Appl Biochem Biotechnol (2012) 167:20682075 DOI 10.

1007/s12010-012-9750-4

Improvement in the Purification Process of the Capsular Polysaccharide from Haemophilus influenzae Type b by Using Tangential Ultrafiltration and Diafiltration
Silvia Maria Ferreira Albani & Mateus Ribeiro da Silva & Mickie Takagi & Joaquin Cabrera-Crespo

Received: 2 February 2012 / Accepted: 22 May 2012 / Published online: 5 June 2012 # Springer Science+Business Media, LLC 2012

Abstract Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122263 and 294480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP 100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up. Keywords Haemophilus influenzae serotype b . Cocamidopropyl betaine . Sodium deoxycholate . Tangential ultrafiltration . Enzymatic hydrolysis

Introduction Haemophilus influenzae type b (Hib), a Gram-negative bacterium, is responsible for infectious diseases such as meningitis and pneumonia in children under the age of 2 [1]. Capsular polysaccharide type b (PSb) produced by this bacterium is considered the virulence factor during invasive infections since it confers increased resistance to host defenses, and used as antigen in the vaccine formulation; however, the natural response for polysaccharide is poor in infants and does not induce long-term T-cell memory. On the other hand, PSb attached chemically to a carrier protein shows to be immunogenic and induces a long-term T-cell memory and its potential as protective immunogenic has moved efforts to improvement of the polysaccharide purification method [2]. The classical procedure used for PSb purification, mainly those described in patents, is based on treatment with cationic detergent, phenol extraction (toxic), and several selective ethanol precipitation which result in laborious, costly, and complex process [36]. For an
S. M. F. Albani : M. R. da Silva : M. Takagi (*) : J. Cabrera-Crespo Instituto ButantanCentro de Biotecnologia, Av. Vital Brasil, 1500, 05503-900 So Paulo, Brazil e-mail: mtakagi@butantan.gov.br

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affordable Hib conjugate vaccine available for the public health system, to attend lowincome countries, the downstream strategy is to recover and purify the PSb to achieve specifications according to World Health Organization (WHO), maximizing yield and minimizing process cost [7, 8]. Our group has successfully developed a simple purification method appropriate to several polysaccharides derived from different pathogenic microorganisms using tangential ultrafiltration, ethanol precipitation, and hydrolytic enzymes digestion [911]. Comparing to the classical method, the use of harsh chemical as phenol was eliminated and ethanol precipitation steps were reduced to two. In this study, a strategy for improving the quality level of purified capsular polysaccharide produced by H. influenzae type b was established, introducing tangential ultrafiltration, detergent treatment, ethanol precipitations steps and extensive enzymatic hydrolysis.

Material and Methods Strain H. influenzae type b strain GB3291 was provided by Brazilian National Center of Meningitis, Adolfo Lutz Institute, Department of Bacteriology, So Paulo, Brazil. The working seed was stored at 70 C [12]. Polysaccharide Production Experiments were carried out in bioreactors Bioflo 2000 (New Brunswick Scientific Co., USA) with 6.5 l. The main cultivation parameters were: pH value controlled 7.5 with NaOH 5 M, temperature 37 C, air supply 1.0 vvm (volume of air per minute per volume of medium), agitation 200800 rpm, and dissolved oxygen tension controlled at 30 % of air saturation. Two different polysaccharide production strategies were considered to ensure robustness in the purification process: batch culture with pulses of glucose (1) and (2), and fed-batch culture with a constant feeding solution rate of 150 ml/h (3) [12, 13]. Polysaccharide Purification The culture broth was centrifuged for the cell separation, and the supernatant was submitted to tangential flow ultrafiltration as described by Takagi et al. [10] with some modifications. The spiral membrane of polyethersulfone of 100 kDa nominal molecular weight cutoff membrane (NMWC) with 0.54 m2 area was used for washing the concentrated sample carried out with six volumes of the following buffers: (1) 25 mM TrisHCl pH 7.5, 150 mM NaCl, 0.1 % (v/v) cocamidopropyl betaine; (2) 25 mM TrisHCl pH 7.5, 0.3 % (w/v) deoxycholate, 2 mM EDTA; and (3) 25 mM TrisHCl pH 7.5; this fraction was named first concentration by tangential ultrafiltration1CTUF100. The concentrated fraction 1CTUF100 was precipitated with two cuts of ethanol, 30 and 80 % (v/v), resulting in fractions soluble EtOH30 and insoluble precipitated 80 % (fraction not quantified), respectively [10]. The polysaccharide was solubilized from the precipitated 80 % fraction with deionized water and the insoluble impurities removed by centrifugation at 17,696g for 1 h at 4 C; this fraction was named as the water soluble. The pH of the water-soluble polysaccharide fraction was adjusted to 7.5 with TrisHCl buffer containing 2 mM MgCl2 and 20 mM NaCl and submitted to the following enzymes

