BioPharmfiles.pharmtech.com/alfresco_images/pharma/2014/08/20/86... · 2018-08-28 · October 2011...

BioPharmINTERNATIONAL

www.biopharminternational.com

The Science & Business of Biopharmaceuticals

Supplement to:

October 2011

Advances in Vaccine

Technologies

GE HealthcareLife Sciences

GE, imagination at work and GE monogram are trademarks of General Electric Company. ReadyToProcess, WAVE BioReactor and ReadyCircuit are trademarks of GE Healthcare companies.

© 2011 General Electric Company – All rights reserved. GE Healthcare Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden

GE04-11. First published October 2011.

ReadyToProcess™

Plug & Play options for flexible vaccine manufacturing

• For virus propagation, WAVE Bioreactor™ system reduces time consuming routines, increases

flexibility and ensures the integrity of your cell culture operations.

• For harvest operations, ReadyCircuit™ assemblies provide a single-use, aseptic flow path for

clarification and purification applications.

Visit www.gelifesciences.com/readytoprocess

www.biopharminternational.com October 2011 Supplement to BioPharm International s3

Vaccines Trends

Challenges and Trends

in Vaccine ManufacturingAn evaluation of the technologies and process parameters needed

to develop a safe, effective, and economically efficient vaccine.

Suma Ray

The biotechnology era has experienced signif-

icant changes in the number of companies

involved in vaccine manufacturing as well as in

the production systems they use. Nevertheless,

challenges in this area are multiple. In the cur-

rent vaccine-manufacturing environment, time to mar-

ket and cost effectiveness are key issues that need to be

addressed in addition to smooth R&D and clinical stud-

ies. Furthermore, scale up and safety are important for

maintaining a successful manufacturing process.

As a result, state-of-the-art technologies to simplify

vaccine development and manufacturing are becom-

ing ever-more crucial. In this article, the authors dis-

cuss these challenges and evaluate the technologies

and process parameters that need to be considered

when developing a safe, effective, and economically

efficient vaccine. The article primarily deals with pro-

phylactic vaccines.

Background

Vaccines are a group of biologics considered to be the

lifeline of the human race. It has been said about vac-

cines that, “With the exception of safe water, no other

modality, not even antibiotics, has had such a major

effect on mortality reduction…” (1).

A good vaccine is one that elicits an appropriate

immune response for the particular pathogen, which

could either be a cell-mediated response to tuberculosis

or an antibody response to a bacterial or viral infec-

tions. It should be safe to use in a variety of patients

and the vaccine itself should not cause disease or

induce adverse effects. A vaccine should offer long-

term protection (ideally lifelong) with one dose and

retain its immunogenicity despite adverse storage con-

ditions before administration. Furthermore, it must be

inexpensive to make and buy (2).

The development and manufacturing of vaccines

must follow four ground rules (3):

• Vaccines must be developed, produced, and deliv-

ered in large volumes.

• Process costs must be kept down.

Me

dio

ima

ge

s/P

ho

tod

isc

/Jo

n B

oye

s/G

ett

y Im

ag

es

Suma Ray is a senior process development scientist at Sartroius Stedim India Private, Ltd., [email protected].

s4 Supplement to BioPharm International October 2011 www.biopharminternational.com

Vaccines Trends

• Development and delivery

times must be short.

• Patient and employee safety

should not be compromised.

The largest market for vaccines

is found in the developing world,

where doses need to be less expen-

sive than those sold in the devel-

oped world in order to have any

chance of getting to the patients

who need them (4). Companies,

therefore, need to weigh the risks of

killed or weakened whole organisms

against the costs of recombinant

vaccine production for products

meant to help a large percentage of

the economically weak population.

As part of the “Elements of

Biopharmaceutical Production”

series (see sidebar), this article pres-

ents an overview of the various

challenges encountered during pro-

cess development for the manufac-

ture of prophylactic vaccines.

cHaLLEngES

Process development for vaccines

can pose unique obstacles for man-

ufacturers. Because most vaccines

are new products, there is no history

or experience to rely on with regard

to how subjects will respond to the

drug. Furthermore, promising pre-

clinical results in animal models are

generally not duplicated when the

therapy is tested in humans. Often,

it is challenging to develop robust

manufacturing processes and to val-

idate quality control assays for these

products because there is a need for

a specific biological assay for each

product (5).

One important difference

between the production of vac-

cines and other biopharmaceuti-

cals is the risk-safety consideration

related to working with pathogens

and pathogenic antigens. As with

all biomolecules purified from crude

biological material, the removal of

contaminants (e.g., derivatives from

host cell such as DNA, protein, or

leachables), must be documented.

However, the removal or inactiva-

tion of adventitious viruses remains

a unique challenge (3).

nEW and EXISTIng TrEndS

In VaccInE ManuFacTurIng

The process of vaccine development

begins with a good understanding

of the underlying biology involved.

Egg-based versus cell-culture based

There has been much debate

regarding whether the egg-based

method for manufacturing a viral

vaccine is more effective than

the animal cell-culture based

method. A third method using

virus-like particles (VLPs) is also

in use. This method is similar to

the recombinant-vaccine manu-

facturing process in terms of

cloning and expressing recombi-

nant proteins.

Although the egg-based viral vac-

cine manufacturing process is an

old and familiar technology that

has been practiced for more than 60

years, it has its own complex issues.

Egg-based viral vaccine manufac-

turing, for example requires a large

number of specific pathogen free

(SPF) eggs because the virus needs

to be propagated inside the fertilized

chicken eggs. However, typcially,

one can only produce one or two

doses of vaccine per egg. Here, the

nutrition status of the poultry is also

important because changes in their

diet can dramatically affect the virus

yield from the eggs. The process is

labor intensive and highly suscep-

tible to bacterial contamination. In

addition, individuals who are aller-

gic to eggs might have allergic reac-

tions to such vaccines.

On the contrary, animal cell-

culture driven viral vaccine pro-

cesses are being encouraged by

regulatory authorities, particu-

larly because the process reduces

the lead time from laboratory

to market. Furthermore, during

an emergency, chances of scal-

ing up production capacity of an

egg-based vaccine is dismal con-

sidering the reliance on SPF eggs,

compared with cell culture, which

can be propagated multiple times

from frozen cellbanks. In addi-

tion, the footprint for cell-culture-

based production is considerably

smaller, and processing takes place

in closed systems, thereby reduc-

ing chances of contamination.

However, an important issue

with cell-culture processes is

the tumorigenic and oncogenic

potential of the cells in which

the virus is propagated. Although

highly tumorigenic cell substrates

have never been used in vac-

cine manufacturing, it is impor-

tant to keep in mind that some

Madine-Darby Canine Kidney

epithelial cell (MDCK) sublines

are highly tumorigenic. Such

cell substrates can pose signifi-

cant regulatory challenges (e.g.,

if the cell line harbors oncogenic

or tumorigenic viruses, that virus

could integrate into the recipient’s

genome and cause a tumor). Other

cell-culture concerns include

the oncogenic potential of virus

producing cells due to the pres-

ence of contaminating DNA in

the product. Additionally, the use

of animal-derived components in

the cell-culture media—whether

serum-sourced f rom calf or

trypsin-sourced from porcine—

could be potential sources of ani-

mal viruses. The International

Conference on Harmonization

(ICH) Q5A guideline on the qual-

ity of biotechnology products,

therefore, recommends using

serum-free, chemically defined

media for cell culture and geneti-

cally engineered trypsin to ensure

the absence of any animal-derived

component in the raw materials

used in manufacturing.


Vaccines Trends

Use of VLPs in vaccine

development

During the past few years, the

use of VLPs for the manufactur-

ing of vaccines is becoming more

popular. For example, the H1N1

influenza vaccine developed

jointly by Novovax and Cadila

Pharmaceuticals uses VLPs. The

advantages of a VLP-based vaccine

platform is that it uses a recom-

binant-vaccine technology with

a baculovirus expression system

and, as a result, one does not need

to handle a live pathogenic virus.

This process rules out the need for

a containment facility. There are

no safety concerns and the process

is easily scalable to large quantities

and more economical in terms of

facility, materials, labor, and util-

ity costs (6). When using recom-

binant technology, one can select

the exact genetic match for hem-

agglutinin, neuraminidase, and

matrix proteins of the circulating

virus strain. The entire process of

manufacturing, from cloning to

development and release, takes

about 12 weeks (6). Other VLP-

derived vaccines include Merck’s

Gardasil, which protects against

human papillomavirus types 6, 11,

16, and 18.

