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A strategy of combining prefiltration and chromatography using composite cryogels for large-scale separation of biotransformation compounds from crude high-cell-density broth Junxian Yun, Hao Wu, Jie Liu, Shaochuan Shen, Songhong Zhang, Linhong Xu, Kejian Yao, and Shan-Jing Yao Ind. Eng. Chem. Res., Just Accepted Manuscript • Publication Date (Web): 18 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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A strategy of combining prefiltration and chromatography using

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composite cryogels for large-scale separation of biotransformation

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compounds from crude high-cell-density broth

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Junxian Yun†, Hao Wu†, Jie Liu†, Shaochuan Shen†, Songhong Zhang*†, Linhong Xu‡, Kejian

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Yao†, Shan-jing Yao**§

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†

State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering,

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Zhejiang University of Technology, Hangzhou 310032, China ‡

Faculty of Mechanical & Electronic Information, China University of Geosciences (Wuhan), Wuhan 430074,

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China §

College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

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* Corresponding author 1:

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E-mail address: [email protected] (S.H. Zhang)

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** Corresponding author 2:

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E-mail address: [email protected] (S.-J. Yao).

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ABSTRACT: The separation of interesting biomolecules or compounds from high-cell-density

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suspensions has received intensive attentions in biotechnology industry. In recent years, monolithic

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cryogels have been suggested as a novel class of chromatographic adsorbents for the direct

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separation of biomolecules from such crude microbial feedstocks. However, the preparation of

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large-scale monolithic cryogels for industrial applications and the direct separation of compounds

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from high-cell-density broths in industrial scale are challenge tasks. In this work, a strategy for the

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separation of biotransformation compounds from high-cell-density broths was developed by

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combination of the prefiltration of microbial cells using large-scale cryogels as the filter medium

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and the chromatography followed using large-scale ion-exchange cryogels as the adsorbents.

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Composite cryogel disks (diameter of 143 mm) of poly(2-hydroxyethyl methacrylate) embedded

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with SiO2 nanoparticles (pHEMA−SiO2) and their anion-exchange supports grafted with

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N,N-dimethylaminoethyl methacrylate (pHEMA−SiO2−DMAEMA) were prepared successfully. As

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an example, the separation of cytidine triphosphate (CTP) from a crude high-cell-density broth

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containing 233 g/L Saccharomyces cerevisiae cells was carried out by the prefiltration of cells using

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the cryogel bed packed with the large-scale pHEMA−SiO2 disks followed by the anion-exchange

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chromatography using the packed bed of the large-scale pHEMA−SiO2−DMAEMA cryogel disks.

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The results showed that more than half amount of the yeast cells have been removed successfully

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from the crude broth by the prefiltration using pHEMA−SiO2 cryogel disks, and the CTP with a

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high purity of 98.2% and the recovery of 98.3% were achieved by the anion-exchange

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chromatography using pHEMA−SiO2−DMAEMA cryogel disks from the dissolved feedstock

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filtrated thereafter, indicating that the present strategy is effective and these large-scale composite

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cryogels could be potentially applied as the interesting filter media and chromatography adsorbents

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in industrial biotechnology and downstream processes.

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Keywords: cryogel; bioseparation; filtration; scale-up; cytidine triphosphate.

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1. INTRODUCTION

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The increasing demand for the isolation of interesting molecules from high-cell-density microbial

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fermentation broths and cultures has promoted the investigations of new separation methods and

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novel adsorbents. Monolithic cryogels are one class of such example materials used as the

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adsorbents for the chromatographic separation of compounds from unclarified feedstocks. These

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interesting sponge-like polymer supports have supermacropores with sizes from several to hundreds

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of microns and thus high permeability and porosity, which permit the convective mass transfer of

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compounds and free passage of microbial cells.1–10 Small or lab scale monolithic cryogels can be

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prepared conveniently via cryo-polymerization of aqueous gel-forming monomers or suspensions in

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small columns or molds under freezing conditions, as demonstrated by numerous researchers as

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examples reviewed in references. 1–10 However, the preparation of a monolithic cryogel with large

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diameter and height is difficult since it is needed to rapidly remove a great deal amount of heat from

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the relative big volume of gel-forming solutions or suspensions. Therefore, the direct scale-up of

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monolithic cryogels is a challenge work. Moreover, the large-scale separation of target compounds

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from high-cell-density culture suspensions using monolithic cryogels is also a difficult task.

