VIRUS PURIFICATION METHOD USING APATITE COLUMN
20230212530 · 2023-07-06
Assignee
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2770/32651
CHEMISTRY; METALLURGY
B01D15/08
PERFORMING OPERATIONS; TRANSPORTING
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
[Problems] The present invention provides a purification method capable of more effectively removing the contaminants with physical characteristics similar to the virus from the composition containing the virus, while preventing the denaturation of the virus, than conventional methods.
[Means to solve problems] A purification method for removing a contaminant from a composition containing a virus particle and the contaminant, the purification method comprising: a preparation step of preparing the composition containing a virus particle and a contaminant; a first adsorption step of adsorbing the virus particle on a first adsorbent composed of a calcium phosphate compound by contacting the composition with the first adsorbent; a first elution step of eluting the virus particle from the first adsorbent to obtain a first eluate; a second adsorption step of adsorbing the virus particle on a second adsorbent composed of a calcium phosphate compound by contacting the first eluate with the second adsorbent; and a second elution step of eluting the virus particle from the second adsorbent to obtain a second eluate; one of the first elution step and the second elution step comprising pH gradient elution, and the other comprising salt concentration gradient elution.
Claims
1. A purification method for removing a contaminant from a composition containing a virus particle and the contaminant, the purification method comprising: a preparation step of preparing the composition containing a virus particle and a contaminant; a first adsorption step of adsorbing the virus particle on a first adsorbent composed of a calcium phosphate compound by contacting the composition with the first adsorbent; a first elution step of eluting the virus particle from the first adsorbent to obtain a first eluate; a second adsorption step of adsorbing the virus particle on a second adsorbent composed of a calcium phosphate compound by contacting the first eluate with the second adsorbent; and a second elution step of eluting the virus particle from the second adsorbent to obtain a second eluate; one of the first elution step and the second elution step comprising pH gradient elution, and the other comprising salt concentration gradient elution.
2. The purification method according to claim 1, wherein the pH gradient elution comprises a step of changing a pH value of a solution supplied to the first adsorbent from a first pH value to a second pH value, the first pH value is acidic, and the second pH value is neutral or alkaline.
3. The purification method according to claim 2, wherein the first pH value is 6.0 or lower, and the second pH value is 7.5 or higher.
4. The purification method according to claim 1, wherein the salt concentration gradient elution comprises a step of changing a salt concentration of a solution supplied to the second adsorbent from a first concentration to a second concentration, the first concentration is 10 mM or less, and the second concentration is 100 mM or more.
5. The purification method according to claim 1, wherein the first elution step comprises the pH gradient elution, the first adsorbent comprising fluoroapatite, and the second elution step comprises the salt concentration gradient elution, the second adsorbent comprising hydroxyapatite.
6. The purification method according to claim 5, wherein the first elution step is conducted before the second elution step.
7. The purification method according to claim 1, wherein the virus particle is a poliovirus particle.
8. A method for producing a virus particle, the method comprising steps of preparing a composition containing a virus particle and a contaminant, and removing the contaminant from the composition by the purification method recited in claim 1.
9. The purification method according to claim 2, wherein the virus particle is a poliovirus particle.
10. A method for producing a virus particle, the method comprising steps of preparing a composition containing a virus particle and a contaminant, and removing the contaminant from the composition by the purification method recited in claim 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0061] The preferred embodiments of the present invention will be described below.
[0062] Adsorbents for use in the pH gradient elution are preferably usable even under acidic conditions. Being usable even under acidic conditions means that even when the pH value of the solution loaded on the column is lower than 7.0, the adsorbents are substantially neither dissolved nor decomposed, exhibiting their separation functions.
[0063] It is preferable that the adsorbents for use in the pH gradient elution are mainly composed of fluoroapatite represented by formula: Ca.sub.10(PO.sub.4).sub.6(F).sub.2. The fluoroapatite preferably has a spherical particle shape. As the fluoroapatite of a spherical particle shape, for example, CFT (ceramic fluoroapatite) available from Bio-Rad Laboratories, Inc. may be used.
[0064] The pH gradient means that the pH value is changed from the first pH value to the second pH value, the first pH value being acidic, the second pH value being neutral or alkaline.
