ASSESSMENT OF ION EXCHANGE CHROMATOGRAPHY …

www.wjpr.net Vol 4, Issue 07, 2015.

1101

Priya et al. World Journal of Pharmaceutical Research

ASSESSMENT OF ION EXCHANGE CHROMATOGRAPHY

PARAMETERS FOR DESIGNING AN OPTIMUM PURIFICATION

PROFILE OF L-ASPARAGINASE FROM PSEUDOMONAS

FLUORESCENS

Hrishikesh Mungia, Rohan Dighe

b and V.G.Shanmuga Priya

a*

aDepartment of Biotechnology, KLE Dr M S Sheshgiri College of Engineering and

Technology, Belgaum (India).

bSerum Institute of India Limited Pune(India).

ABSTRACT

Purification is a vital step in the downstream processing of any

industrially important compound. The purification profile must be

designed by considering the efficacy of each step along with

economics involved. The current study is based on designing an

optimum purification profile for L-asparaginase, comprising

ammonium sulphate fractionation, diafiltration and ion exchange

chromatography. Maximum enzyme recovery in the first step was

observed at 60% saturation of ammonium sulphate. The removal of

ammonium ions and concentration of the sample was carried out using

a 30 kDa poly ether sulphone diafiltration membrane. The optimization

of purification profile was mainly concentrated on assessment of static

and dynamic binding capacity of Seralite SRC 120 ion exchange resin

to L-asparaginase. Optimum adsorption of enzyme to resin was

observed at pH 4 within 70 minutes. The adsorption pattern was best explained by Langmuir

isotherm. The adsorbed enzyme was eluted by using 0.2 M NaCl. The analysis of Height

Equivalent to Theoretical Plates (HETP) shows 77% column performance, indicating the set

parameters are optimum for performance of the column.

KEYWORDS: L-asparaginase, Purification, optimization, ion exchange chromatography,

adsorption, HETP.

World Journal of Pharmaceutical Research SJIF Impact Factor 5.990

Volume 4, Issue 7, 1101-1114. Research Article ISSN 2277– 7105

Article Received on

30 April 2015,

Revised on 25 May 2015,

Accepted on 14 June 2015

*Correspondence for

Author

V.G.Shanmuga Priya

Department of

Biotechnology, KLE Dr

M S Sheshgiri College of

Engineering and

Technology, Belgaum

(India).


1102


INTRODUCTION

The enzyme L-asparaginase finds immense applications in cancer therapeutics and has

shown promise for Acute Lymphoblastic Leukemia (ALL) treatment in children.[1]

Tumor

cells are unable to synthesize asparagine, thus increasing the asparagine requirement of

leukemic cells and consequently their dependency on circulating asparagine,[2]

L-

asparaginase transforms L-asparagine into L-aspartate and ammonia via a covalently bound

intermediate involving a β-aspartyl enzyme.[3]

This deprives the leukemic cell of circulating

asparagine, leading to cell death. A number of microorganisms have been studied for

production of L-asparaginase including Escherichia coli,[4]

Serratia marcescens,[5]

Enterobacter cloacae,[6]

Pseudomonas stutzer,[7]

and Erwinia sp.[8]

Downstream purification of proteins involves a capture step, resulting the removal of

impurities and in concentration of product. Ion-exchange chromatography has proved to be a

very important tool in downstream purification,[9]

The popularity of ion-exchange

chromatography is due to its widespread applicability, high resolving power, high capacity,

simplicity and ease of control in preservation of biological activity during processing,[10]

These could be achieved by studying the relationship between the physicochemical properties

of preparative ion-exchange resins and chromatographic protein retention, thus offering a

potential to guide for selection process in adsorbent design.[11]

The design of an optimum purification profile for L-asparaginase comprises ammonium

precipitation, removal of salt, filtration and concentration of loading sample. These steps are

equally important as they help in reducing the impurity level in the sample carried on to the

ion-exchange column. It also helps in eliminating smaller molecules like peptides and other

proteins. It is to be ensured that while designing the overall purification profile the enzyme

stability and activity are maintained by selecting optimum conditions with respect to pH,

temperature and concentration of ions.

