GE Healthcare

Purification
technologies
for vaccines
and vectors

19
Meeting the challenges of modern vaccine
and vector purification
Like protein-based drugs, vaccines and vectors are scrutinized
by regulatory authorities that demand stringent standards of
safety and efficacy. The biopharmaceutical manufacturing
companies themselves also place high demands on the
products they make: their challenges include bringing safe
biopharmaceuticals to the market quickly and on budget.
GE Healthcare is fully-equipped to satisfy all such demands.
Our chromatography products are used in the purification of
90% of all FDA-approved biopharmaceuticals, including new
vaccines, and some 70% of all proteins at laboratory scale.
Proven skills and know-how upstream
and downstream
Our laboratory, pilot and production-scale microcarrier cell
culture, chromatography, and membrane filtration systems
are in constant use around the world. For example, industrial
cell culture with Cytodex™ microcarriers has proven to be
a reliable and cost-effective means for manufacturing both
human and animal healthcare viral vaccines.
Our R&D, technical back-up and problem-solving expertise
are available to help at every stage. Downstream, we are well
aware of the purification requirements of vaccines and viral
vectors. Their different sub-type properties, high molecular
weights, complex surface properties, and fragile structures
pose special challenges, as does their often narrow window of
pH stability. When modifications to traditional protein-based
approaches are required, we have the solutions you need.
To meet the challenges of the large vaccine and vector pipeline,
plus a significant worldwide need for new and better products,
GE Healthcare is committed to supporting the production and
purification of biomolecules.
In this presentation, we highlight technologies for the
purification of:
• Human recombinant subunit vaccine
• Human viral vaccine
• Plasmid DNA for human and animal gene therapy and for
DNA vaccination
• Viral vectors for human gene therapy
• Animal viral vaccine

Production hall for the large-scale manufacture of influenza vaccine.

(Reproduced with kind permission of Baxter Biosciences Corp).

Cover
Surface illustration of influenza virus. The red spikes represent haemagglutinin,
the yellow box-like features neuramindase, and the green surface peeping
through between the spikes the viral envelope.
Vaccines like influenza and hepatitis are powerful tools used to limit the number
of serious viral infections and pandemics. Viral vectors like adenoviruses and
adeno-associated viruses are vehicles being developed for delivering genetic
material to target cells in gene therapy.
Copyright Russell Kightley Media, www.rkm.com.au


Strategies for the purification and production
of vaccines and vectors
Recent decades have witnessed a large increase in the
immunological and technological strategies employed for
manufacturing vaccines. Many early developments were
focused on improving the safety, efficacy and supply of
traditional prophylactic vaccines such as hepatitis and
influenza. However, most efforts have been directed towards
developing new human vaccines for diseases not previously
treatable. In addition, attention has also swung from purely
replacement strategies towards preventative strategies for
non-infectious diseases such as Parkinson’s disease, as well
as for therapeutic use, e.g. cancer.
Veterinary vaccines are also experiencing an upsurge in
interest, especially for major problems areas such as foot and
mouth disease, Newcastle’s disease, and avian influenza.
Modern approaches of using viral vectors as vehicles for
delivering genetic material to the target cell in gene therapy
are contributing to the renewed focus on vaccines. Combine
this development with higher safety demands imposed by the
FDA (US Food and Drug Administration) and EMEA (European
Medicines Agency) and the need for better production and
purification processes becomes clear.
Processing improvements in vaccine
manufacture
A number of opportunities present themselves for manufacturers
wanting to deliver safer, more efficacious and cost-effective
vaccines.
Microcarrier culture systems are an attractive means of
growing mammalian cells. The technique is well-proven
in vaccine production, where it lowers culture medium
and serum costs, decreases labor and reduces the risk of
contamination. Within downstream processing, ultrafiltration
and liquid chromatography achieve high recoveries of pure
and safe final products.
All these technologies share many vital attributes. They are
robust, economical and easy to scale up. Furthermore, they
are supported by a full range of products and services all the
way from start-up to routine production in full compliance
with regulatory requirements.

Large-scale liquid chromatography and membrane separation systems

offer vaccine manufacturers many opportunities to improve the
safety, efficacy and cost-effectiveness of human and veterinary
vaccines, as well as plasmid DNA products intended for gene therapy.