[0065] From the viewpoint of increasing the retention volumes of viruses, the first pH is preferably 3.0 to 7.0, more preferably 3.5 to 6.5, further preferably 4.0 to 6.0, and most preferably 4.5 to 5.5.
[0066] the second pH is preferably 7.4 to 9.0, more preferably 7.6 to 8.8, further preferably 7.8 to 8.6, and most preferably 8.0 to 8.4.
[0067] It is preferable that the adsorbents for use in the salt concentration gradient elution are mainly composed of hydroxyapatite represented by formula: Ca.sub.10(PO.sub.4).sub.6(OH).sub.2. The hydroxyapatite preferably has a spherical particle shape. As the hydroxyapatite of a spherical particle shape, for example, CHT TYPE1 and CHT TYPE2 available from Bio-Rad Laboratories, Inc. may be used.
[0068] A salt used for eluting viruses by the salt concentration gradient is preferably a phosphate buffering agent. That is, an eluent for eluting viruses is preferably a phosphate buffer solution. The phosphate buffer solution includes, for example, an aqueous solution containing phosphate such as sodium phosphate, potassium phosphate, lithium phosphate and ammonium phosphate. In addition, the buffer solution may be a 2-morpholinoethanesulfonic acid (MES) buffer solution or a sulfate such as sodium sulfate.
[0069] The salt concentration gradient is preferably to increase the salt concentration in the eluent from the first concentration to the second concentration.
[0070] The first concentration is preferably 1 mM to 50 mM.
[0071] The second concentration is preferably 80 mM to 270 mM.
[0072] When the phosphate buffer solution is used as the eluent, the phosphate buffer solution may further contain other salt than phosphate. The other salt may be a salt of alkali metal or alkali earth metal. The other salt may include NaCl, KCl, NH.sub.4Cl and MgCl.sub.2, and is preferably NaCl.
[0073] The other salt concentration may be 0.1 M or more and 4.0 M or less, and is preferably 0.2 M or more and 2.5 M or less, more preferably 0.5 M or more and 2.0 M or less, further preferably 0.75 M or more and 1.75 M or less, and most preferably 1.0 M or more and 1.5 M or less. The phosphate buffer solution containing the other salt of such concentrations can achieve the separation of the viruses with higher purity.
[0074] From the viewpoint of preventing hydroxyapatite from being dissolved, the pH of the eluent used in the salt concentration gradient elution is preferably 7.0 to 9.0, more preferably 7.05 to 8.0, and further preferably 7.1 to 7.5.
[0075] The removal rate of the double-stranded DNA (dsDNA) by the purification method of the present invention is preferably 97% by mass or more per 100% by mass of the total amount of dsDNA included in the sample before conducting these steps. The removal rate is more preferably 98% by mass or more, further preferably 99% by mass or more, and most preferably 99.9% by mass or more.
[0076] The flow rate of the buffer in the elution processes in the steps 1 and 2 is preferably about 0.1 mL/min or more and 10 mL/min or less, and more preferably 0.2 mL/min or more and 5 mL/min or less, from the viewpoint of reducing the time required for separation operation and reliably separating the target.
[0077] Either of the pH gradient elution and the salt concentration gradient elution may be carried out first. By carrying out both, both protein contaminants and DNA contaminants can be removed. It is preferable that the pH gradient elution is carried out first, and then the salt concentration gradient elution is carried out. The reason is as follows. The DNA contaminants in the sample are excessively adsorbed on the adsorbents in the presence of the other salt such as chloride sodium for use in the salt concentration gradient elution. Therefore, by removing the DNA contaminants in the sample via the pH gradient elution before the sample is used for the salt concentration gradient elution, the amount of the DNA contaminants, which are adsorbed on the adsorbents and hardly separated from virus in the salt concentration gradient elution, is reduced, resulting in higher purity of the virus obtained. Otherwise, the eluate obtained in the preceding elution may be diluted, and then supplied for the next elution.
[0078] The purification method of the present invention is preferably used for a compound having a positively or negatively charged portion. The purification method of the present invention is preferably directed to a positively or negatively charged protein, more preferably directed to RNA viruses such as poliovirus, calicivirus and influenza virus, and DNA viruses such as adenovirus, further preferably directed to viruses that are not substantially deactivated under acidic conditions such as poliovirus.
