Classifications

• • 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
  • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
  • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
  • B01D15/203—Equilibration or regeneration
• • 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
  • B01J20/34—Regenerating or reactivating
  • B01J20/345—Regenerating or reactivating using a particular desorbing compound or mixture
  • B01J20/3475—Regenerating or reactivating using a particular desorbing compound or mixture in the liquid phase
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B08—CLEANING
  • B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
  • B08B3/00—Cleaning by methods involving the use or presence of liquid or steam
  • B08B3/04—Cleaning involving contact with liquid
  • B08B3/08—Cleaning involving contact with liquid the liquid having chemical or dissolving effect

Definitions

• Hydroxyapatite and fluorapatite are used for purification of a wide variety of biomolecules, including proteins, carbohydrates, polynucleotides, and viral particles.
• the present invention provides for a method for cleaning an apatite solid surface following target molecule purification by a non-adsorbing flow through process.
• the method comprises,
• the alkaline hydroxide is selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide.
• the concentration of the alkaline hydroxide is between 0.1 and 1 M. In some embodiments, the concentration of the alkaline hydroxide is between 0.3 and 0.7 M.
• the cleaning comprises contacting the solid surface with a phosphate solution.
• the phosphate solution has a pH at or between 6.5 and 10.0.
• the phosphate concentration of the phosphate solution is at or between 0.1 and 1.0.
• the apatite is selected from the group consisting of hydroxyapatite and fluorapatite.
• the apatite is ceramic hydroxyapatite or ceramic fluorapatite.
• the apatite is a non-ceramic apatite.
• the target molecule is a protein.
• the protein is an antibody.
• the contacting comprises contacting the solid surface with a solution at a pH of between 5.0 and 7.5.
• the apatite solid support is in the form of a column.
• Neutralizing the solid apatite surface refers to treating the surface of the apatite surface such that the solid surface does not contain sufficient hydronium ions to significantly affect (i.e., cause a greater than 0.2 acidic pH shift of) the pH of a subsequent cleaning buffer.
• Antibody refers to an immunoglobulin, composite, or fragmentary form thereof.
• the term may include but is not limited to polyclonal or monoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.
• Antibody may also include composite forms including but not limited to fusion proteins containing an immunoglobulin moiety. “Antibody” may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc and other compositions, whether or not they retain antigen-binding function.
• An "apatite solid surface” refers to fused nanocrystals (ceramic apatite), microcrystals , or compounded microcrystals.
• Ceramic apatites include, but not limited to, ceramic hydroxyapatite (e.g., CHTTM) or ceramic fluorapatite.
• Ceramic apatites are a form of apatite minerals in which nanocrystals are agglomerated into particles and fused at high temperature to create stable ceramic microspheres suitable for chromatography applications.
• Compounded microcrystals include but are not limited to HA Ultragel® (Pall Corp.).
• Microcrystals include but are not limited to Bio-Gel HTP, Bio-Gel® HT, DNA-Grade HT (Bio-Rad) and Hypatite C (Clarkson Chromatography).
• Hypatite refers to a mixed mode solid support comprising an insoluble hydroxylated mineral of calcium phosphate with the structural formula Caio(P0 4 )6(OH) 2 . Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity.
• Hydroxapatite is commercially available in various forms, including but not limited to ceramic, crystalline and composite forms. Composite forms contain hydroxyapatite microcrystals entrapped within the pores of agarose or other beads.
• Fluorapatite refers to a mixed mode support comprising an insoluble fluoridated mineral of calcium phosphate with the structural formula Caio(P0 4 )eF 2 . Its dominant modes of interaction are phosphoryl cation exchange and calcium metal affinity. Fluorapatite is commercially available in various forms, including but not limited to ceramic and crystalline composite forms.
• sample refers to any composition having a target molecule or particle of interest.
• a sample can be unpurified or partially purified.
• Samples can include samples of biological origin, including but not limited to blood, or blood parts (including but not limited to serum), urine, saliva, feces, as well as tissues.
• alkaline hydroxide refers to a metal alkali hydroxide comprising any cation elements in Group I of the periodic table, including, e.g., lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr).