ProcESS dEVELoPMEnT ScHEME

For a cELL-cuLTurE BaSEd VaccInE

Current technologies for vaccine

manufacturing can help ensure

quality and reduce time. Some of

these technologies include ana-

lytical methods such as surface

plasmon resonance, upstream

technologies such as micro-

carrier beads for adherent cell

lines, high-throughput screen-

ing methods, and downstream

technologies such as membrane

chromatography and cross-flow

filtration. Figure 1 outlines the

schematic flow for complete vac-

cine-process development.

Upstream process development

The productivity of large-scale cell

culture can be increased either

by scaling up to larger volumes

with cell densities of 2–3 x 106/mL,

or by intensifying the process in

smaller volumes but with higher

cell densities (up to 2 x 108 cells/

mL). When intensifying cell densi-

ties, more frequent media changes

are needed and perfusion is even-

tually applied (7).

Many alternative technologies

are also available. Cross-linked

dextran beads (i.e., microcarriers),

for instance, provide an extended

surface and a stable environ-

ment for optimal cell growth.

Microcarrier culture of anchorage-

dependent or entrapped cells

reduces volume and thus belongs

to the latter of the options cited

above (8). The technique, in gen-

eral, has many advantages for the

commercial manufacturer. It can

be operated in batch or perfusion

modes during cell culture and it

is well-suited to efficient process

development and smooth scale-

up. Washing and changing culture

media just before viral infection

is easier because there is no virus

inside and one does not have to

follow additional precautions as

required while handling a virus.

The reactors can also be modified

to grow other organisms.

Downstream process development

As evident from Figure 1, the down-

stream processing and purification

steps occupy a major portion of the

vaccine-manufacturing process. As

a result, the use of robust and eco-

nomical steps to develop and opti-

mize parameters for purification is

beneficial. For example, in general,

downstream steps are more expen-

sive to carry out than upstream

steps because of the use of chroma-

tography columns and membranes.

The onus for generating a product

that is absolutely free of pathogens

and other contaminants such as AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

RS

Figure 1: Process-development scheme for vaccine manufacturing.

Clarification

Concentration

Inactivation

UPSTREAM DOWNSTREAM PURIFICATION & FORMULATION

Mediapreparation

Antigengrowth

Contaminantremoval

Formulation/Adjuvants

Preservativestabilizer

Sterilefiltration

Fill andFinish

A SpeciAl AdvertiSing Section


page_header page_header page_header page_header A SpeciAl AdvertiSing Section

Vaccine development and manufacture is one of the most challenging tasks within biopharmaceuticals today. R&D projects are characterized by technical com-plexity, and production processes by methods that can be both inflexible and

costly. But vaccines can also be highly rewarding, and one route to success is to deploy single-use products in order to create more flexible and less costly production processes. Inflexible production methods can put producers at a real disadvantage, especially when millions of doses of new vaccines are needed at short notice. Flexible, single-use platforms, like the ReadyToProcess™ product line, include single-use bioreactors, ready to use chromatography products and self-contained fluid management, filtration and sensing modules. As well as shortening time-lines, they can help secure safety and qual-ity standards wherever production is located.

The production of vaccines against seasonal as well as pandemic influenza virus continues to represent the majority of inactivated and live, attenuated virus vaccine production. The demand for such vaccines continues to grow due to increased aware-

ness of immunization, especially in emerging markets.1 Furthermore, changes in the virus strain from season to season, as well as the location of pandemic breakouts, create

a greater need for flexibility and speed in flu vaccine production.2

ProPagation of influenza virus in single-use bioreactors

Traditional influenza vaccine production uses fertilized hen eggs. This method is labor-intensive and requires large facilities with limited scalability. A switch to cell cul-ture–based vaccine production methods and use of disposable products provides flex-ibility to substantially reduce the time to vaccine clinical trials and approval, effectively decreasing the time-to-market.

Magnusson et al. have previously shown that WAVE Bioreactor™ systems can be used to establish processes to propagate influenza virus in Vero cell culture using dispos-

able products and animal-component–free conditions for cell growth.3 (See figure 1.) They developed an animal-free culture process, which is important from a regulatory perspective in human vaccine production, and it is critical to live virus vaccines because there are limited options for inactivating adventitious virus. They were able to grow these cell cultures in animal-component–free conditions while providing a fast and sim-ple process that enables scale-up to production. A WAVE Bioreactor 20/50 system using a disposable Cellbag™ bioreactor was shown to be a fast and convenient alternative to stainless steel bioreactors because it is easy to set up and no cleaning is required.

Harvest of influenza virus using single-use systems

Following propagation of influenza virus in a cell-based process, the virus must be harvested from the cell culture medium. We have developed a platform harvest process using ReadyCircuit™ single-use assemblies to remove cell debris, microcarriers and host cell contamination (proteins and DNA). ReadyCircuit assemblies comprise bags, tubing, sensors and filters which form self-contained purification modules that maintain an aseptic path and remove time-consuming process steps associated with conventional systems. This process has been verified on influenza virus produced in MDCK and Vero cell lines.

Increased Process Productivity for Influenza Vaccine using Single-Use Solutions

By Jakob Liderfelt and Ann-Christin

Magnusson

Jakob Liderfelt, Scientist

Jakob Liderfelt has five years of experience in process development at a CMO involved in downstream processing of biopharmaceuticals and an additional six years of experience as a scientist at GE Healthcare Life Sciences in downstream process applications for monoclonal antibodies with a focus on disposables and single use.

Ann-Christin Magnusson, Senior Research Engineer

Ann-Christin holds a degree in Biomedical Engineering from Uppsala University. She has more than 20 years of hands-on experience including cell culture, molecular biology and analytical assay development. In her current role, Ann-Christin is responsible for building the technical expertise in cell culture for vaccine manufacturing.

Visit us at:

www.gelifesciences.com/bioprocess

SponSored by

GE Healthcare Life Sciences supports scientists, engineers, and companies working in the discovery, development, and manufacture of biopharmaceuticals, vaccines, and cell-based technologies. We are continuously developing our portfolio of advanced products and services for cell culture, downstream purification and analytics to enable efficient and flexible bioprocessing. GE Healthcare is a unit of General Electric Company with expertise in medical diagnostics, drug discovery, and biopharmaceutical development and manufacture. GE Healthcare’s vision is to develop innovative solutions that help reduce costs, increase access, and improve quality of medical care around the world.



Removal of cell debris and microcarriers using nor-

mal flow filtration

Microcarriers and whole cells were allowed to settle and a ReadyCircuit assembly designed to perform serial normal flow filtration (NFF) using ULTA™ Prime GF 2.0 µm and ULTA Prime GF 0.6 µm filters was employed to further clarify the supernatant. (See fig-ure 2.) The membrane was wetted with phosphate-buff-ered saline (PBS) prior to processing. A peristaltic pump was used to drive the flow, a scale for monitoring of fil-tered volume and a disposable pressure sensor for moni-toring of pressure. Finally, PBS was also used to rinse the filters in order to increase product recovery. Final filter loading was 87 L/m2 and the final inlet pressure was 0.1 bar. Haemagglutinin (HA) yield was >95%.

Concentration of influenza virus and removal of

host cell protein (HCP) and DNA

A second ReadyCircuit assembly designed for cross flow filtration (CFF) using a 500 kD ReadyToProcess hollow fiber was employed to concentrate the virus 22X and to remove HCP and DNA via diafiltration (6 diavolumes). (See Figure 3.) Two peristaltic pumps were used to drive flow rates, two scales to moni-tor volumes and three disposable pressure sensors to monitor pressure drop (ΔP) over the filter cartridge and transmembrane pressure (TMP). Flux control was performed using manual clamp valves. The buffer used for diafiltration and product recovery was PBS. At a filter loading of 57 L/m2 a throughput of 62 L/m2 was achieved with an average flux of 23 LMH. HA yield was 80% over the concentration step, and over both filter steps log reduction values (LRV) of 0.8 and 1.4 were achieved for HCP and DNA, respectively.