[0079] Herein, “being not substantially deactivated” means that the infectivity of the virus-containing sample is not substantially reduced under conditions such as acidic conditions. In the embodiment, the “substantially no deactivated” infectivity of the sample is preferably 70% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more, of its original infectivity.
[0080] Herein, that a composition A is “mainly composed of” a compound B means that the amount of the compound B is 50% by mass or more per 100% by mass of the total amount of the composition A. In the embodiment, this ratio is preferably 70% by mass or more, more preferably 80% by mass or more, further preferably 90% by mass or more, and most preferably 95% by mass or more.
Example
[0081] Here, we report on the design and experimental validation of a sequential liquid chromatographic procedure using ceramic fluoroapatite (CFAp) and CHAp for purification of the Sabin type 2 strain of poliovirus.
[0082] Materials and Methods
[0083] Preparation of Sabin Type 2 Virus-Containing Cell Culture Supernatant
[0084] Vero cells (the American Type Culture Collection, Manassas, Va., USA) were cultured in minimum essential medium (MEM; Thermo Fisher Scientific Inc., Waltham, Mass., USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc.) and L-glutamine (2 mM; Thermo Fisher Scientific Inc.) in 225-cm.sup.2 flasks (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) at 37° C. in 5% CO.sub.2 for 3 days. The medium was then changed to MEM containing 2% FBS and 2 mM L-glutamine, and the cells were grown for a further day. After changing the medium to MEM (63 mL) without FBS, Sabin type 2 virus was inoculated onto the cell monolayer at MOI 0.01 and cultured at 37° C. for 2 days. The cell culture supernatant was then collected, passed through a 0.45-μm filter (polyethersulfone membrane; Thermo Fisher Scientific Inc.) to remove cell debris, and stored without any stabilizer at −80° C. until use. The virus was highly stable under this condition and showed no change in its TCID.sub.50 value during the 4-month storage period (data not shown).
[0085] CHAp and CFAp Columns
[0086] CHT Ceramic Hydroxyapatite, Type II (CHAp; 40-μm particle size) and CFT Ceramic Fluoroapatite, Type II (CFAp; 40-μm particle size) were purchased from Bio-Rad Laboratories Inc. (Hercules, Calif., USA). Both are ceramic-type materials with strict specifications. The particles were packed into empty stainless steel columns (4.6 mm i.d.×35 mm; Sugiyama Shoji Co., Ltd., Kanagawa, Japan) in-house using a dry method.
[0087] Chromatographic Procedures
[0088] Chromatography was performed using a BioLogic DuoFlow™ system (Bio-Rad Laboratories Inc.) with a 10- or 20-mL sample loop at a flow rate of 1.0 mL/min. The samples were loaded onto a CHAp/CFAp column and eluted using a linear concentration gradient of sodium phosphate buffer (NaPB) ranging from 10 mM to 600 mM. The resulting eluate was monitored for ultraviolet (UV) absorbance at 260 and 280 nm and for conductivity. The collected fractions were kept at 4° C. and were immediately used for the evaluations described below. Before use, the running buffers were filtered (0.22 μm). The fraction collector was placed in a biological safety cabinet, and the attached tubes were autoclaved. In addition, the interiors of the pumps, columns, and lines were sterilized with ethanol for disinfection (Amakasu Chemical Industries, Tokyo, Japan), washed with autoclaved ultrapure water followed by 600 mM NaPB, and equilibrated with 10 mM NaPB before use. After the experiments, the column and system were sterilized with 0.5M NaOH for 10 column volumes and washed with autoclaved ultrapure water to remove the alkaline solution.
[0089] Measurement of Infectivity of the Virus-Containing Fractions
[0090] The Sabin type 2 virus titer was obtained by measuring the median tissue culture infectious dose (TCID.sub.50) using a confluent monolayer of Vero cells in 96-well microplates. To prepare the confluent monolayer, Vero cells (100 μL, 1×10.sup.5 cells/mL) were cultured in MEM containing 10% FBS at 37° C. for 1 day. Each fraction obtained by liquid chromatographic separation was 10-fold serially diluted with MEM containing 10% FBS, and the resulting diluents (50 μL) were inoculated into each well (n=3) and cultured for 1 week. Each well of the microplates was then examined under a light microscope CKX31 (Olympus Corporation, Tokyo, Japan) to determine whether cytopathic effects had occurred. Titers were calculated using the Reed-Muench method.