• exemplary alkaline hydroxides include, for example, NaOH, LiOH, and KOH.
• Flow-through mode refers to an operational approach to chromatography in which the chromatography conditions are established so that a target molecule (from a sample) to be purified flows past the chromatography support upon application, while at least some other components of the sample are selectively retained, thus achieving removal of at least some non-target components of the sample.
• the present invention is based, in part, on the surprising discovery that an alkaline hydroxide solution (e.g., NaOH) is useful to neutralize the surface of an apatite solid support following a flow through purification of a target molecule and before a cleaning step.
• an alkaline hydroxide solution e.g., NaOH
• Hydrogen (or hydronium) ions can accumulate on an apatite solid surface following flow through purification of a target molecule.
• a subsequent cleaning step e.g., with a 0.1-1.0 M phosphate solution
• degradation of the column can occur by displacement of calcium ions in the apatite support.
• neutralizing the solid support with an alkaline hydroxide solution prior to the cleaning step one can avoid significant degradation that can otherwise occur to the apatite solid surface.
• the sample containing the target molecule is contacted to the apatite surface in flow-through mode as is known in the chromatography arts.
• the flow through comprises a solution of pH at or between 5.0 and 7.5.
• Exemplary buffers include, e.g., phosphate buffers, optionally also containing sodium (e.g., NaCl). Flow through may be conducted at fast linear flow rates such as 300-600 cm/hr. However slower linear flow rates such as 50-200 cm/hr are also applicable.
• Flow-through mode refers to an operational approach to chromatography in which the buffer conditions are established so that intact non- aggregated target to be purified flows through the chromatography support, while other molecules (e.g., in some embodiments aggregates and other large molecules (including viruses) are selectively retained, thus achieving their removal.
• Flow-through mode conditions can be developed depending on the specific target desired.
• An exemplary flow- through condition is, for example: Condition the column for flow though by sanitizing with 0.5-1.0 N NaOH, wash with 0.2 M sodium phosphate at pH 6.5-7.5, equilibrate the column with flow through buffer (5-20 mM sodium phosphate, pH 5.0-7.5), apply the sample and collect the flow though containing the target molecule, wash the column with 0.3 - 1 column volume of 1 M NaOH, and clean the column with 0.1 - 1M phosphate.
• any flow- through conditions are contemplated for the invention.
• Neutralization occurs after the target has flowed through and optionally been collected.
• the neutralization comprises contacting the apatite surface with a sufficient amount of a solution comprising a sufficient concentration of an alkaline hydroxide.
• Exemplary alkaline hydroxides include, for example, NaOH, LiOH, and KOH, though other alkaline hydroxides can also be used as desired.
• the alkaline hydroxide that neutralizes the apatite solid surface is between, e.g. , 1 -100 mM, l-20mM, 10- 2000 mM, 10-lOOOmM, 10-500mM, 10-200mM, or 10-lOOmM, etc.
• amino functional bases and alkaline carbonates can be used to neutralize the apatite surface as described herein for alkaline hydroxides.
• neutralization can be achieved with any amino functional base (triethyamine, Tris, ammonia, etc.).
• the amino compound may form gas with the cleaning buffer and cause an ammonia-like odor.
• alkaline carbonates e.g., lithium, sodium or potassium carbonate
• C0 2 gas could form in the column causing back pressure.
• a neutral apatite surface will result in a pH change of no more than 0.1 or 0.2 between the input and effluent following neutralization.
• the pH of the cleaning buffer is input at 7.0
• the effluent would not drop to less than 6.8 during cleaning if the surface were neutralized.
• calcium ions in the effluent to determine whether the surface is neutralized. In the presence of released free hydronium ion, apatite releases calcium. Thus, the presence of more calcium in the effluent than was in the input buffer indicates that the surface has not been neutralized.
• An exemplary cleaning solution is a phosphate buffer of about 0.1 -1.0 M and having a pH of about 6.5-10.0.
• the buffer may optionally also include other salts (e.g., KC1, NaCl), though salts are not generally necessary once the surface has been neutralized.
• Ceramic hydroxyapatite examples include, but are not limited to CHT Type I and CHT Type II.
• Ceramic fluorapatite examples include, but are not limited to CFTTM Type I and CFT Type II.
