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Powder Technology 197 (2010) 54-57

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Powder Technology
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Crystallization of porcine insulin with carbon dioxide as acidifying agent

Gisele Atsuko Medeiros Hirata, Andr Bernardo, Everson Alves Miranda
LEBp Laboratrio de Engenharia de Bioprocessos, Departamento de Processos Biotecnolgicos, Faculdade de Engenharia Qumica, Universidade Estadual de Campinas, UNICAMP,
Caixa postal 6066, CEP 13083-970 Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Recent studies on the use of volatile electrolytes such as CO2 in protein precipitation showed that these
Received 25 May 2009 agents are a promising alternative to the conventional acids. This use of volatile electrolytes prevents protein
Received in revised form 17 August 2009 denaturation due to local pH extremes, and saline efuent generation is greatly reduced, as the volatile
Accepted 22 August 2009
electrolyte may be separated and recovered from solution just by pressure release. In this work, insulin was
Available online 1 September 2009
successfully crystallized in the presence of zinc using CO2 as acidifying agent. The crystals obtained were
rhombohedral, a common shape for porcine insulin crystals that contain zinc in their structure, and their
Carbon dioxide average size varied with the mixing applied.
Downstream 2009 Elsevier B.V. All rights reserved.
Porcine insulin
Protein crystallization

1. Introduction dissolve and dissociate in water resulting in ions (carbonate, bicarbonate,

and H+) whose concentrations are dependent on the temperature and
About 70% of the products sold by the process and pharmaceutical pressure of the system, causing a reduction in pH. Under depressurization
industries are solid [1]. In the pharmaceutical industry crystallization
of the system the anions CO23 and HCO3 pass from the liquid phase to the
is an important technological process for particle formation and over vapor phase as CO2 and can be removed from the system and recycled in
90% of all pharmaceutical products, such as tablets, aerosols, capsules, the process. Another advantage of the use of volatile electrolytes is that
and suspensions, generally contain drugs in crystalline form [2]. during adjustment of protein solution pH, extremes in local pH are
Protein crystallization is a technique optimized over many decades to prevented because solubilization of the precipitating agent is slow and
produce crystals with better conditions for X-ray diffraction analysis. more homogeneous, occurring along the whole gasliquid interface. In
Its potential in protein recovery and purication has recently been conventional processes, these extreme pH values can result in the
revealed. The crystal form of protein products has an advantage over denaturation of protein or even a reduction in precipitate purity [4].
the dissolved and amorphous forms, since it has longer storage life and The use of volatile electrolytes in downstream processes of proteins
higher purity [3]. is relatively recent with only a few reports in the open literature. The
Frequently isoelectric precipitation is used in downstream proces- published studies on protein precipitation with volatile electrolytes
sing for separation of proteins from aqueous solutions. This method were carried out with complex protein systems such as milk, soy protein
consists of adjustment of solution pH to the isoelectric point of the extracts [5,6], and the fractionation of protein mixtures [7,8]. The iso-
protein (pI). At the pI, the molecule carries no net electrical charge, electric precipitation with carbon dioxide of a single protein, bovine
reducing the electrostatic repulsion between molecules which results in serum albumin, was investigated by Qi et al. [9]; additional investiga-
precipitation. Since the majority of proteins have a pI in the acid region, tions of the activity of some enzymes in the system formed by ethanol,
conventional acids such as H2SO4 and HCl are commonly used for pH carbon dioxide, and water were presented by Yao et al. [10]. Samadi and
adjustment in these precipitations. After precipitation, the efuent Husson [11] described a process of separation by precipitation with CO2
generated must be treated before its disposal into the environment, a for recovering L-aspartic acid. Tashima et al. [12] were pioneers in
complex process step that can be costly. reporting experimental and theoretical aspects of the precipitation of a
The use of volatile electrolytes such as CO2 is an alternative to the use single protein (porcine insulin) using CO2 and developed a thermody-
of conventional acids in the isoelectric precipitation processes. They are namic model to correlate the experimental data. No crystal formation
called volatile electrolytes since, as exemplied in the case of CO2, they was reported in that work.
The objective of this work was to nd conditions for the
crystallization of a single protein using CO2 as acidifying agent, and
Corresponding author. Tel.: +55 19 3521 3918; fax: +55 19 3521 3890. we successfully crystallized porcine insulin in the presence of zinc in a
E-mail address: (E.A. Miranda). pressurized CO2 stirred tank crystallizer.