Purifying a recombinant subunit vaccine
for human use
Hepatitis B surface antigen vaccine
Hepatitis
Hepatitis means ‘inflammation of the liver’, and the most common
cause is infection with one of five serotypes, hepatitis A, B, C,
D, or E. All of these viruses can cause an acute disease with
symptoms lasting several weeks, including yellowing of the
skin and eyes (jaundice), dark urine, extreme fatigue, nausea,
vomiting and abdominal pain. Some symptoms may persist
for up to a year.
Hepatitis B and C virus can cause chronic infection where the
patient becomes a latent carrier of the virus and many years
later can develop cirrhosis of the liver or liver cancer. As
hepatitis B is the tenth leading cause of death worldwide, it is
considered the most serious of the five serotypes.
Hepatitis B vaccine has an outstanding record of safety and
effectiveness. Since 1982, over one billion doses of hepatitis
B vaccine have been used worldwide. Since 1991, the WHO
has called for all countries to add hepatitis B vaccine to their
national immunization programs.
Purification strategy
It is estimated that approximately 5% of the world’s population
is infected by the hepatitis B virus. In China, perhaps up to 9%
of the population carry the infection and as many as one million
are newly infected every year. To curb this trend, great effort
is being made to produce relatively inexpensive yet effective
vaccines for the non-infected segment of the population.
This goal has been achieved due mainly to the large-scale
production of a recombinant vaccine. The chromatographic
methods used to purify the hepatitis B surface antigen
(r-HBsAg) are both robust and economic.
Purification process
Figure 1 shows a process for purifying r-HBsAg from a Chinese
hamster ovary (CHO) culture supernatant. The entire process
comprises three chromatographic steps, with membrane
filtration for buffer exchange and product concentration.

Cell culture supernatant (CCS)

Centrifugation or micro and

Removal of cell debris
cross-flow filtration

Butyl-S Sepharose 6 Fast Flow Capture of r-HBsAg

Sephadex™ G 25 or Diafiltration Buffer exchange

Removal of endotoxins
DEAE Sepharose 6 Fast Flow
and DNA

Ultrafiltration Product concentration

Sepharose 4 Fast Flow Polishing

Purified r-HBsAg

Fig. 1. Downstream process for purifying recombinant

Hepatitis B virus surface antigen (r-HBsAg) from CHO cells.


The key step uses the hydrophobic interaction medium Butyl-S Comments
Sepharose™ 6 Fast Flow to capture r-HBsAg from extraneous
Butyl-S Sepharose 6 Fast Flow was developed and optimized
proteins and other impurities present in the supernatant. This
in cooperation with a process-scale manufacturer of
removes approximately 97% of the A280-absorbing substances
biopharmaceuticals to purify r-HBsAg from CHO cells. It has
with a recovery of r-HBsAg activity better than 90%. Figure 2
proven its value at the capture stage of the process by removing
shows a typical elution profile obtained after chromatography
the bulk of impurities without stringent requirements for
of a clarified cell culture supernatant on a packed column of
sample conditioning (e.g. adjusting pH, salt concentration).
Butyl-S Sepharose 6 Fast Flow. The purification of the target
This contributes a robust, efficient and cost-effective large-
protein in this initial step is 34-fold.
scale vaccine production.
Analysis of fractions 1, 2, 3 for r-HBsAg activity shows that
In addition, it integrates easily with subsequent chromatographic
the unbound fraction (1) is devoid of r-HBsAg activity and that
steps for intermediate purification and polishing, as well as
fraction 2 contains more than 90% of the applied activity.
with membrane filtration for buffer exchange and product
Following intermediate purification on DEAE Sepharose Fast
concentration.
Flow and final polishing on Sepharose 4 Fast Flow, the overall
purification factor is 800-fold. Support for Butyl-S Sepharose 6 Fast Flow, a BioProcess™
medium, includes validated manufacturing methods, secure
supply and a Regulatory Support File (RSF) to assist process
validation and submission to regulatory authorities.

Further information
Data File Code No.

