Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
  • B01J20/288—Polar phases
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3206—Organic carriers, supports or substrates
  • B01J20/3208—Polymeric carriers, supports or substrates
  • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
  • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J47/00—Ion-exchange processes in general; Apparatus therefor
  • B01J47/014—Ion-exchange processes in general; Apparatus therefor in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds

Definitions

• the present disclosure relates to separation media having at least one flocculant ligand as described herein, covalently attached to a base surface or support.
• the disclosure also relates to separation and/or purification of biological molecules of interest using the separation media of the present disclosure.
• the present disclosure is directed, in part, to separation media compositions, methods and systems for the purification and/or separation of biological substances.
• the present disclosure is directed to novel separation media wherein a base surface is functionalized such that a soluble flocculant (flocculant ligand) is immobilized or covalently attached to the base surface.
• the immobilized flocculant separation media generated therein dramatically improved the removal of impurities, for e.g., high molecular weight and low molecular weight species (including antibody aggregates) due to their selective binding of impurities, under certain conditions also described herein.
• the separation media described herein are capable of separating monomers of a biological molecule from aggregates across a wide range of pH and across a wide range of conductivity.
• the disclosure provides a separation medium comprising:
• the disclosure provides that the at least one flocculant ligand is selected from the group consisting of cationic, anionic, non-ionic and natural flocculants.
• the anionic flocculant further comprises an unsubstituted or substituted aliphatic carboxylic acid, an unsubstituted or substituted aromatic carboxylic acid, an unsubstituted or substituted aliphatic sulfonic acid, an unsubstituted or substituted aliphatic acrylic acid, an unsubstituted or substituted aliphatic thiosulfate, an unsubstituted or substituted aliphatic phosphonic acid, or an unsubstituted or substituted aliphatic phosphoric a fatty acid.
• the unsubstituted or substituted aliphatic groups are either linear or branched, and optionally, comprise one or more double bonds.
• the unsubstituted or substituted aliphatic groups have from 1 to about 30 carbon atoms, preferably from 1 to about 20 carbon atoms, more preferably from about 1 to about 10 carbon atoms, most preferably from about 1 to about 8 carbon atoms.
• the unsubstituted or substituted aliphatic group is a C 1 -C 8 aliphatic acid.
• the unsubstituted or substituted aliphatic group is a C 9 -C 30 aliphatic acid.
• the separation medium is contacted with a solution, a feed or an eluent that comprises one or more ligate species, under operating conditions that allow the binding of at least one ligate species from the solution, feed or eluent, to the separation medium.
• the one or more ligate species is a mixture of biological substances.
• the mixture of biological substances comprises a target molecule and at least one impurity.
• the impurity is an aggregate, or is a product related impurity, or is a process related impurity.
• the target molecule is either a monomeric antibody, a therapeutic peptide or protein, a virus or viral particle, a particular variant of a peptide or protein or antibody or virus or viral particle, or a nucleic acid.
• an aggregate is made of: several antibody monomers, antibodies with higher levels of post translational modifications, an antibody monomer in combination with one or more of the following: an antibody light chain, host cell protein (HCP), protein or viral fragment, antibody fragment, viruses or viral particle, cell culture impurity, cell debris, cell culture media component, other unwanted species.
• HCP host cell protein
• the at least one target molecule is an antibody monomer
• the at least one impurity is an aggregate.
• the separation medium selectively binds an aggregate, and wherein the separation medium has a separation factor (a) of greater than 1.
• the antibody monomer is separated from one or more aggregates with the separation factor (a) of at least about 1.5, preferably of at least about 2.5, more preferably of at least about 4.0.
• the antibody monomer is separated from one or more aggregates with the separation factor (a) of about 1.1 to about 11.
• the separation medium is capable of separating an antibody monomer from the aggregate after single contact with the separation medium.
• the antibody monomer purity is > 90% after contact with the separation medium.
• the antibody monomer recovery is > 85% after contact with the separation medium.
• the antibody monomer purity is from about 95 to about 100%, or preferably, from about 98 to about 100%.
• the antibody monomer recovery is from about 85% to about 100%, or preferably, from about 90% to about 100% after contact with the separation medium.
• the antibody monomer is purified in flow-through mode.
• the separation medium selectively binds an antibody monomer, and wherein the separation medium has a separation factor (a) of less than 1.
• the antibody monomer is separated from one or more aggregates with the separation medium having a separation factor (a) of at least about 0.1 to about 0.9, preferably of at least about 0.3 to about 0.9, most preferably of at least about 0.6 to about 0.9.
• the antibody monomer is purified in bind-elute mode.
• the cationic flocculant ligand is either a primary aliphatic amine, a secondary aliphatic amine, a tertiary aliphatic amine, an aliphatic imine, an aliphatic hydrazide, an imidazole, an aliphatic oxime, an aliphatic hydrazine, an aliphatic hydrazone, a linear polyethyl amine, a polyethyleneimine, a heterocyclic quaternary ammonium, or a cationic polyelectrolyte.
• the cationic flocculant ligand is selected from the group consisting of tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, polydiallyldimethylammonium chloride
• PolyDADMAC poly(N,N-dimethylpiperidinium chloride), poly(N-vinylpyrrolidone) (PVP), copolymers of poly(ethyleneimine), and quaternary aminated polyacrylates.
• the separation medium can selectively bind to an impurity, and/ or, wherein the impurity is a nucleic acid.
• the separation medium can selectively bind to impurities, and/ or, wherein the charged variant are is a glycosylated, a glycated, an oxidized, a deaminated, an acidic, a basic, a phosphorylated, a sialylated or a N-terminal acetylated form.
• the at least one non-ionic flocculant ligand is either a styrene, substituted styrene, polymeric styrenes, an uncharged aliphatic, an uncharged branched aliphatic, a hydrophobic polyester, a hydrophilic polyester, a polyacrylamide, a poly(ethylene oxide), or copolymers thereof.
• the flocculant natural ligand is either a polysaccharide, an amino, imino, ammonium, sulfonium or phosphonium functionalized polysaccharide, a collagen, an anionic protein, a cationic protein, a chitosan, an ininglass, guar gum, a cationic protein from Moringa oleifera seeds or Strychnos potatorum seeds, or an alginate.
• the base surface includes but is not limited to: a resin, bead, sphere, particle, microcarrier, membrane, web, bag, bioreactor, tube, plate, array, flat surface, filter, fiber or a fabric.
• the base surface is porous, non-porous, microporous, woven, non-woven, polymeric, non-polymeric, fibrous or winged.
• the base surface is made up of materials including but not limited to: ceramics, glass, metal, silica, synthetic polymeric materials such as styrenic, acrylate, acrylamide, acrylamide containing one or more polymerizable vinyl groups, polymeric monoliths, etc., natural polymers such as cellulose, lignocellulose or their derivatives, agarose, or a combinations of any of these materials.
• the separation medium is contacted with a solution, a feed or eluent comprises one or more ligate species, under operating conditions that allow the binding of at least one ligate species to the separation medium.
• the ligate species is a mixture of biological substances.
• the mixture of biological substances comprises a target molecule and at least one impurity.
• the impurity is either a product related impurity or a process related impurity.
• the target molecule is either a monomeric antibody, a therapeutic peptide or protein, a virus or viral particle, a particular variant of a peptide or protein or antibody or virus or viral particle, or a nucleic acid.
• an aggregate is made of: several antibody monomers, antibodies with higher levels of post translational modifications, an antibody monomer in combination with one or more of the following: an antibody light chain, host cell protein (HCP), protein or viral fragment, antibody fragment, viruses or viral particle, cell culture impurity, cell debris, cell culture media component, other unwanted species.
• HCP host cell protein
• the at least one target molecule is an antibody monomer
• the at least one impurity is an aggregate
• the separation medium is capable of separating a monomer and at least one aggregate in a pH range of about 2 to about pH 11.
• the separation medium is capable of separating a monomer and at least one aggregate in a solution having a conductivity of about 1 mS/cm to about 200 mS/cm.
