KR102624098B1 - Thrombin mass purification method with improved purity - Google Patents

Abstract

The present invention relates to a method for mass purification of thrombin with improved purity in the production of thrombin, which is produced by purifying prothrombin and then converting the prothrombin to thrombin. The sample in which prothrombin is extracted from serum and converted to thrombin is mixed with 20mM Substitution step with sodium phosphate, pH6.5 buffer: Loading the buffer-substituted sample onto a BPX50/500 SP FF column and purifying it by flowing 20mM sodium phosphate pH6.5 buffer at a rate of 15mL/min; And after completing the purification, it includes the step of eluting thrombin by flowing 20mM sodium phosphate, pH 6.5, 1M NaCl buffer.

Description

{Thrombin mass purification method with improved purity}

The present invention relates to a method for mass purifying thrombin with improved purity in the production of thrombin, which is produced by purifying prothrombin and then converting the prothrombin to thrombin.

Thrombin is a serine protease with a molecular weight of about 34,000 and an isoelectric point of about 7.1, that is, a proteolytic enzyme that exerts its action in the final stage of the blood coagulation process. That is, it affects fibrinogen, which forms fibrin, and thereby induces its blood aggregation action.

For this reason, thrombin is used clinically as a topical hemostatic agent in the field of surgery and as a hemostatic agent for upper gastrointestinal bleeding, etc. in the field of internal medicine.

In vivo, thrombin exists in the form of prothrombin as its precursor and is formed through limited hydrolysis by activating factor Generally, thrombin is manufactured by first extracting and purifying prothrombin from human plasma as a raw material, and then converting the purified prothrombin into thrombin.

Therefore, so far, prothrombin is purified as described above, then prothrombin is converted to thrombin, and thrombin is manufactured through a purification step and other necessary steps, and various modified methods based on this method have been reported. .

For example, contacting plasma treated with citric acid with an anion exchanger, converting the adsorbed prothrombin to thrombin on the anion exchanger, recovering the converted product by elution, and treating the plasma with cold ethanol, There is a known method of treating the prothrombin with an anion exchanger, converting the purified prothrombin into thrombin, and then purifying it by treatment with a cation exchanger.

Among these steps, in addition to the above-mentioned method using thromboplastin, the conversion reaction to thrombin includes, for example, a method using snake venom or a method using a high concentration of citric acid salt.

However, the method of purifying thrombin converted from prothrombin to high purity on an industrial scale is an important step in the manufacturing process, but is not clearly presented.

1. Republic of Korea Patent No. 10-2008-0019583 2. Republic of Korea Patent No. 10-2017-0135828

Therefore, the problem to be solved by the present invention is to provide a method of purifying thrombin converted from prothrombin to high purity on an industrial scale.

In order to achieve the above technical problem, the present invention

Step of extracting prothrombin from serum and replacing the sample converted to thrombin with 20mM sodium phosphate, pH6.5 buffer:

Loading the buffer-substituted sample onto a BPX50/500 SP FF column and purifying it by flowing 20mM sodium phosphate pH6.5 buffer at a rate of 15mL/min; and

After completing the purification, a method of mass purification of thrombin with improved purity is provided, comprising the step of eluting thrombin by flowing 20mM sodium phosphate, pH 6.5, 1M NaCl buffer.

As another way to achieve the above technical challenges. This invention

Steps to extract prothrombin from serum and replace the sample converted to thrombin with 20mM sodium phosphate, pH6.5, 150mM NaCl buffer:

Loading the buffer-substituted sample into a Foresight Nuvia cPrime column and purifying it by flowing 20mM sodium phosphate, pH6.5, 150mM NaCl buffer at a rate of 15mL/min; and

After completing the purification, a method of mass purification of thrombin with improved purity is provided, comprising the step of eluting thrombin by flowing a 20mM Tris-Cl, pH 8.0, 0.5M NaCl buffer.

According to the present invention, thrombin converted from prothrombin can be purified to high purity on an industrial scale.

