This article was downloaded by: [UNAM Ciudad Universitaria]On: 20 December 2014, At: 14:53Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Chemical Engineering CommunicationsPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gcec20

ENRICHMENT AND PURIFICATION OFBITESPIRAMYCIN USING MACROPOROUSRESINJie Xu a , Jiawen Zhu a , Kui Chen a , Yanyang Wu a & Jiji Gu aa Chemical Engineering Research Centre, East China University ofScience and Technology , Shanghai , ChinaPublished online: 12 Jul 2012.

To cite this article: Jie Xu , Jiawen Zhu , Kui Chen , Yanyang Wu & Jiji Gu (2012) ENRICHMENTAND PURIFICATION OF BITESPIRAMYCIN USING MACROPOROUS RESIN, Chemical EngineeringCommunications, 199:10, 1320-1333, DOI: 10.1080/00986445.2012.682322

To link to this article: http://dx.doi.org/10.1080/00986445.2012.682322

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Enrichment and Purification of BitespiramycinUsing Macroporous Resin

JIE XU, JIAWEN ZHU, KUI CHEN, YANYANG WU,AND JIJI GU

Chemical Engineering Research Centre, East China University of Scienceand Technology, Shanghai, China

Bitespiramycin was purified by selective adsorption. The adsorption and desorptionproperties of bitespiramycin on six different macroporous resins (HZ806, HZ816,HZ820, HZ830, XAD16, and SP207) were compared systematically. Accordingto the adsorption capacity and selectivity towards 400-O-isovalerylspiramycin,HZ820 was chosen as the most suitable resin for bitespiramycin purification. Theequilibrium data on HZ820 in a batch system were well described by Langmuirand Freundlich models. The film and pore diffusion model was successfully measuredin batch adsorption kinetics. Dynamic adsorption and desorption experiments werealso performed using a packed column of HZ820 to optimize the separation processof 400-O-isovalerylspiramycin from aqueous solution. After being treated withHZ820, the 400-O-isovalerylspiramycin content increased from 80.4% to 91.7%,and the 400-O-isovalerylspiramycin III content increased from 41.7% to 64.4%.

Keywords Adsorption; Bitespiramycin; Desorption; Macroporous resins

Introduction

Bitespiramycin is a new macrolide antibiotic (Shang et al., 1999; Wang et al., 2003)produced by genetically engineered Streptomyces spiramyceticus transformed withthe 400-O-acyltransferase gene from S. mycarofaciens. Bitespiramycin is a new geneti-cally engineered non-natural antibiotic. There are no similar products on the dom-estic or overseas market. It will not bring resistant bacteria along with it orproduce complete cross-resistant bacteria with other medicines. The level of toxicityis low. With high lipophilicity, it will be absorbed quickly and diffused in the bodysystem strongly and widely if taken orally. A difference from similar products (suchas macrolide antibiotics like azithromycin, and erythromycin) is its minimum inhibi-tory concentration (MIC) value of mycoplasma, chlamydia of 0.064 ug=mL, which isless than that of the macrolide antibiotics. In vitro measured activity is 64 timeshigher than that of erythromycin, it suffers from less resistance, and it has a widerantimicrobial spectrum. In addition, its oral absolute bioavailability averaged91.6%; LD50 is greater than 4500mg=kg with low toxicity.

Address correspondence to Jiawen Zhu, Chemical Engineering Research Centre, EastChina University of Science and Technology, Shanghai 200237, China. E-mail: jwzhu@ecust.edu.cn

Chem. Eng. Comm., 199:1320–1333, 2012Copyright # Taylor & Francis Group, LLCISSN: 0098-6445 print=1563-5201 onlineDOI: 10.1080/00986445.2012.682322

1320

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

Bitespiramycin is a mixture of acetylspiramycin, propionylspiramycin, isobutyr-ylspiramycin, and isovalerylspiramycin, which possess similar molecular structures.The chemical structures of these acylated spiramycins are shown in Figure 1 andTable I. According to the structure-activity relationship of bitespiramycin (Rakhitand Singh, 1974; Wang, 1997), its biological activity is related to the number ofcarbon atoms of the 400 acyl group. Macrolides made by genetically engineeredS. spiramyceticus transformed with the 400-O-acyltransferase gene from S. mycaro-faciens have higher bioactivities and cause more enhanced lipotropy than the freeacid. Their biological activities have the following order: isovaleryl> butyryl>propionyl> acetyl> free. Thus, it is important to separate and purify 400-O-isovaler-ylspiramycin I, II, and III from filtered fermentation broth.