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treatment: endonuclease (Benzonase EC 3.1.30.2Sigma)One unit per milligram of nucleic acid was added and the mixture incubated at 37 C for 2 h under 100 rpm shaking. Afterwards, trypsin type I protease (EC 3.4.21.4Sigma), protease from Aspergillus oryzae type II (EC 3.1.27.3Sigma), and protease from S. griseus type XVI (EC 3.4.24.3Sigma) were sequentially added considering 1 IU/100 mg of protein impurities and the mixture incubated. The activity time for each protease was established according to each activity intervals; enzymes showing greater specific activity acted for 4 h (trypsin and S. griseus type XVI) and enzymes with lesser activity acted for 16 h (A. oryzae type II). The enzymatic hydrolyses were followed by the same preset diafiltration conditions used for the first concentration in the 100-kDa NMWC membrane (50 cm2 Biomax, Lab Scale; Millipore, Bedford, MA, USA), thereby generating the Purified PSbEnz+ 2CTUF100 fraction (Fig. 1). Analytical Procedures The PSb was measured by the modified Bial method using ribose as the standard [14]. The protein (Prt) concentration was measured by the Lowrys method. The nucleic acid (NA) concentration was determined by the absorbance (A) readings at 260 nm [7]. The lipopolysaccharide (LPS), also called endotoxins or pyrogens, present in Gram-negative bacteria causes very grave side effects, and it has to be removed from injectable pharmaceutical products. LPS has a specific sugar, 2-keto-3-deoxyoctanate (KDO), that can be measured by the Osborns method [15]. The PSb purification was analyzed considering the following parameters: concentration of PSb was given in milligrams of PSb per liter of culture broth; recovery of PSb (in percent) concentration of PSb on purification step/concentration of PSb on the supernatant fraction 100; relative purity (RP)milligrams of PSb per milligrams of each impurities; and purification factor (PF)RPstep/RPSupernatant of each impurities, where Prt, NA, and LPS (as KDO) were considered impurities. Purification factor indicates how many times the purity of the PSb was increased in relation to the initial purity of supernatant.
Culture Broth
Washing buffer:
1) Tris-HCl/ NaCl/cocoamidopropyl betaine - (6vol) 2) Tris-HCl/deoxycholate/EDTA - (6vol) 3) Tris-HCl - (6vol)

Supernatant
Retentate
Ppt Cells Ultrafiltrate Culture Medium

CENTRIFUGE FERMENTOR

1CTUF100
PREP-SCALE SPIRAL 0.54 m2 100 KDA NMWC 1) Tris-HCl/ NaCl/cocoamidopropyl betaine - (6vol)

Washing buffer: 2) Tris-HCl/deoxycholate/EDTA - (6vol) H2O

3) Tris-HCl - (6vol) 4) Water (6v)

Etanol 30% 80%

Precipitate 80%

Water soluble

CENTRIFUGE

CENTRIFUGE

CENTRIFUGE

UF 100kDa

Ppt

Spr

Ppt

EtOH 30%

EtOH 80%

Solubilization in water

Enzymatic treatment

LAB-SCALE CASSETTES 150

cm2 100 KDA NMWC

Enz+ 2CTUF100 (Purified PSb)