Medium: Butyl–S Sepharose 6 Fast Flow

Butyl-S Sepharose 6 Fast Flow 11-0026-34
Column: XK 50/20 (packed bed volume = 130 ml)
Further reading (1)
Sample: 300 ml of concentrated CCS (containing approx. 12 mg of rHBsAg).
Ammonium sulfate was dissolved to approx. 0.6 M and the
pH adjusted to 7.0.
A280 nm
1.60

1.40

1.20

1.00

0.80

0.60

0.40

0.20

1 2 3
0.00
0 20 40 60 80 100 120 140 160
Fraction number

Fig. 2. Elution profile obtained after the initial purification step of

r-HBsAg using Butyl-S Sepharose 6 Fast Flow.


Purifying a virus for a human viral vaccine
Human influenza vaccine
Influenza
Influenza is caused by a virus that mainly attacks the upper
respiratory tract. Usually lasting for about a week, the
infection is characterized by sudden onset of high fever,
headache and severe malaise, dry cough, sore throat, and
runny nose. Most people recover without medical treatment,
but for the very young, the elderly and people suffering from
certain medical conditions, influenza poses a serious risk. In
these people, the infection may lead to severe complications
of underlying diseases, pneumonia and death.
Influenza rapidly spreads around the world in seasonal
epidemics and imposes a considerable economic burden in
the form of hospital and other healthcare costs as well as lost
productivity.
Annual vaccination is the principal measure for preventing
influenza and reducing the impact of epidemics. Various
types of influenza vaccines have been available and used
for more than 60 years. Most vaccine is still produced in
embryonated chicken eggs, but concerns about a potential
shortage of eggs in a pandemic situation as well as the risk
of allergic reaction to egg albumin have led to a growing use
of mammalian cell culture systems instead. Such systems
are also better able to meet the higher safety demands being
made by the FDA and EMEA.
Purification strategy
Like all viruses, human influenza virus particles are much
larger than molecules such as proteins and peptides. This
feature can be used to advantage to separate virus from the
fermentation broth. Applicable techniques include membrane
separation, e.g. ultrafiltration, and chromatographic
separation such as gel filtration (size exclusion) in group
separation mode. Both have been combined to develop a
complete downstream process for influenza virus vaccine.
Purification process
Figure 3 summarizes the basic purification process. Following
sample preparation via centrifugation and depth filtration, the
virus-containing supernatant from a Vero cell culture is ready
for purification.

Cell culture supernatant

with human influenza virus

Microfiltration
Removal of cell debris
0.45 µm

mAU
Ultrafiltration Virus concentration and
Hollow fiber cartridge NMWC 750 kDa reduction of impurities (HCP) Sepharose 4 Fast Flow
UV1_280nm
UV2_260nm
Sepharose 4 Fast Flow Removal of HCP

Q Sepharose XL Removal of HCP and DNA

(virus)
Hemagglutinin
activity
Ultrafiltration / Diafiltration Virus concentration
Hollow fiber cartridge NMWC 750 kDa and formulation

Purified human influenza virus time

Fig. 3. A simple downstream process comprising ultrafiltration Fig. 4. Human influenza virus elutes in the void volume on group
and chromatography gives pure virus material that should meet separation with Sepharose 4 Fast Flow.
regulatory requirements.


This treated supernatant is initially pre-purified and Comments
concentrated by ultrafiltration using GE Healthcare hollow
This purification method is fully scalable. The chromatography
fiber membrane cartridges. The retentate containing the virus
columns and membrane cartridges can be cleaned-in-place,
is then purified by gel filtration in group separation mode on
and the whole process can be run within one to two days.
Sepharose 4 Fast Flow. Human influenza virus is found in the
Furthermore, it is well suited to viral vaccines. The open flow
void volume (Fig. 4). Host cell components such as small DNA
path of the hollow fiber cross flow membranes promotes
molecules and proteins are retarded and eluted later during
gentle passage with low shear forces.
isocratic elution.
In addition, human influenza virus flows through the chroma-
Final polishing is done on Q Sepharose XL anion exchanger.
tography columns without binding, which makes the process
Influenza virus is found in the flow-through while DNA
very applicable for sensitive particles. It should work well for
molecules and other negatively-charged host cell components
any strain of human influenza virus, and can thus be
bind on the column (Fig. 5).
considered a generic procedure for virus purification.
Final concentration and formulation is performed using
a second ultrafiltration/diafiltration step on a hollow fiber Further information
membrane cartridge. The results obtained (Table 1) indicate Data File Code No.
that the purified virus should meet regulatory requirements.
Sepharose 4 Fast Flow, Sepharose 6 Fast Flow, 18-1020-52
Q Sepharose XL 18-1123-82

mAU mS/cm

Q Sepharose XL

UV1_280nm
UV2_260nm
Cond

Table 1. Human influenza virus quality.

