Chemical Engineering Science 101 (2013) 631–641

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Chemical Engineering Science

journal homepage: www.elsevier.com/locate/ces

Mechanism and model for solubilization of inclusion bodies

Cornelia Walther a, Sabrina Mayer a, Gerhard Sekot a, Dorota Antos b, Rainer Hahn a,c,
Alois Jungbauer a,c, Astrid Dürauer a,c,n
a
Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria
b
Rzeszow University of Technology, Al. Powstancow Warszawy 6, 35-959 Rzeszow, Poland
c
Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria

H I G H L I G H T S G R A P H I C A L A B S T R A C T

Solubilization of inclusion bodies

does not follow the rules of ordinary
solubilization processes.

Solubilization of inclusion bodies is
controlled by pore diffusion.

Power input and mixing frequency is
not relevant to dissolve inclusion
bodies.

Solubilization of inclusion bodies
can be described by a homogeneous
layer model with a high order of
reaction. Solubilization of inclusion bodies is a diffusion controlled process. After penetration by the solubilization agent protein
diffuses from a densely packed core through pores in a skeleton of insoluble aggregated protein. This process is
described by a homogeneous layer model.

art ic l e i nf o a b s t r a c t

Article history: Inclusion bodies are a source of large quantities of protein with high purity over-expressed in Escherichia coli.
Received 8 May 2013 Efficient solubilization is the initial step in recovering the protein. We visualized solubilization using high-
Received in revised form resolution light and transmission electron microscopy over time and the most common solubilization agents,
11 July 2013
urea and guanidine hydrochloride, as well as sodium hydroxide. Upon solubilization, the inclusion bodies
Accepted 15 July 2013
Available online 24 July 2013
were penetrated by the solubilization agent, shrinking the densely packed cores as the protein diffused out.
Despite the complex process, solubilization could be described by a homogeneous layer model with a high
Keywords: order of reaction. The solubilization of protein aggregates, such as inclusion bodies, does not follow the rules
Inclusion body of ordinary solubilization processes, but is controlled by pore diffusion. Consequently, power input and
Solubilization mixing frequency is not relevant to the process.
Model
& 2013 Elsevier Ltd. All rights reserved.
Mechanism
Diffusion

1. Introduction lab scale and industrial production of biopharmaceuticals, despite the

Expression of recombinant proteins as inclusion bodies yields high
levels with high purity. Therefore, inclusion bodies are often applied in
Abbreviations: GuHCl, Guanidine hydrochloride; IB, Inclusion body; MTP,
Microtiter plate; NIR, Near infrared; TEM, Transmission electron microscopy
n
Corresponding author at: Department of Biotechnology, University of Natural
Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.
Tel.: +43 1 47654 6594; fax: +43 1 47654 6675.
E-mail address: astrid.duerauer@boku.ac.at (A. Dürauer).
additional steps of inclusion body recovery, solubilization, and refold-
ing (Eiberle and Jungbauer 2010; Jungbauer and Kaar 2007;
Middelberg 2002). Solubilization, a crucial step during protein recov-
ery, affects yield and may influence the refolding/oxidation process on
the way to the native state. The underlying mechanism is not
fully understood; protein aggregates, such as inclusion bodies, are
assumed to solubilize in a manner analogous to other solid matter.
Inclusion bodies are porous structures, also referred to as refractile
bodies, in which more than one protein species coexist (Bowden et al.,
1991; Carrió et al., 1998; Carrió et al., 2000; Taylor et al., 1986).

0009-2509/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ces.2013.07.026
632 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641