• a chromatography system comprising; a column, and, enclosed within the column, a separation medium as described above, is described.
• the system comprises a pump for passing a liquid through the separation mediu m at a velocity of about 50 to about 1000 cm/hr, wherein the liquid is a solution, an eluent, or a feed comprising one or more biological substances.
• a method of separating a monomer and least one aggregate comprising;
• the biological substance in the flow-through fraction is collected.
• the biological substance in the flow-through fraction is an antibody monomer.
• the biological substance in the flow-through fraction is an impurity.
• the antibody monomer purity is > 90% after contact with the separation medium, or preferably, the antibody monomer purity is at least about 95% to at least about 100%, or more preferably, from at least about 98% to at least about 100% purity.
• the antibody monomer purity is at least about 95% to at least about 100%, or more preferably, from at least about 98 to at least about 100% purity; and/or, the antibody monomer recovery is at least about 85% to about 100%, or preferably, from at least about 95% to about 100% recovery after contact with the separation medium.
• the method further comprises: iii. eluting one or more bound biological substance to provide a plurality of fractions.
• the method further comprises: iv. analyzing the fractions by size exclusion chromatography.
• the step of passing the solution is carried out at a pH of about 2 to about 11.
• the step of passing the solution is carried out at a conductivity of about 1 mS/cm to about 200 mS/cm.
• the method further comprises regenerating the separation medium.
• a method of purifying a protein of interest from a solution, an eluent, or a feed comprising: i. providing a separation medium according to any one of claims 1-26, or claims 27- 32, or claims 33-44; and ii. passing the solution, eluent, or feed comprising the protein of interest and one or more impurities through the separation medium at a rate sufficient to allow the one or more impurities to bind to the separation medium, is described.
• the protein of interest is in the flow-through fraction.
• the protein of interest is an antibody monomer.
• the antibody monomer purity is at least about 95% to at least about 100%, or more preferably, from at least about 98 to at least about 100% purity; and/or, the antibody monomer recovery is at least about 85% to about 100%, or preferably, from at least about 90% to about 100% recovery after contact with the separation medium.
• the solution, eluent, or feed is a spent cell culture fluid, a solution containing proteins, or a biological fluid.
• the culturing is performed in a flask, a plate, a well, an array, a bioreactor, a disposable container, or a bag.
• At least about 90% to about 100% of an impurity is removed.
• FIG. 1a The Size Exclusion Chromatography (SEC) profile of an antibody input sample (feed) after protein A column purification is shown.
• the purity of the SEC feed is about 90-95% pure, but the antibody monomer (target molecule) still contains impurities like high molecular weight (HMW) and low molecular weight (LMW) aggregate species (the species peaks are labeled as 1, 2, 3).
• HMW high molecular weight
• LMW low molecular weight
• the exemplary novel separation media described in the present disclosure provide unique features that enable the effective removal of HMW and LMW from a given sample or feed.
• FIG. 1b demonstrates the effective removal of aggregate species, including the removal of closely related dimer impurity in a purification run with Flocculant Resin A.
• the panels demonstrate the effectiveness of aggregate removal in F5 (fraction 5) compared to the load, and the subsequent breakthrough fraction (F7).
• the feed (Load) is already 94.3% pure but contains HMW and LMW species.
• Resin A effectively removes closely related, difficult to purify HMW spps (monomer plus additional light chain).
• Exemplary resin A was able to remove close to 100% of the higher molecular weight species.
• FIG. 1c The graph shows the yield - the cumulative monomer recovery (%) vs. the purity (%) of the monomer when purified on Resin A (as discussed in FIG 1b above). The sample purity falls gradually after fraction 5 (which alone, shows 90% recovery).
• FIG. 1d shows the purification of three sample proteins myoglobin (Protein 1), alpha- chymotrypsinogen A (Protein 2), and lysozyme (Protein 3), on Resin A.
• Resin A has greater selectivity for the impurity compared to a control, non-flocculant, cation exchange (CEX) resin
• POROS XS - compare the separation of the impurity peak from the monomer between the two resins, as pointed out by the bold arrow. This suggests that Resin A is a better choice for the removal of impurities than standard ion exchange purification.
• FIG. 1e shows the separation of two very similar proteins on Resin A compared to control, non- flocculant, cation exchange (CEX) resin POROS XS.
• the impurity protein cytochrome C
• the target protein ribonuclease A.
• Resin A has an alternative selectivity profile compared to that POROS XS resin.
• the heat map shows that resin A had the best selectivity for the removal of aggregated species from a Protein A purified antibody solution compared to control resins (see region with dark dots).
• the heat map show that all exemplary resins A, B, C and D showed good selectivity for the removal of aggregated species from a Protein A purified antibody solution.
• the heat map is based on selectivity factor (a) data shown in TABLES 2 and 3. If we compare the heat map across the control resins of 2a and the flocculant resins A-D, we see that the operational window with high selectivity is greater for the flocculant resins than the commercial, non-flocculant resins.
• FIG. 3a shows an expanded Size Exclusion Chromatogram of fractions taken from the purification profile for Resin B is shown.
• FIG. 3b shows the chromatograms of various fractions from the run of FIG 3a.
• the breakthrough fraction there is good removal of most species, with the HMW species being completely removed across the entire run, and other HMW species being reduced.
• FIG. 4a and 4b show purification data from Resins A and Resin C respectively, displayed as antibody breakthrough curves, each at pH 4.5, 5.0 and 5.5.
• FIGs 5a and 5b show elution analysis with 1M Tris buffer at pH 8.5, for Resin A and Resin C respectively.
• Fraction compositions as determined by size exclusion chromatogram (SEC), show the removal of higher molecular weight species through binding to the resins. Selectivity is pH dependent. Tris elution shows significant removal of the dimer species but very little monomer loss. HMW species is bound tightly to the resins, showing excellent selectivity for this species.
• FIG. 6a shows a chromatographic line graph of variants of ovalbumin, when an ovalbumin sample is injected onto and separated using an analytical HPLC column (Thermo Scientific ProPac SAX- 10). Each peak represents a variant ovalbumin (difference in glycosylation, sialylation, etc.) or a group of variants not resolved on this column (labelled peaks 1-6).
• FIGs 6b, 6c, 6d and 6e show the HPLC profile of ovalbumin variants in each fraction as a stacked bar graphs.
• FIG 6b is the fraction from commercial AEX resins POROS XQ (control, non-flocculant AEX resin);
• FIG 6c is from POROS HQ (control, non-flocculant AEX resin);
• FIG 6d is from AEX resin 1 ; and
• FIG 6e is from AEX resin 2.
• FIG. 7 DNA versus protein binding capacity of AEX Resins 1, 2, 3, and 4 versus commercial, non-flocculant, anionic exchange resins - POROS XQ control and POROS HQ control.
• the chromatographic panel shows the variant profile of ovalbumin when injected onto and separated using an analytical HPLC column.
• Base surface or support includes but are not limited to: resins, beads, spheres, particles, microcarriers, membranes, webs, bags including single use bags, bioreactors, tubes, plates, arrays, flat surfaces, filters, fibers, fabrics, etc.
• Each type of supports described herein can be porous, non-porous or microporous, woven or non-woven, polymeric or non-polymeric, fibrous or winged.
• a support or surface can be made of a variety of materials, including but not limited to: ceramics, glass, metal, silica, synthetic polymeric materials such as styrenic, acrylate, acrylamide, vinyl, polymeric monoliths, etc., natural polymers such as cellulose, lignocellulose or its derivatives, agarose, for e.g., SepharoseTM, or a combinations of such materials.
• Flocculant means a chemical entity that induces, or has the potential to cause, precipitation of biological materials such as a “target molecule” described herein upon contact with the flocculant.
• flocculants are thought to hold to the biological material being precipitated by weak physical interactions, which may ultimately lead to separation.
• Exemplary flocculants described include but are not limited to: cationic flocculants, anionic flocculants, non-ionic flocculants, natural flocculants, and combinations thereof, as will be described further below.
