Bind and Elute Mode of Operation

A. Equilibrium buffer:

Sun (US8058407) discloses a method of purifying at least one acidic protein by applying an equilibration buffer of CaCl2 to HA, containing the HA with a load buffer, washing with CaCL2 and eluting with a buffer with 2-50 mM phosphate.

B. Binding/Loading

There are two different types of adsorbing sites on the surface of HA, calcium and phosphate sites. The former appear to be responsible for the binding of acidic groups, carboxyls and phosphates whereas the latter ones for the binindg of basic groups (Bernadi, Biochimica et Biophysica acta, 278 (1972) 409-420). 

Prior to protein loading, the resin is commonly equilibrated with a buffer of the same strenght as the loading buffer and at the same pH (Cummings US 13/205354). 

–Polyethylene glycol and glycine have been used to improve the performance of hydroxyapatite chromatography (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555).

Sheldon (WO/2010/148143) discloses methods of isolating recombinant adeno-associated virus (rAAV) particles by capturing the rAAV particles on an apatite chromatography meidum in the presence of polyethylene glycol (PEG). 

–Ca or Mg ion: Phosphate within the HA can be blocked (or bridged) by Ca ion or Mg ion which provides strengthened interaction between positive charge within a protein (Gorbunoff (Protein Chromatography on Hydroxyapatite Column, Method in Enzymology, Academic Press Inc., 1985, pp370-380) (US Patent Application 12/355,686).

–Phosphate buffer: 

In HA, the column is normally quilibrated, and the sample applied, in a low concentration of phosphate buffer and the adsorbed proteins are then eluted in a concentration gradient of phosphate buffer (Sun US2005/0107594).

Protein loading of a HA column is commonly conducted at pH 6.5 with phosphate buffer at 2mM ot 5mM, conditions that promote the adsorption of protein to the hydroxyapatite surface (Cummings US 13/205354).

–Salts:

In some cases, adsorption is further promoted by the inclusion of minor amounts of NaCl or KCl (Cummings US 13/205354).

Sun, US 2005/0107594 disclose using NaCl with HA for the purification of immunoglobulins and removal of high molecular weight aggregates. In one embodiment, the invention discloses using an elution or load buffer that contains from 1-20 mM sodium phosphate and 0.2-2.5 M NaCl, wherein the elution buffer has a pH from 6.4-7.6. Conditions vary depending on whether a binding or flow-through mode are used. 

Mitsui (EP256836) discloses that the degree of tPA adsorption to HA is governed by pH and/or salt concentration and also varies depending on the MW of tPA. Namely, the binding strenght of tPA to HA becomes greater as the pH decreases or the salt concentration drops. At the same pH and salt concentration, tPA having higher MWs is adsorbed more tightly.

C. Washing 

 E. Elution:

Elution on HA is caused by anions (usually phosphate) which compete for the calcium sites of HA with the carboxyl or phosphate groups of the macromolecules; or by cations (Na+, K+ or, more effectively Ca2+ or Mg2+) which compete for the phosphate groups of HA with the basic groups of proteins ((Bernadi, Biochimica et Biophysica acta, 278 (1972) 409-420). 

Gorbunoff, (Analytical Biochemistry, 136, 425-432 (1984)) discloses elution behavior as a function of their isoelectric point. Basic proteins having isoelectric points above 8 elute with PO4, F-,, Cl-, SCN- and ClO4- at moderate molarities, Proteins with isoelectric points between 7 and 8 elute with PO4, F- and CL- ions but not SCN- or Ca2+. Acidi proteins do not elute with Ca2+ ion. 

–Salts:

(Gorbunoff (Protein Chromatography on Hydroxyapatite Column, Method in Enzymology, Academic Press Inc., 1985, pp370-380) (US Patent Application 12/355,686) as well as other salts such as KCl, NaCL, KAc and NaAc can be used as eluting agent for HA. (Gagnon (232nd National Meeting of the American Chemical Society, Sep 10-14, 2006, Sanfransico, California, USA)  (US Patent Applicaiton 12/355,686).

The effects of different salts on the selectivity of a given apatite are unpreditable. For example, in the absence of phosphate, sodium chloride is unable to elute most IgG monoclonal antibodies from native HA, even at concentrations in excess of 4 moles per liter. When eluted with a combination of lower concentration of salts, such as 0.25M sodium chloride and 50 mM phosphate, IgG is one of the earliest eluting proteins (compare to when just phosphate is used above) (Gagnon, US2009/0186396). 

(a) Phosphate:

Phosphate refers to salts based on phosphorus (V) oxoacids such as, but not limited to, sodium phosphate and potassium phosphate (Gagnon, US2009/0186396). 

Once bound, the most common elution mechaism has been a gradient of increasing phosphate concentration. This would seem to be the most convenient choice of elution bufers since it serves as a displacement agent, disrupting both the COO:Ca+ interaction as well as the NH3+PO4 interaction. The strong calcium affinity of phosphate suspends calcium chelation and coordination iteractions, while its ionic character suspends phosphoryl cation exchange interactions. In exclusively phosphate gradients, IgG is typically one of the latest eluting proteins, using requiring 100-150 mM phosphate (Gagnon, US2009/0186396). 

In the increasing phosphate concentration method, all proteins bound to the column can be eluted and resolved based on the strenght of the interaction with the phosphate group on the hydroxyapatite matrix. Therefore, by using a gradient of increasing phosphate concentration, the most weakly bound proteins bound by the NH3+:P (V interaction (more acidic proteins) will elute earlier than those bound by the COO:Ca+ interaction (more basic proteins). In addition, the elution time of various bound proteins and resolution between them can be altered significantly through changes in the pH of the elution buffer (Mazzola, WO/2009/017491). Elution is accomplished by displacing the non-specific protein-hydroxyapatite pairing with ions such as Ca2+ or Mg2+. Negatively charged protein groups are displaced by negatively charged compounds, such as phosphates, thereby eluting a net-negatively charged protein.

Mitsui (EP256836) discloses a method of separating a mixture of tissue plasminogen activator (tPA) species having different MW by contacting the mixture with hydroxyapatite to adsorb the tPA species and then treating the HA with eluents to elute the fractions sucessively. The eluents are different in pH, salt concentrations or both pH and salt concentrations. In one embodiment the eluent is phosphate. In a second, the eluent is sodium chloride (see salts below).