Host cell proteins n.d.*/dose

(virus) Host cell nucleic acids < 100 ng/dose**

Hemagglutinin
activity Infectivity Same as sucrose-gradient purified
* n.d. = not detectable.
** dose = 45 µg HA antigen (antigen quantified by immunoassay).
time

Fig. 5. The pooled void volume from the Sepharose 4 Fast Flow group
separation step is further purified by anion exchange chromatography
on Q Sepharose XL.


Purifying plasmids for human and animal gene therapy
Plasmid DNA
Gene therapy Purification strategy
Gene therapy aims to cure disease at the genetic level. Genes For gene therapy applications, plasmid DNA should be in
are delivered to cells where they express a therapeutic protein supercoiled form and essentially free from bacterial chromosomal
or peptide that combats the disease in question. Plasmid DNA DNA, RNA, proteins and endotoxins. In addition, the purification
complexes are used as delivery vehicles (vectors) to get the media and process should facilitate smooth and cost-effective
gene to the target cell. transfer to large-scale production. As the most common host
Plasmid DNA intended for clinical applications must meet for plasmid DNA production is E. coli, the real challenge is to
stringent purity requirements. If it is then to be used commercially, remove other nucleic acids, endotoxins and trace contaminants.
the purification method employed must also be suitable for Economy and throughput requirements make certain methods
large-scale GMP-compliant production. less suitable for large-scale purification. While products can
enter Phase I with material purified using techniques that are
not amenable for production-scale use, Phase II/III clearly
Clarified cell lysate
require scalable techniques that are also economical.
Filtration and chromatography are fully scalable, economical
Ultrafiltration and compliant with cGMP. However, the process will have to
Lysate concentration
Hollow fiber cartridge NMWC 100 kDa
take the size of plasmids into account.

Sepharose 6 Fast Flow Removal of RNA

PlasmidSelect
SOURCE 30Q
Ultrafiltration
Hollow fiber cartridge NMWC 300 kDa
Purified supercoiled plasmid DNA
Fig. 6. The three-step chromatographic
process begins with a clarified lysate.
Purification process
The production of highly pure plasmid DNA at all scales is critical
to its success as a therapeutic. The three-step purification
Capture of supercoiled described here was developed at the bench-top and transferred
plasmid DNA
to pilot-scale by increasing column diameter and membrane
area accordingly. Starting material is a cultivated E. coli,
Polishing/Removal containing a high-copy number plasmid. It is harvested prior
of traces of endotoxins
to alkaline lysis and then clarified by centrifugation. At pilot-
scale, the clarified lysate is concentrated five times on a
Product concentration hollow fiber ultrafiltration cartridge.
and formulation
Figure 6 illustrates the process flow scheme and Figure 7
shows the chromatograms: 1) RNA removal and buffer
exchange on Sepharose 6 Fast Flow, 2) selective purification

System: ÄKTApilot System: ÄKTApilot

System: ÄKTApilot Sample: Plasmid DNA after group separation Sample: Supercoiled plasmid DNA after
Sample: Clarified alkaline lysate on Sepharose 6 Fast Flow PlasmidSelect
Column: Sepharose 6 Fast Flow, BPG™ 100 Column: PlasmidSelect, FineLINE 70 Column: SOURCE 30Q, FineLINE™ 35
A260
(mAU) mS/cm
A260 A260
1. (mAU) 2. mS/cm (mAU) 3. mS/cm
6000
200
5000 RNA 4000
2500 200
4000 150 2000 200 3000
150
3000 1500 ccc pDNA 150
100 2000
pDNA 100
2000 1000 100
50 1000 50
1000 500 50
oc pDNA
0 0 0 0 0 0
0 1000 2000 3000 4000 5000 0 2000 4000 6000 8000 ml 0 1000 2000 3000 4000 5000 6000 ml

Fig. 7. The key separation is step 2 where PlasmidSelect separates supercoiled plasmid DNA from open circular plasmid DNA and
remaining contaminants such as residual genomic DNA and RNA. oc = open circle, ccc = covalently closed circle.