• a "flocculant ligand" is generated when a flocculant or a flocculant-like chemical entity is covalently attached, coupled, immobilized or functionalized on to any base surface or a support described above. Once of skill in the art would know to use a suitable reactive functional group on the base surface or support to attach the flocculant ligand.
• the immobilized flocculant or flocculant-like ligand is thought to interact with a biological species (the "ligate") in solution, reversibly, by ionic, hydrophobic, hydrogen-bonding, or a combination of these type of interactions.
• Separatation medium means a base surface or support that is functionalized with a flocculant or a flocculant-like ligand.
• a separation medium for e.g., a flocculant
• a functionalized resin is part of a chromatography system comprising; a column, and, separation medium, for e.g., a resin, is enclosed within the column.
• Ligate means any molecule or species which can interact, in a reversible manner, with a support comprising the immobilized flocculant or the flocculant-like ligand.
• Binding refers to the interaction of the ligate with the immobilized flocculant or the flocculant-like ligand; this is generally a reversible reaction which can be disrupted by changes in pH, ionic strength, etc., of the solution.
• the "target molecule,” described herein may be the “ligate”.
• the "impurity” may be the "ligate”.
• Target or "target molecule” means any molecule of interest.
• the target molecule is the molecule that needs to be purified, concentrated, or separated, or isolated, or enriched.
• the target molecule is a biological molecule of interest, for e.g., antibodies, proteins;
• peptides peptides; glycoproteins; lipoproteins, enzymes, nucleic acids (RNA, DNA, etc.); nucleoproteins; viruses; viral fragments; viral capsids; viral antigens; antigenic proteins; cellular markers; cells or particular cell types - for e.g., certain types of T cells; a cellular component or cell parts; organelles and the like;
• the "target molecule” of interest may have pharmaceutical, diagnostic, agricultural, and/or any of a variety of other properties that are useful in commercial, experimental or other applications.
• a "target molecule” of interest can be an antibody or protein therapeutic.
• proteins produced may be processed or modified.
• a protein may be glycosylated.
• the "target molecule” is an antibody.
• the "target molecule” is a monomeric antibody.
• the "target molecule” can be an enzyme, such as lysozyme,
• Impurity or "contaminant” means any unwanted ligate species other than the "target molecule".
• any undesired products in the mixture may include aggregates as described below, host cell proteins (HCP), host cell metabolites, antibody fragments, protein fragments, nucleic acids, endotoxins, viruses or viral particles or viral protein fragments, other impurities from cell culture such as cells and their fragments, cell culture media components, media additives, media derivatives, product related contaminants, lipids, etc.
• Certain contaminants may first be removed by initial steps including, but not limited to, centrifugation, sterile filtration, depth filtration or tangential flow filtration.
• Impurities also mean 'product related impurities' which include but are not limited to charge variants of the target molecule, truncated forms, or aggregates of the molecules (e.g., dimers, trimers, etc.).
• Charge variants may include but are not limited to glycosylated, glycated, oxidized, deaminated, acidic, basic, phosphorylated, sialylated or a N-terminal acetylated forms, of the desired target molecule.
• Impurities could also mean 'process related impurities' which include but are not limited to: host cell proteins, nucleic acids, aggregates (precipitates, cell debris, media components, fragments, etc.).
• Aggregate can be “high molecular weight species” (HMW) or a “low molecular weight species” (LMW).
• HMW high molecular weight species
• LMW low molecular weight species
• antibody “aggregates” refer to higher molecular forms of an antibody formed due to the aggregation of one or more monomeric antibodies into dimers, trimers, tetramers, etc.
• An aggregate may comprise multiple monomers of the desired antibody in the antibody aggregate, or, it may comprise additional antibody light chains, or antibodies with a higher level of post translational modifications, or aggregated host cell proteins (HCP), or aggregated antibody plus protein fragments, or a combination of antibody monomer with one or more contaminants including antibody fragments, protein fragments, host cell proteins (HCP), viruses or viral particles or viral protein fragments, other impurities from cell culture such as cells and their fragments, cell culture media components, or other unwanted ligate species.
• HCP aggregated host cell proteins
• HCP host cell proteins
• viruses or viral particles or viral protein fragments other impurities from cell culture such as cells and their fragments, cell culture media components, or other unwanted ligate species.
• HMW impurities can include aggregates (dimers, trimers, tetramer etc., and/or, antibody monomer with a light chain, and/or antibody with high levels of post translational modifications, and/or aggregated host cell proteins (HCP), and/or aggregated antibody or protein fragments.
• Aggregates can also be "low molecular weight species” (LMW) impurities such as host cell proteins (HCP) or antibody fragments or protein fragments.
• LMW low molecular weight species
• antibody is used in the broadest sense to cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, immunoadhesins and antibody-immunoadhesin chimerias.
• An antibody may include, for example, an antibody of any molecule class, e.g., IgG 1 , IgG 2 , etc. that is to be purified from a mixture containing contaminants.
• an “antibody fragment” includes at least a portion of a full length antibody and typically, an antigen binding or variable region thereof; for e.g., they include Fab, Fab', F(ab') 2 , and Fv fragments; single-chain antibody molecules like camelid antibodies; diabodies; linear antibodies; and multispecific antibodies formed from engineered antibody fragments.
• the term “monoclonal” antibodies indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies described herein include "chimeric" and
• humanized antibodies and “human” antibodies, which can be isolated from various sources, including, e.g., from the blood of a human patient or recombinantly prepared using transgenic animals.
• “Monomer” in the context of antibodies refers to a monomeric antibody.
• “monomer” in a non-antibody context may mean the monomeric form of any biological molecule of interest (target), say, of a heteromeric protein or enzyme.
• Selectivity is a term that is used to describe the preferential binding of one ligate species over another on any base surface. Selectivity can also be expressed as a separation factor (a), which is a measure of the preferential selectivity of a separation medium for either an impurity or a target molecule, depending on the operating conditions.
• a separation factor
• Separation factor (a) K p impurity / K p target molecule.
• the target molecule is an antibody monomer
• the impurity is an "aggregate”. This embodiment is also seen in Example 1 where Equation 1 is also expressed as,
• Separation factor (a) K p aggregate / K p monomer.
• selectivity describes the separation of solutes in a chromatographic run, and refers to the overall chromatographic profile (retention time, separation, elution order, etc.) for a series of ligates.
• "Recovery” refers to the amount of purified ligate material obtained compared to the amount of ligate available in the original feed. In a certain embodiment, recovery is the amount of purified ligate capable of being eluted from a chromatographic column relative to the amount of ligate available in the original feed.
• flocculants may also cause reduction of product related impurities such as aggregates and may under certain conditions lead to the inactivation of viruses. Resulting harvests are of reduced complexity and lead to simplified purification operations and potentially a reduced number of operations to product molecules of sufficient purity for human therapeutic use.
• Soluble caprylic acid has been used as a flocculant to flocculate proteins.
• a method for a purifying protein of interest using soluble CA is described in US application US20120101262A, hereby incorporated by reference in its entirely.
• the final concentration of soluble caprylic acid used to the purification mixture was generally between about 0.05 and 5% (v/v), or in certain embodiments, the contaminant precipitate is allowed to form between soluble CA and target protein for between about 30 to 120 minutes after addition of the caprylic acid (e.g., between about 30 to 60 minutes). Due to the hydrophobic character of CA, it may bind to the antibody protein of interest and be carried through the purification process as a contaminant. Hence, additional steps may be needed to remove the
• the pH of the solution needs to be adjusted to less than pH 5 prior to adding soluble CA, which might cause antibody aggregation thereby increasing the number of impurities in the end-product.
• Caprylic acid has also been used as a wash solution in chromatography operations to specifically remove or precipitate process and product impurities before elution of the target molecule.
• its relatively poor solubility in aqueous solution limits the operation ranges within manufacturing processes.
• temperature fluctuations as might be encountered during transfer of manufacturing operations from one plant to another across the globe, challenges process robustness through changing solubility.
• the CA and other flocculants described herein are surface bound (immobilized); which means that a flocculant like CA need not be removed, or the CA/ or flocculants have a far lesser chance of contaminating the target protein of interest.