A conventional separation method for bitespiramycin is solvent extraction. You(2007) studied the solvent extraction of bitespiramycin in detail. In general, the yieldof solvent extraction is lower than 30% (the yield of adsorptive separation is about50%). The product quality was unstable. Alternatively, adsorptive separations have

Figure 1. Chemical structures included in bitespiramycin.

Table I. Types and structures of bitespiramycin

Chemical name R1 R2

Spiramycin II COCH3 HSpiramycin III COCH2CH3 H400-O-acetylspiramycin II COCH3 COCH3

400-O-acetlyspiramycin III COCH2CH3 COCH3

400-O-propionylspiramycin III COCH2CH3 COCH2CH3

400-O-isobutyrylspiramycin II COCH3 COCH(CH3)2400-O-isovalerylspiramycin I H COCH2CH(CH3)2400-O-isovalerylspiramycin II COCH3 COCH2CH(CH3)2400-O-isovalerylspiramycin III COCH2CH3 COCH2CH(CH3)2

Enrichment and Purification of Bitespiramycin 1321

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

had widespread use in the purification of antibiotics and other small molecules. Forexample, macroporous resins have been successfully used in the separation ofadipoyl-7-amino-3-deacetoxycephalosporanic acid from fermentation broth. Twoother examples include the separation and purification of erythromycin A from itsisomers and the separation of cephalosporin C (Xie et al., 2001; Sun, 2009; Li,2007). In particular, spiramycin has been successfully purified using macroporousresin (Gu and Ren, 1991). Bitespiramycin fermentation liquors contain more than20 components with similar chemical structures, which makes separation difficult.To our knowledge, there have been no previous reports using macroporous resinto separate and purify bitespiramycin. In this article, an efficient method of separat-ing bitespiramycin from aqueous solution and enriching 400-O-isovalerylspiramycinis described.

Methods

Chemicals and Reagents

Bitespiramycin was provided by Tonglian Pharmaceutical Co., Ltd. (Shanghai,China). The contents of 400-O-isovalerylspiramycin I, II, and III were 12.6%,27.8%, and 41.6%, respectively. Distilled water was provided by the ShanghaiTonglian Research Institute (Shanghai, China). Methanol was of high-performanceliquid chromatography (HPLC grade), and other chemicals were of analytical grade.All solutions were buffered with 0.2M phosphate hydrate (pH 6.0). The pH of bite-spiramycin fermentation approaches 6.0. Therefore, in this study, the samples wereall in this condition. A further study will investigate the effects of pH in detail.

Adsorbents

Macroporous resins, including HZ806, HZ816, HZ820, and HZ830, were providedby Huazhen Co., Ltd. (Shanghai, China), XAD-16 was purchased from Rohmand Haas (USA), and SP207 was purchased from Organo Mitsubishi Chemicals(Japan). The resins were soaked in 95% ethanol, shaken for 24 h, and then washedwith acetone, ethanol, and distilled water, alternately. Finally, they were dried at313K and kept in desiccators for use.

Batch Adsorption Equilibration

Static adsorption experiments of bitespiramycin on macroporous resins were per-formed as follows: 0.5 g samples of resins together with 100mL of 1.2mg=mL buf-fered bitespiramycin solution were added to a flask and shaken (220 rpm) for 20 hat 293K. All visual experiments were repeated three times for accuracy.