The architecture of inclusion bodies is described as sponge like In the reaction layer with
organization combining a nonfunctional amyloidal fibrous scaffold
dm
and native/native-like protein filling this matrix. While the amyloid ¼ Akp ðC S cd Þp ð1Þ
dt
structured protein fibers are correlated to the hardly soluble part, the
native/native-like structures are the easily extractable fraction of the In the diffusion layer with
inclusion bodies (Cano-Garrido et al., 2013; Morell et al., 2008; dm
¼ Akd ðC S cÞ ð2Þ
Peternel and Komel 2011). Currently, the solubilization of inclusion dt
bodies is empirically described and the obtained data only valid for the where m is the mass of the inclusion body [kg], A the inclusion
studied experimental conditions; a general conclusion cannot be body surface [m2], kp and kd are the kinetic constants of the
derived (Freydell et al., 2007). A predictive model based on the reaction rates in the corresponding layer [m/s], p is the pseudo-
mechanism of solubilization is the basis for engineering the whole order of the reaction, CS is the protein concentration at equilibrium
process. In addition, a model for predicting solubilization time and the at the solid surface [kg/m3], cd is the protein concentration at the
yield of different solubilization conditions is required for scaling up/ interface of the reaction and diffusion layers [kg/m3], and c is the
scaling down and process optimization. protein concentration in bulk liquid [kg/m3].
Therefore, the question arises of whether models developed for the As the protein concentration cd at the interface is unknown,
dissolution of pharmaceutical tablets can also be used to describe both kinetic equations were combined into a homogeneous layer
inclusion body solubilization. Costa and Sousa Lobo (2001) gave an model so that all necessary concentrations can be determined.
overview of several models for the dissolution profiles of solid On the basis of the determined solubilization kinetics and micro-
pharmaceutical dosage forms. The polymorphic form, crystallinity, scopic images, the model was expressed as:
particle size, solubility, and amount in the dosage form were found to
influence the release kinetics (Salomon and Doelker 1980). Costa and dm
¼ AkðC S cÞξ ð3Þ
Sousa Lobo (2001) described Zero Order Kinetics, First Order Kinetics, dt
the Weibull model, the Higuchi model, the Baker–Lonsdale model, the where k is the lumped kinetic constant [m/s] and ξ is an empirical
Hixson–Crowell model, the Korsmeyer–Peppas model and the Hop- exponent for the driving force.
fenberg model for dissolution of dosage forms. Harland et al. (1988) Eq. (3) can be rewritten as follows:
and Yang et al. (1996) described release mechanisms based on
dm dVρ dV dρ dr 4 dρ
hydrodynamic swelling and Hsu et al. (2009) and Huang et al. ¼ ¼ρ þV ¼ ρð4πr 2 Þ þ πr 3 ð4Þ
dt dt dt dt dt 3 dt
(2012) described a shrinking core model for dissolution of spherical
particles. where ρ is the particle density [kg/m3], V is the volume of a
These models describe dissolution from tablets, whereas the spherical inclusion body [m3], and r is the radius of the inclusion
solubilization of inclusion bodies has different preconditions. Inclusion bodies during solubilization [m]. Since the variations of the
bodies are biological material containing high-density protein aggre- particle density with time are expected to be insignificant
gates with a matrix of insoluble nonfunctional amyloid protein and compared to changes of the particle radius, the contribution of
soluble native/native-like protein as filling, both of the same species. the second term in Eq. (4) can be neglected. Therefore, combining
The soluble fractions of the protein in the inclusion bodies consist of Eqs. (3) and (4) yields:
macromolecules that exhibit partially developed secondary structural dr
elements (Carrió et al., 2000; Khan et al., 1998; Oberg et al., 1994; 4ρπ r 2 ¼ AkðC S cÞξ ð5Þ
dt
Singh et al., 2009). These structures undergo certain changes during
For spherical particles, A is 4πr2. Therefore:
the solubilization process. In contrast, pharmaceutical tablets usually
comprise smaller molecules that do not undergo any changes in dr k
¼  ðC S cÞξ ð6Þ
structure during dissolution and are usually incorporated in support dt ρ
material of different species. The solubilization process of inclusion The local concentration in the bulk phase can be calculated as
bodies is more complex than that of solid dosage forms because the follows:
solubilization agent has to penetrate the whole system before solubi-

3
3 π R ρ 3 π r ρ n
4 4 3
lization starts. Also, due to the different sizes of molecules involved,
the diffusivities are in different ranges. c¼ ð7Þ
Vf
In the present study, we analyzed the process of inclusion body
solubilization using several techniques. SDS-PAGE, light micro- where n is the number of particles, Vf is the liquid volume [m3],
scopy, and transmission electron microscopy were applied to gain and R is the average radius of inclusion bodies before solubiliza-
deeper understanding on the macroscopic and microscopic levels. tion [m]. Noting that:
The kinetics was determined by monitoring the decrease in m0 c0 V f
turbidity and increase of protein in solution over time. None of n¼ ¼ 3
ð8Þ
mi
3π R ρ
4
the previously established models were able to characterize this
process properly. Therefore, we developed a model of higher order where m0 is the total mass of the inclusion body before solubilization
to predict the solubilization of inclusion bodies. [kg], mi is the mass of the single particle [kg], and c0 is the maximal
concentration of protein available for solubilization [kg/m3]. Eq. (7)
can be solved to obtain Eq. (9):
2. Theoretical background !
R3 r 3
c¼ c0 ð9Þ
Fig. 1 schematically depicts the model of inclusion body R3
solubilization. We simplified the solubilization process to two This expression can be substituted into Eq. (6) to obtain a valid model
steps: the solubilization and conformational changes of the for the solubilization of inclusion bodies:
protein that occur in the inner reaction layer and the subsequent
! !ξ
release of the protein in the outer diffusion layer. Both processes,
dr k R3 r 3
which are triggered by two different concentration-driving forces, ¼ CS  c0 ð10Þ
dt ρ R3
can be represented by the following kinetic equations.
 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641 633

Fig. 1. Schematic depiction of the underlying mechanism for the established model: (a) inclusion body agglomerate prior to solubilization; (b) single inclusion body upon
solubilization with the equilibrium protein concentration at the solid surface Cs, the protein concentration at the interface of the reaction and diffusion layer cd, and the
concentration of protein in the bulk solution c; (c) cross-section of the inclusion body upon solubilization, illustrating the diffusion path of the solubilized protein.