[0091] Other Evaluations
[0092] The concentrations of double-stranded DNA (dsDNA) and proteins were determined using the Quant-iT™ PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific Inc.) and the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the liquid chromatographic fractions were concentrated using a ultrafiltration (Ultracel YM-10; Millipore Corporation, Billerica, Mass., USA) and applied to 15% polyacrylamide gel (c-PAGEL; ATTO Corporation, Tokyo, Japan). Each protein band was visualized by silver staining, and the molecular weight was calculated by the Gel Doc™ EZ Imager (Bio-Rad Laboratories Inc.).
[0093] Results
[0094] Two-Step Procedure for the Purification of Sabin Type 2 Virus
[0095] We initially used one-step CHAp chromatography to purify live attenuated Sabin type 2 virus. However, this proved unsuccessful because both protein and DNA contaminants could not be separated from the virus fraction using this method. Therefore, we attempted to introduce separation by CFAp chromatography as the first step before conducting CHAp chromatography. The strategy of this two-step chromatographic procedure is outlined in
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[0097] CFAp (ceramic fluoroapatite)
[0098] CHAp (ceramic hydroxyapatite)
[0099] NaPB (sodium phosphate buffer)
[0100] Step 1: pH gradient elution of Sabin type 2 virus from a CFAp column
[0101] We separated three viruses [poliovirus Sabin type 2; dengue virus type 1 (Hawaii) (Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan); and influenza virus NYMC X-181 (National Institute for Biological Standards and Control, Hertfordshire, UK)] on a CHAp column by elution with a similar concentration gradient of NaPB at different pH values (
[0102] The contents of the experiments in
[0103]
[0104] (A) Chromatogram obtained under the following conditions:
[0105] Column, CFAp;
[0106] sample (volume), cell culture supernatant containing Sabin type 2 virus (5 mL);
[0107] column wash, 10 mM sodium phosphate buffer (NaPB; pH 6.4, 10 mL) and 300 mM NaPB (pH 6.4, 20 mL);
[0108] equilibration, 300 mM NaPB (pH 5, 15 mL);
[0109] elution, linear pH gradient at 300 mM NaPB from pH 5 to pH 8.2 for 10 mL; and
[0110] wash after separation, 300 mM NaPB (pH 8.2, 5 mL) and 600 mM NaPB (pH 8.2, 10 mL);
[0111] blue line, ultraviolet (UV) absorbance at 280 nm;
[0112] black broken line, conductivity;
[0113] red line, infectivity in median tissue culture infectious dose (TCID.sub.50); and
[0114] purple line, double-stranded DNA (dsDNA) contents.
[0115] “Fr. A” was pooled for the evaluation.
[0116] (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
[0117] The cell culture supernatant and pooled Fr. A were concentrated 10-fold and 30-fold, respectively, by ultrafiltration using a molecular weight cutoff of 10,000. The molecular weights of the marker proteins are given in kDa.
[0118] Step 2: Separation of Sabin Type 2 Virus from Double-Stranded DNAs by CHAp
[0119] Effect of NaCl Addition to the Mobile Phase
[0120] We next examined whether a high concentration of NaCl in the mobile phase of Step 2 would allow double-stranded DNA to be removed from the virus fraction based on the knowledge that excess sodium ions on the phosphate site of hydroxyapatite decrease the electrostatic repulsion between the phosphate site and DNA, causing an increase in the relative affinity of the calcium site for DNA. To evaluate this, we separated two viruses (influenza virus NYMC X-181 and feline calicivirus) in the presence of 0 to 1.5M NaCl. We found that the retention volume of double-stranded DNA increased with increasing NaCl concentrations in the running buffer, whereas the retention volume of the virus particles decreased (
[0121] The contents of the experiments in
[0122]
[0123] Column, ceramic hydroxyapatite (CHAp);
[0124] sample (volume), cell culture supernatant containing Sabin type 2 virus (5 mL);
[0125] buffer pH, 7.2;
[0126] column wash, 10 mM sodium phosphate buffer (NaPB; 9 mL);
[0127] equilibration, 10 mM NaPB with NaCl (14 mL);
[0128] elution, linear concentration gradient from 10 mM to 600 mM NaPB with NaCl for 30 mL; and
[0129] wash after separation, 600 mM NaPB (10 mL).
