EP0701566A1 - Tampon de fixation a haute teneur en sel pour la purification d'immunoglobulines avec un ligand d'affinite synthetique - Google Patents

Tampon de fixation a haute teneur en sel pour la purification d'immunoglobulines avec un ligand d'affinite synthetique

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Publication number
EP0701566A1
EP0701566A1 EP94921883A EP94921883A EP0701566A1 EP 0701566 A1 EP0701566 A1 EP 0701566A1 EP 94921883 A EP94921883 A EP 94921883A EP 94921883 A EP94921883 A EP 94921883A EP 0701566 A1 EP0701566 A1 EP 0701566A1
Authority
EP
European Patent Office
Prior art keywords
gel
igg
avid
pbs
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94921883A
Other languages
German (de)
English (en)
Inventor
Randal A. Goffe
Yuan Shi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UniSyn Technologies Inc
Original Assignee
UniSyn Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by UniSyn Technologies Inc filed Critical UniSyn Technologies Inc
Publication of EP0701566A1 publication Critical patent/EP0701566A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation

Definitions

  • This invention relates to the affinity chromatographic purification of immunoglobulins. More particularly, the invention relates to a high salt buffer which increases the binding capacity of synthetic affinity chromatographic gels of the kind described in United States patent 4,582,875. BACKGROUND OF THE INVENTION
  • affinity gels such as protein A, protein G and AVID-AI®, a synthetic gel available from UniSyn Technologies, 14272 Franklin Avenue, Tustin, CA.
  • Such gels are used as a chromatographic matrix, as a particulate slurry and in other ways known to or within the skill of the art.
  • AVID-AL has been found to bind antibodies whose immunoglobins do not bind with high affinity to protein A or protein G. See,e.g., Ngo (1990); Narinesingh (1991) and Khatter (1991) . Moreover, AVID-AL has a lower mass than protein A or protein G with consequent greater resistance to acid, base, organic solvent, proteolytic enzyme and autoclaving treatment.
  • Hydrophobic interaction impacts inter se repulsion which adversely impacts in two ways the affinity gel absorption of IgG molecules dissolved in an aqueous medium. First, aggregation of unbound molecules is constrained or inhibited. Second, bound molecules constrain or inhibit attraction by an affinity gel of as yet unbound molecules.
  • the efficiency of affinity gel immunoglobin purification procedures is mediated by the establishment within an aqueous medium of a dissolved salt concentration defined by two parameters.
  • a first parameter defines a lower limit of said concentration effective to modify the hydrophobic interaction of immunoglobin molecules and thus reduce inter se repulsion.
  • any concentration of dissolved salt higher than that of conventional PBS is useful in the invention.
  • a second parameter defines the upper limit of salt concentration which must be lower than that which would result in indiscriminate precipitation of the desired immunoglobin and other proteins as a consequence of the known "salting out" effect.
  • affinity gel purification yields high purity immunoglobins in good yield from contaminated aqueous media such as cell culture supernatants.
  • the salt concentration of such media is adjusted to a value in excess of that conventional with PBS and below that which would result in the precipitation or salting out of the desired IgG molecules with consequent mediation of the hydrophobic interaction between the IgG molecules and reduction of inter se repulsion.
  • PBS PHYSIOLOGICAL SALINE SOLUTION
  • PBS means an aqueous solution 0.01 molar in KH2P04; 0.01 molar in Na2HP04 and 0.14 molar in NaCl having an ionic strength of 0.26.
  • Figure 1 depicts generalized chemistry for the production of affinity gels more fully described in U.S. patent 4,582,875.
  • Figure 2 depicts the effect of various salts on the binding of purified human IgG to AVID-AL and rProtein A gels.
  • the binding capacity is shown as a percentage of the binding capacity obtained with KCl and assayed at a total buffer ionic strength of 1.28 M at pH 7.0.
  • Figure 3 depicts the relationship between the amount of IgG bound on AVID-AL gel and the concentration of purified human IgG in physiological saline solution (PBS) containing different concentrations of Na2S ⁇ 4 (the saturation curves) .
  • Figure 4(a) depicts the isolation of IgG from human serum by AVID-AL gel. PBS loading and washing conditions are indicated.
  • Figure 4(b) depicts the isolation of IgG from human serum by AVID-AL gel with PBS 0.75 molar of sodium sulfate. Loading and washing conditions are indicated.
  • gel volume was 0.5 mL.
  • Human serum was diluted 8.9-fold with PBS binding buffer per se or 0.75 M in Na2S ⁇ 4 to provide an IgG concentration of about 2 mg/mL in solution and applied at the flow rate of 0.25 mL/min at room temperature. Fractions of 1.0 mL were collected. Elution was carried out with neutral elution buffer, pH 7.4.
  • Figure 5 depicts the polyacrylamide gel electrophoresis (PAGE) sodium dodecyl sulfate (SDS) gradient (8-25%) in non-reducing conditions of fractions obtained from human serum in Figures 4(a) and 4(b).
  • Lanes 3-5 correspond to Figure 4(a) , lanes 6-8 to Figure 4(b) .
  • Figure 6 depicts the SDS-PAGE electrophoretic analyses for estimating the purity of IgG from human serum as a function of different sodium sulfate concentration.
  • FIG. 1 schematically illustrates some of the chemistry relevant to the synthesis of these gels.
  • X ⁇ is a nucleophile
  • PFP is pentafluoropyridine
  • DMAP is a nucleophile
  • AVID-AL (UniSyn) binds a wide range of immunoglobins under PBS conditions.
  • the bound IgG can be eluted by a neutral elution buffer at physiological pH (pH 7.4).
  • the AVID-AL R (UniSyn) used in the following examples was a commercial product from manufacturing Lot #3-4-1.
  • a commercial rProtein A gel was provided by RepliGen (Cambridge, MA) .
  • Solutions of purified human IgG containing a concentration of 12.5 mg/mL were purchased from The Binding Site (San Diego, CA) .
  • Human serum was obtained from Sigma (St. Louis, MO) .
  • Cell culture media (RPMI-1640) and fetal bovine serum (FBS) were obtained from Mediatech (Herndon, VA) and Summit Biotechnology (Greeley, CO) , respectively. All other chemicals were of analytical grade.