Figure 1: SDS-PAGE step by step of prothrombin sample collection.
Figure 2: SDS-PAGE to confirm thrombin activation
Figure 3: Buffer substitution for SP purification of thrombin
Figure 4: Experiment for determination of SP purification buffer
Figure 5: Experiment for establishing SP tablet washing conditions
Figure 6: Confirmation of purification using the final SP purification conditions
Figure 7: Nuvia cPrime Purification Buffer Determination Experiment
Figure 8: Nuvia cPrime tablet washing buffer concentration determination experiment
Figure 9: Nuvia cPrime tablet washing buffer concentration determination experiment
Figure 10: Buffer replacement of samples before SP purification and Nuvia cPrime purification
Figure 11: Protein quantification of SP purified and Nuvia cPrime purified fractions.
Figure 12: SDS-PAGE analysis of SP purified and Nuvia cPrime purified fractions.
Figure 13: HPLC analysis of SP purified and Nuvia cPrime purified fractions.
Figure 14: Storage buffer replacement of SP purified and Nuvia cPrime purified fractions.
Figure 15: HPLC analysis of SP purified and Nuvia cPrime purified fractions.

Hereinafter, specific details for carrying out the present invention will be described in detail.

I. Purpose of experiment

In order to purify pure thrombin from plasma samples, an experiment was conducted to construct a method of concentrating and separating prothrombin from plasma and converting prothrombin to thrombin to purify activated thrombin.

The experimental method was described by Z Ding et al. A Rapid simplified Purification of Bovine thrombin: Preparative Biochemistry. Vol.25, p21-28, 1995 paper and Krishna Kumar Turaga et al. Rapid Purification of high purity thrombin and preparation of a novel hemostat for clinical purpose : Indian J. Hematol. Blood Transfus Vol.24, p54-58, 2008 was conducted based on the paper, and during the experiment process, the method introduced in the paper was specified and a new method was established to make the experiment easier or improve.

II. Experiment result

1. Sample preparation before thrombin purification

go. Plasma collection

To remove the white precipitate that appears when the frozen citrated plasma is thawed, it was centrifuged at 4°C at 2,500 rpm for 15 minutes and the supernatant was collected.

me. From Plasma Prothrombin extraction

1) To the 500mL of plasma obtained above, 60mL of 1M BaCl2 was added one drop at a time for 20 minutes, stirred slowly, and left for 30 minutes.

2) Centrifuged at 4,000g for 15 minutes at 4℃ to collect the precipitate, which is Barium-Prothrombin complex. Washed the precipitate with 1mM sodium citrate, pH 7.5 buffer, and centrifuged at 4,000g for 15 minutes at 4℃ to remove the supernatant. This process was repeated three times until the supernatant became transparent.

all. Prothrombin hydrolyzing

Two methods were attempted to dissolve the sediment sample obtained above.

The first is a method of obtaining prothrombin bound to barium by adding 1L of 100mM citrate pH6.8 buffer based on 250mL of plasma. At this time, a saturated solution of ammonium sulfate is added and stirred at 4℃ for more than 1 hour to precipitate the protein, and then the precipitate is dissolved. Carried out.

However, at this time, a problem occurred where the sediment did not dissolve well, so the second method was tried.

Second, 25 mL of 0.2M Na2EDTA, pH 7.4 buffer was added to remove barium from the barium-prothrombin complex and drop thrombin. The sample obtained in this way was replaced with 50mM Tris, 2mM CaCl2, 150mM NaCl, and pH7.5 buffer (thrombin activity buffer) using 30kDa UFP.

Buffer replacement was performed four times, 1L at a time, with more than 4L of active buffer per 400mL of sample, and a final sample of 400mL was obtained. Protein patterns were confirmed through SDS-PAGE analysis of each step of the sample obtained through the experiment so far (see Figure 1).

Starting with citrated plasma (1), the supernatant (2) and precipitate (3) obtained by centrifuging this, the supernatant (4) obtained by centrifugation after treatment with BaCl2, the washing solution obtained after washing the precipitate (5), and the precipitate with EDTA. After dissolving in buffer, the obtained sample (6) was sequentially loaded onto a 14% SDS-PAGE gel and stained with CBBR.

Samples 1, 2, 4, and 5 were loaded without boiling by adding 1uL of 5X reducing sample buffer to 4uL of sample, and samples 3 and 6 were loaded by mixing 8uL of 5X reducing sample buffer in 32uL of sample and boiling for 10 minutes. In lane 6, the 70kDa protein expected to be prothrombin appeared dark, and the protein around 50kDa, which appeared darkest in the plasma sample, was significantly removed.