0032-5910/$ see front matter 2009 Elsevier B.V. All rights reserved.
G.A.M. Hirata et al. / Powder Technology 197 (2010) 54-57 55

Fig. 1. Scheme of the experimental apparatus for the crystallization of proteins with CO2.

Fig. 2. Photomicrographs of porcine insulin crystals in 50 mM NaHCO3 and 0.4 mM ZnCl2 under pressurized CO2 at 5 C. Equilibrium pH runs: R1 (magnetic stirring), pH 6.50;
R2 (magnetic stirring), pH 6.50; R3 (impeller stirring), pH 6.37; R4 (impeller stirring), pH 6.35; and R5 (impeller stirring), pH 6.46.
56 G.A.M. Hirata et al. / Powder Technology 197 (2010) 54-57

2. Materials and methods biological microscope (model Tim-108, Opton, Brazil) with a magni-
cation from 40 to 1600.
2.1. Materials

Porcine insulin (96.6% pure, 0.55% Zn) was donated by Biobrs 3. Results and discussion
(Brazil). Sodium bicarbonate (100% pure) was obtained from J. T. Baker
(Mexico); carbon dioxide (>99.8% pure), from White Martins Gases The attempts to crystallize insulin at 5 mg/mL were not successful in
Industriais (Brazil); and zinc chloride, from Vetec Qumica Fina (Brazil). generating crystals due to gelation of the insulin solutions. However, a
Ultra-pure water was obtained using Milli-Q equipment from Millipore lower insulin concentration (1.84 mg/mL) produced relatively large and
(USA). All the other reagents were of analytical grade. well-formed crystals (Fig. 2). Although the crystal sizes obtained in the
experiments were different, the crystal shape was similar to that of the
2.2. Equipment rhombohedral zincinsulin crystals. This rhombohedral form was also
observed by Schlichtkrull [14], who reported that crystal shape is
The equipment for protein crystallization with volatile electrolytes dependent on the species of insulin. For example, porcine and human
(Fig. 1), was the same as that used by Tashima et al. [12]. The insulin form rhombohedra, but sheep and bovine insulin appear to be a
equipment has a cylindrical pressure vessel in stainless steel with a twin formation.
340 mL internal volume, a diameter of 5.5 cm and a thermal jacket for The reduction in insulin concentration in the liquid phase with
liquid phase temperature control (accuracy of 1.0 C). The solution time indicated the new phase formation a solid phase observed
pH was measured to an accuracy of 0.03 by a high-pressure glass after separation of precipitate and supernatant by centrifugation
electrode TBX567 (ABB, Carlson City, USA). Pressure was measured to (Fig. 3a). During the acidifying process, the CO2 is dissociated in
an accuracy of 0.15 bar by the digital transducer PSI-420 (0 to solution, causing the reduction in pH, decreasing the protein solubility
60 bar) and indicated on the instrumentation panel PLN-2 (both and favoring the crystallization processes (Fig. 3b). These concentra-
devices by Zurich Indstria e Comrcio, Brazil). The solution could be tion and pH proles are similar to the ones reported by Tashima et al.
mixed by either a magnetic stirrer (3.8 cm long with a diameter of [12]. In that paper, the precipitation of this same insulin did not lead to
1 cm) or an impeller with four paddles at 45 angles with agitation protein denaturation. Thus, no loss of activity is expected in this case.
rotational speed from 120 to 720 and from 0 to 300 rpm, respectively. Crystal size varied with type of mixing system, magnetic stirrer or
The impeller was located 2.2 cm from the bottom of the reactor and it impeller (Table 1). In the case of the magnetic stirrer, the crystal size
has a diameter of 2.7 cm. The magnetic bar did not come in contact was smaller than it was in the case of the impeller (average sizes of
with the surface of the bottom of the reactor during stirring since it
was kept about 1 cm from the bottom of the reactor by a thin structure
in order to minimize crystal breakage.