In the rare cases where alternatives to phosphate as a primary eluting salt have been discussed in the literature, suggestions have included calcium chloride, citrate and fluoride salts, but without mention of sulfates (Gagnon, US2009/0186396). 

—-Phosphate + chloride salts: Some applications elute HA with combinations of phosphate and chloride salts. Chlorides preferentially elute the phosphoryl cation exchange interaction while having relatively little effect on calcium affinity interactions Gagnon (US 2009/0186396). 

———–Phosphate + calcium cloride (CaCL) or calcium sulfate (CaSO4):  

Jensen (US2009/0047723) discloses elution of FVII protein using either an appropriate combination of pH and phosphate or calcium in the form of calcium chloride or calcium sulfate).

Cummings (US 13/205354) discloses that the deteriorated of HA during protein elution can be mitigated by eluting adsorbed proteins by the use of an elution buffer that contains a combination of calcium and phosphate ions. Calcium ion can be supplied by an calcium salt such as calcium chloride that is soluble in the elution buffer. Phosphate ion can also be supplied from any phosphate salt such as sodium phosphate that is soluble in the elution buffer. In certain cases, sodium chloride is also included in the elution buffer for enhanced desorption of the protein.

———Phosphate + sodium chloride (NaCL):  Sun (US2005/0107594)  discloses HA for the purification of immunoglobulins using an elution buffer or load buffer that contains from 1-20 mM soium phosphate and from 0.2 to 2.5 M NaCL, wherein the elution buffer or load buffer has a pH from 6.4 to 7.6.

Cummings (US201101782760 discloses an elution buffer (0.55 M NaCl, 5 mM sodium phosphate at pH 6.5).

(b) sodum chloride (NaCL): Gagnon (US2009/0186396) discloses purification of a biomolecule by binding in the presence of calcium, convertion to native apatite and elution in a soidum chlorida gradient.

(c) calcium chloride (CaCL2):  Elution with Ca2+ is characterized by a drastic difference between the elution behavior of proteins as a funciton of their isoelectric point. All acidic proteins, as well as those with isoelectric points between 7 and 8, do not elute with Ca2+ ion even at 3 M sale. Basic proteins, to the contrary, elute at a Ca2+ molarity of 0.001-0.003 (Gorbunoff, Analytical Biochemistry, 136, 425-432 (1984).

—-CaCl2 + sodium sulfate: Gagnon (US2009/1086396) discloses binding DMA to HA and elution with 3 mM CaCL2, 1.0 M sodium sulfate, pH 6.7. 

 (d) sulfate: refers to salts based on sulfur (VI) oxoacides such as sodium sulfate and ammonium sulfate (Gagnon, US2009/0186396).

–Amino Acids:

——-Monocarboxylic zwitterion: refers to a molecule containing a single carboxyl moiety and at least one moeity with a positive charge. Examples include amino acids glycine, proline, lysine and histidine.  Gagnon (US 2009/0186396) discloses elution in the presence of a monocarboxylic awitterion.

Flow-through Mode of Operation

Vedantham (US 2003/0166869) teaches a method for separating a protein from another using HA in which the protein does not bind to HA but the other protein(s) does. In one embodiment, the protein is an antibody and the other protein is Protein A. 

a. Loading buffer: Sun (US2005/0107594A1) discloses antibody purification using a flow-through mode for HA with a load buffer containing 0.2 to 2.5 M NaCl at slightly acidic to slightly basic pH. The antibody preparation is then allowed to flow through a HA, while impuritied such as HMWA bind to the column. The column is optionally washed to allow additional purified antibodies to flow through.

Overload and Elute Mode

Nadarajah ((Nadarajah, US14/355818 (US2014/0301977)) teaches a method for purifying a polypeptide such as an antibody by a) loading the composition onto a mixed mode chromatography material such as CaptoAdhere in an amount in excess of the dynamic binding capacity of the material for the polypeptide, b) eluting the polypeptide under condtiions where contaminants remain bound and c) pooling fractions comprising the polypeptide in the chromatography effluent from steps a0 and b). In some embodiments, the partition coefficient of the chromatography material for the polypeptide is greater than 30 or greater than 100. 

Column Considerations

1. Column Dimensions: Sun, US2005/0107594 discloses many types of column diameters which can be used with cHA. 

Calcium-derivatized apatities

The inoic state of HA columns can be varied by changing the ionic nature of the buffers with which they are equilibrated. Thus, equilibration with PO4 buffer should make the surface of the column negative due to complexation with HA-C12+ stes. Equilibration with CaCl2 should have the opposit effect, while NaCl should lead to a neutral column surface (Gorbunoff, Analytical Biochemistry 136, 440-445 (1984). 

Native hydroxyapatite and fluorapatite can be converted to calium derivatized forms by exposure to soluble calcium in the absence of phosphate. This converts P-sites into secondary C-sites, abolishing phosphoryl cation-exchange interactions, increasing the number of C-sites, and fundamentally altering the selectivity of the apatite support. Small alkaline proteins typified by lysozyme (13.7-14.7 Kda, pI 10.7) fail to bind to calcium derivatized apatites, but most other proteins bind so stronly that even 3M calcium chloride is inadequate to achieve leution. Other chloride salts also fail to achieve elution. Calcium derivatized apatites are restored to their native forms by exposure to phosphate buffer, at which point they may be eluted by methods commonly applied for elution of native apatite supports. (Gagnon, US 2009/0186396). 

Ceramic hydroxyapatite (CHT or cHA): 

Ceramic hydroxyapatite refers to forms of the respective minerals in which nanocrysals are agglomerated into particles and fused at high termperature to create stable ceramic microspheres. New production procedures developed in the 1980s resulted in HPLC compatible HA beads. A major breakthrough in this context was the development of a sintering process for the production ofceramic HA variants. The ceramic material was mechanically stable and combined excellent flow properties with high particle porosity, while essentially maintaining the retention properties of the Tiselius HA. Sun (US 2005/0107594) teaches that ceramic hydroxyapatite was a known chromatography medium with the advantages of providing for durability and for a fast flow rate (¶6).

