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CN5173 Term Paper

Comparison on downstream processing of Hepatitis A


Name: Wang Lin

Matric Number: U075467H

Submission Date : 1/Nov/2010


Comparison on downstream processing of Hepatitis A

------------HAVRIX® and VAQTA®

Hepatitis A is an acute infectious disease of the liver caused by Hepatitis A virus

(HAV) (1). The virus usually spreads through stool or blood, but also widely
spread through contaminated food in undeveloped or developing countries
where hygiene condition is not well regulated. The infection of HAV does not
develop chronic liver disease, however, symptoms such as fever, fatigue,
abdominal pain, nausea, appetite loss, depression etc, may appear two to six
weeks after infection (1). Since Hepatitis A is a self limiting disease, protective
antibodies are developed in response to the first infection and provide lifelong

According to the statistics from WHO (1), 80 -95% of the children below 5 years
old do not develop any symptoms after infection, while the risks of developing
clinical symptoms rise with age. Among older children and adults, 75-90% of the
cases will develop symptomatic diseases after infection. Moreover, the mortality
rate is as high as 2.1% for adults above 40 years old. Therefore, vaccination is
developed to provide protection against the diseases.

Currently, four licensed HAV vaccines, Vaqta®, Epaxal®, Avaxim® and

Havrix®, are available worldwide. All of the four vaccines are safe and effective
for lifelong protection (1). Among the four vaccines, the first three vaccines are
manufactured from cell-culture-adapted HAV propagated in human fibroblasts,
following with purification of cell lysates (2); the fourth vaccine is manufactured
from HAV purified from infected human diploid cell cultures, following with
purification of culture medium supernatant (3). In the following part of the
paper, I will use Vaqta® produced by Merck and Havrix® produced by
GlaxoSmithKline as examples to compare the different downstream processes
of the two methods.

Both Vaqta® and Havrix® consist of attenuated strain of highly purified

Hepatitis A virus inactivated with formalin. With different upstream fermentation
materials and methods, the virus is harvested intracellularly for the former while
extracellularly for the latter. Since this article is only targeted on the
downstream processes, the differences in fermentation processes will not be
discussed here, but brief descriptions of fermentation beer are given below in
order to stress the different objectives of downstream processing.

In extracellular harvesting, the HAV is propagated and continuously released

into the cell culture medium (3). Therefore, the objective is to purify the virus
antigen from the fermentation beer supernatant. On the contrary, in
intracellular harvesting, the infected cells are propagated in the bioreactors,
either in the form of microcarrier coating or a monolayer in the broth 1 (2).
Therefore, the objective in downstream processing is to purify the virus antigen
from the cell lysate.

In the following part, I will summarize the key steps of downstream processes of
Vaqta® and Havrix® respectively, after which an analysis of the two methods
will be discussed.

VAQTA® downstream process flow

 Seperation of insolubles
Before disrupt the cells with detergent, the bioreactors are drained and the cell
monolayer or microcarriers are rinsed with PBS solution to remove the
remaining culture medium (2). Although the literature did not provide detailed
description of this step, in my perspective, the rinsing step can be viewed as
cake washing which produces a concentrated clean cake of cells.
 Cell disruption
The concentrated cells are lysed with detergent Triton X-100, which solubilise
and permeablize the membranes. In the case of microcarrier technology is used
(4), the cell lysate is recycled several times to 5µ m orifices and allow the
microcarriers (MC) and nuclei to settle while the MC free lysate is collected. A
second volume of Triton X-100 is added to the MC beads and recycles through
orifices. The two sets of MC free lysate are pooled together as MC free cell

As technology develops, microcarrier fermentation technology is more preferred as it provides larger
surface area for cell growth on the small microbeads(90 to 250 microns in diameter). If microcarrier
technology is used, an additional step of filtering out the microbeads from lysate is needed before filter out
the virus particles from the cell lysate.
lysate preparation. The repeated cell lysis on MC beads helps minimize the
product loss thus improves product yield.
 Removal of unwanted cell debris from lysate
The MC free lysate preparation consists of HAV and unwanted contaminants
such as cell debris, proteins, nucleic acids etc. The preparation is firstly
subjected to 0.2µ m filtration to remove the small debris (5). Subsequently, the
filter lysate is treated with nuclease, which digested the nucleic acids (5).