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In recent years, pHEMA-based or polyacrylamide (pAAm)-based cryogel disks or discs have

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been prepared successfully in glass or metal molds with the diameters from several to more than

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one hundred of millimeters.4,11–14 These disk-form cryogels could be potentially employed in the

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scale-up of monolith cryogels because large-scale beds with an expected height can be prepared by

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packing these disks together in a column with a large diameter.

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CTP is known as an important nucleoside triphosphate and a precursor for RNA synthesis. This

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high-energy compound has received wide applications in biological and therapeutic areas. In

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industrial process, CTP can be produced by microbial synthesis using cytidine monophosphate

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(CMP) as the main substrate and Saccharomyces cerevisiae as the whole-cell catalysis and enzymes

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resource.15–18 In order to ensure the biosynthesis productivity and efficiency, a large amount of yeast

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cells is always used in industrial biotransformation process and thus the cell density in the crude

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broth is usually high. However, the separation of CTP from such unclarified broth with high density

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of cells is complex, and cost intensive. Anion-exchange chromatography using cryogels has been

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suggested as one of promising effective candidate methods for the direct isolation of nucleoside

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triphosphates from crude microbial feedstocks.14,17,19–21 For the high-cell-density suspensions, the

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entrapment or clogging of microbial cells always observed at the entrance of the cryogel beds,

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which could resulted in the decrease of flow rates and thus the complex chromatographic

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performance and unsatisfied separation efficiency.

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In order to perform the chromatography perfectly by cryogels, it is needed to remove some

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microbial cells from the broth to reduce the cell density to a suitable range before the loading.

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Traditionally, the separation of microbial cells from high-cell-density suspensions can be achieved

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by centrifugation or filtration.22–26 Centrifugation employs equipments of centrifuges or

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hydrocyclones to separate the microorganisms from the crude broth by utilizing the difference in

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densities of the cells and the liquid components, and thus usually gives a clarified supernatant. This

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approach is relatively mature in techniques, but suffers some disadvantages like the high cost and

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the requirements of complex equipments and a large amount of energy. Filtration offers an

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alternative effective way for the separation of cells from crude microbial suspensions. It usually

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employs permeable media as the filter material and several filtration methods like the dead-end

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filtration, cross-flow filtration as well as dynamic filtration with rotating disks or drums, and

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rotating or vibrating membranes, as examples in references, 23–26 have been applied in the removal

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of cells. Among them, the dead-end filtration is one of remaining useful tools for the separation of

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cells due to some advantages like the easy operation, low energy consumed and relatively low cost

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needed. In general, the dead-end filtration employs porous materials or membranes typically with

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pore sizes of sub-microns, which are always close to the sizes of microbial cells and thus, the

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filtration performance was influenced by the morphology, size and shape of the cells.23 The

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blockage by the cells and the filter supports themselves lead to a high filtration resistance, and the

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fouling problems in the filtration process. Therefore, it is of great importance to investigate new

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filter materials. Cryogels have porous structures and low flow resistance and thus could be a new

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choice as the novel dead-end filtration matrices. As yet, however, there is a lack of data regarding

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the cell filtration using cryogels and also the chromatographic separation of CTP in a large or

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industrial scale.

