the group comprising a silica-binding domain, a cellulose-binding domain, or a zeolite-binding domain.