of supercoiled covalently closed circle (ccc) plasmid DNA
from open circle (oc) on PlasmidSelect, 3) endotoxin removal
and polishing by anion exchange chromatography on
SOURCE™ 30Q. All steps are run on ÄKTApilot™ system. Final
concentration and buffer exchange of the pure plasmid is
performed by ultrafiltration.
Table 2 lists purity data for a pilot-scale run. Results show
that the process achieves high purity and meets regulatory
requirements for cGMP production of plasmid therapies. Yield
is also high (Table 3).
Comments
This pilot-scale process meets the need for large quantities of
highly pure plasmids. It is robust, economical, fully compliant
with cGMP, and easy to scale up further to production.
Note that the three media used in the purification, plus a
detailed protocol, are available in the Supercoiled Plasmid
Purification Starter Pack. This pack purifies approximately
10 mg of ‘gene therapy standard’ supercoiled plasmid DNA
per cycle and is thus an excellent starting point on the path
to cGMP production. For some applictions this process can be
modified to produce plasmid DNA of lower quality by simply
removing one or two chromatography steps. The product will
not be as pure, but the cost per gram will be less, which may
be desirable for some uses.
Further information
Data File Code No.
SOURCE 30Q and SOURCE 30S 18-1107-12
PlasmidSelect – Supercoiled Plasmid Purification
Starter Pack 18-1161-73
Poster
Downstream processing of supercoiled
plasmid DNA 28-4057-63
Further reading (2, 3)

Table 2. Final product purity for a pilot-scale run.

ccc plasmid DNA (%), CGE* 95

ccc plasmid DNA (%), AGE** 99

Plasmid concentration (mg/ml) 3.9

Endotoxins (E.U./mg plasmid DNA) 0.05

RNA (µg/mg plasmid DNA) 0.02

Amount of gDNA (µg/mg plasmid DNA) 0.97

Protein content (µg/ml) <51

* Capillary gel electrophoresis.
** Agarose gel electrophoresis.
1
Below detection limit.

Table 3. Pilot-scale yield after each

purification step.*
Yield

Sepharose 6 Fast Flow** 92

PlasmidSelect (%) 72

SOURCE 30Q (%) 98
Ultrafiltration (%) 81
* Calculated using UV absorption (A260)
** pDNA concentration in the ultrafiltration-clarified lysate was measured
with PicoGreen™ fluorescence assay (Molecular Probes Inc).
Supercoiled Plasmid Purification Starter Pack purifies approximately
10 mg of gene therapy standard’ supercoiled plasmid DNA per cycle.
Its component media (Sepharose 6 Fast Flow, PlasmidSelect and
SOURCE 30Q) are available in process quantities, paving the way to
cGMP production.


Purifying a virus for human gene therapy
Viral vectors
Adenovirus and adeno-associated of hemophilia B, cystic fibrosis, alpha 1 anti-trypsin deficiency,
Parkinson’s disease and Canavan’s disease. (During et al., 2001;
virus vectors
Flotte et al., 2004; Kay et al., 2000; Wagner et al., 2002).
Adenoviruses are non-enveloped DNA viruses of which there
are many human and animal-specific serotypes. Human Purification strategy
adenoviral infections are associated with mild respiratory
The molecular weight of adenoviruses and AAV exceeds that
infections and rarely cause serious illness. Most of the adult
commonly seen for proteins. This high molecular weight and
population has some antibodies to adenoviruses.
large hydrodynamic diameter will affect specific aspects of
Adenoviruses are well researched and their use in gene therapy how the feedstock is processed, e.g. its tolerance to shear
is based on several favorable characteristics: (i) they are easy stress and resistance to mass transfer.
to produce in culture; (ii) they infect a broad spectrum of cell
Today’s bead-type chromatography media will not accommodate
types; (iii) the adenoviral genome has a low risk of integration
such large particles within their pore structure; they only allow
into chromosomal DNA; (IV) it can be readily manipulated and
surface binding. The main strategy outlined below utilizes an
mutant or deleted viruses can be prepared; and (v) the virions
ion exchange medium with an increased surface area and
are sufficiently robust.
long, flexible dextran chains for greater ligand presentation.
Adeno-associated viruses (AAV) are small, single-stranded DNA
The production to support the clinical development efforts
viruses of the parvovirus family. There are six human serotypes,
requires scalable and efficient production methods, particularly in
all of which appear non-pathogenic. The main advantage of
the downstream processing. The use of Capto™ chromatography
these vectors for gene therapy is their persistent expression
media for the processing of viral vaccines and vectors combines
of the gene of interest and reduced need for repeated dosage.
high capacity via a long, flexible dextran chain for greater ligand
rAAV is a powerful vehicle to treat serious human diseases. presentation with high flow velocity and low backpressure to
Currently there are clinical studies ongoing for the treatment reduce process cycle times and increase productivity.