• immobilized flocculants are capable of separating proteins of interest from impurities.
• an antibody monomer can be separated from an impurity (aggregate) efficiently, to a greater degree and purity and with greater recovery than previously known with commercial separation products and methods.
• the disclosure relates to immobilization of a molecule typically used as a flocculent to the surface of a solid support.
• the disclosure provides a separation medium comprising a base surface; and at least one flocculant ligand covalently attached to the base surface.
• the disclosure provides a chromatography system comprising a column, and, enclosed within the column, a separation medium comprising a base surface; and at least one flocculant ligand covalently attached to the base surface.
• the separation media described herein can be used in methods for the separation or purification of a target molecule, such as a biological molecule, from contaminants in a mixture.
• the separation media described herein are provided in a system, such as a chromatography system comprising a column, and, enclosed within the column, a separation medium as described herein.
• a range of flocculant ligands are immobilized on a surface to generate separation media.
• the proposed solution was to immobilized surfaces with cationic flocculant ligands, anionic flocculant ligands, non-ionic flocculant ligands and natural flocculant ligands. If any given ligand (cationic, anionic, non-ionic or natural) identified for immobilization is not a flocculant in its soluble form, the present disclosure does not encompass the non-flocculant molecule.
• the flocculant ligand is an "anionic flocculant ligand" that is an aliphatic compound comprising an unsubstituted or substituted aliphatic carboxylic acid, an unsubstituted or substituted aromatic carboxylic acid, an unsubstituted or substituted aliphatic sulfonic acid, an unsubstituted or substituted aliphatic acrylic acid, an unsubstituted or substituted aliphatic thiosulfate, an unsubstituted or substituted aliphatic phosphonic acid, or an unsubstituted or substituted aliphatic phosphoric a fatty acid.
• the unsubstituted or substituted aliphatic groups are either linear or branched, and optionally, comprises one or more double bonds.
• the unsubstituted or substituted aliphatic groups may have from 1 to about 30 carbon atoms, preferably from 1 to about 20 carbon atoms, more preferably from about 1 to about 10 carbon atoms, most preferably from about 1 to about 8 carbon atoms.
• the flocculant ligand can be an aliphatic acid, a branched aliphatic acid, or a substituted aliphatic acid, each having from 1 to about 30 carbon atoms.
• the flocculant ligand can be a fatty acid.
• the flocculant ligand can be an optionally substituted C 1 -C 8 aliphatic acid.
• the flocculant ligand can be an optionally substituted C 9 -C 30 aliphatic acid.
• the flocculant ligand can be an optionally substituted C 1 -C 3 aliphatic acid.
• the flocculant ligand can be an optionally substituted C 3 -C 6 aliphatic acid. In some embodiments, the flocculant ligand can be an optionally substituted C 6 -C 10 aliphatic acid. In some embodiments, the flocculant ligand can be an optionally substituted C 10 -C 15 aliphatic acid. In some embodiments, the flocculant ligand can be an optionally substituted C 15 -C 20 aliphatic acid. In some embodiments, the flocculant ligand can be an optionally substituted C 20 -C 30 aliphatic acid.
• the flocculant ligand can be an arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the substituent group can be a cationic group, an anionic group, or a non-ionic group.
• the flocculant ligand can be an arylalkyl compound having from 1 to about 30 carbon atoms comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 1 -C 8 arylalkyl comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 9 -C 30 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 1 -C 3 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 3 -C 6 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 6 -C 10 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 10 -C15 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 15 -C 20 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the flocculant ligand can be an optionally substituted C 20 -C 30 arylalkyl compound comprising at least one substituent group, such as an amine or a carboxylic acid, that is capable of providing flocculant properties to the compound.
• the cation exchange separation medium (cationic flocculants) described herein can be represented by the following diagrams:
• R can be a functional group as described above, such as a carboxylic acid, amine, sulfonic acid, phosphonic acid, and the like
• R 1 can be H or a substitutent, such as C 1 -C 20 alkyl, C 6 -C 10 aryl, carboxylic acid, amine, sulfonic acid, phosphonic acid, hydroxyl, thiol, carbonyl, and the like
• n can be from 0 to about 30.
• the flocculant ligand is a "cationic flocculant ligand" that is an aliphatic primary aliphatic amine, a secondary aliphatic amine, a tertiary aliphatic amine, an aliphatic imine, an aliphatic hydrazide, an imidazole, an aliphatic oxime, an aliphatic hydrazine, an aliphatic hydrazone, a linear polyethyl amine, a polyethyleneimine, a heterocyclic quaternary ammonium, or a cationic polyelectrolyte, a branched aliphatic primary, secondary, or tertiary amine, or a substituted aliphatic primary, secondary, or tertiary amine, each having from 1 to about 30 carbon atoms.
• a cationic flocculant ligand that is an aliphatic primary aliphatic amine, a secondary aliphatic
• the flocculant ligand can be an optionally substituted C 1 -C 8 aliphatic primary, secondary, or tertiary amine. In some embodiments, the flocculant ligand can be an optionally substituted C 9 -C 30 aliphatic primary, secondary, or tertiary amine. In an exemplary embodiment, the cationic flocculant resins is antimicrobial in nature.
• Exemplary "cationionic flocculant ligands" or polymers include but are not limited to, Tris(2- aminoethyl)amine, Tris(3-aminopropyl)amine, linear polyethyl amines of varying chain lengths, and polyethyleneimine, poly(N-vinylpyrrolidone) (PVP), quaternary aminated polyacrylates, poly(N,N- dimethylpiperidinium chloride), polydiallyldimethylammonium chloride (PolyDADMAC), copolymers of poly(ethyleneimine), and quaternary aminated polyacrylates, etc.
• Other potential solutions include antimicrobials from the personal care industry, like Triclosan can also be used as cationic flocculants.
• anion exchange separation medium anionic flocculant
• anion exchange separation medium anionic flocculant
• a non-ionic flocculant ligand is immobilized to a base surface, and it can either be a styrene, substituted styrene, polymeric styrenes, an uncharged aliphatic, an uncharged branched aliphatic, a hydrophobic polyester, a hydrophilic polyester, a polyacrylamide, a poly(ethylene oxide), or copolymers thereof.
• a natural flocculant ligand is immobilized on a base surface, and it can either be a polysaccharide, an amino, imino, ammonium, sulfonium or phosphonium functionalized polysaccharide, a collagen, an anionic protein, a cationic protein, a chitosan, an ininglass, guar gum, a cationic protein from Moringa oleifera seeds or Strychnos potatorum seeds, or an alginate.
• the base surface can be a solid support having a format known to one of ordinary skill in the art. It will be appreciated that each format provided herein for a base surface comprises a solid support.
• the base surface may include but is not limited to: a resin, bead, sphere, particle, microcarrier, membrane, web, bag, bioreactor, tube, plate, array, flat surface, filter, fiber or a fabric.
• the base surface may be porous, non-porous, microporous, woven, non-woven, polymeric, non-polymeric, fibrous or winged.
• Exemplary base surfaces may include, but are not limited to, chromatographic resins, membranes, porous beads, porous monoliths, winged fibers, woven fabrics, non-woven fabrics, silica, SepharoseTM, porous polyvinylether polymeric beads, non- porous beads and their derivatives, styrenic beads and their derivatives, acrylate beads and their derivatives, acrylamide containing one or more polymerizable vinyl group, and the like.
• the base surfaces provided herein are merely exemplary, and that the identity of the base surface is not particularly restricted.
• base surfaces useful in connection with the present teachings include any base surface that is capable of being modified according to the methods described herein.
• Exemplary base surface may be made up of materials including but not limited to: ceramics, glass, metal, silica, synthetic polymeric materials such as styrenic, acrylate, acrylamide, acrylamide containing one or more polymerizable vinyl groups, polymeric monoliths, etc., natural polymers such as cellulose, lignocellulose or their derivatives, agarose, or a combinations of any of these materials.
• suitable base surfaces include, but are not limited to those describes in US Patent No.