3. Materials and methods Table 1

Autoprotease fusion proteins used for solubilization kinetics.
3.1. Chemicals
Fusion protein Amino acids MWa [Da] pIa Reference

Acetonitrile was purchased from J.T. Baker (Deventer, the EDDIE 168 19149.5 6.59 Achmüller et al., 2007
Netherlands). Hydrochloric acid, ammonia solution, Tween 20, EDDIE-Interferon α2b 333 38400.6 6.34 Q86UP4b
KH2PO4, Na2HPO4, C6H5Na3O7 2H2O, MgCl2 7H2O, CaCl2 2H2O, EDDIE-Fuzeon 204 23582.4 5.67 Morozov et al., 2007c
glucose monohydrate, FeSO4 7H2O, AlCl3 6H2O, ZnSO4 7H2O,
MW, molecular weight; pI, isoelectric point.
Na2MoO2 2H2O, CuCl 2H2O, and H3BO3 were obtained from Merck a
Estimated using the Expasy ProtParam tool. (http://ca.expasy.org/tools/prot-
(Darmstadt, Germany). Uranylacetate dihydrate and glutaralde-
param.html).
hyde solution were obtained from Fluka (Buchs, Switzerland). All b
The fusion protein has been produced at laboratory/pilot scale at Sandoz
other chemicals were purchased from Sigma (Saint Louis, Austria, the target is the therapeutic protein IFNa2b closely related to the reference
MO, USA). in UniProtKB/TrEMBL (http://www.uniprot.org/uniprot).
c
Fuzeons or a Enfuvirtide, a 36 amino acid therapeutic peptide, aa-Sequence:
YTSLIHSLIE ESQNQQEKNE QELLELDKWA SLWNWF.
3.2. Inclusion bodies