Advantages of single use for harvest

Process time for the workflow described above was compared to the same processes using traditional systems. As was the case with virus propagation, utili-zation of single-use harvest systems eliminates set-up, cleaning and sterilization requirements. In this exam-ple, total process time was reduced from 13.5 hours to 5.5 hours by employing ReadyCircuit disposable pro-cess assemblies. In addition, ReadyCircuit assemblies increase process flexibility, since multiple products can be produced using platform with no risk of cross-prod-uct contamination, and increased process safety due to containment of the live influenza virus.

conclusions

We have shown that WAVE Bioreactor systems and ReadyCircuit disposable assemblies can be successfully employed for the propagation and harvest of influ-enza virus produced in cell culture-based systems. We achieved high yield of haemagglutinin and reduced the total time of harvest from 13.5 hours to 5.5 hours. Furthermore, we were able to remove 85% of host cell protein and 96% of DNA during the harvest step, thereby reducing the demands on the downstream purification process to remove these contaminants.

references 1. Shalini Shahani, Market Research Report: Vaccine technologies

and global markets (Wellesley, MA: BCC Research, 2010), 79 2. Shahani, Market Research Report: Vaccine technologies and

global markets, 81 3. Ann-Christin Magnusson, Therese Lundström, Mats Lundgren,

“Optimization of Conditions for Producing Influenza Virus in Cell Culture Using Single-Use Products,” BioProcess International

Industry Year Book 2011-2012, 112-113.

GE, imagination at work and GE monogram are trademarks of General Electric Company. Cellbag, ReadyCircuit, ReadyToProcess, ULTA and WAVE Bioreactor are trademarks of GE Healthcare companies. © 2011 General Electric Company – All rights reserved. First published 2011

1 2 3 4 5 6 7

Tit

er

(lo

g10)

Figure 1: TCID50 values from seven different 2-L infiuenza

batches propagated in a WAVE Bioreactor system (TOH 72 h).

Wast

e, 1L b

ag

Product, 10L bag

3-way TFeed pump

Jumper 2 µm GF 0.6 µm GFPressuresensor

Figure 2: ReadyCircuit assembly for serial flltration using

ULTA Prime GF 2.0 µm and ULTA Prime GF 0.6 µm fllters to

further clarify the supernatant.

NFF filtrateDiafiltration buffer

Pump

3-way T

Pump

Pressure sensor

CFF membranePressure sensor

Pressure sensor

JumperJumper

Harvest bag

Recirculation bag

Perm collection

Figure 3: ReadyCircuit assembly with a 500 kD

ReadyToProcess hollow flber concentrated the virus 22X

and removed HCP and DNA via diaflltration.


Vaccines Trends

host cell protein (HCP), host cell

DNA, or endotoxin, relies on down-

stream steps. If this cost escalates,

the manufacturer could run into

business risks.

The current industry trend

for purification of biological

therapeutics, including vaccines,

involves the use of membrane

chromatography to purify viruses

as well as for polishing applica-

tions. Membrane chromatography

offers many benefits compared

with density gradient ultracentri-

fugation, including the removal

of HCPs, contaminating DNA and

endotoxins. Membrane chroma-

tography compared with centrif-

ugation offers many advantages

(e.g., processing time is faster,

virus yield is higher, cleaning vali-

dation is not needed, the carbon

footprint is smaller) (see Table I).

This method can therefore bring

greater effectiveness to the key

stages of bioprocessing.

The implementation of mem-

brane chromatography for virus

purification, in place of density

gradient ultracentrifugation,

is gaining prominence in many

areas, even though the latter

remains the gold standard among

traditional vaccine manufacturers.

Membrane chromatography

has been successfully used in

multiple applications for virus

purification. In a recent study,

human and equine influenza A

virus in cell-culture supernatant

(i.e., serum-free and serum-con-

taining cultivation) was directly

adsorbed onto Sartobind Q 75

anion-exchanger, and eluted out

by displacement with sodium

chloride (up to 1.5 M, pH 7.0),

which resulted in average yields

of 86% based on hemagglutinin

activity (9). In another instance

a virulent wild type NIA3 strain

of Pseudorabies, grown in por-

cine kidney epithelial (PK15) cell

monolayers was purified using

Sartobind S cation exchanger

membranes (10).

In another study, Sartobind D

membrane adsorber was used in a

larger scale downstream process,

for the purification of rotavirus

VLPs to a clinical grade at 46%

global recovery yield, and with

nearly 100% removal of host bulk

DNA together with approximately

98% of HCPs (11). Sartobind D

was also used for purification of

recombinant baculoviruses that

are widely used as vectors for the

production of recombinant pro-

teins in insect cells (12).

Another robust technology is

cross-flow filtration (using tan-

gential-flow filters), which is best

suited for higher solid contents,

more viscous feed solutions, or in

cases where concentration, recov-

ery, and purification of cells or

target species is desired. This tech-

nology is largely used for concen-

trating and washing feed streams

before chromatography (3).

For vaccine manufacturing,

particularly virus purification,

fully scalable macrovoid-free

hollow-fiber technology applied

to ultrafiltration and microfiltra-

tion offers great advantages in

the virus-purification process due

to its open porous structure. The

open-flow path design of hollow

fibers gently processes cell suspen-

sions and other particulate feed

streams and reduces shear forces,

thereby maintaining the integrity

of the virus. Hence, recovery rates

of the target virus and overall pro-

cess economics are improved (13).

PLaTForM TEcHnoLogIES


One way to eliminate bottlenecks,

especially in downstream-process

development, is to use purifica-

tion platforms. The biopharma-

ceutical manufacturing industry

has communicated timesavings

of 3–8 months using a fast-track

development approach when

technology platforms are applied

in all key aspects of development.

This includes upstream as well as

downstream processing, such as

cell-line development, cell culture,

downstream processing, analyti-

cal concepts, and filling (3). Major

considerations when designing a

manufacturing platform include

the infrastructure, resources and

manufacturing capacity available,

the degree of scalability and pro-

ductivity desired, the time avail-

able for producing the first dose

and the dosing regime. Figures

2 and 3 illustrate some platform

processes for vaccine manufac-

turing in eggs and in animal cell

culture. A complete platform for

downstream processing as noted

in one study includes three steps:

depth filtration, ultra/diafiltration

(UF/DF), and membrane adsorp-

tion for purification of recom-

binant baculoviruses. Global

recovery yields reached 40% at

purities over 98% (12).

Figure 2: A generic process for egg-

based vaccine manufacturing using

platform technologies. SPF is specific

pathogen free. UF is ultrafiltration. DF

is diafiltration.

Procurement of 10-11 days old fertilized SPF eggs

Inoculation and propagation of virus inside the egg

Harvesting of allantoic fluid & clarification

Virus purification using ultracentrifugation/cross flowfiltration/chromatography

Virus Inactivation

Concentration using UF/DF

Formulation/Adjuvants/Preservatives

Sterile filtration

Fill and finish


Vaccines Trends

VaccInE STaBILITY and

SuPPLY-cHaIn ManagEMEnT

Another important parameter to

consider when manufacturing a

vaccine is its thermostability. All

vaccines lose potency over time,

and the rate of potency loss is tem-

perature dependent. Hence, there

is a need to develop, monitor, and

maintain cold-chain systems to

ensure that the potency of vac-

cines is maintained under refriger-

ated conditions (mostly between

2–8 ºC) until the point of use. The

World Health Organizaion (WHO)

recommends that reconstituted

vaccines be kept cold and that any

unused vaccine from a multidose

vial be discarded after 6 h (14). This

is primarily due to the instability of

reconstituted vaccines and also due

to the chances of bacterial contam-

ination because live vaccines do

not contain preservatives (15). New

approaches to develop thermosta-

ble vaccine formulations that are

resistant to damage caused by freez-

ing or overheating are necessary

to eliminate dependence on a cold

chain. Such approaches could have

great economic benefits in terms

of reducing vaccine wastage and

preventing adverse health conse-

quences of administering damaged

vaccines to recipients. Furthermore,

such approaches would improve

the effectiveness of vaccines and

enable delivery of the vaccine to

remote populations (15).