Ceramic hydroxyapatite is available in 2 types, Type I, with a medium porosity and relatively high binding capacity and Type II, with a larger porosity and a lower binding capacity. Either porosity can be used, and the optimal porosity for nay particular protein separation will vary with the proteins or the composition of the source mixture (Cummings, US 13/205354).

1. Type I (Ca5(PO4)3OH)2: Ceramic spherical HA particles have been commercialized since 1983 by Bio-Rad Laboratories Inc under the name CHT®(e.g., CHT Type I and CHT Type II). The binding mechanisms of resins such as type I is based on a mixed-mode interaction with Ca2+ as the positively charged functional group and PO43- as the negatively charged functional group. It has the separation properties of crystalline hydroxyapatite but can be used reproducibly for several hundred cycles at high flow rates and in large column. (US2009/0124790).

Type I has a high protein binding capacity and better capacity for acidic proteins.

2. Type II (Ca5(PO4)3OH)2: Type II has a lower protein binding capacity but better resolution of nucleic acids and certain proteins. The type II material also has a very low affinity for albumin and is especially suitable for the purification of many species and classes of immunoglobulins.

Other Modifications

Surface modification with Hexanoic and Decanoic Acids: 

Tanaka teaches calcium hydroxyapatite Ca10(PO4)6(OH)2 having the application as an adsorbent for protein (mixed mode chromatography matrix) (abstract; p. 31, line 4) modified with hexanoic acid CH3(CH2)4(COOH) (title). Tanaka further teaches that that hexanoic acid is hydrogen-bonded to the surface P-OH groups of the CaHAP (hexanoic acid is linked directly to a hydroxyl-functionalized solid support).

Fluoroapatite: 

Fluoroapatite refers to a mixed mode support comprising an insoluble fluoridated miniral of calcium phosphate with the structural formula Ca10(PO4)F2. Fluorapatite is prepared by fluoridating hydroxyapatite. Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity. Fluorapatite is commercially available in various forms, including ceramic and crystalline composite forms. Fluoroapatite is markedly more stable under acidic pH conditions than hydroxylapatite (US 7,939,643).

I. Ceramic fluorapatite (CFT): Since 2006 ceramic FA has also been commercially available as a chromatographic stationary phase from Bio-Rad under the name ofCFT®(e.g., CFT Type I and CFT Type II).

General Principles (Complex Interactions):

Hydroxyapatite (HA) chromatography of proteins involves the non-specific interaction of the charged amino or carboxylate group of a protein with oppositely charged groups on the hydroxyapatite, where the net charge of the hydroxyapatite and protein are controlled by the pH of the buffer. HA is used for purification of a wide variety of biomolecules, including proteins, phosphoroteins, carbohydrates, polynucleotides and viral particles. The column is usually equilibrated and the sample applied to a buffer that contains a low concentraiton of phosphate and eluted in an increasing gradient of phosphate salts (see operating conditions). In the alternative, some biomolecules may be eluted in an increasing gradient of chloride salts, but both elution formats impose disadvantages. The high phosphate concentration in which antibodies elute has strong buffer capacity that may interfer with subsequent purificaton steps. The high conductivity at which some biomolecules elute in chloride gradients may also interfer with downstream steps. Both situations require either that the eluted biomolecule be diluted or buffer exhanged which has a negative impact on process economics. As a result, HA steps are often placed at the end of a purification process. This tends to eliminate them from consideration as capture steps.  (Gagnon, US2009/0186396). 

Hydroxyapatite chromatography utilizes a calcium and phosphate based inorganic material with the structural formula of Ca10(PO4)6(OH)2, which forms both the matrix and ligand. Chemically reactive sites include pairs of positively charged calcium atoms and triplets of negatively charged phosphate groups. The interactions between hydroxyapatite and proteins are multi-modal, hence its classification as a mixed mode support. One mode of interaction involves metal affinity of protein carboxyl clusters for crystal calcium atoms. Another mode of interaction involves cation exchange of positively charged protein amino residues with negatively charged crystal phosphates. The individual contributions of the 2 mechanisms to the binding and elution of a particular protein can be controlled in part by the choice of salts used for elution. Due to its biosimilar composition, possible “leachables” present no problem. The material is also known to distinguish clearly between native and denatured variants of the same portein as well as between monomers, dimers and aggregates.

Studies have demonstrated that IgG is retained by HA by a combination of calcium metal affinity and phosphoryl cation exchange, regardless of subclass, light chain type or pI. (Gagnon, J Sep Sci, 2009, 32(22): 3857-3865).

Due to the dual functionality of calcium and phosphate groups comprising the matrix, the specific nature of protein interaction is complex. The amino groups on a protein are attracted to the phosphate sites, but are repelled by calcium sites. The situation is reversed for carboxylic groups as they are attracted to the calcium sites, but repelled by the phosphate sites (Mazzola, WO2009017491). Since proteins usually carry both positive and negative charges that vary in number as a function of the pH, the interaction of such molecules with hydroxyapatite becomes complex. On the other hand, this complexity can be exploited to design highly specific conditions for binding and elution in apatite chromatography. To date hydroxyapatite chromatography is often considered as a “last resort” to be used when everything else fails. This is partically due to the complexity of the interaction, which requires more sophisticated method devlopement than, e.g., Protein A affinity chromatography, but also to some of the physico-chemical properties of the hydroxyapatite itself  (Schubert, J. Chromatography A, 1142 (2007) 106-113). 

Although HA has been widely and successfully used, particularly for the separation of nucleic acids, the mechanism of their operation is little understood. The principles of ion-exchange chromatography cannot be applied to HA since the relation between protein affinity and their electrochemical behavior is not very prounouced in the case of HA. It is known that low MW substances such as amino acids, are poorly or not at all bound to HA. Synthetic polypeptides of various MW bind to HA, provided that they have a considerable total charge. A charge, however, is not a sufficient condition. For a given ionic strenght, the adsorption-desorption process is dependent on pH and for a given pH on ionice strenght (Gorbunoff, Analytical Biochemistry 136, 425-432 (1984). 

Unfortunately, the interaction of proteins with hydroxyapatite columns is not clearly understood. Tehrefore, determining conditions for the desired separation cannot be done theoretically. (Makino, J. Chromatographic Science, 27, 1989). 