  • Human IgG was bound to AVID-AL gel by placing 18.0 ⁇ L of purified human IgG in 972.0 ⁇ L PBS (0.02 M phosphate, 0.14 M sodium chloride (pH 7.0) with 10.0 ⁇ L of AVID-AL gel taken from a bulk of 20 mL which was washed first with regeneration buffer (20% methanol, 1% acetic acid, pH 3.4) and then with PBS for equilibration of the gel before using to bind IgG. The binding adsorption was run in a test tube overnight at 4°C with rotating on a Fisher hematology/chemistry mixer (Fisher Scientific, Pittsburgh, PA) .
  • the binding capacity of AVID-AL gel was determined by calculation of the corresponding amount of IgG for mass balance. The same was done with a rProtein A gel as a reference under the same conditions as used to determine the binding capacity of AVID-AL gel.
  • AVID-AL gel Approximately 0.5 mL of AVID-AL gel was packed into a 1.0 mL column.
  • a Rainin peristaltic pump with an eight-channel head (Rainin, Woburn, MA) was used for buffer delivery.
  • the column was washed with regeneration buffer until a baseline was obtained according to the absorbance, A 280 nm given by LKB UV detector.
  • the column was then pre-equilibrated with PBS.
  • a sample of 1.35 mL of prefiltered human serum dissolved in 10.35 mL of binding buffer was pumped through the column with a flow rate of 0.25 mL/min, and 1.0 mL fractions were collected from the start of the loading step.
  • the amount of fed human IgG was about 22 mg.
  • Bound IgG was eluated using neutral elution buffer (composition 20% glycerol and 1.0 M Tris.HCl in DI water, pH adjusted to 7.4 ⁇ 0.2 by 6N NaOH) provided by UniSyn Technologies (Tustin, CA) with a flow rate of 1.0 mL/min. Protein output was measured and the absorbance of each fraction at 280 ran was used to determine its IgG concentration. Between runs the column was washed with regeneration buffer and PBS with minimal to no protein elution seen. Electrophoretic analyses were performed on 8-25% polyacrylamide gradient gels by using the Phast system from Pharmacia. The amount of purified IgG in fractions was confirmed using radial immunodiffusion.
  • Table 1 summarizes the effect of salt type and concentration on the partition coefficient, a, which is defined as the fraction of the total solute (purified human IgG) adsorbed at any instant because the affinity adsorption of purified human IgG to AVID-AL gel represents a partitioning of solute between two phases, liquid and solid.
  • Table 1 shows that the binding capacity of AVID-AL gel improves at higher ionic strengths of Na2S0 4 and (NH4)2S04 with increasing ⁇ .
  • 1/P Ct/(K a P b ) - 1/K a
  • P is the IgG concentration in free solution at equilibrium with AVID-AL gel
  • Pfc is the IgG bound onto the AVID-AL gel (mg/ml gel)
  • K a and C ⁇ can be estimated from double reciprocal plots of the respective binding saturation curves and the values are summarized in Table 2.
  • the dissociation constants for purified human IgG in PBS obtained under equilibrium conditions were 0.048, 0.022, 0.0058, 0.0052, and 0.0046 mg/mL, for Na2 ⁇ 4 concentrations of 0, 0.25, 0.50, 0.75, and 1.00 M, respectively.
  • Example I The results from Example I were confirmed by performing experiments in which 0.5 L of AVID-AL gel was packed in a 1.0 L chromatographic column.
  • the chromatograms shown in Figures 4(a) and 4(b) are for such columns, wherein human serum was diluted by either PBS (regular binding buffer) or PBS containing 0.75 M of Na2S ⁇ 4 as an improved binding buffer.
  • PBS regular binding buffer
  • PBS containing 0.75 M of Na2S ⁇ 4 as an improved binding buffer.
  • This operation yielded 8.0 mg and 14.6 mg bound IgG on the column, respectively.
  • the bound IgG can be eluted using neutral elution buffer with pH of 7.4.
  • the binding capacity for AVID-AL in the high salt buffer was 26.2 mg IgG/mL gel, which was 2.1-fold higher than those of the run using PBS as a binding buffer.
  • Binding Capacity (mg/mL gel) 12 . 4 26 . 2 Table 4 is a comparison of the binding capacity for IgG between AVID-AL gel when used with PBS buffer and a high salt binding buffer of the invention. A comparison with Protein A gel is also set forth.
  • the composition of the high salt binding buffer (HSBB) was KH 2 P0 4 0.01 M; Na 2 HP04 0.01 M, NaCl 0.14 M and Na2 ⁇ 4 0.5 M. The buffer is adjusted to physiological pH 7.4 with IN NaOH as necessary.
  • the binding capacity and the recovery for IgG are the function of either IgG concentration in species or loading speed of sample passed through the gel column.
  • Table 4 unambiguously demonstrates that the immunoglobin hydrophobicity modifying effect of the buffer compositions of this invention combines with the specificity of the AVID-AL affinity ligand in a very unique way. Specifically, both high yield and high purity product is obtained. This result contrasts sharply with the indiscriminate salting out of all proteins present in a cell culture supernatant or the like.
  • This example shows the effect of Na2S ⁇ 4 concentration in high salt binding buffer on binding capacity of human IgG and the purity of IgG eluted from 0.5 mL Avid AL gel.
  • FIG. 6 depicts the SDS-polyacrylamide gradient gels on 8-25% in the Phast Gel System were shown below:
  • Lane 1 Lane 1 and 9) Molecular weight marker reference proteins Lane 2) Unbound protein after human serum binding by using PBS Lane 3) Unadsorbed protein after column wash using PBS Lane 4) Eluted human IgG by using Neutral
  • Binding Capacity 2.8 mg/mL 8.3 mg/mL This invention provides a novel and significant improvement in techniques for the purification of immunoglobin solutions, particularly cell culture supernatants by affinity chromatography in which a synthetic ligand or gel is utilized.
  • aqueous buffer solutions containing sulfate, acetate, chloride potassium or sodium ions in a concentration sufficient to enhance the binding of a synthetic affinity gel for an immunoglobin for an immunoglobin are provided.
  • the buffers of the invention preferably comprise aqueous solutions which are at least 0.1 molar, preferably 0.5 to 2.0 molar in sodium, potassium or ammonium sulfate, acetate or chloride.
  • a particularly preferred buffer is 0.25 to 0.5 molar in sodium sulfate.
  • Another preferred buffer is PBS which is 0.1 to 2.0 molar in sodium sulfate.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