[0130] Lines are the same as in
[0131] TCID.sub.50, median tissue culture infectious dose.
[0132] Sequential Two-Step Chromatographic Purification of Sabin Type 2 Virus
[0133] We performed the two-step sequential procedure (Steps 1 and 2 described above) to isolate Sabin type 2 virus from the cell culture supernatant. A representative case is shown in
[0134] The virus particles were separated by pH gradient elution on a CFAp column (
[0135] The contents of the experiments in
[0136]
[0137] (A) Purification of Sabin type 2 virus by pH gradient elution on a ceramic fluoroapatite (CFAp) column (Step 1).
[0138] Column, CFAp;
[0139] sample (volume), cell culture supernatant containing Sabin type 2 virus (10 mL);
[0140] wash, 10 mM sodium phosphate buffer (NaPB) (pH 6.4, 9 mL) and 300 mM NaPB (pH 6.4, 20 mL);
[0141] equilibration, 300 mM NaPB (pH 5, 15 mL);
[0142] elution, linear pH gradient from pH 5 to pH 8.2 at 300 mM NaPB for 10 mL; and
[0143] wash after separation, 300 mM NaPB (pH 8.2, 5 mL) and 600 mM NaPB (pH 8.2, 10 mL).
[0144] Lines are the same as in
[0145] Fr. B was pooled and further purified in Step 2.
[0146] (B) Removal of double-stranded DNA (dsDNA) from the Fr. B fraction by NaPB elution with 1M NaCl on a ceramic hydroxyapatite (CHAp) column (Step 2)
[0147] Column, CHAp;
[0148] sample (volume), Fr. B obtained in (A) diluted 6.7-fold with 0.9% NaCl (17 mL);
[0149] buffer, pH 7.2;
[0150] column wash, 10 mM NaPB (13 mL);
[0151] equilibration, 10 mM NaPB (14 mL) with 1 M NaCl;
[0152] elution, linear concentration gradient from 10 mM to 187 mM NaPB with 1 M NaCl for 10 mL; and
[0153] wash, 600 mM NaPB (10 mL).
[0154] Fr. C was pooled for further evaluation.
[0155] (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the pooled fraction obtained through the two-step purification process
[0156] The pooled Fr. C fraction obtained in (B) and the cell culture supernatant were concentrated 100- and 10-fold, respectively, by ultrafiltration using a molecular weight cutoff of 10,000. The sizes are given in kDa on the left. Arrows indicate the major proteins in Fr. C.
[0157] TCID.sub.50, median tissue culture infectious dose.
Discussion
[0158] When manufacturing vaccines for use in clinical trials, preclinical studies, or commercial medicines, the aim is to minimize impurities and maximize the recovery of the vaccine during purification. The impurities are typically DNA and proteins derived from host cells, components of the cell culture medium, and/or some ligands released from purification process. In this study, we successfully purified Sabin type 2 virus directly from the cell culture supernatant without using any concentrating or buffer exchange processes, achieving the mean recovery rate in TCID.sub.50 of 58.7%±30.0%, and the mean removal rates of proteins and double-stranded DNA of 99.95±0.006% and 99.99%±0.003%, respectively, (three independent experiments).
[0159] In Step 1, the cell culture supernatant was loaded directly onto a CFAp column and eluted with a pH gradient. In Step 2, the resulting virus fraction was diluted with 0.9% NaCl, loaded on a CHAp column, and eluted with an NaPB gradient in the presence of a high concentration of NaCl. Because both Steps 1 and 2 were highly reproducible, the viral fraction could be obtained without the need for any detection processes. This retention-volume-dependent fractionation should reduce the time and cost required to make vaccines. Furthermore, although the procedure currently contains multistep and offline purification processes, it should be possible to automate all the purification steps in the future. This two-step chromatographic purification is also easier to scale up than other current procedures because it was constructed using fully scalable processes (
[0160] Abbreviations in
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[0162] CFAp, ceramic fluoroapatite;
[0163] CHAp, ceramic hydroxyapatite;
[0164] NaPB, sodium phosphate buffer; and
[0165] PV, poliovirus.