Cleaning of HA surfaces

Cummings (US2012/0192901) teaches a method for cleaning an apatite solid surface by neutralizing the apatite solid surface with an alkaline hydroxide such as NaOHand then cleaning the apaptite solid surface such as with a phosphate solution. The invention is based on the discovery that hydrogen (or hydronim) ions can accumulate on an apatite surface following flow through purificaiton of a tareget molecule. If one performs a cleaning step without first neutralizing the column, degradation of the column can occur by displacement of calcium ions in the apatite support.

Cation Mixed Mode (Anion exchangers)/Cation with hydrophobic interaction:

Positively charged hydrophobic ligands belong to the group of anion exchanger mixed mode (for example Capto MMC) (for example Capto MMC) (Melinda, US 20170058019)

Ligands comprising at least one acidic moeity such as a carboxyl group and also comprising at least one hydrophobic moeity such as a phenyl ring or an aliphatic hydrocarbon chain can be used (Gagnon, US 8,188,242).

A method of synthesising a multi-model cation exchanger media is disclosed in WO 03/24588.

(Johansson, J. Chromatogr A, 1016(1), 2003, pp. 35-49) discloses various ligands used in the synthesis of different multi-modal cation-exchangers.

Operating Conditions:

Washing/elution:

—ethylene glycol + Inorganic salt: 

Falkenstein (US13/883243, published as US Paent No: 9422329; see also US Patent Application No: 15/216099, published as US Patent 10377794 and US Patent Application 16/778,128, published as US 20200165297) discloses washing an antibody from a multimodal weak CEX such as Capto MMC or Streamline CST using ethylene glycol and an inorganic salt such as sodium chloride, potassium chloride and ammonium chloride. In some embodiments, a binding buffer comprising an inorganic salt and denaturant such as gaunidinium hydrochloride or urea is applied to the multimodal weak CEX.

Specific Types of Cation-Mixed Modal Resins:

Weak-Type Cation Mixed Mode:

–Capto-MMC: A mixed-mode chromatography support which exploits a combination of cation exchange and hydrophilic interaction functionalities is Capto-MMC. Capto-MMC is a weak cation-exchanger with a phenyl group for hydrophobic interactions and an amide group for hydrogen bonding.

Capto MMC is a multimodal cation exchanger and has a multimodal ligand that may interact with target molecules in several different ways. It contains a carboxylic group and thus its feautures partily resemble those of a weak cation exchanger. However, in addition, several other types of interactions are involved, including hydrogen bonding and hydrophobic interaction (GE Healthcare Data File 11-0035-45AA).

Liao (US 13/654574, published as US 2013/0102761) discloses a mixed mode chromatography that combines cationic exchange and hydrophobic functionalities with a large pore support matrix. Specifically, the mixed mode chromatography medium comprising a ligand which includes a hydrophobic group (aromatic and substituted (alkyl groups such as hexyl) aromatic groups such as a phenyl and biphenyl groups) joined to either a carboxyl or sulfogroup (—SO3H) by a peptide containing chain. A ligand contaning the linkage -C(O)-NH-CH2- between a carboxylic functionality as the weak cation exchange group and a phenyl functionality as the hydrophobic group is benzoylamino acetic acid. In embodiments in which the ligand is a benzamidoacetic acid or a 2-benzamidoethanesulfonic acid, a particularly convenient linkage between the phenyl ring of the ligand and the matrix is one in which the amine group of the linkage is bonded to the phenyl ring at a para-position relative to the carbonylamino acetic acid group in the case of the benzamidoacetic acid or to the carbonylaminoethylsufonic acid group in the case of the 2-benzmidoethanesulfonic acid. An example of a compound that can form both the ligand and at least part of the linkage is 4-aminobenzamidoacetic acid (also known as para-aminohippuric acid) and also 2-(4-aminobenzaido)ethanesulfonic acid. To couple a pendant amine-contianing ligand to a matrix with exposed vicinal diols, the diols can be oxidized to aldehyde groups and the aldehyde groups can then be couple to amine groups to form secondary amino linkages.

Synthetic Resins:

–-Methacrylate: Melter (J of Chromatography A, 1200 (2008) 156-165) disclose retention behavior of C-terminal MAb vartiants on Fractogel EMD COO- (s) which is a methacrylate based plymeric resin with carboxyl functional groups.

Cation, anion, and hydrophobic interaction:

Examples include ABxTM which is a mixed-mode ion exchanger designed for the purification of antibodies (see BAKERBOND ABx). 

Cation exchange and hydrophilic interaction:

Examples include Capto-STM which is a strong cation exchange and is a trademark of GE Healthcare Bio-Sciences.

Cation exchange + hydrophobic interaction + hydrogen bonding and pi-pi bonding:

Examples include Capto-MMCTM which is a multimodal cation exchange.

Hydroxyapatite (HA) See “hydroxyapatite”

Hydrophobic charge induction chromatography (HCIC): See “affinity chromatography”. 

Anion exchange and hydrophilic interaction: (see Anion Mixed Mode)

Anion and hydrophobic: (see Anion Mixed Mode)

Cation with hydrophobic interaction: See Cation mixed mode   

Cation, anion, and hydrophobic interaction: See Cation mixed mode  

Commercial examples include ABx. Ma (US14208043)

Cation exchange and hydrophilic interaction: See Cation mixed mode  

Commercial examples include Capto-MMC. Ma (US14208043)

Cation exchange + hydrophobic interaction + hydrogen bonding and pi-pi bonding: See Cation mixed mode 

Commercial examples include Capto-MMC. Ma (US14208043)

Affinity Chromatography + Charged species (e.g., Anion or Cation)

Affinity + AEX Groups:

Bian (US2007/0207500) disclsoes a method of coupling an affinity ligandsuch as Protein A or protein G to a support with an associative group that interacts with the solid support. The associative group can be a positive or a negative charge. Bian exemplfies reacting Sepharose 4B agarose beads with positively charged assocaitive groups (quaternary amine ligands).