• ceramic hydroxyapatite and ceramic fluorapatite refer to roughly spherical porous particles of any average diameter, including but not limited to about 10, 20, 40, and 80 microns. The choice of hydroxyapatite or fluorapatite, the type, and average particle diameter can be determined by the skilled artisan.
• non-ceramic types of apatite solid surfaces can also be used according ot the invention.
• non-ceramic solid apatites include but are not limited to compounded microcrystals (e.g., HA Ultragel® (Pall Corp.)) and microcrystals (e.g., Bio-Gel HTP, Bio-Gel® HT, DNA-Grade HT (Bio-Rad) and Hypatite C (Clarkson Chromatography)).
• the chemical environment inside the column is typically equilibrated. This can be accomplished, for example, by flowing an equilibration buffer through the column to establish the appropriate pH; conductivity; identity, molecular weight, and other pertinent variables.
• the sample preparation is also equilibrated to conditions compatible with the column equilibration buffer. In some embodiments, this involves adjusting the pH of the sample preparation prior to loading.
• the sample preparation is contacted with the column.
• the sample preparation can be applied at a linear flow velocity in the range of, for example, about 50-600 cm/hr. Appropriate flow velocity can be determined by the skilled artisan.
• the invention is practiced in a packed bed column, a fluidized/expanded bed column and/or a batch operation where the support is mixed with the sample preparation for a certain time.
• an apatite support is packed in a column.
• the apatite support is packed in a column of at least 5 mm internal diameter and a height of at least 25 mm.
• Another embodiment employs the apatite support, packed in a column of any dimension to support preparative applications.
• Column diameter may range from less than 1 cm to more than 1 meter, and column height may range from less than 1 cm to more than 30 cm depending on the requirements of a particular application. Appropriate column dimensions can be determined by the skilled artisan.
• the mixed mode column can optionally be cleaned, sanitized, and stored in an appropriate agent, and optionally, re-used.
• one benefit of the neutralization solution of the present invention is that degradation of an apatite column can be avoided or delayed.
• the methods of the invention can be used to purify essentially any target molecule in a complex sample.
• the target molecule to be purified is a component of a biological sample. Examples of such components include but are not limited to proteins, lipids, sugars, carbohydrates, viral particles, amino acids, nucleic acids, and can include combinations thereof, e.g., a lipidated or glycosylated protein, or mixtures thereof.
• samples to which the method is applied include unpurified or partially purified biomolecules from natural, synthetic, or recombinant sources.
• Unpurified samples can be derived from, e.g., plasma, serum, ascites fluid, milk, plant extracts, bacterial lysates, yeast lysates, or conditioned cell culture media.
• partially purified samples come from unpurified preparations that have been processed by at least one chromatography, ultrafiltration, precipitation, other fractionation step, or any combination thereof.
• An exemplary target molecule is an antibody (including but not limited to a monoclonal antibody and/or antibody fragments) or other peptide or polypeptide.
• the chromatography step or steps can employ any method, including but not limited to size exclusion, affinity, anion exchange, cation exchange, protein A affinity, hydrophobic interaction, immobilized metal affinity chromatography, or mixed-mode chromatography.
• the precipitation step or steps can include, for example, salt or PEG precipitation, or precipitation with organic acids, organic bases, or other agents.
• Other fractionation steps can include but are not limited to crystallization, liquid:liquid partitioning, or membrane filtration.
• Ultrafiltration can include direct concentration of the sample and/or diafiltration.
• a chromatography column of apatite is equilibrated with 5 mM sodium phosphate buffer, pH 6.5.
• a sample solution of equilibration buffer or sample buffer containing a target molecule is applied to the apatite column and purification of the target molecule achieved by flowing it through the apatite.
• Adsorbed hydrogen ion is neutralized by eluting the column with sufficient amount of a strong base such as sodium, potassium or lithium hydroxide.
• the apatite is cleaned with a phosphate buffer of sufficient concentration to elute adsorbed biological compounds such as DNA, basic proteins and endotoxin, etc.

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• Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Analytical Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Solid-Sorbent Or Filter-Aiding Compositions (AREA)
• Treatment Of Liquids With Adsorbents In General (AREA)
• Peptides Or Proteins (AREA)

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