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In this paper, a strategy of integrating the prefiltration of microbial cells and the chromatography

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followed using large-scale composite cryogels as the filter media and chromatographic adsorbents

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were proposed for the separation of CTP from a crude high-cell-density yeast broth. Large-scale

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pHEMA−SiO2 cryogel disks for the filtration and anion exchange pHEMA−SiO2−DMAEMA

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cryogel disks for the chromatography were prepared by the cryo-polymerization approach. A

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cryogel bed packed with pHEMA−SiO2 cryogel disks was employed to remove yeast cells from the

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crude feedstock, and another cryogel bed packed with pHEMA−SiO2−DMAEMA disks was used to

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isolate CTP by chromatography from the dissolved feedstock filtrate thereafter. Properties of these

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cryogel disks, the filtration performance as well as the chromatographic performance of the cryogel

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packed beds were investigated experimentally.

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2. MATERIALS AND METHODS

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2.1. Materials. Saccharomyces cerevisiae (CICC 1508) was from China Center of Industrial

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Culture Collection. Ammonium persulfate (APS, 98%), N,N,N’,N’–tetramethylethylenediamine

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(TEMED, 99%), 2-(dimethylamino) ethyl methacrylate (DMAEMA, 98%), poly(ethyleneglycol)

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diacrylate (PEGDA, 99%, Mn~258 g/mol), were from Sigma-Aldrich (Steinheim, Germany).

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Tetraethyl orthosilicate (TEOS, 99%), 2-hydroxyethyl methacrylate (HEMA, 97%) and other

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chemicals were analytical grade from local sources.

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2.2. Preparation of large-scale pHEMA composite cryogel disks embedded with SiO2

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nanoparticles. Monodispersed SiO2 nanoparticles with the mean size of about 364 nm were

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synthesized by two-step hydrolysis and condensation of TEOS in a mixture of ethanol, water and

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ammonia solution.19,27,28 Typically, 2 mL TEOS was added in 27.5 mL solution of ethanol and

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ammonia (10:1, v/v) in 5 min, stirred and maintained at 30℃ for 12 h. The obtained mixture was

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used as the seed and added into 100 mL solution of ethanol and ammonia. Then 7.5 mL TOES was

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added in 55 min, stirred and maintained at 30℃ for 12 h. Finally, SiO2 nanoparticles were obtained

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by centrifuge after washing with ethanol and deionized water.

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The large-scale composite cryogel disks were prepared by cryo-polymerization of the aqueous

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gel-forming mixture containing SiO2 nanoparticles under freezing conditions, as similar as that for

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the preparation of those small-scale monolith cryogels.17,19–21 The preparation of each disk was

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achieved in a stainless steel mold with the inner diameter of 143 mm, the height of 20 mm and the

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wall thickness of 2 mm, as same as that used in our previous work.13,14 The mold was assembled

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together with the top and bottom slabs (thickness of 2 mm). The inlet and outlet tubes with the inner

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diameter of 8 mm were connected to the mold for the injection and discharge of fluid. For the

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preparation of each cell-filtration cryogel disk, about 270 mL the aqueous suspension containing of

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90.8 g HEMA, 27.4 g PEGDA and 1.2 g SiO2 was stirred, pre-cooled to about 4℃, and then 33 mL

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aqueous solution containing 0.47 g APS and 33 mL aqueous solution containing 0.47 g TEMED

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were added. The obtained suspension was pumped into the mold for cryo-polymerized at -15℃ for 8

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h followed by maintaining at about -20℃ for one night. After thawed the cryogel disk was washed

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with enough water for further experiments. The large-scale composite cryogel disks for the

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chromatographic separation of CTP were similar but prepared with the suspensions containing

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gel-forming agents of low concentrations, i.e., the aqueous suspension of the same volume

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containing 38.1 g HEMA, 11.4 g PEGDA, 1 g SiO2, 0.396 g APS and 0.396 g TEMED was used in

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each preparation. The obtained cryogel disks of pHEMA−SiO2 were grafted by 0.5M DMAEMA

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with the initiator solution of 0.056 M K5[Cu(HIO6)2] and 1 M NaCl (2:1, v/v) at the temperature of

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53℃ for 2 h, as similar as those for other large-scale ion-exchange cryogel disks.17,29 The

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pHEMA−SiO2−DMAEMA cryogel disks were obtained by washing the cryogel with 0.1 M HCl

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and deionized water for further use.