The adsorbed amounts of bitespiramycin, qe, were calculated from:

qe ¼ ðC0 � CeÞ � V=W ð1Þ

where qe is the equilibrium solid phase concentration (mass solute=mass sorbent); C0

and Ce are the initial and equilibrium concentrations of the liquid phase (masssolute=volume solution), respectively; V is the volume of the liquid phase (volume);and W is the weight of the adsorbent (mass).

1322 J. Xu et al.

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

Resin selectivity is defined as the difference in adsorption capacities for differentcomponents. The selectivity value was obtained from:

K ¼ Cother=qother � q=C ð2Þ

where K is the selectivity of 400-O-isovalerylspiramycin; q and C are the equilibriumconcentrations of 400-O-isovalerylspiramycin in the solid and liquid phase, respect-ively (mass solute=mass sorbent and mass solute=volume solution); and qother andCother are the equilibrium concentrations of other components except for 400-O-isova-lerylspiramycin in the solid and liquid phase, respectively (mass solute=mass sorbentand mass solute=volume solution).

Equilibrium adsorption isotherm experiments on the selected resin were conduc-ted by contacting 100mL of sample solutions at different concentrations (0.40–1.20 g=L) with 0.5 g of the resin, and shaking it for 20 h at 293, 303, and 313K,respectively.

In adsorption of biological compound research, the adsorption isotherms areoften described by the Langmuir model or the Freundlich model. As the originalassumptions of the Langmuir isotherm were used in the derivation, each site foradsorption is equivalent in terms of adsorption energy, and there are no interactionsbetween adjacent adsorbed molecules. Once all the adsorption sites are filled, nofurther adsorption can occur (Sun et al., 2008). The Langmuir model can beexpressed by the following mathematical formula:

qe ¼ qmCe=ðKd þ CeÞ ð3Þ

Here, Kd is an experimentally determined equilibrium constant for the adsorp-tion process.

The Freundlich model is an empirical equation. The Freundlich equation isexpressed by:

qe ¼ KfC1=ne ð4Þ

where Kf and n are the Freundlich constants; Kf is an indicator of the adsorptioncapacity; and the exponent n is dimensionless and relates to the magnitude of theadsorption driving force. Favorable adsorption corresponds to values of n less than1, while a value of n greater than 1 indicates unfavorable adsorption (Jia and Lu,2008; Du and Yuan, 2007; Gao et al., 2007).

Kinetics of Batch Adsorption

The rate of adsorption was determined by analyzing the bitespiramycin solution con-centration in a batch system over time. The experiments were initiated by adding0.25 g of adsorbent to 100mL of 1.20 g=L buffered bitespiramycin solution at 293,303, and 313K, respectively. The adsorption vessel was agitated at 220 rpm in athermostatic shaker.

The adsorption process will lead us to three basic approaches related to thediffusion inside the adsorbent particle: (a) solid phase diffusion, (b) pore diffusion,and (c) a combination of both. In this work, the film and pore diffusion model

Enrichment and Purification of Bitespiramycin 1323

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

(Otero et al., 2005; Ramos et al., 2004; Li et al., 2003; Costa and Rodrigues, 1985)was chosen to represent the adsorption on the macroporous resin.

The film and pore diffusion model uses the following assumptions:

The adsorption process is isothermal,The adsorption process is represented by a equilibrium isotherm given by q¼ f(Cp, i)

at the pore wall interface,The adsorbent is made of a porous material into which the solute must diffuse in a

manner described by effective pore diffusivity Dp,Nass transfer to the surface of the adsorbent is governed by a film model character-

ized by a mass transfer coefficient kf,Surface diffusion in which the adsorbate moves directly between adsorption sites

without interim desorption into the liquid phase occurs at a negligible rate,and a term to describe this process is unnecessary,

The adsorption particles are spherical, with uniform size and density.

The mass balance equation inside the particle is given by

ð1� epÞ@q

@tþ ep

@Cp;i

@t� epDp

1

R2

@

@RR2 @Cp;i

@R

� �� �¼ 0 ð5Þ

where Cp, i is the solute concentration in the particle pores, q is the adsorbed phaseconcentration, R is the particle radial coordinate, and t is the time variable. At anyparticle radial position R, the adsorption equilibrium isotherm q¼ f(Cp, i) holds.