2.3. Experimental methods: porcine insulin crystallization

The protocol for crystallization of insulin used in this work was

based on a large-scale (commercial) animal insulin protocol in which
two temperatures 15 and 5 C were used. First, insulin was
dissolved in 100 mL of 10 mM HCl and then 125 mL of 100 mM
NaHCO3 and 25 mL of water were added to produce a solution with a
nal protein concentration of 1.84 mg/mL. The solution was ltered
with a 0.2 m syringe lter Minisart 16534 (Sartorius, Germany), and
71 l of 20% zinc chloride (1.47 M) was added to the solution. The
reactor vessel previously equilibrated at 15 C was charged with this
protein solution and closed. The solution was then kept under stirring
at 120 rpm for the runs carried out with the magnetic stirrer and at
65 rpm for the runs with the impeller until the solution temperature
reached 15 C. The pH was adjusted by slow addition of CO2. The CO2
feeding was interrupted when the desired pH was reached and the
pressure (maximum of 3.5 bar) was allowed to fall to 1 bar in 1 h by
CO2 release. The solution was then kept at 15 C for 4 h longer, the
stirring was stopped, and the temperature set to 5 C for 1940 h. The
reactor was then opened after the slow release of pressure, the liquid
phase and crystals were separated by centrifugation, and the size of
crystals and the insulin concentration in solution determined.

2.4. Analytical methods

2.4.1. Determination of the protein concentration

The protein concentration in the solution was determined by
absorbance at 280 and 320 nm according to Gehle and Schgerl [13]
using a DU 640 spectrophotometer (Beckman Instruments, USA).

2.4.2. Crystal analysis Fig. 3. Crystallization of porcine insulin in 50 mM NaHCO3 and 0.4 mM ZnCl2 in a CO2
atmosphere at 5 C: a) insulin concentration and b) pH of solution as functions of time.
The crystal size distribution was analyzed with quasi-elastic light Equilibrium pH runs: , pH 6.50 (R1, magnetic stirring); , pH 6.50 (R2, magnetic
scattering Mastersizer S equipment (Malvern, UK) for the size range of stirring); , pH 6.37 (R3, impeller stirring); , pH 6.35 (R4, impeller stirring); and , pH
0.05 to 900 m. Crystal morphology was observed with a trinocular 6.46 (R5, impeller stirring).
G.A.M. Hirata et al. / Powder Technology 197 (2010) 54-57 57

Table 1
Porcine insulin crystallization with CO2 in NaHCO3 solution.

Crystallization runs R1 R2 R3 R4 R5

Mixing system Magnetic stirrer Impeller

pH5 C 6.50 6.50 6.37 6.35 6.46

t5 C (h) 19 19 19 19 40
Average size (m) 7.59 0.34 10.56 0.34 16.73 0.43 14.80 0.43 19.58 0.81
10%a 1.36 5.31 5.50 5.50 3.48
90%b 11.78 16.43 28.75 28.23 41.72
Cf (mg/mL) 0.31 0.35 0.17 0.15 0.25
Yc (%) 83 81 91 92 86

Initial Zn concentration = 30 g/mL.

t5 C: time the solution was kept at 5 C.
pH5 C: nal pH at t5 C.
Cf: nal protein concentration.
10% of sample particles were below the average size.
90% of sample particles were below the average size.
calculated insulin recovery for initial protein concentration of 1.84 mg/mL.

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