However, protein bands of various sizes were also colored together.

la. Thrombin activation

First, an experiment was conducted to determine the concentration of snake venom treatment to convert prothrombin to thrombin.

The snake venom used is Snake Venom from Echis carinatus (Cat.# V8250-100MG) from Sigma-aldrich and Echis from Latoxan. carniatus multisquamatus Using two types, venom with a concentration of 1mg/mL is added to 1mL of sample at 10uL (1:100), 5uL (1:200), 3,3uL (1:300), 2,5uL (1:400), 2uL (1) :500) and shaken at 37℃ for 3 hours.

To confirm activation, 8uL of 5X reducing sample buffer was mixed with 32uL of the sample, boiled for 10 minutes, loaded at 40uL each, electrophoresed, and stained with CBBR (see Figure 2).

As a result of the above experiment, thrombin activation was confirmed from Venom treatment from 1:100 to 1:500, and the thrombin conversion amount for each venom ratio seemed to be similar, but it was decided to process at a ratio of 1:200 in future experiments to ensure reliable results.

For the next purification step, 250 mL of sample was treated with snake venom at a ratio of 1:200 and shaken at 37°C for 3 hours.

2. Thrombin purification

go. Cation Ion Exchange Chromatography Tablets ( CIEX : SP sepharose )

The determination of purified resin was carried out by referring to papers reported through previous research.

Krishna Kumar Turaga et al. (2008) purified thrombin by performing phenyl purification after SP purification.

1) Since the composition of the buffer used during SP purification was not described in detail, purification was started by making sodium phosphate, pH6.0 buffer and pH6.5 buffer based on the estimated pI value of thrombin.

The column used was HiTrap SP FF, 5mL (Code #. 17-5157-01) provided by GEHC.

In the first purification, 10 mL of the sample activated with venom was replaced with 20mM sodium phosphate, pH6.0 buffer, and in the second purification, the same sample was replaced with 20mM sodium phosphate, pH6.5 buffer. Sephadex G-25 resin was used for buffer replacement (see Figure 3).

2) In Figure 3, 10mL of sample was replaced with the first buffer to obtain 26mL of sample, and 20mM sodium phosphate pH6.0 buffer was flowed through the HiTrap SP FF column at a rate of 5mL/min to equilibrate the column.

After flowing more than 5cv of buffer, 26mL of sample was loaded onto the column, and then more than 5cv of 20mM sodium phosphate pH6.0 buffer was flowed.

For protein elution, 20mM sodium phosphate, pH6.0 buffer was linearly gradient eluted to a concentration of 0-1M NaCl, and a total of 100mL of buffer was flowed.

The second SP purification was carried out in the same manner as the first SP purification, only the buffer pH was used differently at 6.5.

The samples obtained through the two experiments, which were the column loading sample, flow through, and elution fractions, were sequentially analyzed by SDS-PAGE (see Figure 4).

Based on the results in Figure 4, it can be seen that more proteins other than thrombin bind to the column when using the pH 6.0 buffer rather than the pH 6.5 buffer, so the SP purification buffer was decided to be pH 6.5 buffer, and the concentration of NaCl It was confirmed that the higher the value, the higher the purity of thrombin.

3) Next, an experiment was conducted to determine the washing conditions for SP purification. Based on purification steps 1 and 2 above, the sample was buffered with 20mM sodium phosphate pH6.5 buffer, and 50mL of the sample was buffered to obtain 95mL. All processes were carried out in the same manner as purification No. 2 above, and in the elution step, elution was carried out by increasing the concentration by 10% from a buffer without NaCl to a buffer containing 1M NaCl, resulting in 10%B, 20%B, and 30% B. %B, 40%B, 50%B, and 100%B, step gradient elution was performed. Fraction samples from each stage were analyzed by SDS-PAGE (see Figure 5).

Based on the experiment in Figure 5, it was confirmed that proteins other than thrombin were mainly separated on the column up to 20% B condition, and then thrombin came out starting from 30% B.

Based on the above results, it was decided to wash under 20% B conditions and elute thrombin under 50% B conditions, and proceeded with the next experiment (see Figure 6).