For adsorption in a stirred tank, the rate of change of the bulk concentration isgiven by:

dCL;i

dt¼ � 3nkf

Rp;iV

� �ðCL;i � Cp;iÞjR¼Rp;i

ð6Þ

With the boundary and initial conditions:

t ¼ 0;CP;i ¼ CP;ið0;R;ZÞ ð7Þ

R ¼ 0;@Cp;i

@t¼ 0 ð8Þ

R ¼ Rp;i@Cp;i

@R¼ ki

epDpðCL;i � Cp;iÞjR¼Rp;i

ð9Þ

Herein, an orthogonal collocation on finite elements method was used to discre-tize the partial spatial derivates (Equation (5)). Then, the partial differential equa-tions were converted to a system of ordinary differential equations. The ordinarydifferential equations were solved by MATLAB 7.

Dynamic Adsorption and Desorption

According to the static experiment results, the proper resin was selected for thedynamic adsorption and desorption experiments. A certain amount of adsorbent(8.0 g) was poured into the packed chromatography column (1 cm ID) at room

1324 J. Xu et al.

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

temperature. The resin depth was 15 cm. The chromatography column was equili-brated with a buffer solution (pH 6.0). The buffered solution containing bitespiramy-cin was then pumped into the bed at one of several flow rates (1.2 bed volume (BV)=h,1.8 BV=h, and 2.4 BV=h) with a BT00-300T constant-flow pump (Lange Constant-Flow Pump Corp., China) for a sufficient period to give at least 100% breakthrough.After reaching adsorptive saturation, the column was eluted with organic solvent(n-hexane, petroleum ether, ethyl acetate, acetone, or methanol). The volume ofdesorption solutions was 6BV. The desorption solutions were analyzed by HPLC(Agilent, 1200 series).

Analytical Methods

The concentration of bitespiramycin was determined using a 752(s) UVultraviolet-visible spectrophotometer (Lengguang Tech Co., Ltd., Shanghai, China)at a wavelength of 483 nm, according to the sulfuric acid color development reactionmethod (You et al., 2008; Wu et al., 1998).

Bitespiramycin samples were analyzed by HPLC. Chromatographic separationwas carried out using a Hypersil BDS-C18 reversed-phase column (4.6� 250mm,5 mm, Elite). The mobile phase consisted of 44% (v=v) phosphate buffer (0.5%,w=w) and 56% (v=v) methanol. The flow rate of the mobile phase was 1.0mL=min.The chromatograms were examined at 231 nm.

Percent Recovery of Bitespiramycin

The percent recovery of bitespiramycin was expressed by the following equation:

Y ð%Þ ¼ Cd � Vd

ðC0 � CaÞ � Va� 100 ð10Þ

where Y is the percent recovery of bitespiramycin (%); Ca is the concentration ofbitespiramycin in the liquid phase percolation through the column (mass=volume);Va is the volume of liquid phase percolation through the column; Cd is the concen-tration of bitespiramycin in the selected eluent (mass=volume); and Vd is the volumeof the selected eluent.

Selectivity

Ki, selectivity in the desorption processes, compares the contents of the object inproducts and feed solution according to the following formula:

Ki ¼ai

ð1� aiÞ� ð1� ai0Þ

ai0ð11Þ

where ai and ai0 are the contents of 400-O-isovalerylspiramycin or 400-O-isovalerylspir-amycin III in eluent and feed solution, respectively.

Enrichment and Purification of Bitespiramycin 1325

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

Results and Discussion

Adsorption Capacities and Selectivity

As shown in Table II, HZ820 had the highest adsorption capacity of bitespiramycin,while HZ806 showed the highest selectivity adsorption capacity of 400-O-isovaleryl-spiramycin, among the six resins studied.

Acetylspiramcin, propionylspiramycin, isobutyrylspiramycin, and isovalerylspir-amycin are all low polar adsorbates. Their polarities have the following order: free>acetyl> pripionyl> butyryl> isovaleryl.