In this study, a purification profile is designed in accordance with the stability of enzyme.

The profile is focused on optimization of chromatography parameters by evaluation of pH,

adsorption time, adsorption isotherm, static binding capacity, HETP parameters and dynamic

binding capacity. Thus, the study helps in estimating and increasing the efficiency of the

matrix used for purification.


1103


MATERIALS AND METHODS

Microbial strains: Pseudomonas fluorescens NCIM2639 was acquired from National

Collection of Industrial Microorganisms (NCIM), Pune, India, and maintained on nutrient

agar slants (pH 7.4).

Biochemical assay of L-asparaginase

L-Asparaginase activity was determined by the method of Shirfrin et a, [12]

In brief, 0.1 ml

of 189 mM L-asparagine was added to 1.0 ml of 50 M Tris-hydrochloride buffer (pH 7.4), the

volume was made up to 2 ml by distilled water. The reaction was quenched after 30 min by

the addition of 100 ul of 1.5M trichloroacetic acid (TCA). 200 ul supernatant was diluted in

deionized water, followed by the addition of 0.5 ml Nessler’s reagent to a total volume of 5

ml. The liberated ammonia was determined spectrophotometrically at 436 nm. The

concentrations of ammonia were determined from a standard curve with ammonium sulfate

as the source of dissolved ammonia. 1 I.U (International Unit) of L-Asparaginase is equal to

1 µmole of ammonia liberated from L-Asparagine per minute.

Production and recovery of L-asparaginase enzyme

The optimum production of L-asparaginase from Pseudomonas fluorescens was carried by

using optimized production condition as described in an earlier study.[13]

The cell-free

supernatant from the culture was tested for enzyme activity and protein concentration. The

supernatant was further subjected for ammonium sulphate precipitation.

Ammonium sulphate precipitation

Finely powdered ammonium sulphate was added to the cell-free supernatant till 20%

saturation.. After 2 h, the mixture was centrifuged and the supernatant was brought to 40%

saturation of ammonium sulphate.. The step was repeated for 60, 80 and 100% saturation.

Precipitates obtained were dissolved in 10 mM Phosphate buffer pH7.

Stability of Crude enzyme

The stability of enzyme activity was examined by incubating crude enzyme in the pH range

2-10 for 24 h. The enzyme activity was estimated at every 4 h interval.

Removal of salt and Concentration of protein

Microfiltration


1104


The solution obtained after ammonium sulphate precipitation was pretreated to remove

impurities and prevent clogging of Diafiltration membrane. The sample was subjected to

micro-filtration using a micro-filtration assembly with membrane pore size of 0.45 μ m. The

enzyme activity, volume and protein content were analyzed for the filtrate after micro-

filtration.

Diafiltration

An polyether sulphone ultra filtration assembly with a membrane molecular weight cut off of

30 kDa was selected. Cross flow filtration technique was applied for salt removal and protein

concentration. The removal of ammonium ions was confirmed by using monograph

method.[14]

The enzyme activity, volume and protein content were analyzed for the retentate

after diafiltration.

Optimization of Ion exchange chromatography

L-Asparaginase isolated from Pseudomonas stutzeri has a pI of 6.38.[7]

Hence a cation

exchange chromatography was used for the adsorptive purification of L-Asparaginase from

the crude fermentation broth subjected to ammonium precipitation and diafiltration. Based on

economics Seralite SRC-120 ion exchange resin was selected for study.

Batch Studies

Batch studies were carried out to determine the adsorption equilibrium time, nature of

adsorption isotherms, static binding capacity of the resin and the optimal binding and elution

conditions. This data helps in carrying out the purification in column mode with efficiency.