Fig. 1 Flow diagram of PSb purification. The fractions considered in the purification table are highlighted in bold

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Results and Discussion Polysaccharide produced by H. influenzae b is a linear heteropolymer made of repetitive units of D-ribitol-(1-1)- D-ribose-3-phosphate, hydrophilic, negatively charged, polydisperse, and an extracellular polymer with high molecular mass (0.302106 kDa), resistant to nucleases and proteases. The purification strategy is usually based on the differences in the physicochemical properties between PSb and its impurities: (1) Proteins have different and not disperse molecular mass, however most of them present in the microorganism are less than 150 kDa and sensitive to proteases. (2) Nucleic acids can display low and high molecular mass and can be sensitive to shear stress, enzymatic hydrolysis by nucleases, and can be precipitated with ethanol. (3) LPS is the major component of outer membrane of Gram-negative bacteria, consisting of a lipid and polysaccharide joined by a covalent bond. In aqueous solutions, LPS can form micelles and/or vesicles of high molecular mass. The hydrophobic interactions in the lipid region can be broken with detergent cocamidopropyl betaine and sodium deoxycholate. On the other hand, EDTA chelates the divalent ions present in the polysaccharide moiety of the LPS; it became unstable, and the combination of both effects generates monomers with low molecular mass 1020 kDa [1618]. Microbial polysaccharides have gained relevant interest for the biotechnological application, and it is commercially valuable with several applications in the medical, veterinary, pharmaceutical, and cosmetic field, such as hyaluronic acid, heparin, and chondroitin sulfate [1921]. For commercial purpose each product must follow appropriate regulatory rules in order to guarantee the quality. For the pharmaceutical products, all producers should follow requirements according to WHO [7], Food and Drug Administration (FDA), or the Unites States/European pharmacopeia. The polysaccharide specifications for H. influenzae type b are based on the WHO guidelines considering that the RP regarding the proteins and nucleic acids in the final product (PSb) must be 100 [7]. In the previous work described by Takagi et al., the yield of purified polysaccharide was 68 %, however the required purity of PSb related to Prt was not attained [11]. In order to improve the purity level concerning mainly to protein, an extensive washing in the presence of two detergents and chelating agent was introduced in the first step (1CUTF100) and last step of purification (Enz+ 2CUTF100). Purification of PSb from different fermentation strategies was shown in the Tables 1, 2, and 3. The bulk of impurities was eliminated in the first step of purification in the ultrafiltrate fraction. Figure 2a, b illustrates that the significant elimination bulk of impurities relates to protein of 6980 % and nucleic acids of 5296 %, and Fig. 2c emphasizes the removal of LPS (KDO) in the CUTF100 step after cocamidopropyl betaine and deoxycholate inclusion in the washing buffer. Almost 7295 % of LPS (KDO) were eliminated in this first step comparing with 16 % of elimination in the previous work represented in this work by experiment 4, in which the elimination of LPS was distributed in every purification step. Hydrophobic interactions in the lipid region of the LPS were broken by using cocamidopropyl betaine, a zwitterionic detergent, and sodium deoxycholate, an anionic one. On the other hand, EDTA chelates the divalent ions present in the polysaccharide fraction of the LPS causing it to be unstable and generate monomers with low molecular mass [2225]. The two selective ethanol precipitation steps, despite its own mild effect in the final purity, were necessary to reach the purity specification. In previous experiments, purification of polysaccharide using enzymatic treatment without ethanol precipitation steps and the introduction of enzymatic hydrolysis (with or without heat treatment) before the two ethanol precipitation steps did not accomplish the required purity (data not shown). The efficiency of the enzymatic treatment was improved when the sample containing polysaccharide was previously treated with ethanol. The cleavage of hydrophobic interactions among aggregates