[0166] Two-step chromatographic purification may also be useful for purifying other poliovirus strains such as Salk and Sabin types 1 and 3, as well as other nonenveloped viruses including hepatitis A virus, norovirus and feline calicivirus. However, this process does have some methodological limitations because Step 1 uses a low pH that will disrupt some viruses. Consequently, it may not be applicable for the purification of flaviviruses, which are unstable at acidic pH values. In addition to their role in vaccine production, infective virus purification techniques also play an important role in the field of gene therapy. In particular, there is an urgent clinical need for the large-scale purification of adenovirus because this virus is considered one of the most suitable platforms for gene transduction. Several other gene therapies have also yielded promising results for the use of virus vectors that express clinically relevant genes. For example, oncolytic adenovirus has been drawing attention recently as an antitumor medicine, and dozens of studies using this virus are now registered in Clinical-Trials.Gov (https://clinicaltrials.gov/), a database of clinical studies conducted around the world. This new purification method should be appropriate for adenoviruses that are resistant to acidic purification conditions.
[0167] Supporting Information
[0168]
[0169] Chromatograms of Sabin type 2 virus-containing cell culture supernatant separated on a ceramic hydroxyapatite (CHAp) column at different pH values
[0170] Separation occurred at buffer pH values of (A) 6.4, (B) 7.2, and (C) 8.2.
[0171] Column, CHAp (40 μm);
[0172] sample (volume), cell culture supernatant containing Sabin type 2 virus (5 mL);
[0173] column wash and equilibration, 10 mM sodium phosphate buffer (NaPB; 11 mL);
[0174] elution, linear concentration gradient from 10 mM to 300 mM NaPB for 20 mL; and
[0175] wash after separation, 600 mM NaPB (8 mL).
[0176] blue line, ultraviolet (UV) absorbance at 280 nm;
[0177] black broken line, conductivity;
[0178] red line, infectivity in median tissue culture infectious dose (TCID.sub.50); and
[0179] purple line, double-stranded DNA (dsDNA) contents.
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[0181] Chromatograms of dengue virus type 1-containing cell culture supernatant separated on a ceramic hydroxyapatite (CHAp) column at different pH values
[0182] Separation occurred at buffer pH values of (A) 6.4, (B) 7.2, and (C) 8.2.
[0183] Column, CHAp (40 μm);
[0184] sample (volume), cell culture supernatant containing dengue virus type 1 (10 mL);
[0185] column wash and equilibration, 10 mM sodium phosphate buffer (NaPB; 10 mL);
[0186] elution, linear concentration gradient from 10 mM to 300 mM NaPB for 30 mL and from 300 mM to 600 mM for 8 mL; and
[0187] wash after separation, 600 mM NaPB (5 mL).
[0188] Lines are the same as in
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[0190] Chromatograms of influenza virus NYMC X-181 containing cell culture supernatant separated on a ceramic hydroxyapatite (CHAp) column at different pH values
[0191] Separation occurred at buffer pH values of (A) 6.5, (B) 6.8, and (C) 7.5.
[0192] Column, CHAp (40 μm);
[0193] sample (volume), cell culture supernatant containing influenza virus NYMC X-181 (5 mL);
[0194] column wash and equilibration, 10 mM sodium phosphate buffer (NaPB; 15 mL);
[0195] elution, linear concentration gradient from 10 mM to 600 mM NaPB for 30 mL; and
[0196] wash, 600 mM NaPB (5 mL).
[0197] Lines are the same as in
[0198]
[0199] Chromatograms of influenza virus NYMC X-181 containing cell culture supernatant in the presence of NaCl
[0200] The buffer contained (A) 0 M, (B) 0.14 M, (C) 0.5 M, and (D) 1 M NaCl.
[0201] Column, ceramic hydroxyapatite (CHAp);
[0202] sample (volume), cell culture supernatant containing influenza virus NYMC X-181 (5 mL);
[0203] buffer pH, 7.5;
[0204] column wash and equilibration, 5 mM sodium phosphate buffer (NaPB) with NaCl (15 mL);
[0205] elution, linear concentration gradient from 5 mM to 600 mM NaPB with NaCl for 30 mL; and
[0206] wash after separation, 600 mM NaPB (5 mL).
[0207] Lines are the same as in