Optimization of binding pH

Sodium acetate buffers (10 mM, pH 3-6) were used to optimize the protein binding to the

resin. 1 ml of resin suspension equilibrated in each pH was added to 9 ml of diluted

diafiltered sample and incubated at 25°C for 2.5 h at 100 rpm. The equilibrium samples were

immediately assayed and the partition coefficients were calculated by using equation 1 as

given by Manera et al,[15]

f = Q/C …… (1)

where f = partition co-efficient, Q and C are enzyme equilibrium activities at equilibrium of

adsorbed and free enzyme respectively. Optimum pH for adsorption was determined using a

graph of partition co-efficient v/s pH,


1105


Determination of adsorption equilibrium time

The adsorption equilibrium time was determined as discussed by Makino et al,[16]

3ml of

resin suspension equilibrated in 10 mM sodium acetate buffer pH 4 was mixed with 27 ml

diafiltered enzyme solution with two different initial L-asparaginase concentrations, 24.8

U/ml and 10.1 U/ml. The mixtures were incubated at 25°C for 2.5 h at 100 rpm. The aliquots

from each mixture were assayed every 10 min. Equilibrium time was estimated from a graph

of ratio of final concentration to initial concentration v/s time was plotted.

Adsorption isotherm studies

To determine the adsorption isotherm models and the parameters that best fit the process,

enzyme binding to resin was measured at different enzyme concentrations.. Initial and final

enzyme activities were measured. The isotherm indicates the nature of adsorption and was

determined by plotting a graph of Q vs C.

Optimization of elution conditions

The elution of bound L-Asparaginase was carried out by varying the ionic strength of the

elution buffer. 1 ml of resin suspension equilibrated with 0.01 M Sodium acetate buffer at pH

4 was added to 9 ml of clarified fermentation broth containing L-Asparaginase and incubated

for 10 minutes. Supernatant from each tube was discarded and the resin was washed with

wash buffer. The supernatant was then discarded, and 5 ml of elution buffer containing

varying concentrations of NaCl from 0.2 to 1 M was added to each of the tube. The

supernatant was then assayed for L-Asparaginase activity and total protein concentration.

Column studies

HETP and Dynamic binding capacity analysis

The selected resin Seralite SRC-120 was packed in column with a bed height of 10 ± 0.2 cm.

The column was equilibrated with 15 column volume of 0.01 M Sodium acetate buffer pH 4.

Concentrated diafiltered sample was loaded on the column and the column effluent fractions

were collected at a flow rate of 1.0 ml/min. L-Asparaginase activity was analyzed (U/ml) for

all the fractions . A breakthrough curve was plotted as effluent enzyme activity (U/ml) vs.

cumulative volume (ml) for enzyme adsorption. The dynamic capacity of resin and column

performance was determined by evaluation of height equivalent to theoretical plate (HETP)

value. The HETP analyses were carried by using breakthrough data. A comparative analysis

was carried out by comparing the theoretical HETP with actual HETP data to give a clear

picture on column efficiency.


1106


Purification of L-asparaginase on column

The Seralite SRC-120 was packed in column with bed height of 10 ± 0.5 cm. The column

was equilibrated with 15 column volumes of 0.01 M Sodium acetate equilibration buffer at

pH 4. Concentrated diafiltered sample with a volume of 20% breakthrough volume was

loaded. The column was washed with 3 column volumes of 0.01 M Sodium acetate buffer at

pH 4 to remove any unbound proteins. The elution of bound L-Asparaginase was carried out

using the selected elution buffer with optimum molarity of salt concentration. The elution

fractions were collected at a flow rate of 1 ml/min. Collected fractions were assayed for L-

Asparaginase activity and total concentration of protein.

RESULTS AND DISCUSSION

Production and recovery of L-asparaginase enzyme

During the production of L-asparaginase enzyme from Pseudomonas fluorescens, a

maximum enzyme activity of 206 U/ml was recovered from the culture supernatant The

maximum recovery of enzyme activity was obtained at 60-80% of ammonium sulphate

precipitation, with 1.5 fold increase in activity and with a purification factor of 4.41 (Table

1).