In addition to ensuring vac-

cine stability, optimizing the

vaccine supply chain through

quality management in vaccine

manufacturing plays a key role

in safe and effective vaccine

manufacturing. In this context,

performing a risk analysis for the

manufacturing-process param-

eters that affect product quality,

as well as an assessment of each

validation step, including clean-

ing protocols, monitoring of the

air filters, and assurance of steril-

ity of the source materials, are

also crucial to a quality manage-

ment process (16).

rEguLaTorY ISSuES


Vaccines are generally (although

not always) prophylactic biomol-

ecules, therey making their devel-

opment and commercialization

complex. A set of basic regulatory

criteria from the WHO applies

to vaccine manufacture, regard-

less of the technology used to

produce the products. Licensure

of a new vaccine is based on the

demonstration of safety and

effectiveness, and the ability to

manufacture in a consistent man-

ner. The manufacturer facilitates

the development and evaluation

of new vaccines by anticipating

and addressing the regulatory

issues involved. General regula-

tory issues that are applicable to

other biologicals, such as detec-

tion of adventitious agents and

improved test methods that are

reliable and sensitive, are valid

for vaccines as well. Additional

vaccine-specific issues include

determining correlates of pro-

tection necessary for evaluating

efficacy, improving assays for

potency, or finding animal mod-

els that can be used for the eval-

uation of efficacy when human

clinical trials are not feasible or

unethical (17).

Although the use of animal-

cell culture for manufacturing

viral vaccines is the current prac-

tice, regulatory challenges tied to

this protocol are extensive. The

WHO requires additional reports

to ensure safety of the popula-

tion receiving vaccines gener-

ated out of cell culture. Required

tests include confirming tumori-

genicity, checking for extrane-

ous agents of the cell substrate,

residual HCP and residual DNA,

equivalence of cell culture and

egg-based vaccines (i.e., antigenic

characterization by cross-reactiv-

ity of specific antisera and animal

protection studies).

Membrane chromatography Density gradient ultracentrifugation

High virus titers; up 1013 VP/ml Yield of up to 106 VP/ml only

10–20 × faster (~ 2h) Time consuming (~ 36h)

Single-use (no validation required) Requires validation against batch to batch carryover contamination

Non toxic and does not require the removal of any residual contaminants

Requires removal of toxic material like CsCl2 or sucrose from finished product

Removes host cell protein (HCP) and contaminating DNARequires additional step for removal of other host-cell related contaminants

Operates on a plug-and-play mechanism with single-use concept and (no maintenance required)

High maintenance

Small footprint in line with process analytical technology (PAT) and quality-by-design (QbD) principles

Footprint and in-process (i.e., PAT) control are challenges

Table I: Comparison between the use of membrane chromatography and density gradient ultracentrifugation for

virus purification.


Vaccines Trends

THE nEEd For SIngLE-uSE In

VaccInE ManuFacTurIng

Generally speaking, single-use

technologies reduce bottlenecks

in manufacturing environments.

Implementation of single-use

technologies in vaccine manu-

facturing should be the gold

standard. Single-use steps elimi-

nate the need for cleaning and

validation, which automatically

eliminates any kind of cross-

contamination, while maintain-

ing the aseptic path. Apart from

eliminating cleaning and vali-

dation, the reduced set-up time

associated with these systems is

crucial to meeting the time-sen-

sitive demands of vaccine man-

ufacturing. Single-use process

steps can be easily assembled

and are quickly configurable,

thereby reducing downtime and

capital investment in facility and

equipment.

Aseptic processing has always

received attention from regu-

lators in the biopharmaceutical

sector because of the high risk of

microbial contamination that can

affect patient health, particularly

when the molecule is a prophy-

lactic material. Traditionally, asep-

tic processing is done in critical

or controlled areas, depending on

the risks associated with certain

steps in the manufacturing pro-

cess (16).

Interestingly, discarding a

device, without having to prove

that it has been sufficiently

cleaned, is one step that both

regulatory authorities as well as

manufacturers seem happy to

embrace (18). This approach is

especially beneficial for vaccines,

because vaccinating large, healthy

populations, carries much greater

risks than treating relatively small

groups of people with conven-

tional drugs.

For manufacturers attempting

to minimize the risks of vaccine-

batch contamination, single-use

technologies provide an impor-

tant avenue for enhanced safety.

Single-use systems that the sup-

plier presterilizes and bundles

together can further simplify

set-up. With fewer opportuni-

ties for operator error, single-

use technologies can improve

safety and production economics.

Some single-use products, such

as bags, also save space, because

they lie flat and can be stacked

before use. Unlike permanent

storage tanks, single-use bags are

typically ordered on an as-needed

basis to avoid excess unused

equipment (18).

Although single-use technolo-

gies can play a prominent role in

egg-based vaccine manufacturing,

in the areas of filtration, storage,

and at connection points, their

use multiplies in cell-based appli-

cations. More specifically, single-

use technologies can be used for

media preparation, clarification,

and cell harvesting in upstream

processes as well as in buffer prep-

aration, capture, and polishing

chromatography steps, purifica-

tion and filling in downstream

processes. Single-use bioreactors

are an example of the increas-

ingly growing role of single-

use technologies in upstream

processing (18). A recent study

demonstrated the successful devel-

opment of a complete single-use

downstream process by imple-

menting depth filtration, UF/DF,

and membrane chromatography

for the purification of recombi-

nant baculoviruses (12).

Finally, environmental con-

cerns have been cited as key

motivators for moving toward

single-use systems in bioman-

ufactur ing (19). This bel ief

stems f rom the observat ion

that although single-use sys-

tems require significant energy

to produce them and gener-

ate plastic waste, the amount

of energy and water consumed

in the production of water-for-

injection and steam used in

clean-in-place/steam-in-place

operations can more than off-

set the waste issue. When waste-

to-energy plants are considered

for disposal of the plastics, the

environmental benefits of sin-

gle-use technologies are further

enhanced (20). By one estimate,

the commutes of the plant

employees account for more

than 50% of the total carbon

emissions associated with bio-

manufacturing (19).

The above observations demon-

strate that single-use technologies

make it possible to develop and

scale up processes quickly and facil-

itate manufacturing by reducing

the cleaning-validation burden.

concLuSIon

Despite the advances in vaccine

manufacturing across the globe,

Figure 3: A generic process for

animal cell culture based vaccine

manufacturing using platform

technologies. HCP is host cell protein.

Animal cell culture propagation to host virus

Inoculation and culture of virus inside the host

Harvesting of virus and clarification of bulk

Virus purification using ultracentrifugation/cross flowfiltration/chromatography

Removal of contaminants like HCP and DNA using chromatography

Virus

Concentration using UF/DF

Formulation/Adjuvants/Preservatives

Sterile filtration

Fill and finish


Vaccines Trends

regulatory, technical, and manu-

facturing hurdles still stand in

the way of companies seeking

to take a candidate product to

the clinic and eventually to mar-

ket. The identification of suitable

vaccine candidates is only one

of many hurdles involved in the

translation of a vaccine candidate

from the bench to the clinic (21).

Identifying suitable antigens,

adjuvants, and delivery methods

are just the beginning of vaccine

development (22). Public demand

for safe and effective vaccines

continues. In addition, regula-

tory requirements have led to an

emphasis on well characterized,

safe vaccines (21).

The need for well defined,

single-use platform processes

to reduce complexities, ensure

safety, and maintain timelines to

market, therefore, is only grow-

ing. Additionally, because process

development provides a techno-

logical foundation for manufac-

turing, analytical methods and

assay development for character-

ization and potency determina-

tion must be part of a company’s

approach. Such an approach helps

to lay the groundwork for success-

ful commercialization (22).

rEFErEncES

1. S. Plotkin, W. Orenstein, P. Offit,

Vaccines (Saunders, Philadelphia, 5th

ed. 2008).

2. C. Scott, supplement to BioProcess Intl.

6 (6) s12–s18 (2008).

3. S. Fetzer, supplement to BioPharm Intl.

21 (1) s1–s6 (2008).

4. C.P. Steffy and L. Rosin, BioProcess Intl.

2 (4), s48–s57 (2004).

5. P. Holland-Moritz, BioPharm Bulletin

(June 2006).

6. S. Srivastava, Vaccine World Summit

March 1–3 (2011).

7. S.S. Ozturk, Cytotechnology 22, 3–16

(1996).

8. A. Heuer, The Bridge Editor’s Note 36 (3)

3 (2006).

9. B. Kalbfuss et al., Jrnl. of Membrane Sci.

299, 251–260 (2007).

10. A. Karger, J. Schmidt, and T.C. Metten-

leiter, Jrnl. Virol. 72, 7341–7348 (1998).

11. T. Vicente et al., Jrnl. of Membrane Sci.

311, 270–283 (2008).