The adsorption capacity and selectivity of an adsorbent are affected by its sur-face area, pore volume, average pore diameter, and polarity (Shi and Shi, 2008). Thephysical properties of the resins studied are summarized in Table III. The resinHZ820 had the largest surface area and a relatively larger pore volume comparedto the other macroporous resins, which is favorable for the mass transfer process.HZ806 is a weakly polar resin, while the others are all nonpolar resins; therefore,its adsorption capacity was lower than the others. There were no obvious differencesin selectivities among the six types of resins studied.

According to the adsorption capacity and selectivity of 400-O-isovalerylspiramycindata, HZ820 was a suitable resin to separate bitespiramycin and purify 400-O-isovaler-ylspiramycin. Therefore, HZ820 was used for further experiments.

Adsorption Isotherms

The experimental adsorption data are shown as Figure 2. The estimated para-meters are summarized in Table IV and show that the correlation coefficients of

Table II. Effects of resins on adsorption of bitespiramycin

Adsorbent

HZ806 HZ816 HZ820 HZ830 XAD16 SP207

Adsorption capacity(Kg=Kg)

0.0576 0.1397 0.1923 0.1332 0.1721 0.1391

K 1.29 1.13 1.12 1.22 1.03 1.04

Table III. Physical properties of the test macroporous resins

Tradename Polarity

Particlediameter (mm)

Surface area(m2=g)

Average porediameter ðAoÞ

Pore volume(mL=g)

HZ806 weak-polar 250 600 90 1.30HZ816 nonpolar 380 950 86 1.60HZ820 nonpolar 380 950 150 1.72HZ830 nonpolar 380 800 200 1.80XAD16 nonpolar 700 800 150 1.82SP207 nonpolar 600 600 110 1.30

1326 J. Xu et al.

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

the Langmuir and Freundlich models were both high. The correlation coefficientsof the Langmuir model were higher than those of the Freundlich model, because ingeneral the adsorption energy on nonpolar polymeric sorbents is homogeneous(Kondo et al., 2006). The adsorption capacity was enhanced as the temperatureincreased from 293 to 313K. The dissociation constant Kd decreased with increas-ing temperature, which indicates that rising temperature benefits the adsorptionprocess. Therefore, the adsorption of bitespiramycin on HZ820 is a thermonega-tive process. This is consistent with the rules of solvent replacement (Geng,1998). Adsorption of solute is accompanied by desorption of solvent. When bite-spiramycin binds to the adsorbent, more water molecules are desorbed from theadsorbent due to the relatively larger size of bitespiramycin molecules, while thedesorption of water molecules is an endothermic process. Temperature increaseis beneficial for adsorption. However, a too high temperature affects the stabilityof fermentation and increases energy consumption in the production process.Therefore, the operation temperature should be not too large a deviation fromroom temperature.

The Freundlich model provides that values of n are less than 1. This means thatadsorption can take place easily. Thus, the adsorption process between bitespiramy-cin and HZ820 resin is a favorable process.

Figure 2. Langmuir adsorption isotherm for bitespiramycin on HZ820 at 293, 303, and 313K.

Table IV. Estimated parameters for Langmuir and Freundlich isotherm models

Langmuir Freundlich

Temperature (K) qm(g=g) Kd R2 Kf 1=n R2

293 0.1588 0.0519 0.9879 0.1779 0.2581 0.9784303 0.1701 0.0354 0.9720 0.1973 0.2438 0.9508313 0.1831 0.0302 0.9511 0.2106 0.2106 0.9336

Enrichment and Purification of Bitespiramycin 1327

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

Adsorption Kinetics

The bitespiramycin adsorption rate data are depicted in Figure 3. The adsorptionamount increased with contact time. In the first 240min, the adsorption amountincreased sharply, and after 240min, a steady-state approximation emerged. In thepharmaceutical industry, it is essential to determine the sorbent contact time foroptimal manufacturing efficiency.