The proteins were produced by Npro fusion technology in E. coli.
Using this technology, target molecules are over-expressed in
fusion with a tailor-made mutant of the autoprotease Npro from
classical swine fever virus as described previously (Achmüller
et al., 2007). Upon refolding, the fusion partner is released from
the C-terminal end of the autoprotease by self-cleavage, leaving
the target protein with an authentic N-terminus. This process
enables high-level production of recombinant peptides and
proteins in E. coli without the need for chemical or enzymatic
removal of the fusion tag (Achmüller et al., 2007; Dürauer
et al., 2010). These autoprotease fusion proteins are deposited
predominantly in inclusion bodies. For the present studies, experi-
ments are shown for an Npro fusion protein containing Fuzeon
(a peptide) and Interferon α2b (a protein) (Table 1).
3.3. Recombinant protein expression and inclusion body isolation
The recombinant proteins were over-expressed in E. coli BL21
(DE3) from a pET30a plasmid (Novagen, Madison, WI, USA) contain-
ing the corresponding gene. E. coli fed batch cultivation was
performed with a semisynthetic batch medium and subsequent
C-limited fed-batch medium on the 30 L scale at 37 1C and pH 7.0.
634 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641
The wet cell paste was harvested using a disk centrifuge
(Pathfinder PSC 1-06-177; GEA Westfalia Separator Group, Oelde,
Germany) and resuspended by ultra turrax (IKA, Staufen, Ger-
many) in 50 mM Tris, 50 mM NaCl, and 0.02% Tween at pH 8.0 to
reach a dry matter of 30 g/L. The slurry was passed twice through
a Panda 2K homogenizer (GEA Niro Soavi S.p.A., Italy) at a pressure
of 1000 bar. Inclusion bodies were separated by the disk centrifuge
and the resulting pellet washed twice with 20 mM Tris, 0.5 M
NaCl, and 0.02% Tween at pH 8.0. After centrifugation, the pellet
was resuspended using the ultra turrax. The pellet was then
washed once with 0.5 M sodium chloride. After each washing
step, the inclusion bodies were separated using the disk centri-
fuge. The final pellet was resuspended in water to obtain a 40%
inclusion body suspension and stored at  20 1C.
The EDDIE-Interferon α2b was kindly provided by Sandoz
GmbH (Kundl, Tirol, Austria).
3.4. Analysis by light microscopy
For analysis by light microscopy, the solubilization agent was
added to resuspended inclusion bodies in microtiter plates and the
progress of solubilization recorded over time at 50  magnifica-
tions using a Leica DMI 6000 CS microscope (Leica Microsystems
CMS, Wetzlar, Germany). The microtiter plates were not shaken
during recording when placed on the microscope. 15 images
per second were recorded during solubilization. Recording time
depended on the specific protein and solubilization agent. The
brightness and contrast levels of all microscopic pictures were
adjusted to the same level using Corel PHOTO-PAINT X5.
3.5. Analysis by transmission electron microscopy
For analysis by TEM, samples of inclusion body suspension
were drawn at distinct time points during solubilization. The
solubilization was stopped by adding a 10% glutaraldehyde solu-
tion for 15 min, resulting in a final glutaraldehyde concentration of
5%. Subsequently, solid fractions were separated by centrifugation
for 60 s at 13,200 rpm and 21 1C (Centrifuge 5415R, Eppendorf,
Hamburg, Germany). The supernatant was discarded and pellets
resuspended in water.
For negative staining, 300-mesh copper grids (Christine Gröpl
Electron Microscopy, Tulln, Austria) coated with Pioloform film,
stabilized by carbon evaporation, and freshly glow discharged
were floated for 2 min, face down, on the surface of approximately
10 mL of a suspension of inclusion bodies in water. For cross-
linking, the grids were floated for 15 min in 2.5% glutaraldehyde
solution. The samples were subsequently stained with 1 drop of a
1% uranylacetate solution in water for 30 s. After removing the
excess stain by blotting the grid with filter paper, it was air-dried
as described by Hayat and Miller. (1990). The negatively stained
specimens were analyzed in a Tecnai G2 transmission electron
microscope (FEI Company, Hillsboro, OR, USA) at 120 keV.
3.6. Immunogold labeling of solubilized inclusion bodies
Sample preparation was carried out as described for TEM
analysis until cross-linking. The grids were washed twice with
phosphate-buffered saline. To block nonspecific binding of anti-
bodies to the grid, it was blocked for 2 h in phosphate-buffered
saline containing 2% bovine serum albumin. The grids were then
incubated with the primary antibody in phosphate-buffered saline
containing 2% bovine serum albumin for one hour. After washing
three times with Tris-buffered saline containing 0.5 g/L Tween 20,
the secondary antibody labeled with 10-nm gold nanoparticles in
phosphate-buffered saline containing 2% bovine serum albumin
was applied for 1 h. After the incubation, the grids were washed
three times with Tris-buffered saline containing 0.5 g/L Tween 20,
once with phosphate-buffered saline, and three times with water.
Finally, negative staining with uranylacetate was performed as
described earlier for the TEM experiments.
3.7. Determination of solubilization kinetics in MTP
Prior to the experiments, inclusion bodies were lyophilized and
stored at 4 1C. Adequate quantities of these dried powders were
measured for each experiment. The starting concentration c0 for
the experiments was 10 mg/mL lyophilized inclusion bodies.
Previous experiments have shown that lyophilization of the
inclusion bodies does not significantly influence the solubilization
kinetics (Dürauer et al., 2009). Nevertheless, to simulate the
conditions of aqueous suspensions used in industrial scale, inclu-
sion bodies were resuspended in 0.2 mL water by orbital shaking
in 2 mL reaction vials for 1 h. The concentration of solubilization
agent was adjusted by taking the dilution factor of the inclusion
body suspension into account. Three frequently used chemicals
were investigated: urea (4 and 8 M), guanidine hydrochloride
(GuHCl, 2 and 5 M), and NaOH (100 mM). The solubilization
process was started by adding 1.8 mL of the solubilization agent.
Four to eight 180-mL aliquots, along with two aliquots of blank
solubilization agent, were transferred to a microtiter plate (trans-
parent flat bottom, low absorbance, Greiner, Germany) immedi-
ately after the addition of the solvent. The absorbance at 600 nm
was measured immediately on a multifunctional microtiter plate
reader (GENiosPro, TECAN, Männedorf, Switzerland), calculating
an average value of ten reads per well with 5 s of shaking prior to
each measurement. Between measurements, the microtiter plate
was placed on an orbital microtiter plate shaker (Thermomixer™
comfort MTP, Eppendorf, Hamburg, Germany) at 550 rpm and
21 1C. In parallel, the same amount of inclusion bodies was
suspended in water and four 180-mL aliquots transferred to the
same microtiter plate for measurements. After 2 h, solubilization
was stopped in all wells by centrifugation at 13,200 rpm and 21 1C
for 5 min and the supernatant clarified by filtration through a
0.22-mm filter (Millipore, Billerica, MA, USA). For solubilization
employing NaOH, no kinetics was determined. The solubilization
agent was added to the inclusion bodies, and then after 30 s
50 mM MTG was added. After an additional 90 s, solubilization
was stopped by centrifugation for 30 s at 13,200 rpm and 21 1C
and the supernatant clarified by filtration through a 0.22-mm filter.
For each experiment an aliquot of the clarified protein solution
was immediately analyzed by reversed-phase (RP)-HPLC to calcu-
late the amount of fusion protein in the solution.
The solubilization kinetics was calculated as described pre-
viously (Dürauer et al., 2009) For conversion of the decrease in
turbidity into the increase in fusion protein in solution, the
absorbance curve determined at 600 nm was mirrored. To calcu-
late the curve of the fusion protein concentration in solution, the
highest value after mirroring was normalized to the protein
concentration determined by RP-HPLC analysis. The protein con-