• the support must be a reactive support and that reactive group should be capable of undergoing rapid, direct covalent coupling with a given flocculant or flocculant-like chemical entity to form the "flocculant ligand" or result in flocculant derivatized supports.
• surface hydroxyl groups on the base resin react in an SN2 reaction.
• one end of the reaction would comprise a nucleophilic group while the other end would comprise an electrophilic (leaving) group.
• electrophiles include halides (for e.g., bromine and chlorine), tosylate, mesylate, esters including NHS esters, carbonates, carbamates and the like.
• halides for e.g., bromine and chlorine
• tosylate mesylate
• esters including NHS esters
• carbonates carbamates and the like.
• nucleophile-electrophile pair, or entities could be either on the base surface or on the "flocculant ligand", but the nucleophile-electrophile entities must be present for the reaction to occur or for covalent coupling to occur.
• exemplary functional groups on a flocculant include, but are not limited to: leavings groups, such as halides, including bromine and chlorine, tosylate, mesylate, and the like; or electrophilic groups, such as esters, including NHS esters, carbonates, and the like; or nucleophilic groups, such as amines, thiols, hydroxyl, and the like.
• compositions and methods of the present disclosure are capable of separating a monomer and an aggregate to a greater degree and purity than previously known commercial separation products and methods.
• separation of a target molecule from impurities including the separation of monomers from aggregates, in particular of a mAb from antibody/ impurity aggregates, can be measured by the separation factor (a) explained below.
• the separation factor can be used to determine if a particular molecule binds in high quantity or preference to a solid support as described herein, such as a bead or resin comprising a flocculant ligand covalently attached thereto, than does another molecule.
• the separation factor can be used to determine if a monomer of an antibody binds in high quantity or preference to a solid support as described herein, such as a bead or resin comprising a flocculant ligand covalently attached thereto, than does an aggregate of the same antibody.
• the separation factor can also be used to determine if an aggregate of an antibody binds in high quantity or preference to a solid support as described herein, such as a bead or resin comprising a flocculant ligand covalently attached thereto, than does a monomer of the same antibody.
• the separation factor (a) is greater than or equal to 1 indicating a preference for binding of aggregate compared to monomer.
• the separation factor (a) is greater than or equal to about 2. In some embodiments, the separation factor (a) is greater than or equal to about 2.5. In some embodiments, the separation factor (a) is greater than or equal to about 3. In some embodiments, the separation factor (a) is greater than or equal to about 4.
• the separation factor (a) is greater than or equal to about 5. In some embodiments, the separation factor (a) is greater than or equal to about 6. In some embodiments, the separation factor (a) is greater than or equal to about 7. In some embodiments, the separation factor (a) is greater than or equal to about 8. In some embodiments, the separation factor (a) is in the range of about 2.5 to about 11. In some embodiments, the separation factor (a) is in the range of about 4 to about 9.
• the separation factor (a) is less than or equal to 1 indicating a preference for binding of monomer compared to aggregate. In some embodiments, the separation factor (a) is from about 0.1 to about 1. In some embodiments, the separation factor (a) is from about 0.1 to about 0.3. In some embodiments, the separation factor (a) is from about 0.3 to about 0.6. In some embodiments, the separation factor (a) is from about 0.6 to about 0.9.
• the flocculant modified solid support may be utilized in several modes, including but not limited to bind and elute and flow through applications. Bind and elute applications can be applied when the target molecule and potentially its impurities bind to the solid support and are differentially separated by the action of an elution buffer. In the bind and elute mode of operation it is also possible that the impurities do not bind, or are not as strongly bound as the target molecule and are removed from the solid support by a flowing a wash buffer over the solid support. In some embodiments, a bind and elute mode of operation can provide a separation factor (a) of less than or equal to 1, indicating a preference for binding of monomer compared to aggregate.
• a separation factor a
• a separation factor (a) of less than or equal to 1 will indicate that the mAb is bound to a separation medium of the present disclosure, while the aggregate of the mAb is removed from the solid support by a flowing a wash buffer over the solid support.
• a bind and elute mode of operation can provide a separation factor (a) of greater than or equal to 1, indicating a preference for binding of aggregate compared to monomer.
• the separation factor (a) in a flow through mode of operation is greater than or equal to about 2.
• the separation factor (a) in a flow through mode of operation is greater than or equal to about 2.5.
• the separation factor (a) in a flow through mode of operation is greater than or equal to about 3.
• the separation factor (a) in a flow through mode of operation is greater than or equal to about 4.
• the separation factor (a) in a flow through mode of operation is greater than or equal to about 5.
• the separation factor (a) in a flow through mode of operation is greater than or equal to about 6. In some embodiments, the separation factor (a) in a flow through mode of operation is greater than or equal to about 7. In some embodiments, the separation factor (a) in a flow through mode of operation is greater than or equal to about 8. In some embodiments, the separation factor (a) in a flow through mode of operation is in the range of about 2.5 to about 11. In some embodiments, the separation factor (a) in a flow through mode of operation is in the range of about 4 to about 9.
• recovery of target molecule can be up to 100%. In some embodiments, recovery of target molecule can be up to 99%. In some embodiments, recovery of target molecule can be up to 98%. In some embodiments, recovery of target molecule can be up to 95%. In some embodiments, recovery of target molecule can be up to 90%. In some embodiments, the purity of the recovered target molecule can be up to 100%. In some embodiments, the purity of the recovered target molecule can be up to 99%. In some embodiments, the purity of the recovered target molecule can be up to 98%. In some embodiments, the purity of the recovered target molecule can be up to 95%. In some embodiments, the purity of the recovered target molecule can be up to 90%.
• Target molecules or molecules of interest are molecules of interest
• Target molecules can be any biological molecule of interest, for example, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, immunoadhesins and antibody-immunoadhesin chimerias.
• An antibody may include, for example, an antibody of any molecule class, e.g., IgG 1 , IgG 2 , etc. that is to be purified from a mixture containing contaminants.
• an “antibody fragment” includes at least a portion of a full length antibody and typically, an antigen binding or variable region thereof; for e.g., they include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules like camelid antibodies; diabodies; linear antibodies; and multispecific antibodies formed from engineered antibody fragments.
• the term “monoclonal” antibodies indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies described herein include "chimeric" and
• humanized antibodies and “human” antibodies, which can be isolated from various sources, including, e.g., from the blood of a human patient or recombinantly prepared using transgenic animals.
• Exemplary target molecules may also include but are not limited to, proteins, enzymes, RNA, DNA, antibodies, cell parts, organelles, and the like.
• the target molecule can be an enzyme, such as lysozyme, chymotrypsinogen, ribonuclease A, and the like.
• the target molecule can be a protein, such as bovine serum albumin (BSA), cytochrome C, and the like, the target molecule is either a monomeric antibody, a therapeutic peptide or protein, a virus or viral particle, a particular variant of a peptide or protein or antibody or virus or viral particle, or a nucleic acid.
• BSA bovine serum albumin
• Solid supports that are built as chromatography resins, beads or particles can be used in column format, as is conventional practice, or in batch mode, such as in containers, bioreactors, bags, wells, flasks, etc., that are usually equipped with mechanisms for stirring. Solid supports such as membranes or filters would typically be manufactured for use in filter devices of which there are many designs. [00139] In some embodiments it is likely that conditions can be identified to effectively inactivate viruses. These cases are highly desirable, as they eliminate the need for a viral inactivation hold and thus would be specifically amenable to continuous operations.
• the separation media described herein will be exposed to certain conditions based on the desire application.
• the separation medium is stable when contacted with an acidic or a basic solution, or at least one solvent.
• the at least one solvent can be any solvent known to one of skill in the art for use in chromatography applications.
• solvents include, but are not limited to, aliphatic hydrocarbons, aromatic hydrocarbons, alcohols, ketones, aldehydes, glycol ethers, esters, glycol ether esters, and halogenic solvents.
• the at least one solvent can be any common solvent, such as methanol, ethanol, acetone, ethylene glycol, acetonitrile, water, acidic solutions, basic solutions, and the like.