Table1: L-asparaginase enzyme recovery from fermented broth by fractional

ammonium sulphate precipitation.

The validation of ammonium sulphate precipitation was carried out by direct use of

ammonium sulphate to cause direct precipitation instead of fractional precipitation. The

enzyme recovery is as shown in Table 2.The direct precipitation helped in better enzyme

recovery. 71.1% of enzyme was recovered from crude fermented broth by direct increase in

ammonium salts concentration to 60% as compared to 31% of enzyme recovery obtained by

40-60% by individual fractional increase in ammonium salt concentration.

(NH4)2SO4

fractionation

ml

Enzyme

activity

(U/ml)

Total

Activity

U

Protein

mg/ml

Total

Protein

mg/ml

Specific

activity

U/mg

Purification

factor

Recovery

%

Crude 0 % 50 206.63 10331.5 0.76 38 271.88 1 100

0-20 % ------- ----- ----- ------ -------- ------- -------

20-40 % 10 210 2100 0.62 6.2 338.7 1.24 20.32 40-60 % 10 240.2 2402 0.43 4.3 558.6 2.05 23.24 60-80 % 10 290.2 2902 0.39 3.9 744.1 2.85 28.08


1107


Table2: L-asparaginase enzyme recovery from fermented broth by direct increase in

concentration of ammonium sulphate precipitation.

Stability of Crude enzyme activity

The stability of enzyme activity for 24 h in various pH conditions is as shown in Fig. 1. The

enzyme activity is highly stable between pH 5-6. The enzyme activity was unstable at pH 2

and 9. There is a gradual decrease in activity over time period when incubated in pH 3. The

decrease in activity is more rapid in pH 8 as compared to pH 3.

Figure1: Stability of enzyme activity in varying pH incubated for a time interval of

24hrs

Removal of ammonium ions and Concentration of protein

Diafiltration

The ammonium precipitated solution was subjected to microfiltration in order to prevent

clogging of diafiltration membrane. The filtrated sample was further subjected to

diafiltration. As a result there is an increase in purification factor and recovery of enzyme

(Table 3). Diafiltration increased the recovery of enzyme by 1.27 fold and the purification

factor by 1.45 fold. The total removal of ammonium ions was observed after 7 cycles of

diafiltration as confirmed by monograph method.

(NH4)2SO4

fractionation

ml

Enzyme

activity

(U/ml)

Total

Activity

U

Protein

mg/ml

Total

Protein

mg/ml

Specific

activity

U/mg

Purification

factor

Recovery

%

Crude 0 % 50 206.63 10331.5 0.96 48 215.23 1 100

0-80 % 20 280.63 5612.6 0.56 5.6 1002.25 4.65 54.32


1108


Table3: Assessment of enzyme activity and protein concentration after removal of

ammonium ions by filtration techniques

Fraction ml Enzyme

activity U/ ml

Total

activity U

Protein

mg/ml

Total

protein mg

Specific

activityU/mg

Purification

factor

Recovery

%

Micro filtered

broth 20 277.63 5552.6 0.55 5.5 1009.56 4.69 53.74

UF Retentate 5 1418.5 7092.5 1.21 4.84 1465.39 6.81 68.49

Optimization of Ion exchange chromatography parameters by batch studies

Optimization of binding pH

The assessment of enzyme activity for unbound enzyme to the resin was used to estimate the

partition co-efficient. The partition co-efficient indicates the efficacy of resin at a particular

pH. Fig. 2 shows the binding pattern of enzyme to resin with respect to pH. The maximum

binding was observed at pH 4.

Figure2: Binding efficacy of Seralite SRC-120 at different pH

Determination of adsorption equilibrium time

The equilibrium time assists in studying the time taken by the enzyme to bind to the matrix.