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Properties of the pHEMA−SiO2 and pHEMA−SiO2−DMAEMA cryogel disks were characterized

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by measuring the microstructure, water permeability and porosity, according the methods reported

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in references.30–33

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2.3. Filtration of Saccharomyces cerevisiae cells from crude high-cell-density broth by

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large-scale pHEMA−SiO2 cryogel disks. Saccharomyces cerevisiae (CICC 1508) cells grown on

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yeast peptone dextrose (YPD) agar plates (1% yeast extract, 2% peptone, 2% dextrose and 2.0%

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agar) were transferred to YPD medium and the fermentation was achieved at 30℃. When the optical

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density (OD560) of the suspension was about 2.0, the cells were harvested from the broth by

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centrifugation at 6500 rpm for 6 min and washed with 0.9% NaCl solution. In order to enhance the

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passage of endoenzymes and CTP through the cell membrane, the yeast cells were treated by the

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freezing-thawing process, i.e., maintained at about -23℃ for one night and then thawed at room

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temperature. The freezing-thawing process was carried out three times. The obtained cells were

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employed in the biotransformation step.

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About 863 g treated yeast cells were transformed into 3L substrate solution containing 60 mM

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CMP, 100 mM KH2PO4, 12 mM MgSO4 and 150 mM dextrose, and the biotransformation was

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carried out at 35℃ for about 90 min. Then the suspension was heated to 90℃ and maintained for 20

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min to stop the reaction. The obtained suspension was used in the filtration test.

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The large-scale cryogel filtration system used in this work was shown in Figure 1. Five

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pHEMA−SiO2 cryogel disks were packed into a glass cylinder with the inner diameter of 140 mm

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and height of 200 mm. The glass cylinder was connected to a stainless steel vessel with the inner

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diameter of 164 mm and height of 250 mm filled with compressed low-pressure N2 for the supply

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of displacing gas at a constant pressure and a glass vessel for the supply of microbial suspension by

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a peristaltic pump. The filtration of cells was conducted under the constant-pressure mode by

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maintaining the gas pressure at about 0.015±0.005 MPa. About 3900 mL transformed yeast

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suspension was used in the filtration. Typically, the suspension was pumped into the glass cylinder

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and then the valve was switched to introduce N2 into the cylinder to displace the suspension passing

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through the pHEMA−SiO2 cryogel bed at a constant pressure. The effluent samples were collected

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at the time interval of about 1-2 min and the flow rates were measured by the sample volume and

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the time interval. The solid content in each sample was tested by the weight of cells and particulates

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like denatured proteins centrifuged from the sample. The effluent filtrate was collected and used in

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the following chromatographic experiments.

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Figure 1.

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2.4. Chromatographic isolation of CTP from the filtrate by the packed bed of large-scale

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pHEMA−SiO2−DMAEMA disks. The large-scale cryogel bed was prepared by packing

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pHEMA−SiO2−DMAEMA disks into a glass column with the inner diameter of 140 mm. The

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packing procedure was similar as that for polyacrylamide-based cryogel disks in our previous

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work.14 About 280 mL glass beads with diameter of 5 mm were packed on the top of the cryogel

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bed to enhance the uniform fluid distribution at the column inlet. The total bed height was about

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800 mm and the height of glass beads was 18 mm. Chromatographic isolation of CTP from the

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broth filtrate was carried out at a constant flow velocity of 0.80 cm/min for the loading process and

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0.68 cm/min at the wash and elution steps, respectively. The broth was diluted five folds with 0.01

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M HCl and used as the loading feedstock. The cryogel bed was equilibrated with 0.01 M HCl and

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then 9.98 L diluted filtrate broth was loaded onto the cryogel bed. The column was washed with

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0.01 M HCl and eluted with 0.02 M NaCl in 0.01 M HCl followed by 0.1 M NaCl in 0.01 M HCl,

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respectively. The column effluent was recorded over time and collected fractionally for further

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analysis. Column cleaning was performed with 1 M NaCl in 0.01 M HCl. The chromatography

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processes were monitored using a flow-through UV spectrometer at 254 nm by the flow branching

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from the main effluent stream.