Engstrand (WO 2006/043895) discloses separaing antibodies using a multi-modal serpation matrix to adsorbe undersired compounds while the antibodies remain free in the liguid where the multi-modal matrix includes first groups such as N,N-dimethylbezylamine which are capable of itneracting with negatively charged sites on the target compounds and second groups such as Protein A (Sepharose 6 FF) which are capable of at least one interaction other than charge-charge interaction with the target compound. 

Affinity + CEX Groups:

Minakuchi (US 15/022890, published as US 2016/0237113; see aslo US 14/424,718, published as US 9,890,191) discloses a method for purifying a target substance such as an immunogloublin using one integrated column with two different carries, a first carreir having an affinity ligand such as Protein A which has affinity for the immunoglobulin and a second column having a cation exchange group where the column is an integrated column having a first column having the 1st carrier or a second column having the 2nd carrier or is a single column having a mixture of the first and second carrier, and passing an eluate through the column to elute the target substance/antibody. 

Nakamura (JP2010133734) discloses a carboxylation carrier for affinity chromatography having a carboxylic acid group containing a thiol group and a ligand containing a primary amino group fixed to the carboxylation carrier by an amide bond. The carboxylated carrier is produced by first introducing an eopoxy group into a carrier for chromatography such as by using epichlorohydrin, reacting the epoxy group with a compound having a thiol group and a carboxylic acid group. Any material capable of introducing an epoxy group can be used such as a natural polymer carrier, a synthetic polymer carrier or an inorganic carrier. A ligand such as Protein A containing a primary amino group is immobilized on the carobxylated carrier by an amide bond using a dehydrating condensation agent. 

See also Ion Exchange Chromatography and particularly types of resins

Mixed Mode Chromatography involves the use of solid phase chromatographic supports that employ multiple chemical mechanisms to absorb proteins or other solutes. Examples include chromatographic supports that exploit combinations of two or more of the following mechanisms: anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding and metal affinity. Mixed mode chromatography supports provide unique selectivities that cannot be reproduced by single mode chromatography methods such as ion exchange, but method development is complicated, unpredictable, and may require extensive resources, as exemplified by hydroxyapatite which is a crystalline miner of calcium phosphate (US7999085).

Multi-modal chromatography is a general term that encompasses all forms of separations in which multiple chromatographic mechanisms are deliverately used. This can be accomplished by attaching two or more chemically different ligands that interact with the sammple molecules in different ways. It can also be achieved by utilizing one type of ligand able to interact with the target molecule through different intermolecular forces. (Johansson, J. Chromatogr A, 1016(1), 2003, pp. 35-49).

Advantages and Disadvantages

Mixed mode chromatography provides unique selectivities that cannot be reproduced by single mode chromatography methods such as ion exchange. It provides potential cost savings, longer column lifetimes and operational flexibility compared to affinity based methods. However, the development of mixed mode chromatography protocols can place a heavy burden on process development since multi-parameter screening is required to achieve their full potential. Method development is complicated, unpredictable, and may require extensive resources to acheive adequate recovery due to the complexity of the chromatographic mechanism. (Nti-Gyabaah, US14/355014). 

unpredictability:

Mixed mode represent a broad and increasing diversity of ligands that exploit the combined functions of two or more chemical mechanisms. The influence of their primary mechanisms can be demonstrated fairly easily, for example, electrostatic and hydrophobic interactions, but the practical contributions and control of secondary functionalities are poorly understood, including metal coordination, pi-pi bonding, hydrogen bonding, and van der Waals forces. These introduce a strong element of unpredictability that is compounded by variations in ligand density and physical configuations among ligands of similar chemical character. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70)

Mixed-mode chromatography/ multi modal chromatography or in connection with a specific procedrue “hydrophobic charge induction chromatography” involves interaction of at least two principles; hydrophobic interaction and ion exchange or metal affintiy itneraction and ion exchange. Mixed-mode chromatography provies less predictable selectivities that cannot be reproduced by a single mode chromatogrpahy method such as ion exchange of hydrophobci interaction chromatography. Positively charge hydrophobi ligands below to the group of anion exchanger mixed-mode (for example CaptoAdhere) and the negatively charged ligands below to teh caiton exchanger mixed-mode (for example Capt MMC). Some mixed-mode media have zwitterionic character (for example Bakerbond ABx). Other mixed-mode media possess hydorphobic ligands which are ionisalbe and convert form uncharged to postivley charged by lowering the pH (for example MEP HyperCel). Finally, hydroxyapatite and fluroapatite media hav emroe complex mixed-mode fucntions by possessing positively charged calcium ions and negatively charged phosphate groups). (Zoltan, US 20220194981)

Types of Mixed Mode Chromatography Media/Supports See outline

Particular Types of Proteins Purified

Antibodies: See “antibody purification” and “mixed mode”

Prion Proteins:

Gilljam (WO2009/024620) discloses using a multimodal chromatographic material for the purification of a target protein by setting buffer conditions so that the protein is bound whereas prions do not bind followed by eltuion of the target protein suing an elution buffer with alcohols such as mono or dihyroxyalkanols (e.g., ower aliphatic alcohols such as methano, ethano, propanol) or by admending the pH or ionic strenght of the buffer. In one embodiment the elution buffer contains ethylene glycol (45-55%) and/or sodium chloride.

See also filtration membranes using graft chains with functional groups (under “filtration”)

The use of ligand “tentacles” or “extenders” to improve protein binding capacity and modify resin selectiveity involves placing a ligand on polymer chains coupled to a base matrix such as by grafting, and extend away from the base matrix surface. Ligand extenders typically create greater binding capacity because th eextnders increase ligand availability where target molecule binding exceeds taht of a monolayer adsorption on the surace. (Soice, WO 2012/015379).

Processes for Attachment of Graft Chains

Hermanson (“Immobilized Affinity Ligand Techniques” discloses chemically modifying a matrix so that the product of the process will react to form a coalent bond with a ligand of chocie. Numberous activation chemistries are described. 