Purification processes
Figure 8 outlines an ion exchange-based, adenovirus-specific
purification scheme staring with a clarified cell lysate. The
Clarified cell lysate
medium, Q Sepharose XL, provides fast, scalable isolation of
adenovirus and other viruses for gene therapy and vaccine

Microfiltration 0.65/0.2 µm Removal of cell debris

Ultrafiltration Medium: Q Sepharose XL

Virus concentration
Hollow fiber cartridge NMWC 300 kDa Sample: Pre-treated Ad5-CMV-NTR

mAU mS/cm

Q Sepharose XL Capture of Ad5 viral vector 600 UV1_280nm

UV2_260nm
500
Cond
Sepharose 4 Fast Flow Polishing 400

300

Ultrafiltration Product concentration 200

Hollow fiber cartridge NMWC 300 kDa and formulation
100

0
Purified Ad5 virus 0 100 200 300 400 ml

Fig. 8. Downstream processing of adenovirus, Ad5. Fig. 9. Purification scheme for adenoviruses based on the
(Reproduced with kind permission of Cobra Biomanufacturing, UK.) Q Sepharose XL ion exchanger. (Reproduced with kind
permission of Cobra Biomanufacturing, UK.)

10
applications. The time-savings of this chromatographic
purification compared with traditional centrifugation are
significant; just a few hours compared with up to twenty-four,
and the recovery is higher. In addition, scale-up to the production
of clinical batch sizes is straightforward. Figure 9 shows a
typical chromatogram.
In the particular case of AAV, Capto™ MMC and ViralQ
chromatography media have been used in the outlined
downstream processing scheme (Fig. 11) for the purification
of AAV 4 eGFP viral vectors derived from HEK293 cell lysate.
Figure 10A shows the intermediate purification of the viral
particles using Capto™ MMC after initial capturing by either ion
exchange chromatography or filtration. Applying a step-wise
gradient, a highly selective elution of the virus could be achieved.
For the subsequent polishing (Fig. 10B) Capto™ ViralQ has
been used. The viral fraction eluted in a very sharp and
distinct peak. The recovery for both chromatographic steps
was 86% (+/- 18%, n=5) and 79% (+/- 9%, n=3) respectively.
(Data are kindly provided by INSERM U649, Laboratoire de
Therapie Genique, CHU de Nantes, France;)
Comments
As more viral-based gene therapy and vaccine products move
into late-stage clinical development, the need for fast and
scalable methods for purifying viral vectors is bound to increase.
This applies especially to adenovirus. The media highlighted in
these two processing schemes have all the attributes to fulfill the
need for safe, robust and economic manufacture. Both schemes
also integrate production-efficient membrane processing.
Further information
Data File Code No.
Q Sepharose XL 18-1123-82
Capto Q, S, DEAE 11-0025-76
Capto MMC 11-0035-45
Poster Code No.
Rapid adenovirus purification using
Q Sepharose XL 18-1178-07
Further reading (4)
Capto MMC
Capto Q, S, DEAE

mAU mS/cm
A

3500
250
Cell lysis Virus retrieval
3000
200
2500
2000 Wash AA V4 150
Normal flow filtration Removal of cell debris
1500
100
1000
50 Ultrafiltration
500 Virus concentration
Hollow fiber cartridge NMWC 300
0 0
0 10 20 30 40 50 60 min
Capture Capture
IEX chromatography/filtration

B mAU AA V4 mS/cm
140
Intermediate
250 Purification of AAV
120 Chromatography on CaptoTM MMC

200 100
Polishing Polishing
150 80
CIP Chromatography on CaptoTM ViralQ

100 60

40 Ultra-/Diafiltration Virus concentration

50
Hollow fiber cartridge NMWC 300 and formulation
20
0
0
0 20 40 60 80 100 min Purified recombinant AAV 4eGFP

Fig. 10. (A) Intermediate purification of AAV4 on Capto MMC. Fig. 11. Chromatography based scheme for purifying AAV4.
(B) Polishing of AAV4 on Capto Q.