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2.5 Analysis. Quantitative analysis of the purity of CTP was performed by HPLC in a 1260

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Infinity equipped with a photodiode array detector (1260 VL) and a ZORBAX SB-C18 column (5

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µm, 4.6 mm×150 mm).19 Mobile phase was the aqueous solution of methanol and 0.6% (v/v)

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phosphate (6:94 v/v, pH 6.6 titrated with triethylamine). The detection was at 271 nm with the flow

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rate of 0.5 mL/min, injection volume of 20 µL, and column temperature of 30℃.

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3. RESULTS AND DISCUSSION

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3.1. Properties of large-scale pHEMA−SiO2 and pHEMA−SiO2−DMAEMA composite

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cryogels. The formation of pHEMA−SiO2 composite cryogel disks in the large-scale stainless steel

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mold follows the same mechanisms as that for the preparation of monolithic cryogels in glass

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columns with small diameters.19 For the filtration application purpose, the pHEMA−SiO2 cryogel

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matrix was prepared by cryo-polymerization of the aqueous gel-forming mixture containing SiO2

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nanoparticles with the monomer and cross-linker weight concentration of 35%, while for the

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chromatographic application the pHEMA−SiO2−DMAEMA cryogel matrix was prepared with the

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aqueous gel-forming mixture with the weight concentration of 15%. In both disk preparations, the

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mass content of SiO2 nanoparticles to the gel-forming agents was maintained at about 2 %, which

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was preferred in the preparation of cryogels with satisfied mechanical strength.19

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Figure 2 shows scanning electron microscopy (SEM) images of a pHEMA−SiO2 cryogel disk for

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the filtration test, while the images of pHEMA−SiO2−DMAEMA disks were not shown here due to

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the same microstructures as those monolithic cryogels with the diameter of 10 mm prepared with

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the same concentration of gel-forming agents in a small-scale column.19 As can be seen, the

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microstructures within the pHEMA−SiO2 cryogel disk were also very similar as those in the

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small-scale pHEMA-based and polyacrylamide-based monolith cryogels.29–38 However, the

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gel-skeleton within the pHEMA−SiO2 cryogel disk prepared with the monomer and cross-linker of

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35% was much thicker than cryogels prepared with those of 15%,19 although the pore sizes seemed

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to be very close in both of these cryogels, indicating that the present disks have strong skeleton and

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thus could be expected to have relatively high mechanical strength for the filtration runs.

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Water permeabilities of pHEMA−SiO2 and pHEMA−SiO2−DMAEMA disks were determined by

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Darcy’s equation based on the data of flow rate vs. pressure drop measured on the beds packing

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with 2 to 4 disks, as similar as those in previous work.13,14 The permeabilities of pHEMA−SiO2 and

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pHEMA−SiO2−DMAEMA disks were 1.21×10-12 m2 and 1.68×10-12 m2, respectively. Porosities of

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these disks were also estimated by measuring the content of free water within a given cryogel disk

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sample at a certain volume. The obtained values of pHEMA−SiO2 and pHEMA−SiO2−DMAEMA

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disks were 52.4% and 73.9%, respectively. The porosity of pHEMA−SiO2−DMAEMA cryogels for

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chromatography was close to those of pHEMA cryogels without embedded nanoparticles prepared

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under similar conditions,13 while the porosity of pHEMA−SiO2 cryogels for filtration was lower

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than those of other pHEMA cryogels. The reason is that the total concentration of monomer and

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cross-linker (about 35%) for the present cryogel disks was higher that those usually used in the

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preparation of pHEMA cryogels (about 12-20%), which consequently resulted in the formation of

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smaller pores within the gels and thus the low porosity.

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Figure 2.