centration in solution was calculated as the percentage of total
protein available based on the initial concentration of lyophilized
inclusion bodies.
3.8. Reversed-phase HPLC
RP-HPLC was performed using a TSKgel Super-Octyl column
(4.6  50 mm2, 2 mm, 110 Å) (TOSOH BIOSCIENCE, Germany). A buffer
system of 0.1% (v/v) trifluoroacetic acid (TFA) in water as buffer A and
0.1% (v/v) TFA in acetonitrile as buffer B was used. Solubilization
samples were injected directly. Elution was performed with linear
gradients of 2–50% for EDDIE-Fuzeon and 25–60% for EDDIE-
Interferon α2b. Detection proceeded at two wavelengths, 214 nm
 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641
and 280 nm, to allow proteins and buffer components to be
distinguished. Calibration curves were established for all fusion
proteins to allow for quantification of the protein in solution.
3.9. Modeling procedure
Eqs. (9) and (10) were solved numerically to calculate the
change in the protein concentration in the liquid phase over time.
The equations were implemented into an optimization procedure
that minimized the sum of the square differences between
simulated experimental concentration profiles.
3.10. Determination of particle size distribution
The particle size distribution of inclusion bodies before and
after solubilization was determined using the LUMiSizer™ (LUM
GmbH, Berlin, Germany). In this analytical centrifuge that recorded
the space and time extinction profile, near infrared light illumi-
nates the sample and transmitted light is detected to determine
the settling velocity. Employing the Stokes equation, the particle
size can be obtained based on the distance covered by the
particles. The particle diameter x [m] is calculated as follows:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 
18ηF rm
x¼ ln ð11Þ
ðρρF Þω t m 2 r0
where ηF is the dynamic viscosity of the fluid in [mPas], ρP is the
density of the particles in [kg/m3], ρF is density of the fluid [kg/m3],
ω is the angular velocity [m/s], rm is the distance [m] traveled by
the particle during time tm in [s] from the starting point r0 in [m].
The dynamic viscosity of the fluid before and after solubilization
was determined by a cone and plate viscometer (Brookfield,
Middleboro, MA, USA).
To determine the particle size prior to solubilization, the
measurements were performed in a 30/70 (v/v) glycerol/water
mixture to avoid fast settling. After solubilization, measurements
were performed in the corresponding solubilization agent.
4. Results
First, we investigated the solubilization mechanism by mon-
itoring the change in inclusion body morphology throughout the
solubilization process using light and transmission electron micro-
scopy (TEM). The yield and rate of kinetics were determined via
the decrease in turbidity and increase of protein in solution. Based
on these results, we developed a model for predicting the process
of inclusion body solubilization.
4.1. Analysis of solubilization by microscopic techniques
The morphological changes of inclusion bodies upon solubili-
zation were followed by microscopy. Buffer changes were inves-
tigated for two model proteins under three solubilization
conditions with the most common solubilization agents: urea,
guanidine hydrochloride (GuHCl), and NaOH.
A series of light microscopy images of the solubilization of
EDDIE-Interferon α2b in 100 mM NaOH over a time course of 60 s
is shown in Fig. 2. The microtiter plates were not shaken during
image recording. Prior to solubilization, the inclusion bodies in
aqueous suspension were compact black agglomerates up to
600 mm in diameter (Fig. 2(a)). The inclusion body morphology
changed over the time span of 60 s (Fig. 2(b)–(f)). Immediately
after the addition of NaOH, translucent layers were observed
around the residual compact inclusion bodies. Upon solubilization,
the translucent areas progressed and the cores shrunk. Some cores
also evidently consisted of agglomerates of inclusion bodies,
635
as smaller areas demarcated themselves over time. After more
than 10 min of incubation with the solvent, the distinct inclusion
bodies and agglomerates were no longer distinguishable. The
inclusion bodies deliquesced and the solubilized protein diffused
into the solution. The same characteristics were visualized under
other solubilization conditions; an increasingly translucent layer
around a shrinking core of densely packed protein was observed.
Solubilization of the inclusion bodies was also visualized by
TEM. In water, the inclusion bodies were densely packed objects
with average diameters of 250–500 nm (Fig. 3(a)), which corre-
sponds with the dimension of a single inclusion body (Margreiter
et al. 2008; Singh and Panda 2005). During the solubilization
process, gray branches formed where the protein diffused out at
certain positions from the inclusion bodies (Fig. 3(c) and (d)).
Depending on the solubilization agent, the branches were different
sizes and shapes. A barrier layer (white borderline) with small pores
was observed in images of advanced solubilization (Fig. 3(e)).
The barrier was hidden by the black core in images taken at the
beginning of the process (Fig. 3(b) and (c)). This barrier is found
either intact around the whole inclusion body or broken with only
parts of it visible. After solubilization progressed, the inclusion
bodies were no longer completely filled with protein for solubiliza-
tion conditions resulting in high yields of more than 90% solubilized
protein (Fig. 3(e)). Similar images were recorded for various model
proteins and solubilization conditions. Fig. 3(f) shows the solubiliza-
tion of inclusion bodies which have been subsequently incubated
with autoprotease antibodies and gold-labeled secondary antibo-
dies. The gold nanoparticles selectively marked the fusion protein
diffusing out of and present in the inclusion bodies (black dots).
Thus, the gray branches observed in Fig. 3(c), (d), and (f) contain the
fusion protein over-expressed in the inclusion bodies. These obser-
vations, together with the solubilization kinetics, were the basis for
the development of a model.
4.2. Kinetics and model for solubilization of inclusion bodies
The solubilization kinetics were determined in a high-
throughput system on the m-scale (Dürauer et al. 2009). They
were calculated from the turbidity change over time and the
maximum protein in solution at equilibrium for inclusion bodies
containing two different fusion proteins, EDDIE-Fuzeon (Fig. 4(a))
and EDDIE-Interferon α2b (Fig. 4(b)), under four different condi-
tions (2 M/5 M GuHCl, 4 M/8 M urea). Significant differences in the
rate and yield of solubilization are characteristic of the individual
protein and solubilization conditions. Depending on the condi-
tions, fast kinetics could reach equilibrium within 2–15 min.
To establish the model for the solubilization of inclusion bodies,
we assumed that the process consists of two steps: solubilization
and conformational change of the protein and its subsequent
release. So we modeled two layers, a reactive and diffusive layer
(Fig. 1). The concentration at the interface is unknown; thus, both
descriptive terms were combined into one in a homogeneous layer
model, and consequently, all necessary concentrations (initial
concentration c0, concentration in bulk liquid c, and concentration
at equilibrium Cs) could be determined. Knowing these concentra-
tions, the kinetic constant k and an empirical exponent ξ can be
calculated by the model. The solubilization of inclusion bodies can
be described by a higher order reaction. The model enabled an
accurate approximation of the inclusion body solubilization
kinetics (Fig. 4).
To confirm the underlying assumption of the model that
inclusion bodies shrink over time during solubilization, the parti-
cle size was determined prior to and after solubilization for some
solubilization conditions. The density of the inclusion bodies was
assumed to be approximately 1.33 kg/m3 (Margreiter et al. 2008;
Singh and Panda 2005). The fluid density was calculated prior to
636 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641