The data will help to set the flow parameters during column studies giving sufficient time for

binding of enzyme. The enzyme took 70 min to reach adsorption equilibrium (Fig. 3). . The

data indicates the equilibrium time is independent of concentration, and also provides basis

for carrying out adsorption isotherm studies.


1109


Figure 3: Adsorption equilibrium of Seralite SRC-120 for varying enzyme

concentration.

Adsorption isotherm studies

The isotherm studies were carried out in order to know the binding efficiency of L-

asparaginase to Seralite SRC-120. One of the bases for adsorption is the equilibrium

distribution of L-asparaginase between two phase’s i.e. Seralite SRC-120 and buffer. This

equilibrium is represented as an equation called isotherm. The enzyme analyses were carried

out to understand the nature of isotherms. From Fig. 4 it can be interpreted that the nature of

isotherm belongs to Type I adsorption isotherm and the nature was best explained by

Langmuir isotherm (Equation 2).

Adsorption isotherm equation

Q= Qmax.C/ [Kd + C]…….. (2)

Where Kd = Langmuir isotherm constant. Qmax= maximum matrix binding capacity. The

maximum adsorption capacity and Kd was determined by plotting a graph of 1/Q Vs 1/C (Fig.

5). The maximum capacity of the matrix (Qmax) for L-asparaginase was found to be 142.85

U/ml with a Kd of 7.42. The value of Kd indicates that 7-8 molecules of enzyme adhere to one

molecule of matrix.

Figure4: Langmuir isotherm for L-asparaginase


1110


Figure5: Linearization of Langmuir isotherm for L-asparaginase

Optimization of elution conditions

Of the different concentrations of NaCl tested for L-asparaginase elution, it was observed that

a concentration of 0.2 M NaCl in 0.01 N Sodium acetate buffer at pH 4, gave the maximum

elution of bound L-asparaginase. 54.92% enzyme was recovered with a purification factor of

1.79 (Table 4).

Table4: Optimization of elution conditions for L-asparaginase adsorbed on Seralite

SRC120

Sample Total

Enzyme (U)

Protein

(mg)

Specific activity

(U/mg) Purification factor

% Enzyme

recovery

Bound 113 1.63 69.32 1 100

0.1 M 42.2 0.60 70.33 1.01 37.34

0.2 M 62.06 0.50 124.12 1.79 54.92

0.3 M 50.12 0.44 113.9 1.64 44.35

0.4 M 48.29 0.49 98.55 1.42 43.29

0.5 M 46.08 0.61 75.54 1.09 40.77

COLUMN STUDIES

HETP and Dynamic binding capacity analysis

Breakthrough curve is important in chromatography for studying the effects of mass transfer

during loading and for measuring the dynamic capacity of the column. Thus, a breakthrough

curve gives an estimate of the height equivalent to theoretical plates (HETP) and the overall

mass transfer from the mobile phase to the adsorbent phase under real operating conditions.

The Enzyme breakthrough cure is as shown in Fig. 6. The evaluation HETP analysis is based

on the following data and equations:


1111


Height of resin bed (L) =10cm, Radius of column (r) =0.5cm, Volume of resin VC =15.71cm2,

Initial concentration (Co) =211U/ml, Area under Curve (AUC) =

(Fig. 6).

Total enzyme loaded during breakthrough (W)= Ci.(VE – VB)………. (3)

Enzyme adsorbed during breakthrough (Wa) = (W- AUC)…………... (4)

Fraction of CE adsorbed f = (Wa/W)… ……………………………....(5)

Length of adsorption zone ( La)= L.(VE-VB)/ [VE-(1-f).(VE – VB )]…. (6)

Number of transfer unit (NTU)= 16.[VR / (VE-VB)]2…………………… (7)

Height equivalent to theoretical plate (HETP) = La/ N…………………. (8)

Utilization of Theoretical Efficiency UTE%= (Htheoretical / Hactual) 100%... (9)

1. Estimation of theoretical HETP

a. Exhaust volume VE= 0.95.Co=22.95ml.

b. Break through volume VB=0.05.Co=14.45 ml

c. Retention volume VR=0.5.Co=18.45 ml.