Two standard methodologies for grafting polymer extenders have been developed for creating surface extenders on porous substrates such as those used in chromatogrpahy for protein separation and the like: 1) grafting of monomers form a support via a surface radical (“grafting monomers from”), and 2) grafting a preformed polymer to a support via an activating group (“grafting polymers to”). (Soice, WO 2012/015379)

Covalent ligand attachment to a carrier is typically achieved by the use of reactive functionalities on the solid support matrix such as hydroxyl, carboxyl, thiol, amino groups, and the like. In order to enhance the binding capacity of the matrix, a linking arm (linker) is ofter provided between the ligand and carrier. Such linkers will physically distance the ligand from the carrier, wehreby the target compound is allowed to interact with the ligand with minimal interference form the matrix. The use of linkers in the synthesis of chromatography matrices requires the use of a functional reagent having at least one functional group capable of reacting with a functional group on the surface of the matrix to form a covalent bond therewith; and at least one functional group capable of reacting with a functional group on the ligand to form a covalent bond therewith (Axen, US 2008/0237124 A1 and WO 2007/027139).

Shinohara 13/381,129 teaches a method for purifying an antibody monomer by passing the antibody solution containing antibody aggregates through a porous membrane having a hydrophobic porous substrate, a hydrophilic molecular chain which can have a backbone that is a polymer of at least one monomer slected from glycidyl methacrylate, glycidyl acrylate, glycidyl sorbate, glycidyl itacolate and glycidyl maeate, which is immobilized on the surface of pores of the porous substrate, and a side chain off the molecular chain. In one embodiment a functional group selected from a propylamino group, an isopropylamino group, a diethylamino group, a triethylamino group and a tripropylamino group is bonded to 60-97% of the side cahins held by the molecule chain and 3-40% of the side chains held by the molecular chain have a diol group. 

Cation Exchange Groups or Linkers with Cation Exchange Groups

Sulfphonate-functionaled groups: Axen (US 2008/0237124 and WO2007/027139) discloses a method for the manufacture of a sulphonate-functionalized carrier for cation exchange by providing a polysaccharide carrier having available hydroxyl groups (OH groups) and reacting these OH groups with vinyl sulfphonate (CH2=CH-SO3) to provide a sulfphonate-functionalized cation exchanger. In an advantegeous embodiment, the carrier comprises cross-linked polysaccharides which is done by substituting a part of the OH groups on the polysaccharide, gelling the polysaccharide solution to provide a carreir and then cross-linking the polyscharride gel by reacting OH groups of the polysaccharide. Improvided rigidity is acheived by adding a bifunctional cross-linking agent such as epicholorohydrin, allylbromide and allylglycidyl ether, having one active site such as halides, epoxides, methylol groups and one inactive site and allowing OH groups of the polysaccharide to react with the active site of the cross-linking agent, and the inactive side of the cross-linking agent is activated subsequent to the gelling which activated site is then reacted with the OH groups of the polysaccharide gel to cross-link the gel. In one embodiment, the method comprises a step of providing the polysaccharide gel with extenders such as hydrophilic polymers such as polysaccharides like dextran between carrier such as agarose or dextran and ligand.

Anion Exchange Groups or Linkers with Anion Exchange Groups 

Sulfonic acid and diethylamino groups: 

Kim (J. Membrane Science 117 (1996) 33-38) teaches grafting of an epoxy group containing polymer chain (epoxy group containing vinyl monomer (glycidyl methacrylate, GMA) onto a porous polyethylene membrane of hollow fiber form by applying radiation induced graft polymerization. Some of the epoxides of the poly-GMA graft chains can therafter be converted into a functional group  such a diol group (e.g., diethylamine (DEA)), ion-exchange group, and chelate forming group for specific adsorpition of a target protein.  

Kiyohara teaches grafting two different types of monomers, acrylic acid (AAc) and glycidyl methacrylate (GMA) onto a porous polyethylene hollow fibre by radiation indicued graft polymerization. The carboxyl group fo the AAc grafted hollow fibre was then reaction with N-hydroxysuccinimide to produce a succinimide group as an activated group. Phenylalamine (Phe) as a pseudobiospecific affinity ligand was reaction with the GMA fibre. 

Lee (WO 02/085519) discloses engrafting polymeric brushes such as glycidyl methacrylate to base materials and immobilzing functional groups to the brushes. Ogasawara (JP 012300A, published 2/19/99) also discusses an anion exchange porous holow fiber membrane for removing impurities from a mixture of a desired protein having a graft chang/branch polymer which is polymerized in the substrate.

 Koguma (Biotechnol. Prog. 2000, 16, 456-461, 2000) also teaches a porous hollow fiber membrane for protein recovery having graft chains extended form the pore surface and anion exchange groups introduced into the polymer chains.

Okamura (J. Chromatography A, 953 101-109 (20020 also teaches frafting of an epoxy-group containing polymer chain onto a hollow fiber form of a prorous polyethylene membrane by the immerision of the electron beam irradiated trunk/base polymer in glycidyl methacrylate. Subsequently, the epoxy groups produced were converted into sulfonic acid and diethylamino groups.  

Okamura (J Chromatogr A 2002 Apr 12, 953(1-2), 101-9) discloses grafting of an epoxy group containing polymer chain onto a hollow fiber form of a porous polyethylene membrane by the immersion of an electron beam-irradiated trunk polymer in glycidyl methacrylate diluted with methanol and 1-butanol. Subsequently the eopxy groups produced were converted into sulfonic acid (-SOI) and diethylamino groups (-N(C2H5).

Shirataki (US8,653,246 and US13/572983) also disclose a porous hollow fiber membrane such as polyethylene or polyvinylidene fluoride having a graft chain which are polyers of glycidyl methacrylate on a pore surace and an anion exchange group fixed to the graft chains. The graft chain has a graft rate of from 10-90% and the graft chain has 70% or more of epoxy groups replaced with the anion exchange group. Shirataki also teaches methods of purifying a protein by performing filtration using the membranes. 

See also Graft or Side Chains/extenders/linkers (Reactive Monomers) 

The carrier or substrate which is covered in another section (i.e., polysaccharide, polypeptide or silica) is modified by a synthetic polymer which is covalently bonded to the substrate through surface reacstive groups of the substrate, for example, surface hdyroxy groups of the polysaccharides, surface amino groups of the polypeptides and surface hydroxy or SiOH groups of the silica. The polymer which modifies the substrate is either a homopolymer or copolymer. An essential feature of the polymerizable compound is that it must contain a group capable of covalently bonding with the surface reactive group of the substrate (coupling group) and also contain either an ionizable chemical group or a group capable of transformation to an ionizable chemical group which provides the ionic exchange (ion exchange group)  (Hous, US 4,639,513). 