Looking at Figure 6, a slight thrombin band can be seen at 20%B, but it was confirmed that thrombin was mainly eluted at 50%B. As protein bands other than thrombin were confirmed in the 50%B fraction, phenyl purification was performed as mentioned in the reference paper to increase purity.

me. Mixed Mode Chromatography Tablets (HIC- CIEX : Nuvia cPrime )

Based on the reference paper, when SP purification and phenyl purification were performed sequentially, proteins other than thrombin were eluted together. In order to purify with higher purity and increase the simplicity of the experiment, the two properties of CIEX and HIC were combined together. Eggplant was purified with Nuvia cPrime.

1) To determine the buffer pH to be applied to Nuvia cPrime, a preemptive experiment was conducted using sodium citrate pH6.0 buffer and sodium phosphate pH6.5 buffer. The column used was Biorad's Foresight Nuvia cPrime, 5mL (732-4742), and was purified at a speed of 5mL/min.

For the sample, 10 mL of the activated sample was buffer-substituted with each of the two buffers above.

The first Nuvia cPrime purification used 20mM sodium citrate, pH6.0, 150mM NaCl buffer, and the column was equilibrated by flowing more than 5cv before the experiment. After loading the sample, more than 5cv of 20mM sodium citrate pH6.0 and 150mM NaCl buffer was flowed. For protein elution, a linear gradient elution of 20mM Tris-Cl, pH8.0, and 0.5M NaCl buffer was performed in a 20mM sodium phosphate, pH6.0 buffer, and a total of 100mL of buffer was flowed.

The second Nuvia cPrime purification was carried out in the same way as the first purification, and only the buffer pH was used differently: 20mM Sodium phosphate, pH6.5, and 150mM NaCl. The samples obtained through the two experiments, which were the column loading sample, flow through, and elution fractions, were sequentially analyzed by SDS-PAGE (see Figure 7).

Looking at the results in Figure 7, the Flow Through value is higher and the number of eluted peaks is reduced when using the pH 6.5 buffer compared to the pH 6.0 buffer, confirming that proteins other than thrombin are less bound to the column when using the pH 6.5 buffer. The purification buffer was determined to be a pH 6.5 buffer, and the protein seen around 20 kDa in the eluate after SP purification or phenyl purification was excluded from the flow through in Nuvia cPrime purification, and the protein seen in the elution fraction could be separated from the thrombin fraction. It has been confirmed that

Therefore, an experiment was conducted to determine the column washing conditions.

2) Based on the previous experiment, 110 mL obtained by buffer substitution of 50 mL of the activated sample with 20mM sodium phosphate, pH 6.5, 150mM NaCl buffer was loaded on the column, and 20mL was added with 20mM Tris-Cl, pH 8.0, 0.5M NaCl buffer. It was eluted by increasing the concentration by %. In the end, step gradient elution was performed at concentrations of 20%B, 40%B, 60%B, 80%B, and 100%B, and each fraction was analyzed by SDS-PAGE (see Figure 8).

Proteins other than thrombin were eluted up to the 20~60%B fraction, and thereafter thrombin was mainly identified in the 80%B and 100%B fractions. In the 80%B fraction, in addition to thrombin, proteins of approximately 60 kDa and proteins of approximately 20 kDa were identified, and in the 100%B fraction, only thrombin was seen, while the amount of thrombin tended to be quite low.

In order to purify thrombin with high purity, the washing concentration was further refined and the process was repeated.

280 mL obtained by buffer replacement of 130 mL of activated sample with 20mM sodium phosphate, pH 6.5, 150mM NaCl buffer was loaded on the column, and the washing concentration was progressed from 65%B to 80%B at 5%B intervals (see Figure 9). .

At 60%B concentration, most proteins other than thrombin appear to be eluted, and at 65%B, proteins seen around 20kDa are additionally eluted, but thrombin is also eluted, and no proteins other than thrombin are seen in fractions from 70%B to 80%B. It was confirmed that in the 100% B fraction, a weak band was visible around 30 kDa below thrombin. It was not visible in the 100% B fraction of Figure 9, but it was presumed that the invisible band appeared in this experiment because the fraction was more concentrated.

Accordingly, it was determined that washing the thrombin Nuvia cPrime tablet to 60% B and eluting with 100% B would reduce thrombin loss and obtain a highly pure sample.

The fraction obtained through Nuvia cPrime purification was confirmed to be of higher purity than the fraction obtained through phenyl purification after previous SP purification, but the yield of thrombin did not appear to be high.