12. T. Vicente et al., Gene Therapy 16,

766–775 (2009).

13. J. Tal, J Biomedical Sci. 7, 279–291

(2000).

14. WHO, Temperature Sensitivity of

Vaccines, http://whqlibdoc.who.int/

hq/2006/WHO_IVB_06.10_eng.pdf,

accesses Sept. 6, 2011.

15. D. Chen and D. Kristensen, Expert Rev.

Vaccines 8 (5), 547–557 (2009).

16. E. Boer, supplement to BioProcess Intl. 6

(10), 38–40 (2008).

17. N.W. Baylor, supplement to BioPharm

Intl. 20 (8) (2007).

18. H. Pora, BioPharm Intl. 19 (6) (2006).

19. L. Leveen and S. Cox, presentation

at IBC’s 5th International Single-Use

Applications for Biopharmaceutical

Manufacturing (San Diego, 2008).

20. D. Newman and S. Walker, presentation

at IBC’s 5th International Single-Use

Applications for Biopharmaceutical

Manufacturing (San Diego, 2008).

21. G. Healy, Microbiologist 3, 28–30 (2006).

22. C. Scott, supplement to BioProcess Intl.

9, 3–42 (2010).

acknoWLEdgMEnT

T he aut hor wou ld l i ke to

acknowledge colleagues from

S a r to r iu s S t e d i m B io te c h ,

Mahesh P rashad and Frank

Meyeroltmanns for shar ing

their knowledge and design-

ing the gener ic schemes for

platform processes in vaccine

manufac tur ing. The author

appreciates the support of Dr.

Uwe Gottschalk, vice-president

of pur i f icat ion technologies

at Sar tor ius Stedim Biotech.

The author would a lso l ike

t o a c k n o w l e d g e A n u r a g

S . R a t h o r e , P h D, a f a c -

u lt y member at the Indian

I n s t i t ute o f D e l h i , I nd i a ,

for h is ed itor ia l a ss i stance

w i t h t h i s p ap e r . R a t ho r e

i s a memb er of B ioPha r m

I n t e r n a t i o n a l ’ s E d i t o r i a l

Advisory Board and the author

of the journal’s series on the

Elements of Biopharmaceutical

Production (see below). BP

Previous articles in the series include:

1. Modeling of Microbial and Mammalian Unit Operations

2. Scaling Down Fermentation

3. Optimization, Scale-up, and Validation Issues in Filtration

4. Filter Clogging Issues in Sterile Filtration

5. Lifetime Studies for Membrane Reuse

6. Modeling of Process Chromatography Unit Operation

7. Resin Screening to Optimize Chromatographic Separations

8. Optimization and Scale-Up in Preparative Chromatography

9. Scaling Down Chromatography and Filtration

10. Qualification of a Chromatographic Column

11. Efficiency Measurements for Chromatography Columns

12. Process Validation: How much to Do and When to do it

13. Quality by Design for Biopharmaceuticals: Defining Design Space

14. Quality by Design for Biopharmaceuticals: Case Studies

15. Design Space for Biotech Products

16. Applying PAT to Biotech Unit Operations

17. Applications of MVDA in Biotech Processing

18. Future Technologies for Efficient Manufacturing

19. Costing Issues in the Production of Biopharmaceuticals

20. Economic Analysis as a Tool for Process Development:

To view these articles, visit BioPharmInternational.com/elements.

Elements of Biopharmaceutical Production


Vaccines DNA Vaccines

DNA Vaccine DeliveryDevelopment of the ideal DNA vaccine requires the

optimization of delivery strategies and plasmid vectors.

Rocky cRanenbuRgh

A DNA vaccine is a plasmid produced in

Escherichia coli that contains an antigen

gene controlled by a promoter that func-

tions only in animal cells, usually the

cytomegalovirus promoter. The DNA vac-

cine plasmid must be delivered to antigen-presenting

cells (APCs) of the host, where the antigenic pro-

tein is expressed, processed, and presented to the

immune system. This approach works efficiently in

mice and other animal models when DNA is delivered

by intramuscular injection, and DNA vaccines have

been licensed for veterinary applications, yet no DNA

vaccine has been approved for use in humans. A key

problem is achieving the delivery of sufficient plas-

mid to the APCs. This article discusses the approaches

designed to achieve delivery, including a focus on the

use of live bacterial vectors.

Intramuscular InjectIon

Since DNA vaccination was developed in the early

1990s, the most common method for immunization

has been intramuscular injection of DNA. The DNA

is usually dissolved in water or an isotonic saline

solution, with the inclusion of an adjuvant if neces-

sary. The aim is to get the plasmid into APCs, which

in muscle tissue are primarily dendritic cells. There,

the DNA is transported to the nucleus for expres-

sion, and the resulting polypeptide is processed

and presented on the cell surface in a major histo-

compatibility complex (MHC). DNA vaccination

can therefore produce a protective CD8+ cytotoxic

T lymphocyte (CTL) response via MHC Class I (1).

Also important are bystander effects, whereby anti-

gens expressed and released from adjacent myocytes

(i.e., muscle cells) are taken up by APCs, thus trig-

gering antibody production through MHC Class II.

This effect enables DNA vaccines to be developed to

target a wide range of bacterial, viral, and parasitic

diseases, in addition to the rapidly expanding field

of cancer immunotherapy.

The first licensed DNA vaccine was West Nile-

Innovator DNA, approved in 2005 for the immu-

nization of horses against West Nile Virus. It was

given in two intramuscular doses 2–4 weeks apart,

then as a single dose annually (2). However, this

vaccine has now been discontinued by Pfizer fol-

lowing their acquisition of the developers, Wyeth’s

Fort Dodge Animal Health. Also in 2005, Novartis

Animal Health (Basel, Switzerland) gained approval

for Apex-IHN, a vaccine against infectious hemato-

poietic necrosis virus in salmon, which encodes a

viral glycoprotein and is administered by a single

intramuscular dose of 10 μg DNA (3).

Despite promising results in animals, DNA vac-

cine efficacy has been disappointing in human

clinical trials. This is generally because of a much

lower specific immune response generated by

injected plasmid DNA alone, and has led to the

adoption of a prime-boost vaccination strategy

whereby the plasmid DNA injection is followed

by a boost with the same antigen gene in a viral

vector. This heterologous boosting strategy has sig-

nificantly increased the specific immune response.

Commonly-used attenuated viral vectors include

those based on modified vaccinia ankara (derived

from the smallpox vaccine), lentivirus (mainly

derived from HIV), and adenoviruses of primate

origin. Viral vectors are expensive and complex to

manufacture compared with DNA, thus eliminat-

ing the advantages of the DNA vaccine approach

such as lower cost and simple, generic production

processes. An additional disadvantage is the poten-

tial of a host immune response directed against

the vector. Development of the ideal DNA vaccine

therefore requires the optimization of delivery

strategies and plasmid vectors.

Intramuscular electroporatIon

A traditional approach for inserting DNA into micro-

bial and animal cells in culture has been by the

Rocky cRanenbuRgh, PhD, is head of molecular biology at Cobra Biologics, Stafforshire UK, [email protected].



application of an electric pulse,

which creates transient pores in

the cell membrane and allows

entry of the negatively charged

DNA molecules. Electroporation

has been adapted from its in vitro

single-cell origins to enhance in

vivo delivery to tissues follow-

ing intramuscular injection, and

enables a significant increase

in transfection efficiency and

immune response over injection

alone. The disadvantages include

pain at the injection site due to

the electric pulse, and the require-

ment for specialized electropora-

tion devices. The most common

approaches use a disposable grid

consisting of injection and elec-

trode needles attached to the

device that provides the elec-

tric pulse. The DNA solution is

injected, needle electrodes are

inserted through the skin and into

muscle tissue, and an electric field

applied to facilitate DNA entry

into dendritic cells and myocytes.

Ichor Medical Systems (San

Diego, CA) has developed the

TriGrid Delivery System (TriGrid)

a hand-held electroporat ion

device that has been demon-

strated to increase DNA vaccine

delivery efficiency by as much as

1000-fold over injection alone.