• HPLC high performance liquid chromatography
• solid phase extraction purification from either a harvested cell culture fluid, a cell culture supernatant, or a conditioned cell culture supernatant, a cell lysate, or a clarified bulk; from wherein a target biomolecule or protein is purified; or, sample prep.
• the first chromatography can be selected from an affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, or mix-mode chromatography.
• the second chromatography can be ion exchange chromatography, hydrophobic interaction chromatography, a mix- mode chromatography.
• the second chromatography can be positive-charged membrane chromatography or hydrophobic interaction membrane chromatography.
• the load material may be a clarified or an unclarified feed, or a pool from an affinity purified step, an ion exchange step, from HIC purification, from mixed mode purification, from reverse phase column, or from any other chromatography step.
• the load material concentration may be any concentration that allows the molecule to be soluble at 0.1 mg/mL to >100 mg/mL.
• the pH of the load solution may be adjusted between about pH 1 and about pH 14 before loading on to the chromatographic column with a separation medium as described herein, such as a caprylic acid derivitized resin.
• the pH of the load solution may be adjusted between about pH 1 and about pH 7 before loading on to the chromatographic column with a separation medium as described herein, such as a caprylic acid derivitized resin.
• a separation medium such as a caprylic acid derivitized resin.
• the pH of the elution solution may be adjusted between about pH 3 and about 13, or between pH 3 and about pH 7 before the proceeding step, or, the solution may be adjusted before taking the solution to the next chromatographic step.
• the elution mixture may not be subjected to additional
• the buffer composition ranges can be protein dependent. For example, elution may be carried out using salt to displace monomer, or by pH change of at least 1 pH unit, or a combination thereof.
• Use of additives such as polymers, alcohols, or amino acids may be used to control the degree of secondary or tertiary interaction or a combination thereof.
• impurities are bound and the target molecule flows through with zero to minimal interaction with the resin.
• both the impurity and the target molecule bind to the surface, and the target molecule or the impurity can be selectively eluted through a change in salt concentration, salt type, pH, through the use of additives.
• both the impurity and the target molecule bind, and the target molecule is then displaced by the impurity or by a competitive additive (displacer).
• a competitive additive dislacer
• the materials and methods described herein can be applied to lab scale sample preparation (such as 10 ⁇ L to 50 mL bed volume). For use in semi preparative scale sample preparation (10 mL to 1 L bed volume). In some embodiments, the materials and methods described herein can be applied to large scale purification above 1L bed volume.
• the separation medium is capable of separating a monomer and an aggregate of the same biological molecule in a pH range of about 2 to about 11. In some embodiments, the separation medium is capable of separating a monomer and an aggregate of the same biological molecule in a pH range of about 4 to about 8. In some embodiments, the separation medium is capable of separating a monomer and an aggregate of the same biological molecule in a pH range of about 5 to about 7. In some embodiments, the separation medium is capable of separating a monomer and an aggregate of the same biological molecule in a pH range of about 4.5 to about 7.5.
• the buffer conductivity can be about 0 mS/cm to about 200 mS/cm. In some embodiments, the buffer conductivity can be about 0 mS/cm to about 30 mS/cm. In some embodiments, the buffer conductivity can be about 10 mS/cm to about 30 mS/cm. In some embodiments, the buffer conductivity can be about 1 mS/cm to about 20 mS/cm.
• the buffer conductivity can be about 30 mS/cm to about 135 mS/cm. In some embodiments, the buffer conductivity can be about 30 mS/cm to about 75 mS/cm. In some embodiments, the buffer conductivity can be about 50 mS/cm to about 135 mS/cm. It will be further appreciate that the conductivity can be expressed in terms of buffer concentration. In particular, higher ion concentration, provides higher conductivity. Conversely, lower ion concentration, provides lower conductivity.
• the buffer for use in connection with the present disclosure is not particularly limited, and can be any buffer known in the art for use in separation or purification of biological molecules.
• the buffer can be aqueous NaCl in a concentration of about 5 mM to about 2M. In some embodiments, the buffer can be aqueous NaCl in a concentration of about 5 mM to about 1M, 5 mM to about 500 mM, 5 mM to about 300 mM. In some embodiment, the buffer can be aqueous NaCl in a concentration of about 5 mM, about 25 mM, about 75 mM, about 100 mM, about 200 mM, about 300 mM, about 500 mM, about 1M, about 1.5 M, about 2M.
• Bioprocessing chromatography resins are ubiquitous in the manufacture of biotherapeutics and are essential for the removal of product and process related impurities. In this segment once resins are selected, they are qualified and processed and filed with the FDA. With increasing numbers of new molecule modalities, resins with differentiated resin functionalities are needed. We have created novel resins with unique surface chemistries (flocculant ligands) and generated a new line of flocculant ligand functionalized purification resins that clearly distinguish from any of the existing commercial purification products available.
• Example 1 Exemplary Cationic Exchange (CEX) Flocculant Resins
• CA precipitation method is exemplified in Biotechnology and Bioengineering, Vol. 109, No. 10, 2012, 2589-2598.
• CA precipitation method is exemplified in Biotechnology and Bioengineering, Vol. 109, No. 10, 2012, 2589-2598.
• no attempts have been made so far to immobilize caprylic acid or any other flocculant on to resins to generate purification resins, or to use immobilized flocculant resins for protein purification.
• any anionic ligands such as an unsubstituted or substituted aliphatic carboxylic acid, an unsubstituted or substituted aromatic carboxylic acid, an unsubstituted or substituted aliphatic sulfonic acid, an unsubstituted or substituted aliphatic acrylic acid, an unsubstituted or substituted aliphatic thiosulfate, an unsubstituted or substituted aliphatic phosphonic acid, or an unsubstituted or substituted aliphatic phosphoric a fatty acid, may be used as a ligand to couple to any base surface.
• any anionic ligands such as an unsubstituted or substituted aliphatic carboxylic acid, an unsubstituted or substituted aromatic carboxylic acid, an unsubstituted or substituted aliphatic sulfonic acid, an unsubstituted or substituted aliphatic acrylic acid, an unsubstituted
• the unsubstituted or substituted aliphatic groups are either linear or branched, and optionally, comprise one or more double bonds.
• the unsubstituted or substituted aliphatic groups can have from 1 to about 30 carbon atoms, preferably from 1 to about 20 carbon atoms, more preferably from about 1 to about 10 carbon atoms, most preferably from about 1 to about 8 carbon atoms.
• cationic exchange flocculant resins were generated by coupling anionic flocculants such as unsubstituted or substituted C 1 -C 8 aliphatic acids to base surfaces.
• anionic flocculants such as unsubstituted or substituted C 1 -C 8 aliphatic acids
• the CEX flocculant resin comprised caprylic acid.
• the base surface is a resin or a bead.
• the base surface can include and not be limited to: a resin, bead, sphere, particle, microcarrier, membrane, web, bag, bioreactor, tube, plate, array, flat surface, filter, fiber or a fabric.
• compositions and methods using CEX flocculant resins are capable of separating a monomer and an aggregate to a greater degree and purity than previously known commercial separation products and methods.
• separation of a target molecule from impurities including the separation of monomers from aggregates, in particular of a mAb from antibody/ impurity aggregates, can be measured by the separation factor (a) explained below.
• HMW impurities can include aggregates (dimers, trimers, tetramer etc, and/or, antibody monomer with a light chain, and/or antibody with high levels of post translational modifications, and/or aggregated host cell proteins (HCP), and/or aggregated antibody or protein fragments.
• LMW impurities can include host cell proteins (HCP) or antibody or protein fragments.
• exemplary flocculant resins were prepared as follows. The flocculant caprylic acid (octanoic acid) was immobilized on to a POROS chromatography resin for evaluation of intended use for purification of biomolecules.
• this example describes immobilization of bromo-caprylic acid to the POROS resin by activation of surface hydroxyl groups and an S N 2 substitution reaction.
• the resin could have equally been prepared by coupling hydroxy-caprylic acid / or its derivative to an expoxide activated POROS resin.