Fig. 2. Light microscopy images of inclusion bodies containing recombinant EDDIE—Interferon α2b: (a) aqueous inclusion body suspension prior to solubilization;
(b–f) solubilization over a time course of 60 s in 100 mM NaOH; scale bar 250 mm.
 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641 637

Fig. 3. Transmission electron microscopy images of inclusion bodies before and during solubilization: (a) aqueous inclusion body suspension of EDDIE-Interferon α2b prior
to solubilization, scale bar 200 nm; (b) solubilization of EDDIE-Interferon α2b after 15 min in 2 M GuHCl, scale bar 200 nm; (c) solubilization of EDDIE-Fuzeon after 5 min in
5 M GuHCl, scale bar 100 nm; (d) solubilization of EDDIE-Interferon α2b after 2 min in 100 mM NaOH, scale bar 1 mm; (e) solubilization of EDDIE-Fuzeon after 2 min in
100 mM NaOH, scale bar 200 nm; and (f) solubilization of EDDIE-Interferon α2b after 2 min in 100 mM NaOH, immunogold-labeled, scale bar 100 nm.
638 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641
Fig. 4. Solubilization kinetics of EDDIE-Fuzeon and EDDIE-Interferon α2b:
(a) experimentally determined (symbols) and fitted (lines) solubilization kinetics
of EDDIE-Fuzeon and (b) EDDIE-Interferon α2b under various conditions. The data
were fitted to Eq. (10). Error bars, s.d.; n ¼2.
solubilization. As so far no in situ measurement for the density
change of the inclusion bodies throughout the solubilization is
available, the density difference between inclusion bodies and
fluid could not be determined. Therefore, the particle size at
equilibrium was calculated using the same values as before
solubilization.
Although the observed particle size distributions were broad,
the size of the inclusion bodies decreased during solubilization
(Fig. 5(a)–(d)). Furthermore, the decrease in the radius was greater
for better solubilization conditions, meaning that greater
decreases in particle radius were determined for higher yields of
protein in solution. The kinetic constants and corresponding
decreases in radius are summarized in Table 2. The experimentally
measured decrease in the radius was always greater than that
predicted by the model.
5. Discussion
The mechanism of inclusion body solubilization was elucidated
by microscopy. Light microscopic investigation of inclusion bodies
in aqueous suspension prior to solubilization revealed densely
packed agglomerates up to 600 mm in diameter. The average size
of single inclusion bodies ranges from 200–500 nm, but they can
grow to more than 1 mm (Bowden et al. 1991; Margreiter et al.
2008; Singh and Panda 2005). Therefore, dimensions of up to
600 mm indicate that some of the inclusion bodies are packed in
agglomerates, unordered clusters of single inclusion bodies. This
was confirmed by light microscopy of solubilization over time,
which revealed multiple smaller areas emerged from the bigger
agglomerates. The analysis also showed that, during the solubili-
zation process, the densely packed inner cores of protein shrink as
the solubilized protein diffuses to an outer layer, and subsequently
into free solution. The translucent layer observed around the black
cores (i.e. the inclusion bodies) was interpreted as the zone where
proteins slowly diffused from the inclusion body.
The presented images reflect the solubilization process in a
non-agitated state, whereas in practice the process is carried out in
a stirred tank. Agitation of the solubilization agent will support the
film mass transfer from the outer layer (diffusion layer) to the free
solution. Because the kinetics constants were independent of the
applied mixing frequencies on the m-scale, we concluded that the
rate-controlling step of the solubilization kinetics is the diffusion
from the core through the outer layer.
The TEM images support our model on the single particle level.
The solubilized protein clearly diffused through certain “pores” in
a barrier layer surrounding the inclusion bodies. The number and
dimensions of these pores enabling the diffusion of the solubilized
protein out of the inclusion bodies is characteristic of the solubi-
lization condition used and the individual inclusion bodies. When
inclusion bodies are solubilized with 100 mM NaOH, the protein
diffuses through numerous pores on the entire surface immedi-
ately after contact with the solubilization agent (Fig. 3(d)–(f)). In
contrast, the incubation of the same inclusion bodies with 5 M
GuHCl only shows some pores are “open” for protein diffusion
from the inclusion bodies (Fig. 3(c)). For even weaker solubiliza-
tion conditions, such as 2 M GuHCl, almost no pores are present
(Fig. 3(b)). Solubilization of inclusion bodies by NaOH reaches
equilibrium within 120 s of incubation. Further incubation would
lead to protein degradation. Thus, only the yield of solubilized
protein in solution, not kinetics, was determined for this condition.
Numerous images were taken during solubilization with urea
showing similar mechanism of the process. Nevertheless, none
of them is shown due to low quality caused by crystallization of
the chaotrope on the coated copper grids.
All results obtained with microscopic techniques are in good
agreement with recently published data of Cano-Garrido et al.
(2013) who proposed the architecture of inclusion bodies as
matrix of amyloid like structured protein filled with native /
native-like protein showing different solubilization properties. It
seems likely that the described insoluble amyloid scaffold corre-
sponds with the “barrier layer” that could be observed during
advanced solubilization under strong conditions. Therefore, we
conclude that the “barrier layer” can be assumed as appearance of
the matrix due to solubilization of the filling protein. Accordingly,
the observed pores are the gaps of the matrix.
The kinetics of individual inclusion bodies has been determined
since the fermentation conditions and type of recombinant protein
were shown to influence their structure and porosity (Margreiter
et al., 2008).
Findings from RP-HPLC of the protein in solution at equilibrium
agreed with the assumption that the obtained kinetics depend on
the number and dimension of pores, as solubilization with
100 mM NaOH showed the highest yield of all conditions used,
whereas 2 M GuHCl solubilized the lowest amount of protein.
Overall, the observed kinetics determined via the decrease in
turbidity and increase of protein in solution for diverse solubiliza-
tion conditions showed significant differences in the rate and yield
of protein in solution for individual proteins.
The Shrinking Core model would suggest itself to describe the
inclusion body solubilization process. Such processes are charac-
terized by a fast irreversible reaction inside the solid phase at a
sharp reaction front. This interface moves into the particles,
 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641 639