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3.2. Filtration performance of crude high-cell-density broth in the packed bed of large-scale

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pHEMA−SiO2 cryogel disks. The filtration of Saccharomyces cerevisiae from the crude broth was

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carried out in a batch manner. Due to the high cell density of the broth (233 mg/mL), some yeast

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cells were entrapped on the top surface of the cryogel bed to form the cell cake. However, other

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cells moved and passed through the bed into the filtrate bulk without being removed from the broth.

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In order to test if yeast cells in the filtrate could be further removed by the pHEMA−SiO2 cryogel

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bed with the cell cake, the refiltration was performed by employing the filtrate suspension collected

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from the former cycle as the loading broth in the next run. The total runs of three, i.e., Run 1, Run 2

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and Run 3, were conducted in this work. In each run, the filtration was operated in batch manner by

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displacing the suspension at a constant pressure using the gas until the interface of the gas and the

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suspension reached the positions about 6 to 40 mm above the cell cake surface to ensure no gas was

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injected through either the cell cake or the cryogel bed. Then the fresh suspension was supplied by

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the pump and the filtration continued. Three to four times of the suspension supply were used in

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each runs. Figure 3 shows examples of the filtration bed of pHEMA−SiO2 cryogel disks, the

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suspension broth, the cell cake, as well as the morphology of the cryogel disks and their

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cross-sections after the filtration test.

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Figure 3.

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Figure 4 shows the variations of cell content in the filtrate with the increase of the accumulated

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filtrate volume and the filtrate superficial flow velocity through the bed during the whole filtration

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process. As can be seen, the cell content decreased from 233 mg/mL in the crude broth to an

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averaged value of 129 mg/mL in the filtrate after Run 1. The results indicated that about half

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amount of the yeast cells in the crude broth (55%) were entrapped by the pHEMA−SiO2 cryogel

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bed only by one filtration. However, the averaged cell contents in the filtrates after Run 2 and Run 3

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were 122 and 133 g/L, respectively, indicating that there is no obvious enhancement of the cell

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removal efficiency by refiltration of the filtrates through the given cryogel bed. The reason is that

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the porous cell cake structures were formed during the filtration of Run 1. The flow channels or

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pores within the cell cake, similar as those supermacropores in the cryogel bed, were available and

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effective for the passage of most residual yeast cells with the flow of broth during the next two runs,

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and thus very small amounts of cells could be entrapped during Runs 2 and 3.

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From Figure 4, it is also seen that the filtration velocity during the refiltration runs was lower that

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that in Run 1 and decreased with the increase of refiltration cycles. The mean superficial velocities

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of filtrates observed in Run 1, Run 2 and Run 3 were 1.50, 0.96 and 0.58 cm/min, respectively,

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indicating that the filtration productivity decreased. In fact, the suspension contained cells and

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particulates produced in the heat treatment procedure after the biotransformation. During the

273

refiltration process, the migration of cells and these tiny solids within the pores of both the cell cake

274

and the cryogel bed could further decrease the effective flow space and increase the filtration

275

resistance probably due to the partial blocking or the redistribution of these particulates, and thus

276

caused the decrease of the flow rates.

277

Further increasing the filtration pressure (> 0.025 MPa) induced the collapse of the cell cake and

278

the redistribution as well as the passage of those particulates through the cryogel bed. Therefore, a

279

rather low linear velocity or low pressure should be used to avoid collapse of the filtrate cake. The

280

suitable filtration pressure was below 0.02 MPa in the present work. The filtrate from the above

281

filtration procedures was settled for about three hours and then sedimentation of additional amounts

282

of cells occurred, which resulted in the cell content of about 20 mg/mL in the final filtrate. It should

283

be noted that the agglomerates of those tiny solids were enhanced due to the interactions among the

284

yeast cells and other particulates during the breakthrough of the suspensions through the cryogel

285

bed. Therefore, the cell agglomeration gave further contributions to the cell separation from the

286

suspension bulk following the gravitational settling mechanisms and thus reduced the final cell

287

content in the filtrate bulk. It is also noted that the cell cake can be regenerated easily by the

288

counter-current washing for the re-use purpose. Therefore, these cryogels are reusable and the

289

regeneration procedure is easy.