• the POROS resin had a pore mode of -1,000 ⁇ , and was manufactured and coated with a polymeric coating containing a high density of hydroxyl groups.
• Resin evaluation Evaluation of the resin comprised measurement of the ionic capacity of the resin as a measure of the surface density of the caprylic acid. This was determined by packing of a chromatography column, treating the resin in the column with a dilute acid solution to ensure the hydrogen form of the surface and titration with a dilute solution of sodium hydroxide. Assessment of the volume of sodium hydroxide needed to achieve 50% breakthrough as noted by a pH meter determines the ionic capacity of the resin. In this example the ion capacity was ⁇ 64 ⁇ m ⁇ /mL.
• High throughput screening was used to determine static binding capacity and to map potential operating parameters that could be useful for purification of a therapeutic molecule such as a monoclonal antibody (mAb).
• mAb monoclonal antibody
• the equilibrium binding capacity of a mAb was assessed by contacting the mAb in an appropriate buffer solution with the resin that had been conditioned with the same solution, incubating the resin/mAb combination with gentle agitation, and after an incubation time of ⁇ 1 hour, recovering the supernatant solution for measurement by UV at a wavelength of 280 nm.
• CEX flocculant resin was compared to that of commercially available weak (carboxy methyl) cation exchangers (competitor 1 CM and competitor 2 CM), and a commercially available strong cation exchanger (POROS XS). There is a predictable wider operating range than is observed for the commercially available resins. Comparison of the Static Binding Capacity (SBC) for different resins at representative pH and salt concentrations. Table 1 shows data for Resin A; data for Competitor 1 resin; data for Competitor 2 resin; data for POROS XS.
• SBC Static Binding Capacity
• pH can be varied over a range of pH 2.0 - 9.0, and the salt concentrations can be varied from 0.0 M to 1.5 M salt in order to achieve a desirable SBC.
• the data predicts a wide operational space for the removal of aggregate from the mAb (monoclonal antibody) examined in this test.
• Resin A/ or other derivative flocculant resins had higher selectivity for aggregates under certain operating conditions than commercially available weak carboxy methyl cation exchangers (competitor 1 CM and competitor 2 CM) and a commercially available strong cation exchanger (POROS XS) used under the same conditions (also see Tables 2 and 3 below, which show high a values or higher selectivity for aggregates at given pH and salt concentration).
• This exciting finding was further examined by purification experiment of the same mAb. Under static binding conditions, we are looking at the ratio of bound and unbound monomer and aggregate. The higher alpha, the more aggregate was bound in comparison to the monomer.
• purification resin type A showed significant increase in selectivity for aggregates compared to POROS XS and competitor resin over a wide range of conditions. Under certain conditions, resin type A bound almost the same amount of aggregates as POROS XS resin, while only binding approximately one quarter of the monomer. Thus, resin type A was a great candidate for flow through chromatography to purify monomer, possibly even overload chromatography.
• FIG. Id Conditions: Column dimensions: 4.6mmxl00mm; Total protein load ⁇ 1.2mg; Sample proteins studied were: ⁇ 0.2mg Myoglobin (Protein 1, mwt 16.9kDa, pi 7.36); ⁇ 0.5mg Alpha- Chymotrypsinogen A (Protein 2, mwt 25.6kDa, pi 8.52); -0.5 mg Lysozyme (Protein 3 mwt 14.4kDa, pI 11.35). Separation based on size exclusion would be indicated by elution in order of molecular weight or size (cytochrome c ⁇ lysozyme ⁇ chymotrypsinogen).
• Example 2 Selectivity Evaluations of Cation Exchange (CEX) Flocculant Resins A, B, C, D
• aliphatic flocculant chains of varying lengths were conjugated to porous resins.
• the resins differed in their hydrophobicities as follows: A >>> B >> C > D, where A is comparatively the most hydrophobic, compared to B and C with intermediate hydrophobicity, compared to prototype D with the least hydrophobicity.
• Resins A, B and D were prepared using the same methods described in Example 1, whereas Resin C was prepared using a slight variation of Example 1.
• the separation factor (a) is greater than 1, indicating a preference for binding of aggregate compared to monomer.
• the separation factor can be used to determine if a monomer of an antibody binds in high quantity or preference to a solid support as described herein, such as a bead or resin comprising a flocculant ligand covalently attached thereto, than does an aggregate.
• selectivity factors higher than 2.0 and 4.5 respectively are shown in bold, which show conditions where aggregates including very closely related HMW species, bind preferentially to the flocculant resin columns. This region has high selectivity for aggregates.
• the pure monomer is not expected to bind to the resin and thus the monomer can be collected in flow through mode.
• the separation factor (a) is greater than or equal to about 2. In some embodiments, the separation factor (a) is greater than or equal to about 2.5. In some embodiments, the separation factor (a) is greater than or equal to about 3. In some embodiments, the separation factor (a) is greater than or equal to about 4. In some embodiments, the separation factor (a) is greater than or equal to about 5. In some embodiments, the separation factor (a) is greater than or equal to about 6.
• the separation factor (a) is greater than or equal to about 7. In some embodiments, the separation factor (a) is greater than or equal to about 8. In some embodiments, the separation factor (a) is in the range of about 2.5 to about 11. In some embodiments, the separation factor (a) is in the range of about 4 to about 9.
• pH can be varied over a range of pH 2.0 - 11.0, preferably over a range of pH 2.0 - 9.0 under normal use conditions; the salt can be varied from concentrations of 0.0 M to 1.5 M salt in order to achieve a desirable separation factors.
• Kp is the partition coefficient for either the aggregate or the monomer.
• FIG. 2a shows a heat map comparison of the exemplary separation medium Resin A versus non- flocculant, control resins POROS XS, X-CM and Y-CM. If we take a look at the heat map across the benchmark, non-flocculant resins, the operational window with high selectivity is greater for the flocculant resins than for the commercial, non-flocculant resins.
• FIG 2b shows a heat map comparison of an exemplary separation media - Resins A, B, C and D with each other.
• the resins show a range of ligand hydrophobicities: Resin A >>> B >> C > D, which A being the most hydrophobic resin.
• All resins A-D display increased selectivity for aggregates with selectivity factors >> 1, over a broad range of pH and salt concentrations.
• the flocculant-ligand resins showed greater aggregate selectivity across a range of hydrophobicities, across broader modes of operation (pH and salt concentration).
• the separation factor (a) is less than 1. In some embodiments, the separation factor (a) is from about 0.1 to about 0.9. In some embodiments, the separation factor (a) is from about 0.1 to about 0.3. In some embodiments, the separation factor (a) is from about 0.3 to about 0.6. In some embodiments, the separation factor (a) is from about 0.6 to about 0.9.
• selectivity factors of lesser than 0.9 are shown in bold, which are conditions where monomers bind preferentially to the flocculant base surfaces, resins or columns than any aggregate or any other HMW/ LMW species. In such conditions, the monomer may be purified in bind-elute mode.
• the separation factor (a) is 1, which means that neither the monomer nor the aggregate species can be separated effectively. Therefore, for selectivity or separation of monomer from aggregate, or ligate from solution, or target molecule from impurity, the operating conditions must be manipulated such that the selectivity factor is either >1 (when aggregate/ impurity binds, thus monomer/ target can be purified in flow-through mode), or the selectivity factor is ⁇ 1 (when the monomer/ target binds, thus the monomer/ target can be purified in bind-elute mode).
• One of skill in the purification art would know to manipulate such modes of operation and to fine tune purification conditions in order to achieve the binding/ separation of a desired target.
• pH can be varied over a range of pH 2.0 - 9.0; the salt can be varied from concentrations of 0.0 M to 1.5 M salt in order to achieve a desirable separation factors.
• K p is the partition coefficient for either the aggregate or the monomer.
• the selectivity factor, alpha is greater than 1 the aggregate preferentially binds to the resin compared to the monomer. Under these conditions, flow through mode of purification is preferred. In order to obtain high purity, the selectivity factor should be maximized. A selectivity factor of greater than 2.5 is desired whereas a selectivity factor of 4.5 is preferred.