There are four main types of ion exchange resins differing in their functional groups: strongly acidic (typically, sulfonic acid groups, e.g., sodium polystyrene sulfonate or polyAMPS); strongly basis (quaternary amino groups, for example, trimethylammonium groups, elg. polyAPTAC); weakly acidic (mostly, caroxylic acid groups); weakly basis (primary, secondary, and/or ternary amino groups, e.g., polyethylene amine). There are also specialized types: chelating resins (iminodiacetic acid, thiourea and many others)/ Bill (US Patent Applicaiton No: 14/365,449, published as 10/364268).

Methods of Coupling the Polymer to the Carrier/Substrate

Different methods for immobilization of functional groups include physical adorption (non-covalent bridges such as ionic and hydrogen bonds, hydrophobic interactions and van der Walls forces), immobilization via reactive groups, aminopropyltriethoxysilan e bridges, glutaraldehyde, or bis(sulfosuccinimidyl) suberate activation, or via aldehyde groups, phosphoramidite groups, peptide groups, binding through biotin or avidin, protein A or G, attachment via metal-carrying media such as chelate-forming iminodiacetate groups, copper ions, nickel ions, ferric or ferrous ions, zinc ions, magnesium ions, coavlent attachment of oxidized groups as for example to oxidize the carbohydrate moieites in an antibody’s Fc region with periodate to form aldehyde groups, which are then chemically bound to hydrozide-activated solid supports such as agarose. (WO 02/085519).

The polymerizable compound may have a group capable of reacting with a hydroxy group of the polysaccharide with the formation of a covalent bound. Such polymerizable compounds are defined for example in US 4,070,348 to Kraemer. Hydroxy reactive groups are preferably activated carobxy groups known from peptide chemistry or O-alkylating agents such as alkyl halide or epoxide groups such as acrylic and methacrylic anydrides. The polymerizable compound may be one whcih does not react directly with hydroxy groups of the polysaccharide but rather are covalently coupled to the polysaccharide indirectly, via a bridge compound. This is the case when  a polysaccharide of the carrier is first chemically activated as by oxidation and reacted with a compound having as for example an epoxy group ro a vinyl groups.  When the substrate to be modified is a polypeptide then the comonomer conatins vinyl unsaturation to promote polymerization and or copolymerization and also contains a coupling group which is capable of covalently bonding to the polypeptide cahin through amino  gropus of the polypeptide chain. Typical groups capable of so reacting include glycidyl groups such as glydicyl acrylate and methacrylate and N-methylol groups such as N-acrylamide. When the substrate to be modified is silica or a siliceous material, then comonomer contains both vinyl unsaturation for polymerization purpsoes and a group capable of coupling to the hydroxy or SiOH surface groups. Typical monomers include the glycidyl group contianing monomers with glycidyl acrylate and methacrylate preferred.  The polymeriable comonomer will vary depending on the ultimate use of the carrier and may contain any of the well known ionizable chemical groups such as compounds containing a viny or vinylidine group and a carboxylic acid, a carboxylate salt, a carboxylate ester, a carboxylic acid amide, a secondary or tertiary amine, a quaternary ammonium, a sulfonic acid, a sulfonic acid ester, a sulfonamide, a phosphorice or phsophonic acid or a phosphoramide group. (Hou, US 4,639,513). 

Types of Functional Groups/Ligands

1. Anion-Exchange Groups:

Anionically dissociating groups which can be immobilized include quaternary ammonium salts and primary, secondary and tertiary amino or amido groups such as an amino group, a methylamino group a  diethylamino group.

2. Cation-Exchange Groups:

Cationically dissociating groups which can be immobilized include carboxyl group, a sulfone group, a phosphate group, a sulfoethyl group, a phosphomethyl group and a carbomethyl group (WO 02/085519).

Cation exchange groups are roughly divided into a weak cation exchange group such as a carboxyl group and a strong cation exchange group such as a sulfonic acid group. An adsorbent haivng a weak cation exchange group has a drawback in that the surface charge of the adsorbent changes as the pH of the mobil phase changes, with the result that a binidng capacity to a protein such as an antibody changes. Accordingly, if an adsorbent having a weak cation exchange group is used for separation of an antibody, the reproducibility of the separation becomes poor and the recovery rate of the antibody may decrease. (Koguma, US9643173)

Sulfone/sulphonate ligands (S groups): Sulphonate ligands, known as S groups, are commonly used strong cation-exchange groups. In some cases, cush exchangers are named by the group formed by the functional group and its linker to the carrier; for example SP cation exchangers are S groups linked by propyl to the carrier (WO2007027139).

S groups can for example be introduced into at least a part of reactive functional groups (e.g., hydroxyl groups) possessed by a porous cellulose gel.  Matsumoto (US 13/201,647) describes such a procedure. First, a sulfonation agent such as sodium 3-chloro-2-hydroxypropanesulfonate and sulfonic acid having epoxide such as 1,2-epoxyethanesulfonic acidis put into a reaction container. Next, the dried porous cellulose gel is added to the sulfonation agent to cause a reaction.  

 Axen (WO2007/027139) also discloses manufacturing a chromatography materix by providing a polysacharide carrier having OH group and reacting the hydroxyl groups with vinyl sulphonate to provide a sulphonate-functionalized cation exchanger.  In one embodiment, a bifunctional cross-linking agent having one active site and one inactive site to the polysacharide carrier having an OH group is added such that the hydroxyl groups of the polysaccharide reactive with the active site of the cross-linking agent. After the polysaccharide solution is geled by cooling, the inactive site of the cross-linking agent is activated such that it can react with hydroxyal groups of the polysaccharide gel to cross linke the gel. Then the remaning hydroxyl groups are reacted with vinyl sulphonate to provide the sulfphonated-functionalized (S-functionalized) cation exchanger. In another embodiment, extenders may be provided between the carrier and ligand (see ligand extenders).

