Improvement in the Purification Process of the Capsular Polysaccharide From Haemophilus Influenzae...

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Improvement in the Purification Process of the Capsular

Polysaccharide from Haemophilus influenzae Type bby 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 ultrafiltra-

tion 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 122∼263 and 294∼480, respectively,

and compatible with the specifications established by the World Health Organization for Hibvaccine, RP≥100. These results make this process simple, cheaper, efficient, environmen-

tally 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 infec-

tious 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 [3 – 6]. For an

Appl Biochem Biotechnol (2012) 167:2068 – 2075DOI 10.1007/s12010-012-9750-4

S. M. F. Albani : M. R. da Silva : M. Takagi (*) : J. Cabrera-CrespoInstituto Butantan — Centro de Biotecnologia, Av. Vital Brasil, 1500, 05503-900 São Paulo, Brazile-mail: [email protected]



affordable Hib conjugate vaccine available for the public health system, to attend low-

income 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 ultrafil-

tration, ethanol precipitation, and hydrolytic enzymes digestion [9 – 11]. Comparing to the

classical method, the use of harsh chemical as phenol was eliminated and ethanol precipi-

tation 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, intro-

ducing tangential ultrafiltration, detergent treatment, ethanol precipitations steps and exten-

sive 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, São 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 200 – 800 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 Tris – HCl pH 7.5, 150 mM

NaCl, 0.1 % (v / v ) cocamidopropyl betaine; (2) 25 mM Tris – HCl pH 7.5, 0.3 % (w/ v )

deoxycholate, 2 mM EDTA; and (3) 25 mM Tris – HCl pH 7.5; this fraction was named first

concentration by tangential ultrafiltration — 1CTUF100.

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

17,696× g 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 Tris – HCl

buffer containing 2 mM MgCl2 and 20 mM NaCl and submitted to the following enzymes

Appl Biochem Biotechnol (2012) 167:2068 – 2075 2069



treatment: endonuclease (Benzonase® EC 3.1.30.2 — Sigma) — 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 3.4.21.4 — Sigma), protease from Aspergillus oryzae

type II (EC 3.1.27.3 — Sigma), and protease from S. griseus type XVI (EC 3.4.24.3 — Sigma)

were sequentially added considering 1 IU/100 mg of protein impurities and the mixtureincubated. 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 PSb — Enz+ 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 Lowry’s method. The nucleic acid (NA)

concentration was determined by the absorbance (A) readings at 260 nm [ 7]. The lipopoly-

saccharide (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 Osborn’s 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 purifi-

cation 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.

UltrafiltrateCultureMedium

Retentate

1CTUF100

Supernatant

Culture

BrothWashingbuffer:

PptCells

Etanol

EtOH 30% EtOH 80%

80%

Ppt

Precipitate80%

Spr

H2O

(Purified PSb)

Ppt

Water

soluble

Enzymatictreatment

Washing buffer:

UF 100kDa

FERMENTOR

CENTRIFUGE

CENTRIFUGE CENTRIFUGE CENTRIFUGE

PREP-SCALE

SPIRAL 0.54 m2

100 KDA NMWC

Enz+ 2CTUF100

1) Tris-HCl/ NaCl/cocoamidopropyl betaine - (6vol)2) Tris-HCl/deoxycholate/EDTA - (6vol)3) Tris-HCl - (6vol)

1) Tris-HCl/ NaCl/cocoamidopropylbetaine - (6vol)2) Tris-HCl/deoxycholate/EDTA - (6vol)3) Tris-HCl - (6vol)4) Water – (6v)

30%

LAB-SCALE

CASSETTES 150 cm2

100 KDA NMWC

Solubilizationin water

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

2070 Appl Biochem Biotechnol (2012) 167:2068 – 2075



Results and Discussion

Polysaccharide produced by H. influenzae b is a linear heteropolymer made of repetitive

units of D-ribitol-(1-1)-β D-ribose-3-phosphate, hydrophilic, negatively charged, polydis-

perse, and an extracellular polymer with high molecular mass (0.30∼2×106

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 10∼20 kDa [16 – 18].

Microbial polysaccharides have gained relevant interest for the biotechnological appli-

cation, and it is commercially valuable with several applications in the medical, veterinary,

pharmaceutical, and cosmetic field, such as hyaluronic acid, heparin, and chondroitin sulfate

[19 – 21]. 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 bare 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 69∼80 % and nucleic acids of 52∼96 %, and Fig. 2c emphasizes the removal of LPS (KDO) in the CUTF100 step after cocamidopropyl betaine and deoxycholate inclusion

in the washing buffer. Almost 72∼95 % 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 cocamido-

propyl 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 [22 – 25].