Figure 3 contains plots of the simulation results and the experimental curves.Table V lists the best fit values for the external fluid-phase transport mass transfercoefficient kf and the effective particle diffusion coefficient Dp. The bitespiramycinabsorption profile in the thermostatic shaker was modeled using the equilibrium con-stants derived from the Langmuir isotherm. The fit between the film and porediffusion model and experiment data is excellent.

The liquid film mass transfer coefficient kf and the effective particle diffusioncoefficient Dp increased with increasing temperature. This result indicates that risingtemperature benefits the adsorption process. And the rate-limiting step may be intra-particle diffusion (Dp<< kf).

Dynamic Breakthrough Curve on HZ820 Resin

HZ820 was chosen as the optimal packed absorbent. Here, the flow rate was con-sidered in the kinetics experiments. The concentration of feed solution was

Figure 3. Time evolution of bitespiramycin adsorption on HZ820.

Table V. Estimated parameters for the model fitted to theuptake of bitespiramycin versus time

T (K)

Parameters 293 303 313

kf=(10�6m � s�1) 1.2086 1.7261 1.9505

DP=(10�11m2 � s�1) 2.7298 3.1601 3.7429

1328 J. Xu et al.

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

1.2mg=mL, similar to the concentration during fermentation. The ratio of the heightof the packed adsorbent to the inner diameter of the chromatographic column wasset to 15 to meet the piston flow conditions (Quinones and Guiochon, 1998). Theflow rates were 1.2, 1.8, and 2.4 BV=h, respectively.

The experimental curves are shown in Figure 4. The adsorption performance ofbitespiramycin indicates that varying the flow rate from 1.2 to 2.4 BV=h makesalmost no difference to particle diffusion. Therefore, 2.4 BV=h was chosen as theflow rate for further experiments for experiment efficiency.

Elution Process

After adsorptive saturation, several organic solvents were screened for use in thedesorption experiments in order to select a proper eluent. The desorption flow ratewas 1.2 BV=h and the volume of eluent was 6BV. The elution process results usingvarious eluents are shown in Table VI. As shown in Table VI, polarity is a key factor

Figure 4. Dynamic breakthrough curves of bitespiramycin using HZ820 in fixed beds atvarious flow rates.

Table VI. Results of elution process using various eluents

Eleunt

n-HexanePetroleum

etherEthylacetate Acetone Methanol

Polarity 0 0.01 4.3 5.4 6.4Content of 400-O-isovalerylspiramycin (%)

91.4 91.7 83.8 86.2 85.1

Content of 400-O-isovalerylspiramycin III (%)

66.1 64.4 48.8 49.5 44.9

Yield of bitespiramycin (%) 57 75 92 94 100

Enrichment and Purification of Bitespiramycin 1329

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

affecting selectivity and percent recovery. As polarity decreased, the selectivity of400-O-isovalerylspiramycin increased, while the recovery decreased. Thus, consideringthe product quality and recovery rate, petroleum ether was selected as the appropri-ate eluent in further experiments.

Dynamic Desorption Curve on HZ820 Resin

Petroleum ether was used to elute bitespiramycin. The following flow rates wereinvestigated: 1.2, 1.8, and 2.4 BV=h. During the elution process, fractions were

Figure 5. Dynamic desorption curves of bitespiramycin on HZ820 at various flow rates.

Figure 6. Dynamic desorption curve and selectivity on HZ820 using petroleum ether as theeluent. The instantaneous bitespiramycin concentration in eluent (the elution curve of bitespir-amycin) is plotted in the left y axis, and the selectivities (Ki) are plotted in the right yy axis.

1330 J. Xu et al.

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

collected every 0.5 BV. By analyzing the mass and contents of the collected fractions,the dynamic desorption curve could be acquired.

The desorption curves are shown in Figure 5. The lower desorption flow rateproduced more concentrated products, so desorption at a low rate is much moreefficient than desorption at a high rate when the same volume of eluent is used(Ramos et al., 2004). Thus, 1.2 BV=h was selected as the lowest desorption flow rate.