 Isolation of HAV using adsorption capture column

Typically, after the nuclease treatment, the lysate is feed to the anion-exchange
adsorption column containing Toyopearl 650M resin. Then column is washed
with 0.03M NaCl and sodium phosphate buffer, and eluted with 0.35M NaCl
solution. This step creates the largest concentration increase in the downstream
process (5).
 PEG precipitation to purify HAV
Prepared PEG and NaCl are added in the column elution solution to achieve 5%
PEG and 0.5M NaCl concentrations in the final solution. HAV is precipitated after
agitation followed by incubation on ice. After that, centrifugation is carried out
and the aqueous phase can be removed (5).
 Further purification by solvent extraction
The HAV pellet is resuspended in PSB with PNE and extracted with chloroform
solvent (6). This step results in precipitation of cell derived proteins at the
solvent water interface. After removal of the cell derived proteins, the solvent
phase is reextracted with PNE buffer.
 Size exclusion by chromatography
The final polishing step is the ion exchange and size exclusion chromatography,
which further excludes impurities and collects the purified virus bulk product
 Inactivaton
The purified virus bulk is inactivated by formaldehyde followed with adsorption
on adjuvant Aluminium hydroxide. After diluting the product with Aluminium
hydroxide, the formaldehyde is removed by repeated settle and decant. The
final vaccine product is then packaged into desired forms (2).
HAVRIX ® downstream process flow

For Havrix®, who harvest virus extracellularly, the downstream process is much
simpler. It can be divided into three main stages, namely, concentrating the
HAV harvested supernatant, nuclease and protease treatment, isolation and
purification of HAV product (7).

 Concentrating Hepatitis A virus from the supernatant (7).

Firstly, supernatant is separated from the cells. Since this step can be simply
achieved by either filtration or centrifugation, there is no detailed description
found in the literature. Next, the virus is separated from the cell debris in the
cell culture supernatant by low speed centrifugation (7). After removing the
cellular debris, suitable size of ultrafiltration followed by diafiltration are
 Protease and nuclease treatment
The concentrate from the previous step consists of some residual host cell
nucleic acids and contaminating residual proteins. Therefore, Benzonase is
added and incubated for 3 hours digest the remaining nucleic acids.
Subsequently, protease is added to digest the residual cellular proteins for 24
hrs (7).
 Isolation of HAV product
Finally, the purified virus concentrate are obtained from a sing pass of
diafiltration on a membrane with smaller pore size for the protease and
nuclease treated solution (7).
 Inactivation follows the same procedure as the VAQTA vaccine.
Analysis on the downstream processing methods

Although very different manufacturing processes are used in Vaqta® and

Havrix®, both vaccines have the same active components, show the same
degree of purity and efficacy in protection. It is pretty obvious that the
downstream processes of Vaqta® are more complicated and time-consuming
than Havrix®. It is necessary to use detergent to release the intracellularly
produced virus into the lysate. Besides, it involves a lot of cellular contaminants
removal and detergent removal during the product purification. However, the
extracellular method has its own limitations as well. The challenge associated
with the extracellular harvesting method is the low efficiency of HAV antigen
release of virus into the culture supernatant (7). Typically, there is only 30% of
infectious virus in extracellular. Therefore, the method to concentrate the large
supernatant volume is having much lower yield comparing to the intracellular
harvest method. Nevertheless, if the cost and efficiency on the downstream
process of extracellular method can justify the low overall yield, it still can be as
profitable as the intracellular method.

In conclusion, Vaqta® and Havrix®, which have similar components, can be

produced from different upstream methods thus requires very different
downstream processing procedures. Although the choices of downstream
processes are more or less constrained by the feed characteristics, the
engineers are still flexible in designing an optimized process to produce
profitable drugs for the company, based on product quality requirements,
equipment availability and economic constraints.


1. World Health Organization, Department of Communicable Disease

Surveillance and Response. Hepatitis A. World Health Organization/CSR.
[Online] 7, 2000. [Cited: 30 10, 2010.]

2. Hagen, A., et al. Development, Preparation, and testing of VAQTA, a

highly purified hepatitis A vaccine. Bioprocess Engineering. 2000, Vol.

3. Meyer, Heidi, et al. Method of Large scale production of Hepatitis A

Virus. US 6,885,535,B2 US, 15 Feb, 2005.

4. Leu, Frank S and Seifert, Douglas B. Hepatitis A culture process. US

6,194,210,B1 US, 27 Feb, 2001.

5. Hagen, Anna J, Oliver, Cynthia N and Sitrin, Robert D. Optimization of

Poly(ethylene glycol) Precipitation of Hepatitis A Virus Used to Prepare
VAQTA, a Highly Purified Inactivated Vaccine. Biotechnol.Prog. 1996,
Vol. 12.

6. Hagen, A J, Oliver, C N and Sitrin, R D. Optimization and scale up of

solvent extraction in purification of hepatitis A virus.
Biotechnol.Bioeng. 1997, Vol. 56.

7. Tauer, C, et al. Method of production of purified Hepatitis A Virus

particles and vaccine preparation. US2003/0124511 A1 US, 3 7, 2003.