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




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 con-

tributed in the elimination of the impurities in the both steps of ethanol treatment — experi-

ments 1 – 3 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 1 – 3)

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 1 Purification of the PSb produced by H. influenzae type b in batch culture with glucose pulse(experiment 1)

Fraction PSb(mg/l)

PSbrecoverya

(%)

Protein(mg/l)

RP b

Prt PFc

Prt NA(mg/l)

RP b

NAPFc

NAKDO(mg/l)

RP b

KDOPFc

KDO

Supernatant 782 100 2537 0.3 1 153.1 5 1 7.1 112 1

1CTUF100 751 96 464 1.6 5 61.8 12 2 0.9 751 7

EtOH30 746 95 222 3.4 11 85.4 9 2 0.7 746 7

Water soluble

690 88 219 3.1 10 17.8 38 8 1.3 690 6

PurifiedPSb

184 24 0.7 262.9 853 0.5 368 72 0.1 1840 16

a PSb recovery (in percent)0PSb amount×100/PSb in supernatant amount

b Relative purity — calculated as milligrams of PS per milligram of impurities, where impurities are proteins(Prt), nucleic acid (NA), and KDOc Purification factor — calculated as RPstep/RPSupernatant for each of the impurities

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(%)

Protein(mg/l)

RP b

Prt PFc

Prt NA(mg/l)

RP b

NAPFc

NAKDO(mg/l)

RP b

KDOPFc

KDO

Supernatant 511 100 518.9 1 1 123.3 4 1 3.8 128 1

1CTUF100 244 48 163.0 2 2 16.5 15 4 0.2 1220 10

EtOH30 254 50 13.7 18 19 4.0 64 15 0.1 nd nd

Water soluble

200 39 6.7 29 30 3.4 67 16 0.1 2000 16

Purified

PSb

48 10 0.4 122 123 0.1 480 116 0.01 4800 38

nd not determineda PSb recovery (in percent)0PSb amount×100/PSb in supernatant amount b Relative purity — calculated as milligrams of PS per milligram of impurities, where impurities are proteins(Prt), nucleic acid (NA), and KDOc Purification factor — calculated 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 con-

siderably this cloudiness; nevertheless, the precipitation with 80 % ethanol jointly with

enzyme treatment and second concentration resulted in end-product solution to be

Table 3 Purification of the PSb produced by H. influenzae type b in fed-batch culture (experiment 3)

Fraction PSb(mg/l)

PSba

recovery(%)

Protein(mg/l)

RP b

Prt PFc

Prt NA(mg/l)

RP b

NAPFc

NAKDO(mg/l)

RP b

KDOPFc

KDO

Supernatant 1382.6 100 1204.3 1.1 1 1234.3 1.1 1.0 22.90 60.4 1

1CTUF100 787.8 57 204.3 3.9 3.4 47.9 16.4 14.7 1.40 562.7 9.3

EtOH30 617.0 44.6 93.3 6.6 5.8 26.0 23.8 21.2 – nd nd

Water soluble

620.1 44.9 58.7 10.6 9.2 8.5 73.0 65.1 0.67 925,5 15.3

PurifiedPSb

323.9 23.4 1.9 170.5 148.5 1.1 294.5 262.9 0.26 1245.8 20.6

nd not determineda PSb recovery (in percent)0PSb amount×100/PSb in supernatant amount b Relative purity — calculated as milligrams of PS per milligram of impurities, where impurities are proteins(Prt), nucleic acid (NA), and KDOc Purification factor — calculated as RPstep/RPSupernatant for each of the impurities

1 2 3 40

20

40

60

80

100

120 CA B

% E

l i m i n a

t i o n A N

Experiment

1 2 3 40

20

40

60

80

100

120

% E

l i m i n a t i o n L P S

Experiment

1 2 3 40

20

40

60

80

100

120

% E

l i m i n a t i o

n - P r o t e i n

Experiment

1CTUF100 SprEtOH30 Water soluble Enz2CTUF

Fig. 2 Stack bar considering the elimination (in percent) of the impurities in each step. a Protein, b nucleicacids, and c LPS (KDO). 1 – 3 experiments 1 – 3, and experiment 4 is that described by Takagi et al.




transparent and allowed to reach the required purity in relation to main impurities: nucleic

acid — RPPSb/NA (368, 480, and 294.5) and protein — RPPSb/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

10∼24 %, lower than the previous work with 68 %; however, the purity related to proteinwas low if compared with the new process (122∼263). 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 μ g.