290

Figure 4.

14



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Page 16 of 32

291 292

3.3. Separation of CTP by the packed bed of large-scale pHEMA−SiO2−DMAEMA cryogel

293

disks. By the above filtration and sedimentation, the filtrate contained 20 mg/mL cells, 6.94 mg/mL

294

CTP, 1.57 mg/mL CDP, 0.61 mg/mL CMP and 0.59 mg/mL other impurities. In order to decrease

295

the

296

pHEMA−SiO2−DMAEMA cryogel, the filtrate broth was diluted five folds with 0.01 M HCl and

297

then used as the feedstock. The loading broth had the electro conductivity of 7.88 mS/cm and pH

298

2.6. The cell content of the loading broth was low (about 4 mg/mL) and during breakthrough and

299

wash processes those yeast cells had passed freely through the chromatographic bed without

300

blocking due to the supermacropores in the pHEMA−SiO2−DMAEMA disks. In our previous work,

301

the elution of CTP from small scale anion exchange cryogels has been carried out successfully

302

using the 0.02 M NaCl in 0.01 M HCl (elution 1) followed by either 0.5 M NaCl in 0.01 M HCl

303

(elution 2) for the polyacrylamide-DMAEMA cryogel (bed diameter of 16 mm) or 0.1 M NaCl in

304

0.01 M HCl (elution 2) for the pHEMA−SiO2−DMAEMA cryogel (bed diameter of 10 mm).17,19

305

Therefore, for the present large scale chromatography the elution strategy used in the small scale

306

pHEMA−SiO2−DMAEMA cryogel was employed directly.

concentration

of

ions

to

ensure

the

effective

adsorption

of

CTP

by

the

307

In the present chromatography, 864 samples were collected from the loading to the elution 2, i.e.,

308

47 samples at the loading (the volume from 0 to 9.98 L), 171 samples at the wash (9.98 to 35.18 L),

309

266 samples at elution 1 (35.18 to 60.53 L) and 380 samples at elution 2 (60.53 to 105.89 L),

310

respectively. Another 57 samples during the cleaning procedure were also collected. The

311

concentrations and purities of AMP, ADP and CTP in these effluent samples were analyzed by

15


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312

HPLC and the results are shown in Figure 5. Some examples of the detailed HPLC profiles of the

313

loading broth and the effluent samples during the loading, the wash and the elution stages are also

314

shown in Figure 6, in which A271 denotes the recorded signals at 271 nm. From these figures, it is

315

seen that even there are several compounds in the loading broth (Figure 6(a)), the main compound

316

in the effluent during the loading stage was CMP (Figures 5(a, b) and 6(b)) and its concentration

317

reached 0.03 mg/mL, while the maximal concentration of CDP was very low, i.e., 0.002 mg/mL

318

(Figure 5(a)). It should be noted that during the loading stage the effluent contained 67.3% to 87.8%

319

of CMP, 2.7% to 4.1% of CDP and 12.2% to 30% of other impurities, but no CTP (Figure 5(b)),

320

indicating that most of CDP and CTP were adsorbed or remained within the bed. The reason is that

321

the volume ratio of the loaded filtrate broth to the cryogel bed was low (0.81 in this work) and thus

322

the main broth stream did not move through the whole bed. During the wash stage, the unbound

323

CMP and CDP were washed out the bed and thus the breakthrough of CMP and CDP were

324

observed. The maximal of the concentrations of CMP and CDP were 0.091 and 0.16 mg/mL

325

(Figure 5(a)), respectively, which were higher than those in the loading procedure. It is also seen

326

that most amounts of CMP passed through the cryogel bed during the loading and wash steps

327

without being bound (Figures 5(a, b) and 6(b, c)) and the concentrations of CMP in the elution

328

stages were very low (

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