Four electrodes are arranged in

a diamond shape around the

central needle, which is a con-

ventional single-use hypoder-

mic syringe that is inserted into

the device, and an electric pulse

is administered upon injection

(see Figure 1a). Ichor has demon-

strated greatly enhanced potency

of DNA vaccines delivered with

the TriGrid in several animal

models using DNA vaccines that

encode antigens such as anthrax,

hepatitis B virus, and tumor-

associated antigens. The device

is being evaluated in several

clinical trials, and results dem-

onstrating enhanced responses

to a prophylactic HIV DNA vac-

cine delivered with TriGrid com-

pared with conventional needle

injection have been published

recently (4). In addition, Ichor

has shown in animal models that

delivery of DNA encoding the

protein therapeutics erythropoi-

etin and interferon-β may be a

viable alternative to injection of

the recombinant proteins.

A therapeutic plasmid DNA appli-

cation (although not a vaccine)

that was licensed for pigs in 2008

is LifeTide SW, developed by VGX

Animal Health (The Woodlands,

TX) (5). The plasmid produces

growth hormone-releasing hor-

mone and is administered to sows

of breeding age to increase the

number of piglets weaned, and

requires only a single treatment.

Plasmid delivery is by direct injec-

tion into skeletal muscle, followed

by electroporation using a portable

electrokinetic device. The device,

called CELLECTRA, was developed

by Inovio (see Figure 1b) and uses a

sterile, disposable electroporation

needle array. Inovio has also dem-

onstrated 1000-fold greater DNA

delivery compared with injection

alone. The device is equally suited

to DNA vaccine applications, and

users of CELLECTRA have targeted

a range of pathogens including

HIV, Clostridium difficile, hepati-

tis C virus, and malaria parasites.

Inovio has an ongoing Phase II

clinical trial for cervical-cancer

therapy (human papilloma virus

therapeutic vaccine) using the

device. Additional ongoing human

clinical trials include Phase I trials

for prophylactic vaccines for influ-

enza (i.e., avian and seasonal) and

HIV, and a Phase I clinical trial for

HIV therapy.

While ample evidence indi-

cates electroporation enhances

the delivery and immunogenic-

ity of injected DNA vaccines, it

has the drawback of increased

discomfort at the administra-

tion site due to the electrical

pulses. However, initial human

clinical-trial data with intra-AL

L F

IGU

RE

S A

RE

CO

UR

TE

SY

OF

TH

E A

UT

HO

R

Figure 1: Devices for plasmid DNA delivery indluding (a) TriGrid and (b)

CELLECTRA with applicator (for intramuscular electroporation), (c) DermaVax

(for transdermal electroporation) and (d) ZetaJet (for subcutaneous or

intramuscular high-pressure injection).

1a

1b

1c

1d



muscular del ivery indicates

that the electroporation proce-

dure is tolerable for therapeutic

and even prophylactic applica-

tions. Furthermore, continued

improvements to device design

are likely to result in increased

acceptability of the procedure.

A novel approach to delivering

electrical pulses within target tis-

sue that may increase tolerabil-

ity is the generation of electrical

fields by piezoelectricity, as used

in a contactless, noninvasive

device developed by Inovio.

Another interesting alterna-

tive to penetrative electrodes has

been developed by MagneGene

(Lake Forest, CA), which uses

contactless magnetic electropora-

tion. This technique relies on a

strong, rapidly changing mag-

netic field generated by a paddle-

shaped device to induce rotating

electric fields in the host tissue.

This device has been evaluated

in guinea pigs following injec-

tion with reporter plasmid, where

enhanced in vivo gene expres-

sion was demonstrated. Although

the magnetic field causes muscle

spasms, these subside immedi-

ately after use of the device, and

there is no pain following the

procedure (6).

transdermal mIcroneedles

and electroporatIon

Transdermal DNA immuniza-

tion involves the use of arrays

of microneedles, each a few

hundred microns long, to pierce

the barrier of the stratum cor-

neum (i.e., the skin’s outer layer,

typically 10–20 μm thick) and

deliver the vaccine (7). The

skin has a high concentration

of APCs called Langerhans cells,

which makes it an attractive tar-

get. The advantages of micronee-

dles are that they are easy to

administer and cause less pain

at the injection site than does

a conventional needle. There

are various strategies to achieve

vaccination using microneedles.

The simplest involves arrays of

microneedles that are used to

pierce or scarify the stratum

corneum, thus increasing per-

meability to a topically applied

DNA solution. This approach is

suitable for preclinical studies,

but is not scalable. Alternatively,

the microneedles can be coated

with the dried vaccine which dis-

solves following administration.

Hollow microneedles can be used

to inject solutions containing the

vaccine. These can be submillime-

ter arrays or scaled-down needles

in the range of 1–2 mm. An inter-

esting alternative to fixed, dis-

posable microneedles is a solid,

soluble microneedle array that

is either formulated or coated

with the vaccine. This array is

inserted into the skin, where the

microneedles dissolve or degrade,

leaving only the backing to be

disposed of and thus eliminating

contaminated sharps.

Sm a l lp ox DNA v acc i n a -

tion of mice was demonstrated

using the Easy Vax device sup-

plied by Cellectis therapeutics

(Paris), and was the first example

of microneedle-mediated electro-

poration (8). The gun-shaped Easy

Vax combines the electropora-

tion and microneedle approaches,

with cutaneous Langerhans cells

as the targeted APCs. The plasmid

DNA was dried onto the tips of

the eighty-microneedle array and

inserted into the skin to enable

the DNA to dissolve, followed by

the delivery of six electric pulses.

W h i l e t he m ic r o ne e d l e

approach is gaining interest for

delivery of therapeutic and anti-

genic recombinant proteins, only

a few studies in mice have shown

immune protec t ion aga inst

microneedle-administered DNA

vaccines. An additional problem

when translating animal results

to humans relates to the amount

of DNA that can be loaded onto

a microneedle array. A typical

human clinical trial injected dose

of 2–4 mg DNA is already rela-

tively small compared with the

murine equivalent from which

it is extrapolated, yet this would

be prohibitively high to expect

to dissolve intradermally from a

dried formulation on a micronee-

dle array, which leaves liquid

DNA solution injection as the

only viable strategy.

Cellectis therapeutics also sup-

plies the DermaVax electropora-

tor (see Figure 1c), which requires

a DNA solution to be injected

sub-dermally using a hypoder-

mic syringe, followed by elec-

troporation through a series of

pulses from 2 mm needle elec-

trodes attached to the bench-

top DermaVax apparatus. The

DermaVax system induces a 100-

to 1000-fold greater increase in

gene expression over injection

alone and is being used for the

delivery of DNA vaccines against

HIV-1, as well as vaccines against

prostate and colorectal cancers in

human clinical trials (9).

I n o v i o P h a r m a c e u t i c a l s

has developed a transdermal

electroporation device called

CELLECTRA-3P. In pilot studies

in nonhuman primates, Inovio

The most comon

method for

immunization has

been intramuscular

injection of DNA.



demonstrated that intradermal

administration of a smallpox vac-

cine or an H5N1 vaccine success-

fully protected animals from a

lethal challenge of monkeypox

or an H5N1 infection, respec-

tively (10, 11). The device is now

in Phase I clinical trials for a uni-

versal influenza vaccine. Inovio

has also developed a prototype

minimally invasive electropora-

tion device that has been dem-

onstrated for inf luenza DNA

vaccine delivery.

pressure Injectors

Gene guns have been used since

the advent of DNA vaccination

for high-pressure transdermal

delivery of microbeads coated

with DNA (biolistics). PowderMed

(Oxford, UK, acquired by Pfizer)

developed a handheld particle-

mediated epidermal delivery

(PMED) device that uses a high-

pressure helium minicylinder to

fire DNA-coated gold microbeads

into the epidermis. Because the

gold beads enter the cell cyto-

plasm and target more APCs than

does needle injection, greater CTL

responses can be generated for

the same amount of DNA (11).

In one example, PMED was used

to deliver 2 μg of two plasmids

to rhesus macaques: one with an

H1N1 hemagglutinin gene, the

other expressing the adjuvant

GM–CSF. This method gener-

ated good antibody and CTL

responses (12). However, limita-

tion to the microbead approach

is the amount of DNA that can be

coated onto beads, which could

restrict its ability to be scaled up

for human applications.

Merial (Duluth, GA) devel-

oped the pioneer ing canine

melanoma DNA vaccine Oncept,

which was fully approved in

2010. It contains the human

tyrosinase gene, delivered (fol-

lowing surgical tumor removal)

in four doses at two-week inter-

vals, followed by a single dose

every six months. Oncept is

administered using a needle-

free Canine Transdermal Device

developed by Bioject Medical

Technologies (Portland, OR) (13).