Conclusion

In conclusion, the introduction of detergents and EDTA during the first tangential ultrafil-

tration 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

1 2 3 40

5

10

15

20

25

30

50

100

150

200

250

CA B

R P P

S b / P r t

Experiment

Supernatant

1CTUF100

SprEtOH30

Watersoluble

Enz2CTUF100

1 2 3 40

10

20

30

40

50

60

70

100

200

300

400

500

600

R P P S

b / N A

Experiment

1 2 3 40

1000

2000

3000

4000

5000

R P P S b / L P S

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). 1 – 3 experiments 1 – 3, and experiment 4 is that described by Takagi et al. Horizontal dot line refers to the minimum relative purity




ultrafiltration process can be cleaned, stored, and reused repeated times; ethanol used in the

precipitation steps can be recovered by distillation. Moreover, membrane technology asso-

ciated with enzyme treatment is feasible to downstream strategy for industrial manufacture

purposes due to low-energy requirements; easy modification of the critical operation vari-

ables; efficient, environmentally friendly, and relatively easy to scale-up process

Acknowledgments We gratefully acknowledge the financial support from the São Paulo Research Founda-tion (FAPESP 2007/50082-2) and the Butantan Foundation — Brazil. Silvia M.F. Albani received a scholarshipfrom the Secretary of Education of São Paulo State. We also thank Mr. Lourivaldo Inácio de Souza and Ms.Inês do Amaral Maurelli for the technical assistance.

References

1. Shapiro, E. D., & Ward, J. I. (1991). Epidemiologic Reviews, 13, 113 – 142.2. Kelly, D. F., Moxon, E. R., & Pollard, A. J. (2004). Immunology, 113, 163 – 174.3. Gotschlich, E. C., Liu, T. Y., & Artenstein, M. S. (1969). The Journal of Experimental Medicine, 129,

1349 – 1365.4. Yavordios, D., & Cousin, M. (1983). European Patent Office. EP 0 071 515 A1.5. Frasch, C. E. (1990). In A. Mizrahi (Ed.), Advances in biotechnological vaccines (pp. 123 – 145). New

York: Wiley-Liss.6. Hamidi, A., & Beurret, M. (2010). European Patent Office. EP 1 664 319 B1.7. World Health Organization WHO. (2000). Technical Report Series, 897 , 27 – 56.8. Ward, O. P. (1991). Bioprocessing . New York: Van Nostrand Reinhold.9. Tanizaki, M. M., Garcia, L. R., Ramos, J. B., Leite, L. C. C., Hiss, H., Furuta, J. A., et al. (1996). Journal

of Microbiological Methods, 27 , 19 – 23.10. Gonçalves, V. M., Takagi, M., Carmo, T. S., Lima, R. B., Albani, S. M. F., Pinto, J. V., et al. (2007). In A.

V. Mendez (Ed.), Communicating current research and educational topics in applied microbiology

(pp. 450 – 457). Badajoz: Formatex Research Center.11. Takagi, M., Lima, R. B., Albani, S. M. F., Zangirolami, T. C., Tanizaki, M. M., & Cabrera-Crespo, J.

(2008). Journal of Industrial Microbiology and Biotechnology, 35, 1217 – 1222.12. Takagi, M., Cabrera-Crespo, J., Zangirolami, T. C., Raw, I., & Tanizaki, M. M. (2006). Journal of

Chemical Technology and Biotechnology, 81, 182 – 188.13. Silva, M.R. (2010). Dissertation, Universidade Federal de São Carlos.14. Ashwell, G. (1957). Methods in Enzymology, 3, 87 – 90.15. Osborn, M. J. (1963). Biochemistry, 50, 499 – 506.16. Petsch, D., & Anspach, F. B. (2000). Journal of Biotechnology, 76 , 97 – 119.

17. Magalhães, P. O., Lopes, A. M., Mazzola, P. G., Rangel-Yagui, C., Penna, T. C. V., & Pessoa, A., Jr.(2007). Journal of Pharmacy and Pharmaceutical Sciences, 10, 388 – 404.18. Rienstra, M.S., Scattergood, E.R.M., Sitrin, R.D. (1991). United States Patent US5039610.19. Kogan, G., Soltés, L., Stern, R., & Gemeiner, P. (2007). Biotechnology Letters, 29, 17 – 25.20. Lindahl, U. (2000). Glycoconjugate Journal, 17 , 597 – 605.21. Volpi, N. (2007). Journal of Pharmaceutical Sciences, 96 (12), 3168 – 3180.22. Ozbek, S., Muller, J. F., Figgemeier, E., & Stetefeld, J. (2005). Acta Crystallographica D, 61, 477 – 480.23. Chen, Y., Xu, Q., Yang, H., & Gu, G. (2001). Process Biochemistry, 36 , 773 – 779.24. Chen, Y., Chen, Y. S., & Gu, G. (2004). Chemical Engineering Journal, 99, 137 – 143.25. Pinelo, M., Jonsson, G., & Meyer, A. S. (2009). Separation and Purification Technology, 70(1), 1 – 11.


Improvement in the Purification Process of the Capsular Polysaccharide From Haemophilus Influenzae...

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