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Table 1 Purification of the PSb produced by H. influenzae type b in batch culture with glucose pulse (experiment 1) Fraction PSb (mg/l) PSb recoverya (%) 100 96 95 88 24 Protein (mg/l) RPb Prt PFc Prt NA (mg/l) RPb NA PFc NA KDO (mg/l) RPb KDO PFc KDO

Supernatant 1CTUF100 EtOH30 Water soluble Purified PSb

a b

782 751 746 690 184

2537 464 222 219 0.7

0.3 1.6 3.4 3.1 262.9

1 5 11 10 853

153.1 61.8 85.4 17.8 0.5

5 12 9 38 368

1 2 2 8 72

7.1 0.9 0.7 1.3 0.1

112 751 746 690 1840

1 7 7 6 16

PSb recovery (in percent) 0 PSb amount100/PSb in supernatant amount

Relative puritycalculated as milligrams of PS per milligram of impurities, where impurities are proteins (Prt), nucleic acid (NA), and KDO
c

Purification factorcalculated as RPstep/RPSupernatant for each of the impurities

proteins, nucleic acids, and other bacterial debris may be facilitated in the presence of ethanol, exposing them to enzyme attack. The use of nuclease and proteases provided a broad range of possible digestion sites enhancing the breakage of nucleic acids and proteins into small nucleotides and peptides, respectively, and makes possible pass through the pores of the 100-kDa nominal cutoff membrane [9, 10, 25]. Figure 3 shows the contribution of each purification steps on the relative purity for protein, nucleic acid, and LPS(KDO), where the introduction of both detergents has contributed in the elimination of the impurities in the both steps of ethanol treatmentexperiments 13 which are not evidenced in experiment 4 concerning data related to previous work. Figure 3 makes clear that the relative purity, mainly, for the protein (experiments 13) was achieved just after enzyme treatment and the introduction of exhaustive washing with detergents, but in a different way from the previous study (experiment 4).
Table 2 Purification of the PSb produced by H. influenzae type b in batch culture with glucose pulse (experiment 2) Fraction PSb (mg/l) PSba recovery (%) 100 48 50 39 10 Protein (mg/l) RPb Prt PFc Prt NA (mg/l) RPb NA PFc NA KDO (mg/l) RPb KDO PFc KDO

Supernatant 1CTUF100 EtOH30 Water soluble Purified PSb

511 244 254 200 48

518.9 163.0 13.7 6.7 0.4

1 2 18 29 122

1 2 19 30 123

123.3 16.5 4.0 3.4 0.1

4 15 64 67 480

1 4 15 16 116

3.8 0.2 0.1 0.1 0.01

128 1220 nd 2000 4800

1 10 nd 16 38

nd not determined
a b

PSb recovery (in percent) 0 PSb amount100/PSb in supernatant amount

Relative puritycalculated as milligrams of PS per milligram of impurities, where impurities are proteins (Prt), nucleic acid (NA), and KDO
c

Purification factorcalculated as RPstep/RPSupernatant for each of the impurities

Appl Biochem Biotechnol (2012) 167:20682075 Table 3 Purification of the PSb produced by H. influenzae type b in fed-batch culture (experiment 3) Fraction PSb (mg/l) Protein RPb PSba recovery (mg/l) Prt (%) 1204.3 204.3 93.3 58.7 1.9 1.1 3.9 6.6 10.6 PFc Prt NA (mg/l) RPb NA PFc NA KDO RPb (mg/l) KDO

2073

PFc KDO

Supernatant 1382.6 100 1CTUF100 EtOH30 Water soluble Purified PSb 787.8 617.0 620.1 323.9 57 44.6 44.9 23.4

1 3.4 5.8 9.2

1234.3 47.9 26.0 8.5

1.1 16.4 23.8 73.0

1.0 22.90 14.7 65.1 1.40 0.67 0.26 21.2

60.4 562.7 nd 925,5

1 9.3 nd 15.3

170.5 148.5

1.1 294.5 262.9

1245.8 20.6

nd not determined
a b

PSb recovery (in percent) 0 PSb amount100/PSb in supernatant amount

Relative puritycalculated as milligrams of PS per milligram of impurities, where impurities are proteins (Prt), nucleic acid (NA), and KDO
c