Next, in order to more accurately compare the SP purified fraction and the Nuvia cPrime purified fraction, each purification was performed with the same amount of protein sample and SDS-PAGE was performed on one gel.

all. Cation Ion Exchange Chromatography Tablets ( CIEX : SP sepharose ) and Mixed Mode Chromatography purification (HIC- CIEX : Nuvia cPrime ) comparative experiment

The same analytical column used in the previous experiment, HiTrap SP FF 5mL column and Foresight Nuvia cPrime 5mL, was used, and 125mL of sample obtained by activating 1.5L of plasma each was buffered using Sephadex G-25 to match the buffer of each tablet. 210 mL of each sample was loaded (see Figure 10).

SP purification was carried out in the same manner as the previous SP purification, and in the final sample elution step, a washing step was performed at a concentration of 20% B, and then thrombin was eluted at 100% B. The eluted thrombin fraction had a protein OD value of 1177 mAu and an elution volume of 10 mL.

Next, Nuvia cPrime purification was performed, and in the final elution step, 60% B washing was performed and thrombin was eluted with 100% B buffer. The thrombin fraction protein OD value of Nuvia cPrime was 191 mAu, which was lower than SP purification, but the elution volume increased to 30 mL.

The amount of protein in each fraction was measured using the BCA quantitative method. Albumin standard was used as albumin at a concentration of 2mg/mL from Thermo (REF23209), and Bicinchoninic Acid solution (Cat.# B9643-1L) and Copper(II) sulfate solution (Cat.# C2284-25ML) from Sigma were used as coloring reagents. Used. As a result of measuring the absorbance at OD560nm, for the SP purified fraction, the concentration was 1.64 mg/mL and the amount of protein was 14.8 mg, and for the Nuvia cPrime purified fraction, the concentration was 0.24 mg/mL and the amount of protein was 7.2 mg. The SP purified fraction was measured to contain approximately twice as much protein as the Nuvia cPrime purified fraction (see Figure 11).

Next, SDS-PAGE analysis was performed to confirm the purity of the two purified fractions. For comparison at a glance, the SP purified fraction and Nuvia cPrime fraction were loaded onto one gel. Since the starting sample loaded on both tablets was the same, only one sample was loaded, and each Flow Through and Wash fraction was loaded #5 for SP tablets and #3 for Nuvia cPrime tablets. The thrombin fraction of the SP tablet was loaded #11, and the thrombin fraction of Nuvia cPrime was loaded in a total of three fractions #8, #9, and #10 (see Figure 12).

Looking at the protein bands in the SDS-PAGE analysis, as can be seen from the protein quantification values, the band of the SP purified thrombin fraction (#11) is much darker than the bands of the Nuvia cPrime thrombin fractions (#8, #9, and #10). It was found that in addition to the band expected to be thrombin, various proteins were separated together. Fewer protein bands other than thrombin were seen in the Nuvia cPrime fraction than in the SP fraction, but the amount of thrombin itself tended to be less.

HPLC analysis was performed to further confirm the purity of the purified fraction. The column used was Agilent's ZORBAX 300SB-C18 column (880995-902, 4.6 The flow rate is set to 1 mL/min, starting with 90% solvent A and 10% solvent B, 80% solvent A and 20% solvent B after 1 minute, 50% solvent A and 50% solvent B at 11 minutes, and finally 15 minutes later. Measurements were made by setting the solvent gradient to 90% solvent A and 10% solvent B at the minute point. The sample was loaded with 20uL, equivalent to 4ug (see Figure 13).

As a result of HPLC analysis, only 1peak, presumed to be thrombin, was identified in both SP purified and Nuvia cPrime purified fractions, and no differences were identified between the two purified fractions.

Based on the above results, it was difficult to determine the superiority of thrombin purity between SP purification and Nuvia cPrime tablets, but SP purification tended to be much more advantageous in terms of thrombin purification yield.

SP purification and Nuvia cPrime purification fractions were replaced with storage buffer (15.4mM (0.9%) NaCl, 2mM Sodium citrate, 1g/L L-arginine, pH 7.4) for thrombin activity analysis. To replace the storage buffer, the purified fraction was lyophilized, dissolved in 3 mL tertiary distilled water, and then buffer replaced using sephadex G-25 (see Figure 14).

The buffer-substituted sample was quantified for protein using the BCA quantitative method, which is the quantitative method described in the previous experiment.