Fig. 5. Particle size distributions determined by an analytical centrifuge of EDDIE-Interferon α2b and EDDIE-Fuzeon before and after solubilization: (a) EDDIE-Fuzeon in 2 M
GuHCl, and (b) EDDIE-Fuzeon in 5 M GuHCl, (c) EDDIE-Interferon α2b in 5 M GuHCl, and (d) EDDIE-Interferon α2b in 8 M Urea.

Table 2
Conditions and model parameters for the solubilization kinetics of autoprotease fusion proteins compared to the experimental radius decrease.

Solubilization Fusion protein in Kinetic constant k Empirical Residual radius r/R Residual radius r/R
Model protein
condition solutiona [%] [10  5 m/s] exponent ξ (model) (experimental)

2M GuHCl 23.71 4.8 2.26 0.915 0.2064

5M GuHCl 69.75 37542 5.66 0.643 0.0245
EDDIE-Fuzeon
4M Urea 5.28 9.4 2.76 0.983 n.d.
8M Urea 54.85 10431 18.4 0.761 n.d.

2M GuHCl 0.57 1.2 1.33 0.988 n.d.

5M GuHCl 61.09 1342 4.1 0.729 0.0223
EDDIE-Interferon α2b
4M Urea 0.49 27.8 2.02 0.998 n.d.
8M Urea 8.65 21.7 3.06 0.971 0.7751

n.d., not determined; r is the radius at equilibrium, R is the radius prior to solubilization.
a
Corresponds with Cs in [kg/m3] related to c0 ¼10 kg/m3.

separating two distinct zones: a shrinking unreacted core and an
inward advancing product shell (in the case of spherical particles)
(Hsu et al., 2009; Huang et al., 2012). In contrast, no sharp reaction
front was visualized in TEM images of solubilizing inclusion
bodies. Instead, the images revealed branches of diffusing protein.
Furthermore, the shrinking core model was not suitable to
describe a process of such a high order as has been observed for
the inclusion body solubilization process.
Therefore, a model related to the shrinking core model but
adapted to these deviations was established. This model correlates
the increase of the protein mass in solution with the decrease in
inclusion body volume via a decrease in the particle radius over time.
The residual radii at equilibrium are listed in Table 2. Furthermore,
the kinetic constant k for the process, representing the rates of the
process kinetics, is calculated by combining the influence of
the physical “limitation” of the pores in the inner layer and the
subsequent diffusion to the layer boundary. In addition, an empirical
exponent, ξ, is implemented, referring to the “pseudo-order” of the
reaction in the inner layer; it should not be interpreted as the
conventional order of a chemical reaction, as it cannot give an
indication of the number of elementary species involved in the
reaction process. Throughout the solubilization the density of the
particle is varying which was included in the model. Nevertheless,
the maximum density change over time in case of entire solubilization
—starting density of 1.33 kg/m3 and final density of 1.1–1.2 kg/m3
—was estimated to be 7–15% compared to 100% radius decrease.
640 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641