Koguma (US 9643173) discloses a temperature responsive absorbent prepared by immobilizing a copyolymer containing at least N-isoproylacrylamide to a base material surface. N-isopropylacrylamide (Poly(N-isopropylacrlamide) is known to have a lower limit critical temperature of 32C. The carrier introduced into the surface of the polymer greatly changes surface physical properties such as hydrophilicity/hydrophobicity at a critical termpature. Therefore, if this is grafted or applied as a coating to the surface of a packinga getn for chromatography, sample retenetivity can be obtianed depndent upon the temperature. As a result, retention behavior can be controlled by temperature whihout chaning the compoisiton of an elute. The copolymer has at least a strong cation exchange group in an amount of 0.01 to 5 mol% relative to N-isopropylacrylamide. The absorbent is produce by a surface graft polymerization method using a reaction solution containing a monomer having a strong cation group such as sulfonic acid group or a precursor of a strong cation exchange group in a ratio of 001 to 5 mol % relative to N-isopropylacrylamidel. 

Ishihara (US 14/435456) discloses a cation media for purifying an antibody which has a strong cation monomer unit (examples include for Formula I:  2-acrylamide-2-methylpropanesulfonic acid, 2-acrylamideethane sulfonic acid, 2-methacrylamide-2-methylpropanesulfonic acid and 2-methacrylamideethane sulfonic acid; examples for a Formula II include 3-sulfopropyl methacrylate and 2-sulfoethyl methacrylate. ) and a neutral monomer unit or weak cation monomer unit (examples of a neutral monomer include N,N-dimethylacrylamide, N,N-diethalacrylamide, N-tert-butylacrylamide, N-iso-propylacrylamide, acrylamide, N-(methoxymethl)methacrylamide and N-(iso-butoxymethl)methacrylamide) (example of a weak cation monomer unit include acrylic acid, methacrylic acid, sodium acrylate, potassium acrylate, sodium methacrylate and potassium methacrylate).  Examples the comibnation include 2-acrylamide-2-methylpropanesulfonic acid and N,N-dimethylacrylamide OR  2-acrylamide-2-methylpropanesulfonic acid and acrylic acid. Specific examples of crosslinked cellulose partciles that can be used  include a porous cellulose gel. 

Controlled pore glass (CPG) is a media used as a backbone matrix for chromatographic resins. CPG is generally produced form a borosilicate base material that is heated to separate the borates adn the silicates; the borates are leached out from the material, leaving the silica glass with uniform, controlled pores. Thus, CPG is very similar to silica with respect to its surface chemistry. (Ghose, “preparative Protein purification on underivatized silica” PurificationProcess Devleopment, Amgen Inc, 2003).

CPG has anionic silanol groups and CPG acts like a cation exchanger. Proteins have been shown to be well absorbed on CPG at pH 6.0.   (Mizutani, J. Chromatography, 165 (19779) 143-150). CPG is described in US 3,549,524 and 3,758,284.

CPG is widely used in methods for the isolation or purificaiton of nucleic acids and proteins (Wong, US 6,261,497).  CPG has been used for the separation of a number of biologically interesting compounds. In most of these applications, the separation is based mainly on differences in molecular size.  

Controlled pore glass (CPG) is a silicate containing support material chemically cimilar to silica for use in liquid chromatography. It is commercially available from Pierce Chemical Co., Rockford, Il, with average particle diameter of 37-177 microns and average pore size of 40-1000 Angstroms. Crane (US 4,606,825) discloses purifying IgG using CPG bearing non-corss linked covalently bound polyethylenimine functions.

Millipore sells controlled pored glass for chromatography. It is advertised as incompressible and very durable, does not shrink or swell in different solutions and has a narrow pore size distribution coupled with a large internal surface area. It is avalaible from Milipore in a broad range of pore diameters.

Chromatography on controlled pore glass beads (CPG) is attractive because glass beads have the advantages of incompressibility even at high flow rates and chemical resistance to a broad range of eluting agents. (Mecs et al. Arichives of Virology 81, 303-311 (1984) at p. 304 1st ¶).

Modes and Operating Conditions Used

Bind and elute mode:

Bock (Science, 191:380-383 (1976) also discloses the use of absorption chromatography on CPG in combination with chaotropic buffers. The high adsorption affinity of CPG allows rapid concentration and buffer replacement for proteins from large volumes of salts, sugars, or culture media. Selectivity in the elution of proteins is achieved by the use of different chaotropic eluting agents by varying the pH and by changing the ionic strenght. Builder US 5,451,660 discloses a process for selectively separating a polypeptide of interest from components of differing hydrophobicity by passing the mxiture through underivatized silica particles such that the polypeptide adheres to the silica particles, washing to remove impurities and eluting the polypeptide with a buffer comprising an alcoholic or polar aprotic solvent and an alkaline earth, an alkali metal or an inorganic ammonium salt. 

Builder (US5,451,660) discloses separating a polypeptide of interest crom components of differing hydrophobicity by passing the mixtures through underivatized silicon particles such that the p9olypeptide of interest adheres to the silica particiles, washing the particles and eluting the polypeptide of interest with a buffer comprising an alcoholic or polar aprotic solvent and an alkaline earh, an alkali metal or an inorganic ammonium salt.

Rubin (US4,870,163) discloses applying a mixture containing TNF to controlled pore glass 350, washing with several buffers in sequence which removed protein having no TNF activity, then washing with 0.1 M tris-HCL, pH 9.4, containing 0.1 M arginine and eluting with this same buffer.

Silberstein (US 5,322,838) discloses incubation at pH 7.2 with controlled pore glass beads.  Yoshimoto (US 4,789,658) discloses eluting Il-2 fractions (pH 7.6) and then lyophilized to  dryness and chromatographed on controlled-pore-glass beads column. Braude EP 0291728 discloses adsorption onto controlled pore glass beads in presence of neutral pH buffer. 

Flow through mode:  

Hepbildikler (13/651188, published as US 9657054 and US 15/487,248 published as US 2017/0305964) discloses using underivatized controlled pore glass (uCPG) surfaces to selectively bind dimeric and aggregated IgG in a solution at a pH of 5-7.5. The surfaces can be in the form of beads of a solid surface. The uCPG selectively binds dimeric and oligomeric immunoglobins and HMWCs present in the solution. The monomeric immunoglobulin can be recovered from the flow through of a chromatography containing uCPG or from the supernatant of an inucbation of a solution with uCPG.