The elution and selectivity curves at 1.2 BV=h are shown in Figure 6. The instan-taneous bitespiramycin concentration in eluent (the elution curve of bitespiramycin)is plotted in the left y axis, and the selectivities (Ki) are plotted in the right yy axis.The elution curve appears to reach its peak the earliest among the three curves. Thismay indicate that different components have different retention times on HZ820 dur-ing the desorption process due to the different physical and thermodynamic charac-teristics of each component (Gano et al., 2007).

When the adsorption and desorption processes were complete, the 400-O-isova-lerylspiramycin purity and 400-O-isovalerylspiramycin III purity increased sharply(Table VII). In Table VII, the yield (w=w, %) is equal to the mass of product beforeadsorption or desorption divided by the mass of product after adsorption or desorp-tion, and the content of each component (w=w, %) is the mass percent of calculatedcomponent in the sample. The yield and the content of each component are both for

Table VII. Product quality and yield in adsorption and desorption

Initialproduce

Produce afteradsorption

Produce afterdesorption

Content of 400-O-isovalerylspiramycin (%) 80.4 85.0� 91.7Content of 400-O-isovalerylspiramycin III (%) 41.7 46.2� 64.4Yield of 400-O-isovalerylspiramycin (%) — 74.3 81.4Yield of 400-O-isovalerylspiramycin III (%) — 70.3 94.2Y of bitespirmaycin (%) — 70.7 75.0

�The contents of 400-O-isovalerylspiramycin and 400-O-isovalerylspiramycin III after adsorp-tion were calculated by material balance.

Figure 7. Chromatogram of sample solution before separation on a column packed withHZ820. (Figure provided in color online.)

Enrichment and Purification of Bitespiramycin 1331

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

each step and are not cumulative. During the maximum selectivity, the purities of400-O-isovalerylspiramycin and 400-O-isovalerylspiramycin III reached 95.9% and69.1%, respectively. The chromatograms of sample solution before (Figure 7) andafter (Figure 8) treatment are displayed.

Conclusion

The adsorption capacities and selectivities of six different macroporous resinswere investigated by static adsorption of bitespiramycin. HZ820 was chosen as theappropriate adsorbent for further experiments. The bitespiramycin adsorptionsystem obeyed the film and pore diffusion model for the entire adsorption periodand thus supported the assumption that the rate-limiting step may be intraparticlediffusion. The observed profiles suggested a good fit of the experimental data toboth the Freundlich and Langmuir models. Furthermore, the optimal flow ratewas determined via a series of dynamic adsorption and desorption experiments.After enrichment and purification using a packed column of HZ820, the contentof 400-O-isovalerylspiramycin obviously increased.

Acknowledgments

This work was supported by the Science and Technology Commission of ShanghaiMunicipality (Program No. 10431901500).

References

Costa, C., and Rodrigues, A. E. (1985). Intraparticle diffusion of phenol in macroreticularadsorbents: Modeling and experimental study of batch and CSTR adsorbers, Chem.Eng. Sci., 40, 983–993.

Du, X. L., and Yuan, Q. P. (2007). Comparison of general rate model with a new model—artificial neural network model in describing chromatographic kinetics of solanesoladsorption in packed column by macroporous resins, J. Chromatogr. A, 1145(1), 165–174.

Figure 8. Chromatogram of sample solution after separation on a column packed withHZ820. (Figure provided in color online.)

1332 J. Xu et al.

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014

Gano, T., Offringa, N., and Willson, R. C. (2007). The effectiveness of three multi-componentbinding models in describing the binary competitive equilibrium adsorption of twocytochrome b5 mutants, J. Chromatogr. A, 1144(2), 197–202.

Gao, M., Huang, W., and Liu, C.-Z. (2007). Separation of scutellarin from crude extracts ofErigeron breviscapus (vant.) Hand. Mazz. by macroporous resins, J. Chromatogr. B,858(1), 22–26.

Geng, X. P. (1998). Study on the fractions of thermodynamic function changes for bothadsorption and desorption from a liquid-solid system, Thermochim. Acta, 308(1–2),131–139.