A total of 9.4 mg was confirmed in the SP purified fraction at a concentration of 1.02 mg/mL, and a total of 2.9 mg was confirmed in the Nuvia cPrime purified fraction at a concentration of 0.32 mg/mL.

HPLC analysis was performed with thrombin samples substituted with storage buffer. The analysis method was the same as the previous experiment, and 20uL of sample was loaded, corresponding to 6ug (see Figure 15).

As a result of HPLC analysis, a peak corresponding to 95.2% was confirmed at 12.21 min in the SP purified fraction, and in the Nuvia cPrime purified fraction, what appeared to be one peak before storage buffer replacement was divided into two peaks, each at 11.91 min. Confirmed to be 51.7% and 45.8% at 12.29 min.

Based on the above-described details, the present invention provides the following two methods for mass purification of thrombin with improved purity.

The first way is

Step of extracting prothrombin from serum and replacing the sample converted to thrombin with 20mM sodium phosphate, pH6.5 buffer:

Loading the buffer-substituted sample onto a BPX50/500 SP FF column and purifying it by flowing 20mM sodium phosphate pH6.5 buffer at a rate of 15mL/min; and

After completing the purification, it includes the step of eluting thrombin by flowing 20mM sodium phosphate, pH 6.5, 1M NaCl buffer.

Also, the second method is

Steps to extract prothrombin from serum and replace the sample converted to thrombin with 20mM sodium phosphate, pH6.5, 150mM NaCl buffer:

Loading the buffer-substituted sample into a Foresight Nuvia cPrime column and purifying it by flowing 20mM sodium phosphate, pH6.5, 150mM NaCl buffer at a rate of 15mL/min; and

After completing the purification, it includes the step of eluting thrombin by flowing 20mM Tris-Cl, pH 8.0, 0.5M NaCl buffer.

Claims (7)

(a) replacing the thrombin sample converted from prothrombin with 20 mM sodium phosphate buffer at pH 6.5;
(b) loading the buffer-substituted sample onto a cation exchange chromatography column; and
(c) of thrombin comprising the step of performing step gradient elution, which is performed by adding a 1M sodium chloride buffer selected in the range of 10%B to 50%B compared to the pH 6.5, 20mM sodium phosphate buffer. It is a purification method,
The step of performing the step gradient elution is
(i) a washing step to separate proteins other than thrombin from the sample loaded on the cation exchange chromatography column; and
(ii) A method for purifying thrombin, comprising an elution step of eluting thrombin from the washed sample.
According to paragraph 1,
The buffer used in the washing step is a 1M sodium chloride buffer with a pH of 6.5 and 20% B compared to a 20mM sodium phosphate buffer.
According to paragraph 2,
The buffer used in the elution step is a 1M sodium chloride buffer with a pH of 6.5 and 50% B compared to a 20mM sodium phosphate buffer.
(a) replacing the thrombin sample converted from prothrombin with 20mM sodium phosphate, pH 6.5, 150mM sodium chloride buffer;
(b) loading the buffer-substituted sample onto a Mixed Mode Chromatography column; and
(c) Step gradient elution (step), which is performed by adding 20mM Tris-Cl, pH 8.0, 0.5M NaCl buffer selected from the range of 20%B to 100%B compared to the 20mM sodium phosphate, pH 6.5, 150mM sodium chloride buffer. A method for purifying thrombin that includes the step of gradient elution,
The step of performing the step gradient elution is
(i) a washing step to separate proteins other than thrombin from the sample loaded on the multiple mode chromatography column; and
(ii) A method for purifying thrombin, comprising an elution step of eluting thrombin from the washed sample.
According to clause 4,
The buffer used in the washing step is a 20mM Tris-Cl, pH 8.0, 0.5M NaCl buffer, which is 60% B compared to a 20mM sodium phosphate, pH 6.5, 150mM sodium chloride buffer.
According to clause 5,
The buffer used in the elution step is a 20mM Tris-Cl, pH 8.0, 0.5M NaCl buffer, which is 100% B compared to a 20mM sodium phosphate, pH 6.5, 150mM sodium chloride buffer. Method for purifying thrombin.
According to any one of claims 4 to 6,
A method for purifying thrombin, wherein the combined mode chromatography has the properties of cation exchange chromatography (CIEX) and hydrophobic interaction chromatography (HIC).