On décrit des tampons d'affinité qui augmentent la capacité de fixation des gels synthétiques pour chromatographie par affinité. Les tampons de l'invention contiennent généralement des solutions aqueuses de sels capables d'assurer une concentration suffisante en ions sulfate, acétate, chlorure, potassium, sodium ou ammonium pour augmenter la capacité de fixation de ces gels synthétiques, comme représenté sur la figure.
EP94921883A 1994-04-01 1994-04-01 Tampon de fixation a haute teneur en sel pour la purification d'immunoglobulines avec un ligand d'affinite synthetique Withdrawn EP0701566A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1994/003615 WO1995026977A1 (fr) 1994-04-01 1994-04-01 Tampon de fixation a haute teneur en sel pour la purification d'immunoglobulines avec un ligand d'affinite synthetique

Publications (1)

Publication Number Publication Date
EP0701566A1 true EP0701566A1 (fr) 1996-03-20

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EP94921883A Withdrawn EP0701566A1 (fr) 1994-04-01 1994-04-01 Tampon de fixation a haute teneur en sel pour la purification d'immunoglobulines avec un ligand d'affinite synthetique

Country Status (5)

Country Link
EP (1) EP0701566A1 (fr)
JP (1) JPH08511280A (fr)
AU (1) AU7241294A (fr)
NO (1) NO954704D0 (fr)
WO (1) WO1995026977A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5906747A (en) * 1995-11-13 1999-05-25 Biosepra Inc. Separation of molecules from dilute solutions using composite chromatography media having high dynamic sorptive capacity at high flow rates
US8067182B2 (en) * 2005-03-11 2011-11-29 Wyeth Llc Method of weak partitioning chromatography

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4704366A (en) * 1984-06-22 1987-11-03 Bio-Rad Laboratories, Inc. Process for binding IgG to protein A

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9526977A1 *

Also Published As

Publication number Publication date
WO1995026977A1 (fr) 1995-10-12
JPH08511280A (ja) 1996-11-26
AU7241294A (en) 1995-10-23
NO954704L (no) 1995-11-21
NO954704D0 (no) 1995-11-21

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