Furthermore, so far an in situ measurement of inclusion body density the data confirmed the assumption of an overall decrease in the
is not available. Additionally, our data reveal that a density gradient radius used as the basis of the established model.
inside the inclusion body occurs during solubilization which is even
more difficult to assess. Therefore, not to add uncertainties to the
model and due to the fact that the radius decrease is more 6. Conclusion
pronounced than the density change over time the density change
can be neglected in the calculations. The model was established for To summarize, inclusion body solubilization does not follow
describing the solubilization of single inclusion bodies, but light the rules of ordinary solubilization processes. The process is
microscopy images showed that some of the inclusion bodies are controlled by pore diffusion and, as a consequence, power input
packed in agglomerates. Nevertheless, the model is appropriate for and mixing frequency are assumed to be irrelevant for rate and
describing both because it fit all determined kinetics. Therefore, we yield, as shown on the m-scale. The established model enables
conclude that the diffusion of protein out of a single inclusion body is evaluation of the inclusion body solubilization process on a
the limiting step of the overall solubilization process. An exception is numerical level by reducing the morphological observations to a
the solubilization of EDDIE-Fuzeon in 8 M urea. For this condition, the correlation of decrease in particle radius to an increase of protein
measured values of absorbance at 600 nm in the beginning of the in solution as expressed in Eq. 10. Solubilization conditions, such
process (1 and 2 min of solubilization) were lower than at equilibrium, as chaotrope or additive concentration, and process parameters,
which is why the calculated empirical exponent reflecting the yield of such as pressure, temperature, or dissolved oxygen, can be related
protein in solution is out of scale. Similar results were observed for to the rate and yield of the process. Thus, the process can be
some other conditions where fast kinetics with high concentrations of optimized time wise and according to the required yield of protein
solubilized protein caused high viscosity of the protein solution and in solution, which concurrently increases the overall productivity.
hindered proper mixing in the beginning of the process. These This model allows a move forward from an empirically driven
deviations can be circumvented by reducing the initial protein design to engineering this crucial step of protein recovery from
concentration. inclusion bodies.
The determined parameters showed increase of kinetic con-
stant and yield with increasing chaotrope concentration. A mini-
mal deviation was observed for solubilization of EDDIE-Interferon Nomenclature
α2b with 4 and 8 M urea, where the kinetic for the lower
chaotrope concentration was slightly faster compared to the A IB surface (m2)
higher concentration which is reflected in the calculated kinetic c protein concentration in bulk liquid (kg/m3)
constant as listed in Table 2. In special cases the increasing c0 maximal concentration of protein available for
viscosity with increasing chaotrope concentration will cancel out solubilization (kg/m3)
the improved solubilization. cd protein concentration at interface (kg/m3)
The model assumed that the inclusion body agglomerates CS protein concentration at equilibrium (kg/m3)
shrink. This change was confirmed by experimental data obtained k kinetic constant (m/s)
using an analytical centrifuge, but the change in particle size was kp kinetic constant of reaction rate in reaction layer (m/s)
always overestimated by the measured radius decrease compared kd kinetic constant of reaction rate in diffusion layer (m/s)
to the prediction by the model. This discrepancy is explained by m mass of IB (kg)
the calculation method applied for determining the inclusion body m0 total mass of IB before solubilization (kg)
diameter. As described above, the particle size at equilibrium was mi mass of single particle (kg)
calculated assuming the same density difference as at the begin- MW molecular weight (Da)
ning of the process. The median size of the inclusion body n number of particles (-)
agglomerates prior to solubilization, approximately 50–60 mm p pseudo-order of reaction (-)
(mean 20–30 mm) (Fig. 5), is in good agreement with the dimen- r radius of inclusion bodies during solubilization (m)
sions estimated from light microscopy. Therefore, we assume that r0 starting point (m)
the radius of the particles at equilibrium was underestimated, rm distance traveled by particle (m)
possibly due to decreased particle density over time as a result of R radius of inclusion bodies before solubilization (m)
penetration of the solvent into the particle pores. As shown by t time (s)
Taylor et al. (1986), the porosity can be as high as 85%. However, tm traveling time (s)
the density of the protein solution is higher than the solvent V volume of spherical IB (m3)
density itself. The density difference must be assumed to change x particle diameter (m)
over time; therefore, using the same values for calculations in the
beginning and at equilibrium might lead to an underestimation of greek symbols
final particle size. For solubilization of EDDIE-Interferon α2b with
8 M urea, where only 8.65% of protein was solubilized, the change ηF dynamic viscosity of fluid (Pa s)
of density difference is less significant than for conditions solubi- ξ empirical exponent (–)
lizing higher protein yield as shown for 5 M GuHCl. Therefore, the ρ particle density (kg/m3)
underestimation of the final particle size by the sedimentation ρF liquid density (kg/m3)
experiments is less pronounced for this weaker condition. ω angular velocity (m/s)
Furthermore, the refractive index of the inclusion bodies after
solubilization is not uniform throughout the particles. Some
particles, independent of size, may be too translucent to be
detected after solubilization by the NIR light source of the Author Contributions
measuring device and, therefore, will not be measured. Thus, the
determined particle size after solubilization will be an under- C.W. and S.M. determined the solubilization kinetics. C.W. and
estimation of the true value. Even though the experimentally G.S. recorded the TEM images. D.A. set up the model. Data fitting
determined radius decrease would then be overestimated, and particle size measurements were carried out by C.W. C.W. and
 C. Walther et al. / Chemical Engineering Science 101 (2013) 631–641
A.D. recorded the light microscopy images and drafted the manu-
script. R.H., A.D. and A.J. contributed to the overall concept of the
project, A.D. and A.J. revised the manuscript.
Acknowledgments
We acknowledge M. Debreczeny from the Imaging Center of
DBT/BOKU for supporting the light microscopic analysis and the
Department of Nanobiotechnology/BOKU for providing TEM facil-
ities. We thank Sandoz GmbH for providing inclusion bodies
containing EDDIE-Interferon α2b, G. Striedner's group for fermen-
tation of EDDIE-Fuzeon, A. Trefilov for the subsequent isolation of
inclusion bodies and for the contributions of Boehringer-Ingelheim
RCV. This work has been supported by the Federal Ministry of
Economy, Family and Youth (BMWFJ), the Federal Ministry of
Traffic, Innovation and Technology (bmvit), the Styrian Business
Promotion Agency SFG, the Standortagentur Tirol and ZIT—
Technology Agency of the City of Vienna through the COMET-
Funding Program managed by the Austrian Research Promotion
Agency FFG.
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