Gu, J. F., and Ren, A. H. (1991). Studies on extraction of spiramycin by macroreticularadsorbent H-103, Chin. J. Antibiotics, 16(2), 98–102.

Jia, G. T., and Lu, X. Y. (2008). Enrichment and purification of madecassoide and asiaticosidefromCentella asiatica extracts withmacroporous resins, J. Chromatogr. A, 1193(1), 136–141.

Kondo, S., Ishikawa, T., and Abe, I. (2006). Adsorption Science, Chemical Industry Press,Beijing, China.

Li, J. (2007). Study on the Process of the Resin Separation of Cephalosporin C Filtration,thesis, Beijing University of Chemical Technology, Beijing, China.

Li, P., Xiu, G. O., and Rodrigues, A. E. (2003). Modeling separation of proteins by inert coresorbent in a batch adsorber, Chem Eng Sci., 58, 3361–3371.

Otero, M., Zabkova, M., and Rodrigues, A. E. (2005). Comparative study of the adsorption ofphenol and salycilic acid from aqueous solutions onto nonionic polymeric resins, Sep.Purif. Technol., 45(2), 86–95.

Quinones, I., andGuiochon, G. (1998). Extension of a Jovanovic-Freundlich isothermmodel tomuticomponent adsorption on heterogeneous surfaces, J. Chromatogr. A, 796(1), 15–40.

Rakhit, S., and Singh, K. (1974). Stucture activity relationship in sixteen membered macrolideantibiotics, J. Antibiot., 27(3), 221–224.

Ramos, A., Otero, M., and Rodrigues, A. E. (2004). Recovery of vitamin B12 andcephalosporin-C from aqueous solutions by adsorption on non-ionic polymeric adsor-bents, Sep. Purif. Technol., 38(1), 85–98.

Shang, G., Dai, L., and Wang, Y. G. (1999). Construction of a stable bioengineered strain ofbiotechmycin, Chin. J. Biotechnol., 15(2), 105–111.

Shi, Z. Q., and Shi, R. F. (2008). Adsorption Separation Resin in the Study of PharmaceuticalIndustry, Chemical Industry Press, Beijing, China.

Sun, Y. (2009). Study on separation of erythromycin A and erythromycin C with chromato-graphy method, thesis, East China University of Science and Technology, Shanghai,China.

Sun, Y., Zhu, J. W., Chen, K., Zhu, S., and Xu, J. (2008). Mass transfer mechanisms infixed-bed adsorption of erythromycin, Frontiers Chem. Eng. China, 2(4), 353–360.

Wang, Y. G. (1997). Genetic engineering technology in the macrolide antibiotics, World NotesAntibiot., 18(2), 92–98.

Wang, Y. G., Dai, J. L., and Shang, G. D. (2003). Genetic engineering of bitespiramycin strainof Streptomyces WSJ-195, Chinese Patent C.N. 1,405,299.

Wu, J. M., Chen, K., Sun, X. F., Wang, D. Y., and Zhao, Y. B. (1998). Fluorimetric deter-mination of acetylspiramycin by color reaction of sulfuric acid, J. Xiamen Univ. (Nat.Sci.), 37(6), 892–896.

Xie, Y., Van de Sandt, E., de Weerd, T., and Wang, N.-H. L. (2001). Purification of adipoyl-7-amino-3-deacetoxycephalosporanic acid from fermentation broth using stepwise elutionwith a synergistically adsorbed modulator, J. Chromatogr. A, 908(1), 273–291.

You, X. (2007). Separation and purification of bitespiramycin by solution extraction, thesis,East China University of Science and Technology, Shanghai, China.

You, X., Chen, K., Zhu, J. W., and Guo, X. B. (2008). Effect of temperature and pH value onthe stability of bitespiramycin in the process of separation and purification, Chin. J.Pharm., 39(1), 12–14.

Enrichment and Purification of Bitespiramycin 1333

Dow

nloa

ded

by [

UN

AM

Ciu

dad

Uni

vers

itari

a] a

t 14:

53 2

0 D

ecem

ber

2014