Substituting the above values in equation 3-8, we acquire Htheoretical=0.055cm and

NTUtheoretical=83.63.

2. Estimation of actual HETP

From graph (Fig. 6)

a. Exhaust volume VE= 24ml.

b. Break through volume VB=13 ml

c. Retention volume VR= (VE+ VB)/2=18.5 ml.

Substituting the above values in equation 3-8, we acquire Hactual=0.071cm and

NTUactual=67.6.

Number of theoretical plates is a measure of column efficiency (number of theoretical plates

per meter of chromatographic bed) under specified experimental conditions. The more the

number of plates over a certain length, the lower is HETP and higher is the efficiency of the

system. The actual HETP and NTU value estimated was found to be in comparison with

theoretical value for enzyme adsorption, suggesting a good performance of the ion exchange

resin. As a comparison of actual Vs theoretical column performance on a percentage basis,

UTE was estimated using equation 9. It was estimated around 77% and generally varies


1112


between 60 and 90 percent, indicating closeness of actual column performance to the

theoretical prospective. The dynamic capacity was estimated using equation 10.

DBC= CoVB/VC………….. (10)

The DBC was estimated to 174.60 U/ml is high as compared to static capacity for the resin,

the reasons for which could be appropriate volumetric flow rate (1.0 ml/min), good packing

of the column and void fraction as explained by Van Deemter equation.

…….. (11)

Where A, B, and C represent axial dispersion, molecular diffusion, and non equilibrium

effects, viz., solid-liquid film mass transfer, effective intra-particle diffusion and adsorptive

effects, respectively whereas u is the superficial velocity or flow rate m/s.

Figure6: Enzyme break through curve for HETP and dynamic binding analysis. AUC:

Area under the curve represented by the following equation

Purification of L-asparaginase on column

Column purification was operated by setting conditions and parameters of column based on

the above acquired data. The profile yielded a purification of 79.65% of L-asparaginase with

a fold purity of 2.2 (Table 5).

Table5: Purification of L-asparaginase on Seralite SRC-120

Sample Total Enzyme

(U)

Total Protein

(mg)

Specific activity

(U/mg)

Fold

purity

%

Recovery

Diafiltered 546 9.58 57 1 100

IEC 434.9 4.25 90.56 1.58 79.65


1113


CONCLUSION

The study is based on evaluation and optimization of each step involved in purification of

enzyme. The optimized process for recovery of L-asparaginase from fermented broth was

carried by precipitation of enzyme by using 60% saturation of ammonium sulphate.

Diafiltration was used to remove salts and concentrate the enzyme with minimal losses, . The

optimization of purification profile was focused on assessment of operating parameters for

ion exchange chromatography. Optimum adsorption of enzyme to resin was observed at pH

4 within 70 min. The adsorption pattern was best explained by Langmuir isotherm. The

isotherm estimated a static binding capacity of 142.85 U/ml. The adsorbed enzyme was

eluted by using 0.2 M NaCl. The Dynamic Binding Capacity was estimated to 174.60 U/ml

and is high as compared to static binding capacity for the resin. Also, the analysis of Height

Equivalent to Theoretical Plates (HETP) shows 77% column performance, indicating the set

parameters are optimum for performance of column. The column purification was operated

by setting conditions and parameters of column based on the above acquired data. The profile

yielded a purification of 79.65% of L-asparaginase enzyme from the diafiltered sample.

Hence, an optimum purification profile for recovery of L-asparaginase from fermented broth

was established.

REFERENCES

1. Marshall Shannon A, Lazar Greg A, Chirino Arthur J and Desjarlais John R. Rational

design and engineering of therapeutic proteins. Drug Discovery Today, 2003; 8(5): 212-

221.

2. Broome J. D. L-Asparaginase: Discovery and development as a tumor-inhibitory

agent. Cancer treatment reports, 1981; 65(4): 111–114.

