DOI 10.

1007/s10517-017-3964-y
Bulletin of Experimental Biology and Medicine, Vol. 164, No. 2, December, 2017 229

METHODS

Comparison of Different Methods of Purification

and Concentration in Production of Influenza Vaccine
N. N. Asanzhanova, Sh. Zh. Ryskeldinova, O. V. Chervyakova,
B. M. Khairullin, M. M. Kasenov, and K. K. Tabynov
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 164, No. 8, pp. 261-264, August, 2017
Original article submitted March 9, 2017

The overwhelming majority of influenza vaccines are prepared with the use of chicken em-
bryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein
in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of
ovalbumin content is of crucial importance for vaccine production. We compared two methods
of purification and concentration of influenza virus: zonal gradient ultracentrifugation and
combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrica-
tion of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation
protocol by the results of ovalbumin removal (to meet standard requirements).
Key Words: purification and concentration; chromatography; ultracentrifugation; influenza;
vaccine

About 10% population in the world are infected ev-
ery year with influenza virus, the resultant annual
mortality is 250-500 thousand cases. Preventive vac-
cination is the best influenza virus control strategy.
Because of incessant mutations and reassortation of
influenza virus, annual revaccinations have to be car-
ried out. For this reason, the search for new methods
for preparing highly purified influenza vaccines with
low reactogenic activity, without side effects, and
suitable for different age groups, remains an impor-
tant problem.
One of the main stages of vaccine manufacture is
preparation of purified concentrated virus. Tradition-
ally, the material for influenza vaccine production is
prepared on chicken embryos and the virus is routinely
purified by processes including zonal gradient cen-
trifugation [1,2].
Research Institute of Biological Safety Problems, Zhambyl Region,
Gvardeiskii settlement, Kazakhstan Republic. Address for corre-
spondence: anurika@mail.ru. N. N. Asanzhanova
The manufacture of cell culture-based vaccines
[4,8,11] promoted creation of new purification and
concentration protocols [3,6,10], one of their variants
based on the use of chromatographic methods [7,9,13].
Analysis of modern technologies for virus puri-
fication and concentration has shown that the tech-
nological schemes are based on the use of combina-
tions of several methods, due to which the maximum
purification of vaccine from ovalbumin and various
cell components is attained [5]. The main criterion
for evaluating the efficiency of this or that purification
and concentration protocol is the degree of the virus
antigen purity and concentration. The efficiency of a
method is evaluated by the virus output, expressed
in percent of removal of ballast proteins and nucleic
acids [12].
Hence, use of the optimal methods and technolo-
gies for the virus (antigen) concentration and purifi-
cation from contaminants of the culturing system and
other technological processes is expected to rule out
the mechanical destruction of the virus, improve the