• the selectivity factor, alpha is less than 1 the monomer preferentially binds to the resin compared to the aggregate. Under these conditions bind and elute mode of purification is preferred. In order to obtain high purity, the selectivity factor should be maximized. A selectivity factor of lesser than 0.9 is desired.
• selectivity factor Due to molecular diffusion and differences in partition coefficients for minor post translational modifications, a selectivity factor close to 1 may result in reduced purity. Where the selectivity factor is 1, there will be no separation of the target (for e.g., monomer) and impurity.
• FIG. 3a resin B was used as the separation medium, and the expanded Size Exclusion Chromatogram of fractions taken from the purification profile for Resin B is shown (FIG. 3b).
• FIG 3b the chromatograms of various fractions from the run of FIG 3a is shown.
• the top curve shows the starting material before purification, comprising significant amounts of various HMW aggregate species.
• the % recovery of the flow-through antibody is shown below in Table 5.
• Monomer shows excellent recovery (> 90%) in flow through mode at pH 4.5, 5.0 and 5.5.
• FIGs 4a and 4b show purification data from Resin A and Resin C respectively, displayed as antibody breakthrough curves, each at pH 4.5, 5.0 and 5.5.
• the two flocculant ligand types Resin A and Resin C show consistent and effective removal of high molecular weight species HMW3. While the monomer seems to have similar selectivity as HMW2, another high molecular wt. species HMW1 is gradually removed by these resins.
• FIGs 5a and 5b show elution analysis with 1M Tris buffer at pH 8.5, for Resin A and Resin C.
• the resins were loaded with an antibody solution each at pH 4.5, 5.0 and 5.5.
• Resin A was very effective in removing HMW3 completely.
• Tris elution shows significant removal of the HMW1 species and very little monomer loss.
• HMW3 is bound tightly, especially for resin A which shows excellent selectivity for this species.
• Purification resins having inherent antimicrobial and/or bacteriostatic properties allow resin storage in buffer or water. Additionally, if such resins also have specific functionality for capture or polish operations, it would be a significant benefit to customers.
• Antimicrobial and bacteriostatic surface chemistries are known in many industries. We engineered base resins by adding antimicrobial and bacteriostatic flocculant surface chemistries.
• chemistries include but not limited to, molecules such as organic acids, short chain alcohols and benzyl alcohol, etc. which show excellent base stability.
• Exemplary antimicrobial and cationionic flocculant polymers include but are not limited to, Tris(2-aminoethyl)amine, Tris(3- aminopropyl)amine, linear polyethyl amines of varying chain lengths, and polyethyleneimine, poly(N- vinylpyrrolidone) (PVP), quaternary aminated polyacrylates, poly(N,N-dimethylpiperidinium chloride), poly(DADMAC) (Polydiallyldimethylammonium chloride), etc.
• antimicrobials from the personal care industry, including but not limited to, Triclosan, antimicrobial peptides and proteins that destabilize the biological membrane, etc. These chemistries may also result in having unique selectivity's or affinities, for e.g., for biotheraputics.
• AEX resin 1 comprises a poly(styrene-co-divinylbenzene) bead with a modal pore size of around 200 nm and a hydrophilic coating, functionalized with an amine, resulting in a surface comprising primary, secondary and tertiary amines.
• AEX resin 2 comprises a poly(styrene-co-divinylbenzene) bead with a modal pore size of around 100 nm and hydrophilic coating, functionalized with the flocculant poly(DADMAC), resulting in a fully ionized quaternary amine surface.
• AEX resin 3 comprises a poly(styrene-co-divinylbenzene) bead with a modal pore size of around 200 nm and hydrophilic coating, functionalized with a PEI-like polymer resulting in a surface comprising primary, secondary, tertiary amines and quaternary amines.
• AEX resin 4 comprises a poly(methacrylate) bead with a modal pore size of around 500 nm functionalized with an amine resulting in a surface comprising primary, secondary, tertiary amines and quaternary amines.
• AEX resin 5 comprises a poly(styrene-co-divinylbenzene) bead with a modal pore size of around 40 nm and hydrophilic coating, functionalized with the flocculant poly(DADMAC) resulting in a fully ionized quaternary amine surface.
• AEX resin 6 comprises a poly(styrene-co-divinylbenzene) bead with a modal pore size of around 200 nm and hydrophilic coating, functionalized with the flocculant poly(DADMAC) resulting in a fully ionized quaternary amine surface.
• AEX resins described above can also be evaluated for microbial growth inhibition and bioburden testing in a range of non-bacteriostatic solutions.
• ovalbumin was used as a test molecule and was subjected to a bind - elute type purification on the AEX flocculant resins.
• Ovalbumin is a 42.7 kDa protein, comprising 385 amino acids, with a serpin-like structure.
• Commercially available ovalbumin when purified by precipitation contains several modifications or charge variants that can be considered product related impurities. Modifications or charge variants include, but are not limited to glycosylated, glycated, oxidized, deaminated, acidic, basic, phosphorylated, sialylated or a N-terminal acetylated forms, etc. and they result in a mixture of closely related species that need separation.
• the AEX flocculant resins described herein may use pH-based gradients for the resolutions and/or separation of charge variants of the peptide/protein/ antibody/ product.
• Cationic flocculant ligands are typically polyelectrolytes either formed prior to binding to the surface or created while in one or multipole reaction the polyelectrolyte is bound to the surface.
• Copolymer are used to modulate the charge in order to achieve the desired impurity removal selectivity.
• the chromatographic panel shows the variant profile of ovalbumin when injected onto and separated using an analytical HPLC column (Thermo Scientific ProPac SAX- 10). Each peal- represents a variant or group of variants not resolved on this column (labelled peaks 1-6).
• AEX resins 1 and 2 were compared to the commercially available AEX resins (POROS XQ and POROS HQ).
• POROS XQ and POROS HQ commercially available AEX resins
• a feed solution of ovalbumin was loaded onto the resin in column format until 5% break through occurred. At this point an elution gradient from 0 to 1M sodium chloride was applied. Fractions were collected from the start of the gradient. The same method was used for each test. Each fraction was then analyzed by HPLC. Each peak was identified in comparison to the feed and the composition of each fraction determined.
• FIG - 6b, 6c, 6d and 6e show the composition of each fraction as a stacked bar graph.
• AEX resin 1 In the case of AEX resin 1 (FIG 6d), peak components 1, 2 and 3 were not selectively bound to the column and did not appear in the fraction analysis. In contrast, AEX resin 2 (FIG 6e) shows retention of these components into fraction 8 as well as increased retention of other species when compared to commercial AEX resins POROS XQ (FIG 6b) and POROS HQ (FIG 6c) not based on flocculant chemistry. The differences in profiles demonstrate that the AEX resins have differential selectivity of each resin chemistry.
• the binding capacity of the AEX resins was also characterized using a mixture of bovine serum albumin and DNA, to evaluate nucleic acid binding of the AEX resins. It is known that resins with an increase surface area can be expected to bind a higher amount of protein per unit area.
• Figure 7 shows a comparable ratio of binding for POROS XQ and POROS HQ as well as resin 2, indicating comparable ability to remove host cell DNA from the feed solution.
• Resin 1, 3 and 4 show differentiated binding characteristics with an increase capability to remove DNA compared to BSA.
• the flocculant CEX resins of this disclosure can be used in a variety of application, for e.g., a) an analytical separation and/or screening tool; b) for large scale purification, for e.g., polish strategy; c) addition of flocculant resins to stirred tanks to enhance flocculation of impurities (or target, as applicable) for easy separation of a class of ligate species that selectively bind the flocculant resin; d) application of the flocculant chemistries described to new surfaces including but not limited to: hollow fibers, membranes, nanofibers, to a bioreactor including a Single Use bag or container, etc. for novel applications; e) holding a molecule upstream in a container, etc.

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• 2019
  • 2019-02-27 WO PCT/US2019/019902 patent/WO2019169040A1/en unknown
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