3. Bessoumy Ashraf A., Sarhan Mohamed and Mansour Jehan. Production, Isolation, and

Purification of L-Asparaginase from Pseudomonas Aeruginosa 50071 Using Solid-state

Fermentation. Journal of Biochemistry and Molecular Biology, 2004; 37(4): 387-39.

4. Derst C, Wehner A, Specht V and Rohm K. H. States and functions of tyrosine residues

in Escherichia coli asparaginase. European Journal of Biochemistry, 1994; 224: 533-540.

5. Bernard Heinemann and Alma J. Howard. Production of Tumor-Inhibitory L-

Asparaginase by Submerged Growth of Serratia marcescens. Applied microbiology,

1969; 18(4): 550-554.


1114


6. Nawaz M S, Zhang D, Khan A A and Cerniglia C E. Isolation and characterization of

Enterobacter cloacae capable of metabolizing asparagines. Applied Microbiology and

Biotechnology, 1998; 50: 568-572.

7. Manna S, Sinha A, Sadhukhan R and Chakrabarty SL. Purification, characterization and

antitumor activity of L –asparaginase isolated from Pseudomonas stutzeri. Current

Microbiology, 1995; 30: 291-298.

8. Kotzia Georgia A, Lappa Katerina and Labrou Nikolaos E. Tailoring structure–function

properties of L-asparaginase: engineering resistance to trypsin cleavage. Biochemical

Journal, 2007; 404: 337–343.

9. Bo-Lennart Johansson, Makonnen Belew, Stefan Eriksson,Gunnar Glad, Ola Lind, Jean-

Luc Maloisel1, Nils Norrman. Preparation and characterization of prototypes for multi-

modal separation media aimed for capture of negatively charged biomolecules at high salt

conditions. Journal of Chromatography A, 2003; 1016: 21–33.

10. Arne Stabya, Maj-Britt Sanda, Ronni G. Hansenb, Jan H. Jacobsenc, Line A. Andersend,

Michael Gerstenbergd, Ulla K. Bruusa, Inge Holm Jensena. Comparison of

chromatographic ion-exchange resins IV. Strong and weak cation-exchange resins and

heparin resins. Journal of Chromatography A, 2005; 1069: 65–77.

11. Peter DePhillips and Abraham M. Lenhoff. Relative retention of the fibroblast growth

factors FGF-1 and FGF-2 on strong cation-exchange sorbents. Journal of

Chromatography A, 2004; 1036: 51–60.

12. Shirfrin S, Parrott C L, and Luborsky S W. Enzymatic Assay of L-asparaginase. Journal

of Biological Chemistry, 1974; 249: 1335-1340.

13. Hrishikesh Mungi, Ritika Carvalho, Shruti Ilegar,G.M.Ratnamala, and V.G.Shanmuga

Priya. Optimization of L-asparaginase production form Pseudomonas fluorescens by

Response Surface Methodology. International Journal of Current Microbiology and

Applied Sciences, 2014; 3(11): 350-362.

14. Council of Europe -2004. European Pharmacopoeia 5.0: Vol-2.

15. Manera, A. P, Kamimura, E. S, Brites, L. M. and Kalil, S. J. Adsorption of

amyloglucosidase from Aspergillus niger NRRL 3122 using ion exchange resin. Brazilian

Archives of Biology and Biotechnology, 2008; 51(5): 1015.

16. Y. Makino, P. S. C. Lima, F. M. Filho, M. I. Rodrigues. Adsorption of the inulinase from

Kluyveromyces marxianus NRRL Y-7571 on Streamline® DEAE resin. Brazilian Journal

of Chemical Engineering, 2005; 22(12): 539 – 545.

ASSESSMENT OF ION EXCHANGE CHROMATOGRAPHY …

Documents

Transcript of ASSESSMENT OF ION EXCHANGE CHROMATOGRAPHY …