0007­-4888/17/16420229 © 2017 Springer Science+Business Media New York

230 Bulletin of Experimental Biology and Medicine, Vol. 164, No. 2, December, 2017 METHODS
efficiency of the product purification and its output,
reduce the reactogenic activity of the biopreparation,
shorten the stages and total duration of the process,
use the potentialities of multiplexing and advantages
of modulus technology, etc.
We study two methods for influenza virus puri-
fication (zonal gradient ultracentrifugation and com-
bined protocol including ultracentrifugation/diafiltra-
tion and exclusion chromatography) for preparing
seasonal vaccine.
The recombinant strain NIBRG-121xp was puri-
fied and concentrated by chromatography and ultra-
centrifugation and the results were compared.
MATERIALS AND METHODS
Preparation of virus-containing fluid. The study was
carried out on influenza virus strain A/NIBRG-121xp,
N1H1 (NIBSC, London, UK). The virus was cultured
on 10-day chicken embryos for 48 h at 34oC, after
which allantoic fluid was collected and clarified by fil-
tration through capsules with 2.0/1.2 µ pores (Polysep
II Opticap XLT10; Millipore). The material prepared
by this method was used for subsequent purification
and concentration by the chromatographic method and
ultracentrifugation.
Purification and concentration by the chro-
matographic method. Clarified virus suspension was
concentrated on an ultrafiltration device (Millipore)
to 500 ml on a Pellicon NMWL (nominal molecu-
lar weight limit) 300,000 cassette. Dialysis filtration
was carried out in a tangential flow against 15 vol-
umes of buffer 1 (in M: 1 NaCl, 0.01 PBS (pH 7.4),
0.001 EDTA) for removal of low-molecular proteins
and against 9 volumes of buffer 2 (in M: 0.15 NaCl,
0.01 PBS (pH 7.4), 0.001 EDTA) for buffer altera-
tion. After the buffer was changed, the virus was con-
centrated to 6-fold volume. Fractionation of the virus
and the allantoic fluid high molecular weight protein
components (>100 kDa) was carried out by exclusion
chromatography (gel filtration) on CL-6B sepharose
(50×60 column; Pharmacia). The virus was applied
onto the chromatographic column packed with CL-6B
sepharose equilibrated with buffer 2 at a rate of 300
ml/h (1:0.33). Virus elution was monitored by flow UV
detector. The eluate was collected into sterile flasks.
The concentrations of protein, ovalbumin (OVA), and
hemagglutinin (HA) were measured at each stage of
purification.
Purification and concentration by ultracentri-
fugation. Clarified virus material was filtered through
linear sucrose gradient prepared from 0.125 M citrate
buffer (pH 7.8) and 60% sucrose solution, and centri-
fuged at 35,000rpm for 5 h on an eKII flow industrial
ultracentrifuge (Alfa Wassermann). After ultracentri-
fugation, the linear sucrose gradient was collected by
fractions from 0 to 60%, 53.3 ml each. Each fraction
was then tested for hemagglutinating activity. Frac-
tions with maximum activity were pooled for measure-
ment of the protein, OVA, and HA content.
Measurement of OVA. Measurements of OVA
were carried out using Chicken egg Ovalbumin Elisa
Kit, according to manufacturer instruction (Alpha Di-
agnostics International). The measurements were car-
ried out three times for each sample.
Total protein measurement. The concentration
of total protein was measured by Lowry method with
the Sigma reference protein sample. The measure-
ments were carried out three times for each sample.
Measurement of HA by solitary radial immu-
nodiffusion technique (SRIT). In order to evaluate
the antigenic load at various stages and in the end frac-
tion of the product, the content of HA was evaluated
by SRIT [14]. The HA reference sample and antiserum
for NIBRG-121xp were purchased from the National
Institute of Biological Reference Samples and Control
(#09/196, #14/134, London, UK). All measurements
were carried out three times for each sample.
RESULTS
Purification by chromatographic method. Purifica-
tion and concentration of three experimental batches
(10.6-14.4 liters) of recombinant influenza virus NI-
BRG-121xp strain was carried out by ultrafiltration in
tangential flow and diafiltration with subsequent gel
filtration.
Ultrafiltration with subsequent dialysis removed
more than 94.2-95.5% proteins initially present in the
material (Fig. 1). Removal of OVA was slightly less
effective: 92.4-93.3% of initial content.
Gel filtration of partially purified in CL-6B sepha-
rose virus concentrate resulted in elimination of 93.8-
94.6% proteins and 98.4-99.1% OVA (Fig. 1).
The resultant virus concentrates were purified
from 99.64% proteins and 99.88-99.94% OVA present
initially. The content of HA in the resultant prepara-
tion was 102.8-119.5 µg/ml, which was equivalent to
retention of 54.5-68.8% HA.
Purification by ultracentrifugation. Clarified
suspensions of 30-35 liters were concentrated to 550-
630 ml (as a result of 51-66 concentrations) and puri-
fied. After purification the material was collected by
fractions, and each fraction was tested for the virus
content in hemagglutination test. Fractions containing
31-43% sucrose with the highest hemagglutinating
activity of the virus were selected by the results of
hemagglutination test.
Purification by this method led to elimination of
99.95-99.97% OVA and 98.6-99.5% protein. The con-
N. N. Asanzhanova, Sh. Zh. Ryskeldinova, et al. 231

Fig. 1. Chromatographic purification of three batches of virus-containing allantoic fluid, strain NIBRG-121xp. Here and in Fig. 2: purification
at various stages and final values evaluated by Lowry method (protein), ELISA (OVA), and SRIT (HA). The mean values of 3 measurements
are presented. UF/DF: ultrafiltration/diafiltration.

Fig. 2. Purification of three batches of virus-containing allantoic fluid, strain NUBRG-121xp.

tent of HA in this pool was 297.1-401.2 µg/ml, which
was equivalent to retention of 61.7-74.1% HA in the
resultant product (Fig. 2).
Comparison of the data showed no appreciable
differences in the content of HA, proteins, and OVA.
Purification by both methods led to preparation of
purified virus material with 1.62-2.50 µg/ml OVA
(chromatography) and 1.3-3.2 µg/ml OVA (ultracen-
trifugation).
As OVA is the main protein component of the al-
lantoic fluid, its content can surpass 60% of total protein
content. The use of ultrafiltration/diafiltration ensures
concentration of the material paralleled by removal of
a great amount of protein admixtures (this procedure
usually decreased protein content to 20% from its initial
level). However, the multiplexing process involves cer-
tain difficulties, because of high price of the materials.
Zonal ultracentrifugation provides high purifica-
tion of the concentrated product, but is often more
time consuming than chromatography, because of the
pretreatment stage. Its advantage is the possibility of
processing great volumes, when duration, efficiency,
and safety are the key factors. The pilot ultracentri-
fugation process, presented in this paper, is preceded
by 8-12 h of pretreatment; this time does not include
the adjustment procedure, initiation, preparation, and
purification, while concentration by ultrafiltration/di-
afiltration and subsequent chromatography can be real-
ized within 6-10 h.
Comparative results of purification and concen-
tration under experimental conditions of influenza
vaccine manufacture demonstrate the possibility of
creating modern effective multiplexing process for
GMP preparation of influenza vaccines to be used in
influenza epidemic and pandemic.
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