Bioject specializes in the devel-

opment of needle-free injection

systems that use high-pressure

injection to fire a stream of the

DNA vaccine solution through

the epidermis . Thei r latest

device, ZetaJet, is small and

spring-activated for ease of use

in the field, and fires up to 0.5

mL into skin or muscle tissue.

oral delIvery usIng

lIve BacterIal vectors

The oral route for vaccination

avoids needles and reduces the

logistical issues and costs of imple-

menting vaccination programs

requiring trained healthcare pro-

fessionals, which would be par-

ticularly beneficial in developing

countries. Oral vaccination also

stimulates a mucosal immune

response, which is important

because mucosal surfaces are a

more common route of entry for

many pathogens than is the skin.

Much of the research in oral DNA

vaccine delivery has focused live

bacterial vectors (LBVs) derived

from attenuated bacterial patho-

gens Salmonella and Shigella,

enteric species that are able to

replicate the high copy-number

DNA vaccine plasmids normally

produced in E. coli. These attenu-

ated pathogens carry mutations of

biosynthetic or invasive genes that

eliminate their pathogenicity and

ability to persist in host tissues or

the environment.

LBVs that have been ingested

travel from the stomach into the

small intestine. Here, Salmonella

and Shigella invade microfold (M)

cells and enter lymphatic nod-

ules called Peyer’s patches. (see

Figure 2). Once inside the Peyer’s

patches, the bacteria become

targets for (or actively enter)

macrophages, where they are

internalized in membrane-bound

phagosomes. At this point, one

of two strategies is employed:

Shigella escapes from the phago-

some into the cytoplasm, while

Salmonella alters the composi-

Figure 2: Oral recombinant vaccine delivery using live bacterial vectors. (a)

The live bacterial vectors (LBVs) are ingested and travel into the small intestine,

where they invade the lining of the ileum through M cells and enter Peyer’s

patches. (b) The LBVs invade antigen presenting cells and are phagocytosed.

Shigella can escape the phagosome and enter the cytoplasm, while Salmonella

modifies the phagosome and persists until it is digested by fusion with a lysosome.

MHC is major histocompatibility complex.

2a 2b

Gut MHCClass II

MHCClass I

ShigellaAntigensecretion

Salmonella

Phagosome Lysosome

Gutlining M cell

APC

Peyer’s patch



tion of the phagosome to sur-

vive and replicate (14). However,

the precise mechanism of DNA

vaccine delivery using bacterial

vectors is not understood. For

Shigella, the cells are thought

to eventually lyse in the cyto-

plasm, releasing the DNA vac-

cine plasmids which are then

transported to the nucleus by

the host cell. Attenuating muta-

tions that increase the rate of

cell lysis have been shown to

improve DNA vaccine deliv-

ery using Shigella f lexneri. For

phagosome-bound Salmonella,

the potent ia l mechanism is

less obvious, but could be due

to a bystander effect in which

DNA released from host cells

that have undergone apoptosis

due to Salmonella invasion is

carried to APCs in membrane

blebs (14). As with other DNA

delivery routes, once the plas-

mid enters the nucleus, the anti-

gen gene is expressed, and the

protein processed for MHC pre-

sentation. Another advantage

of bacterial vectors is potent

immunostimulatory properties,

due mainly to the lipid A (endo-

toxin) in the cell membranes,

which eliminates the need for

an adjuvant. The direct target-

ing of the mucosal immune sys-

tem means that vector-directed

immune responses are not likely

to inhibit the repeated use of

live bacterial vectors.

Drug development suf fers

from the high attrition rate in

translating promising preclini-

cal results in animal models into

success in human clinical tri-

als. One variable in DNA vac-

cine development is the relative

dose between preclinical and

clinical subjects. For example,

a DNA vaccination by conven-

tional needle injection of a 20 g

mouse typically requires 100 μg

of DNA to generate a protective

immune response. To scale this

to a 70-kg human would involve

injecting 350 mg, which is pro-

hibitive in terms of both the vol-

ume required and the associated

cost (15). A typical vaccine dose

used in human clinical trials

is 2–4 mg DNA, which repre-

sents only 0.5–1% of the scaled

dose. The extent to which the

lack of scalability has affected

human DNA vaccine develop-

ment is debatable, as the first

vaccine approved was effective in

an even larger animal: the horse.

But in contrast, Salmonella-based

approaches use the same dose in

terms of bacteria and plasmid

DNA in humans as in mice. This

similarity allows the murine dose

to be representative, because S.

enterica serovar Typhimurium

in mice mimics the invasion

and pathogenicity of S. enterica

serovar Typhi in humans.

As with Shigella, Listeria mono-

cytogenes has the abi l ity to

invade M cells and escape from

the phagosome, and has been

used to demonstrate DNA vac-

cine delivery, although it cannot

replicate the high copy-number

plasmids that are universally

used as DNA vaccines. E. coli

strains have also been used as

DNA vaccine vectors, having

been genetically modified to

express the invasion and phago-

somal escape proteins from its

pathogenic relatives.

P u b l i c a c c e p t a n c e o f

Salmonella and other patho-

gen-based vectors is important

for this to be a viable strat-

egy, so it is encouraging that

a cur rent l icensed t y phoid

vaccine is a l ive at tenuated

S. enterica serovar Typhi mar-

keted as Vivotif (Crucell, The

Netherlands), which has been

safely administered to more

than 200 million patients over

25 years (16). Other attenu-

ated enteric bacteria have also

shown good safety profiles in

recent clinical trials, and the

increasing use of probiotics has

spread the concept of ingesting

beneficial bacteria. However,

Figure 3: Bile-adsorbing resin for oral capsular delivery of lyophilized enteric

bacteria.

Stomach

Small intestine

Enteric coating

Bacteria rehydrateand recover bileresistance inbile-depleted zone

Capsule dissolvesand releases livebacteria vectors

Dried Live Bacterial Vaccine

Bile AdsorbingResin

Enteric coating dissolves

Water enters freely

Toxic bile acids retardedby Bile Adsorbing Resin



several issues must be over-

come before live bacterial vec-

tors can be commercialized. The

most convenient oral delivery

for adults would use a capsule

containing lyophilized bacte-

ria. An enteric coating protects

against the stomach acid, but

lyophilized bacteria are suscep-

tible to bile when released into

the small intestine. To mitigate

this susceptibility, Cambridge

Universit y in col laborat ion

with Cobra Biologics developed

a bile-adsorbing resin formula-

tion that allows water to pen-

etrate, but adsorbs the bile acids

(17). This formulation enables

the bacteria to rehydrate for

the few crucial minutes that

are required to increase their

resistance to the effects of bile,

before their release as the cap-

sule dissolves (see Figure 3).

Another key issue to address is

plasmid instability in bacterial

cells. The antibiotic-resistance

genes normally used to select

and maintain plasmid-contain-

ing cells are strongly discour-

aged by regulatory authorities

for reasons of biosafety when

used in LBVs. FDA requires

a valid justification and proof

that these genes cannot be

transferred to commensal bacte-

ria (18). As there is arguably no

longer a credible justification for

their use, antibiotic resistance

genes are not used by compa-

nies developing LBV strategies.

Even if they were permitted,

they contribute to a significant

metabolic burden on the bacte-

rial cell due to their constitutive

expression and the high plas-

mid copy-number—the major

factor in plasmid loss in the first

place—and it is not acceptable

practice to dose a vaccinee with

antibiotics. Therefore, antibi-

otic-free plasmid selection and

maintenance systems are essen-

tial for DNA vaccine delivery

using LBVs, but most systems

retain the metabolic burden of

an alternative expressed select-

able marker gene.

To address th is problem,

Cobra Biolog ics appl ied it s

operator–repressor t it rat ion

(ORT) technology to Salmonella

as ORT–VAC. ORT requires an

essential bacterial chromosomal

gene to be controlled by a pro-

moter such as lac or tet. The cell

is unable to grow in the absence

of the chemical inducer because

the repressor protein binds to

t he ch romosoma l operator

and prevents gene expression.

However, when the cell is trans-

formed with a plasmid that also

possesses the operator (a short,

nonexpressed sequence), mul-

tiple copies of the plasmid titrate

the repressor and enable expres-

sion of the essential gene and

cell growth (see Figure 4).