11
Purifying a virus for an animal viral vaccine
Foot and mouth disease vaccine
Foot and mouth disease
Foot and mouth disease (FMD) is a devastating disease of
livestock. Its clinical signs are fever and the formation of vesicles,
mainly in the cavity of the mouth or nose, the interdigital spaces,
and the coronary knots of the hoof. All species of cloven-hoofed
animals are susceptible and the disease is extremely contagious.
Financial losses can be significant. There are direct losses due
to deaths in young animals, loss of milk, loss of meat and a
decrease in productive performance.
Improved FMD vaccines that better control the effects of the
disease are the focus of much work within veterinary vaccines
today. In South America, many countries work to control
the disease with national and regional livestock vaccination
campaigns. When associated with other control measures
such as surveillance and quarantine, vaccination is an
important part of a national eradication program. The example
described here is based on measures being taken within
Argentina’s FMD control program.
Hollow fiber cartridges are typically used
in the production of vaccines for foot and
mouth disease.
12
Purification strategy
In Argentina, veterinary vaccines are traditionally low-priced
products compared with human vaccines. However, the rise
in the price of the country’s premium-grade quality cattle plus
more stringent regulations means that livestock farmers now
spend more money on better quality vaccines. Nevertheless,
production methods must still be easy and relatively in-expensive.
A further characteristic of the South American market is that
vaccine producers often want to include more than one
bacterial or viral vaccine in the same dose. This forces producers
to concentrate the final purified product. In addition, government
regulations are driving vaccine production towards greater
consistency and higher quality. Membrane-based clarification
and concentration are helping achieve these goals in many
manufacturing processes.
Purification process
The Frenkel system and the suspension cell culture of baby
hamster kidney cells (BHK) are two popular methods of
vaccine production in Argentina, and PEG (polyethylene
glycol precipitation) and cross flow filtration are the two main
methods of concentration.
As Frenkel is an older method that involves infecting the Comments
sublingual epithelium of cows’ tongues, concerns about bovine
Improved production and purification methods similar to
spongiform encephalopthy have meant that the focus has
those outlined here are raising the quality and marketability
switched to BHK production, which is considered safer and
of veterinary vaccines in Argentina and other South American
better able to deliver a purer vaccine. Figure 12 outlines a general
countries. Thanks to very large-scale processing (approx.
production and purification strategy for the BHK method.
10 000 liter batch sizes) with increasing levels of automation,
Regarding concentration, PEG precipitation is a very efficient manufacturers now have purer, more efficacious veterinary
method, but it demands lots of space-taking tanks and vaccines readily available at a cost considerably lower than
consumes a great deal of time. Cross flow filtration is quicker that for human vaccines. It should be noted however that
as precipitation is no longer needed to clarify the product, FMD vaccine is produced in a 3A BioSafety area.
and also takes up less plant area due to the smaller footprint
of the equipment. Further information
Clarification can be achieved using 0.2 µm or 0.45 µm Data File Code No.
microfiltration membranes, Lumen E. The average flux is UniFlux membrane filtration systems 18-1177-25
about 20 LMH. Large process-sized cartridges are used, e.g.
Selection Handbook
sizes 85 and 75.
Hollow fiber cartidges and systems for
Concentration by cross flow filtration uses membranes with
membrane separations 18-1165-29
Lumen C and E and an average Flux of 40 LMH. The 500 KDa
membranes deliver a more purified product by allowing the
unwanted, low molecular weight proteins to pass through.
The process is also fast – depending on batch size, 3 to 6 hours
compared with 48 hours for PEG precipitation.

Cell culture

Virus inactivation

Centrifugation

Clarification

Concentration

Formulation

Filling

Fig. 12. Basic outline of the process

steps for the BHK method of FMD
vaccine production.

