3.2. 2D-AEC×SEC experimental operation conditions
Salt concentration, flow rate, pH, and sample volume are considered as
the most important parameters which should be optimized for developing a
proficient 2D-AEC×SEC methodology. Regarding the self-dissociation
feature and highest immunity of FMDV particles reported at pH below 5.6
and also partially on neutral pH (Liang et al., 2014), the mean pH = 7.3
was selected at all of the optimization steps for the initial and
elution buffers in both column dimensions.
As it was already mentioned, co-elution behavior of virus particles and
BSA indicated that the p I values are very close together, and
accordingly the selection of pH =7.3 about three units higher than of
the p I = 4.8 for BSA can guarantee absorption active ingredient
of vaccine to ion-exchange media.
Regarding to theoretical concepts of IEX approach, sample should be
loaded with the minimum ionic strength on the ion exchange media in
order to prevent from competition between the ions in the medium and the
target protein for adsorption on the ion exchanger. Therefore, 1.2
mS/cm, as the electrical conductivity of the initial buffer (Tri-HCl 20
mM with pH = 7.3) was considered as the reference in order to set the
conductivity of primary feed stock. Based on an experimental study, a
salt adjustment for the primary sample could provide a conductivity
equivalent to 1.2 mS/cm as the initial buffer in AEC. In the elution
step, potassium chloride (KCl) was used to adjust the ionic strength of
the applied elution buffer. Accordingly, a buffer of Tris-HCl (20 mM
with p pH = 7.3) containing 500 mM KCl was used in a stepwise gradient
mode for elution absorbed proteins with suitable conductivity.
Considering the Q-Sepharose base media specifications, the mean flow
rates of 3 mL/min and 8 mL/min were selected for loading and eluting
steps the sample, respectively. In general, loading 20–30% of the
total binding capacity of the column should provide the optimal
resolution with gradient elution mode in IEC (Bailly & Tondeur, 1981).
The total protein concentration of 0.473 mg/mL in the feed stock was
determined by Bradford assay and performance of Q-Sepharose matrix was
evaluated for the sample volume in the range of 50-100 mL of the primary
sample in combination with Sup-200 as the final polishing step. Then,
the total fractions obtained from capturing step were analyzed by HP-SEC
as our last work (Bagheri et al., 2018), and virus infectivity assay was
determined by Reed and Muench’s method (Frenz & Horváth, 1985).Fig. 2 represents the elution pattern of primary sample for
minimum sample loading on Q-Sepharose media and Sup-200. After analyzing
the obtained fractions from the first dimension, the highest amount of
FMDV particles was washed out from the column between 120-180 minutes
for the sample loading which was two times more than the maximum loading
in the previous study (Bagheri et al., 2018). Therefore, pooled virus
fraction was concentrated by the concentration factor 10, and then 4 mL
of this concentrated sample was loaded on the Sup-200. The comparison of
elution pattern for 2D-AEC×SEC and 2D-SEC×SEC indicates a completely
same elution pattern for both methods in the second dimension. Since the
minimum sample volume loaded on the first dimension
(Vinj = 50 mL) is about two times more than the maximum
loading capacity in SEC for 2D-SEC×SEC, it seems that AEC is considered
as a better candidate for capturing step to fulfill speed and capacity
as the two most important criteria in this step. Based on the obtained
data, the overall purification time in the first dimension reduced by a
factor of 0.7 in 2D-AEC×SEC than 2D-SEC×SEC. In order to ensure the
efficiency of anion exchange media in the proposed three-step strategy,
the volume of the sample loaded on the resin increased in different
experiments to maximum 100 mL (equal to four times of the related amount
in 2D-SEC×SEC (Bagheri et al., 2018).
Fig. 3 (A ) and (B ) represent the experimental data, as
well as the simulation results of the elution pattern in the primary
feed stock for maximum sample loading on a combination of Q-Sepharose
and Sup-200 media. In the final polishing step for Sup-200, as the
second dimension, the volumetric flow rate decreased to 2.5 mL/min for
obtaining a better resolution between virus particles and NSPs marker,
BSA, due to the higher concentration of the concentrated sample in an
intermediate concentration step. The simulation results for retention
behavior of BSA, virus, rDNA and IgG components in the first and second
dimension were also depicted in Fig. 3 (A ) and
(B ). In AEC the coupled set of Equations. (1), (4), (5) and (6)
incorporating plate model (Eq. 2, considering convection with dispersion
based on plate number), was discretized and solved using finite element
and orthogonal collocation techniques. It is worth mention that the
number of theoretical plates was estimated experimentally from a pulse
injection of acetone and following the UV chromatogram for determination
peak width at half-height and retention time. Accordingly, the height of
theoretical plate (HETP) was calculated as 0.01177 cm. In order to
assess the effect of controlling diffusion versus convection mechanisms,Pe number, as an important parameter affecting the
performance of separation process, was considered and estimated by Eq.
(3). Therefore, for the total height of the bed\(H_{B}=40\ \text{cm}\), internal diameter of the bed column\(\text{ID}\ =\ 1.6\ \text{cm}\) and particle radius of
adsorbent\(\text{\ Rp}\ =\ 45-165\ \text{μm}\), the optimum amount
for flow rate (or Pe number) and eluents concentration for
FMDV particles elution from Q-Sepharose media were studied by
mathematical modeling to explore the most appropriate condition in AEC
column. Acquire data demonstrated that a flow rate equal to 8 mL/min and
a combination of 73% of buffer A (buffer without salt) and 27% of
buffer B (buffer with 0.5 M salt concentration) can be applied as the
most suitable operational condition to elute virus particles from
Q-Sepharose in the first dimension. Accordingly, the concentration of
KCl at the time of departure of the virus peak from AEC column was
predicted equal to 0.129 M. This is nearly close to experimental data as
indicated 0.135 M KCl concentration for FMDV elution from AEC column.
Moreover, as illustrated in Fig. 3 (A ), simulation
results in the first dimension demonstrated that peaks for virus, BSA
and rDNA have interfering areas in the output of the AEC, which is
satisfactory matched with experimental data. Same results for
experimental and simulated investigations can be explained by very closepI values for these components at natural pH. Similarly,
mathematical modeling and simulation process was also applied for total
bed height equal to\(\ H_{B}=60\ \text{cm}\), internal diameter of the
bed column. \(\text{ID}\ =\ 2.6\ \text{cm}\), particle radius of
adsorbent \(\text{Rp}\ =\ 24-44\ \text{μm}\) in the second dimension
for prediction and obtaining optimal flow rate. Eventually a flow rate
equal to 2.5 mL/min was obtained for optimized operation condition and
achieved data in Fig. 3 (B ) showed that the simulation
results are well-matched with the experimental counterparts. Therefore,
this model can be applied to scrutinize the scaling-up issue in term of
geometry (enlarging the cross-section and length of the columns), flow
rate and sample loading capacity in the columns.