ORT–VAC has recently been

applied for successful immuni-

zation using a tuberculosis DNA

vaccine expressing the anti-

gen mpt64 from Mycobacterium

tuberculosis, generating greater

T- ce l l r e sp onse s t ha n t he

injected DNA vaccine in mice

(19). This technique reduced

the pulmonary count of infect-

ing M. tuberculosis following a

challenge. The positive control

was the standard murine dose

of 100 μg of intramuscularly-

injected DNA, but ORT–VAC

Figure 4: Operator–repressor titration for stable maintenance of selectable

marker gene-free plasmids in live bacterial vectors. (a) The natural promoter of

an essential gene is replaced with an inducible promoter and repressor gene,

such that a repressor protein binds to an operator sequence and prevents

essential gene expression and cell growth. (b) When the cell is transformed with

a multicopy plasmid that also contains the operator sequence, the repressor

protein is titrated by binding to the operator, thus enabling essential gene

expression and cell growth.

4a

Repressor protein

Repressor protein

Repressor gene

Repressor gene

Promoter

Promoter

Essential gene

Essential gene

Operator

Operator

Plasmid

Operator

Essential protein

4b



achieved better results despite

delivering only 0.01– 0.1% of

this plasmid dose in 107–10 8

orally administered bacteria.

the current and

Future dna vaccIne market

Only two DNA vaccines (i.e.,

Apex–IHN and Oncept) and

one DNA therapy (i.e., LifeTide)

are currently on the market, all

for veterinary applications. But

despite a slow start, the DNA vac-

cine market is growing steadily,

and the approval of a human

DNA vaccine in the next few

years would be a significant shot

in the arm for the sector. There

is no fundamental reason why

DNA vaccines will not work in

humans, and the success of the

Oncept canine melanoma vac-

cine is significant, because it is

one of only two licensed immu-

notherapeutic cancer vaccines.

Overall sales in the DNA vac-

cine market totaled $141 million

in 2008, and with a compound

annual growth rate (CAGR) of

69.5%, are forecast to increase

to $2.7 billion by 2014 (20).

The value of human DNA vac-

cines was only $12 million in

2008, but it doubled a year later

and is predicted to reach $2.3

billion by 2014 (149.6% CAGR)

(20). In 2008 there were 95 open

clinical trials involving plasmid

DNA; the majority of these were

for anticancer applications (see

Figure 5) (13).

For all recombinant vaccines,

the selection of an antigen that

is able to stimulate a protective,

long-lasting immune response

is the key to success. In addition

to the antigen choice, effective

delivery technologies are essential

for realizing the promise of DNA

vaccines, because there is a limit

to what can be achieved through

engineering of plasmids and adju-

vant formulation. The fact that

two out of the three plasmid-

based medicines on the market

use relatively novel approaches

(i.e., electroporation and high-

pressure transdermal delivery)

demonstrates the capacity for

innovation in this field. Effective

oral delivery would represent a

significant advance. The cost of

DNA vaccines would be greatly

reduced by a simple manufactur-

ing process and no requirement

for a viral boost or specialized

delivery device.

reFerences 1. M.A. Lui, Immunol. Rev. 239, 62–84

(2010).

2. “West Nile-Innovator DNA”, Fort

Dodge Animal Health. E0257D 15M

AP (Nov. 2008).

3. N.C. Simard, “The Road to

Licensure of a DNA Vaccine for

Atlantic Salmon”, www.bion.no/

filarkiv/2010/07/vaccine_simard.

pdf, accessed Aug. 2011.

4. S. Vasan et al., PLoS One 6 (5),

e19252 (2011).

5. K.E. Broderick et al., Hum. Vaccines

7, 22–28 (2011).

6. T. Kardos. “Contactless Magnetic

Electroporation” presented at the

DNA Vaccines Conference (San

Diego, CA, 2011).

7. M.R. Prausnitz et al., Microbiol.

Immunol. 333, 369–393 (2009).

8. J.W. Hooper et al., Vaccine 25,

1814–1823 (2007).

9. A.K. Roos et al., Mol. Ther. 13 (2),

320–327 (2006).

10. L.A. Hirao et al., J. Infect. Dis. 203

(1), 95–102 (2011).

11. D.J. Laddy et al., J. Virol. 83 (9),

4624–4630 (2009).

12. P.T. Loudon et al., PLoS One 5 (6),

PLoS ONE doi:10.1371/journal.

pone.0011021, Jun. 8, 2010.

13. M.A. Kutzler and D.B. Weiner,

Nature Rev. Genetics 9, 776–788

(2008).

14. M. Shata et al., Mol. Med. Today 6,

66–71 (2000).

15. P.M. Smooker et al., Biotechnol

Annual Rev. 10, 189–236 (2004).

16. G. Dietrich, A. Collioud, and S.A.

Rothen. Biopharm Intl. 21 (10) s6–

s14 (2008).

17. A. Edwards and N.K.H. Slater,

Vaccine 27, 3897–3903 (2009).

18. FDA, Early Clinical Trials with Live

Biotherapeutic Products: Chemistry,

Manufacturing, and Control Information.

(Rockville, MD, Sept. 2010).

19. J.M. Huang et al., Vaccine 28,

7523–7528 (2010).

20. J. Bergin, “DNA Vaccines:

Technologies and Global Markets,”

BCC Research (2009). Bp

Figure 5: The number of open Phase I–III clinical trials involving plasmid DNA

in 2008 (13).

Cancer

Cardiovascular

Viral

Neurological

Occular

Other

Phase III

Phase II

Phase I

0 10 20 30 40

DNA clinical trials in 2008

50 60 70 80

Sartorius Stedim BiotechUSA +1.800.368.7178 | Europe +49.551.308.0

Crossfl ow performance meets high-end single-use technologyand automatic process control. The new cGMP-compliantSARTOFLOW® Alpha plus SU is the ideal choice for– Companies with frequently changing production– Contract manufacturers– Research and development applications– Small-scale production cycles

SARTOFLOW® Alpha plus SU is equipped with a gamma presterilized loop. All product-contacting surface areas, such as self-contained UF or MF units, pressure domes, fl ow meters, valves, bags and tubing, provide sterility and the highest process security.

SINGLE-USE TECHNOLOGY

SARTOFLOW® Alpha plus SU. Outstanding crossfl ow fi ltration performance and effi cient single-use technology combined.

www.sartorius-stedim.com/alpha-plus-su

turning science into solutions

Ce

lliG

en

® is a

re

gis

tere

d t

rad

em

ark

of

New

Bru

nsw

ick S

cie

nti

fic.

New

Bru

nsw

ick™

an

d t

he N

ew

Bru

nsw

ick L

og

o™

are

tra

de

ma

rks o

f E

pp

en

do

rf A

G,

Ge

rma

ny.

© 2

011

New

Bru

nsw

ick S

cie

nti

fic

Eppendorf is

your source for

New Brunswick

equipment

‡ 1 - 650 L capacities

‡ Advanced process

control software

‡ Single-Use, Autoclavable

and SIP vessel options

www.eppendorf.com • Email: [email protected]

In the U.S.: Eppendorf North America, Inc. 800-645-3050 • Worldwide: www.nbsc.com

For over 40 years, New Brunswick has

manufactured a wide range of bioreactors

ideal for growing mammalian, insect or plant

cultures in research or cGMP production.

Whether you prefer the convenience of a single-

use system, or a traditional autoclavable or SIP

bioreactor, count on the combined expertise

of New Brunswick and Eppendorf for all your

culture needs.

New Brunswick Bioreactors feature:

‡ Unique impeller designs to optimize growth

and production, including packed-bed basket

impellers for secreted proteins and low-shear

Cell-Lift impellers for microcarrier cultures ‡ Allen Bradley PLC control option available on

SIP systems ‡ Unrivaled support, including training, setup

assistance, and preventive maintenance

packages, plus in-house labs to assist

with process development, scale-up and

optimization

For more information in North America

visit www.eppendorfna.com/nbs

Better Bioreactors. Better Growth.

Cell Culture Bioreactors

BioPharmfiles.pharmtech.com/alfresco_images/pharma/2014/08/20/86... · 2018-08-28 · October 2011...

Documents

Transcript of BioPharmfiles.pharmtech.com/alfresco_images/pharma/2014/08/20/86... · 2018-08-28 · October 2011...