Purification factorcalculated as RPstep/RPSupernatant for each of the impurities

The supernatant fraction containing PSb was clarified; however, the concentrated fraction 1CTUF100 appeared cloudy. Diafiltration using buffer containing detergents reduced considerably this cloudiness; nevertheless, the precipitation with 80 % ethanol jointly with enzyme treatment and second concentration resulted in end-product solution to be

120

120

120

1CTUF100 SprEtOH30 Water soluble Enz2CTUF

100

100

100

% Elimination - Protein

% Elimination LPS
1 2 3 Experiment 4

80

% Elimination AN

80

80

60

60

60

40

40

40

20

20

20

0 1 2 3 Experiment 4

0 1 2 3 Experiment 4

Fig. 2 Stack bar considering the elimination (in percent) of the impurities in each step. a Protein, b nucleic acids, and c LPS (KDO). 13 experiments 13, and experiment 4 is that described by Takagi et al.

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Supernatant 1CTUF100 SprEtOH30 Watersoluble Enz2CTUF100

A
250

B
RP PSb/ N A
600

C
RP PSb/LPS
5000

RP PSb/Prt

500 400 300

200

150

4000

100

200 100 70 60 2000 50 3000

50 30 25 20

40
15

30
10 5 0

1000

20 10 0 1 2 3 4 1 2 3 4 0 1 2 3 4

Experiment

Experiment

Experiment

Fig. 3 Stack bar representing the relative purity of each step on the polysaccharide purification. a Protein, b nucleic acid, and c LPS (KDO). 13 experiments 13, and experiment 4 is that described by Takagi et al. Horizontal dot line refers to the minimum relative purity

transparent and allowed to reach the required purity in relation to main impurities: nucleic acidRPPSb/NA (368, 480, and 294.5) and proteinRPPSb/Prt (262.9, 122 and 170.5). In the first concentration step, the low recovery of PSb in experiments 2 and 3 of 47 and 57 %, respectively, could be related to the different fermentation strategies followed. In addition the quality of the polysaccharide in terms of the mass molecular size can be affected depending on the cultivation condition causing the variation on the PSb recovery during ultrafiltration in 100 cutoff membranes (paper preparation). The recuperation of PSb were 1024 %, lower than the previous work with 68 %; however, the purity related to protein was low if compared with the new process (122263). Purification is a tradeoff between purity and recovery, which in the pharmaceutical products the requirements defined by regulatory agencies have to be fulfilled. The final recovery of the purified polysaccharide is quite low, and our challenge now is to improve it; even so, it is feasible for the application in large scale and attends the countrywide demand whatever for the amount used in each dose of vaccine is 10 g.

Conclusion In conclusion, the introduction of detergents and EDTA during the first tangential ultrafiltration with nominal cutoff membranes (1CTUF100) and the sustained incubation period with hydrolytic enzymes resulted in the final PSb with both high quality and purity. The final yield of PSb is quite low, however the PSb purification process proved to be robust independent of the fermentation strategy used. The membranes used in the tangential flow

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ultrafiltration process can be cleaned, stored, and reused repeated times; ethanol used in the precipitation steps can be recovered by distillation. Moreover, membrane technology associated with enzyme treatment is feasible to downstream strategy for industrial manufacture purposes due to low-energy requirements; easy modification of the critical operation variables; efficient, environmentally friendly, and relatively easy to scale-up process
Acknowledgments We gratefully acknowledge the financial support from the So Paulo Research Foundation (FAPESP 2007/50082-2) and the Butantan FoundationBrazil. Silvia M.F. Albani received a scholarship from the Secretary of Education of So Paulo State. We also thank Mr. Lourivaldo Incio de Souza and Ms. Ins do Amaral Maurelli for the technical assistance.

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