13
Unrivalled insight and support in
downstream purification
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We’ve been solving protein purification problems for almost
fifty years, experience unmatched by anyone else. The know-how
and practical skills acquired during this time are available to
you as part of the support service we offer process developers
and manufacturers. In addition to in-house expertise, we have
a comprehensive network of outside companies and individuals
that can be brought to your assistance.
Fast Trak Education
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experience of downstream purification processes to train your
staff. Fast Trak courses educate researchers, development
and production staff, and management in downstream
processing. We provide hands-on training in column packing,
chromatography and membrane technologies, and process
optimization and scale up for both pilot and production
operations. The special problems that vaccine and viral vector
purification pose are one of our focus areas. Courses are run
at our Fast Trak centers, or they can be arranged at your site
upon request.
Custom Designed Media
The purification needs of vaccines and vectors often require
modifications of the approaches designed for proteins, so
it is not surprising that modifications to standard chroma-
tography media may be needed as well. Custom Designed
Media (CDM) is a GE Healthcare service that develops such
tailor-made products.
CDM media are developed in collaboration with industrial
users for large-scale purification processes. They cover the
major chromatographic techniques and are well documented.
For example, most have Regulatory Support Files.
For more information about our support services for vaccine
producers, visit:
www.gehealthcare.com/protein_purification
or contact your local representative.

Fast Trak Validation

Regulatory authorities require that companies producing
prophylactic or therapeutic vaccines, or other viral products
validate their production processes.
Fast Trak Validation combines our experience of supplying
chromatography and membrane purification systems to the
pharmaceutical industry with our knowledge of regulatory
requirements. The result is a range of services and documents
that will help vaccine manufacturers reduce time to production Process development and column packing
start-up and lower validation costs. courses in Europe.

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Further reading
1. Belew, M. et al. Purification of recombinant hepatitis B surface antigen
produced by transformed Chinese hamster ovary (CHO) cell line grown in
culture. Bioseparation 1, 397–408 (1991).
2. Scale up of a cost-effective and generic purification process for plasmid
DNA. Life Science News Vol 16, 16–17, Code No 11-0003-48.
3. Scale-up of a method for highly pure plasmid DNA. Downstream 38, 20–21
(2004). Code No 11-0012-70.
4. Brument, N. et al. A versatile and scalable two-step ion-exchange
chromatography process for the purification of recombinant adeno-
associated virus serotypes-2 and -5. Molecular Therapy 6, 678–686 (2002).
5. Herzer, S. et al. Key Issues in the process development of purification
procedures for viral vector and plasmid biopharmaceuticals. BioProcessing
Journal 4, 67–72 (2005).
6. Pitarresi, T.M. et al. Purification of plasmid DNA on an anion exchange
column without the use of RNase to process scale. Molecular Therapy 9,
(Supplement 1) 53–54 (2004).
7. Herzer, S. Process design and scale-up of viral vector gene therapy products:
New purification challenges. Cell & Gene Therapy 1, 43–48 (2004).
8. Diogo, M.M. et al. Studies on the retention of plasmid DNA and Escherichia
coli nucleic acids by hydrophobic interaction chromatography.
Bioseparation. 10, 211–220 (2001).
9. Ferreira, G.N. Cabral, J.M. and Prazeres, D.M. Purification of supercoiled
plasmid DNA using chromatographic processes. J. Mol. Recognit. 11,
250–251 (1998).
10. Diogo, M.M. et al. Production, purification and analysis of an experimental
DNA vaccine against rabies. J. Gen. Med. 3, 577–84 (2001).
11. Jendrek, S. and Ekstrom, D. et al. Development of a production and
purification method for type 5 adenovirus. BioProcessing Journal 4, 42–47
(2005).
12. Blanche, F. et al. An improved anion-exchange HPLC method for the
detection and purification of adenoviral particles. Gene Ther. 7, 1055–1062
(2000).
13. Purifying human influenza virus vaccine — towards a generic strategy.
Downstream 37, 20–21 (2004) Code No 11-0008-46.

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For contact information for your local office,
please visit, www.gelifesciences.com/contact
www.gelifesciences.com/bioprocess
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at any time without notice or obligation. Contact your
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© 2007 General Electric Company – All rights reserved.

GE Healthcare Bio-Sciences AB First published Jan. 2007.

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