Cleanroom

English translation of the German original publication

ADCs: Requirements in terms of

GMP and occupational safety
The challenges presented by a new generation of highly active pharmaceutical products
for cancer treatment
Zur Verwendung mit freundlicher Genehmigung des Verlages / For use with permission of the publisher

Richard Denk SKAN AG, Allschwil

Dr Andreas Flckiger F. Hoffmann-La Roche AG, Basel
Correspondence: Richard Denk, Head of Sales Containment, SKAN AG, Binningerstrasse 116, CH-4123 Allschwil;
e-mail: richard.denk@skan.ch

Summary

Key Words

Antibody drug conjugates (ADCs) are a new generation of highly active pharmaceutical
products used, among other things, in the targeted treatment of cancer. For health and
safety reasons, most of these ADCs require containment down to the double or triple-digit ng/m3 range.
Manufacturing ADCs is a new challenge, particularly in aseptic production. Isolators have
been used successfully in this area for many years now. They are, however, also called
upon to provide active personal protection. A contradiction in terms? At first glance, yes.
While the aseptic process must be operated in positive pressure in accordance with Good
Manufacturing Practice (GMP) requirements, personal protection isolators are typically
operated in negative pressure in order to prevent the hazardous substance from escaping.
Special seals on the isolator, innovative filter technology and a well-conceived pressurecascade concept with active mouseholes make it possible to protect both product and
personnel.

1. Introduction: What are

ADCs, and what makes
them so dangerous?
Antibody drug conjugates (ADCs)
are macromolecules that typically
consist of a monoclonal antibody
(MAb) linked to a number of molecules of an antineoplastic agent.
These small-molecule antineoplastic agents are also referred to as
toxins, warheads or payloads.
The bond between the MAb and
the antineoplastic agent is ensured
by a linker. The task of the MAb in
these ADCs is to deliver the payload as accurately as possible to
the tumour, with a view to ensuring
that as little damage as possible is
done to healthy tissue. The linker is
intended to prevent the payload
from splitting off from the MAb before it reaches the tumour.

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Antibody drug conjugates

Containment
Isolator
High-potency APIs
Active mousehole
FiPa filter cartridge

Authors

Richard Denk

Dr Andreas Flckiger

Richard Denk is Head of Sales Containment at

SKAN AG, which is based in Allschwil, Switzerland.
In 2008 he established the panel of containment
experts within ISPE Germany/Austria/Switzerland
(DACH), which published the Containment Manual in November 2015. He is also an author for
Maas & Peither GMP Publishing on the topics of
containment and hygienic design, as well as one of
the authors of the ISPE Oral Solid Dosage Baseline
Guide. He has been focusing on the manufacture of
highly active / highly hazardous substances for
almost 20 years now, and also developed the
Containment Pyramid for use in this area.

Dr Andreas Flckiger, an internist and occupational physician by training, has been Chief Occupational Health Officer at Hoffmann-La Roche for
over 30 years. His area of responsibility includes
occupational toxicology and occupational hygiene,
which is why he focuses closely on the setting of
threshold limits for occupational safety and system
cleaning, and the issues surrounding containment
solutions. He is a member of numerous international professional associations and committees.

TechnoPharm 7, Nr. 1 (2017)

ECV Editio Cantor Verlag, Aulendorf (Germany)

Denk and Flckiger Antibody drug conjugates

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There are currently also ADCs in

the R&D stage in which the small
molecule (e.g. an antibiotic) has no
antineoplastic effect, and these are
then generally less toxic than those
used to treat cancer. In the future,
therefore, it will be necessary to ask
what exactly the drug in the ADC is
in each individual case. Researchers
are also working on constructs in
which MAbs, or parts of them, are
bound to other highly active proteins or to peptides (e.g. cytokines).
These constructs are typically referred to not as ADCs but as fusion
proteins, and they can be just as
toxic as antineoplastic ADCs. What
is more, there are of course peptides,
polypeptides and proteins, such as
interleukins, which are also macromolecules but neither ADCs nor fusion proteins, albeit just as biologically active as the latter.
Thanks to the specificity of the
MAb, antineoplastic ADCs bind selectively to the cancer cell, where
they are incorporated. The bond between the MAb and the toxin created by the linker is broken, and
the payload is able to take effect.
This mechanism makes it possible
to keep the strain on the body
caused by the cancer treatment to
a minimum, helping to significantly
increase its tolerability.
Certain molecules that have been
known for some time but were too
toxic as a conventional therapy i. e.
as unbound small molecules can
now be used as payloads. These molecules include maytansinoids and
auristatins, for example.
The process involved in manufacturing ADCs is divided into three
main stages: the fermentative production of the MAb, the production
of the warhead and the conjugation
of the two elements to create the
ADC. The linker is manufactured
separately, and is coupled either
with the warhead or the MAb before
conjugation. As a rule, each MAb is
loaded with a small number of toxin
molecules during conjugation. The
finished parenteral solution is ultimately available after conjugation,

which is followed by a series of separation processes. This solution is

merely sterile-filtered once or several
times before being filled and placed
on the market as a solution or a lyophilisate.
The biological activity of MAbs is
generally low. While the manufacturing process is demanding from a
technical perspective and in terms
of aseptic conditions, there are no
notable problems with occupational
safety.
Many of the small antineoplastic
molecules used in ADCs are extremely biologically active. They
often have occupational exposure
limits (OELs) in the single-digit
ng/m3 range, sometimes even lower.
Because MAbs transport the toxins
to the tumour in a targeted manner,
however, the quantities required are
typically low. Even on a commercial
scale, volumes range from a few
dozen grams to a maximum of a
few kilograms.
During conjugation even smaller
quantities of toxin are used, i. e. one
batch of toxin can be enough to produce several batches of conjugate
(ADC). The conjugation process results in the dilution of the toxin molecules, thereby reducing the toxicity
of the material to be handled. One
ADC molecule typically contains
just a few percent of toxin. This
means that the OELs of ADCs are
significantly higher than those of undiluted toxins, but nevertheless
often still between 50 ng/m3 and
1 mg/m3. What is more, the parenteral solution consists primarily of
water, which means that the ADC
content of the solution is well below
10 %. If the solution is lyophilised, it
must be assumed that the ADC content of the lyophilisate will be
roughly ten times higher than that
of the reconstituted solution. These
dilution and concentration effects
naturally have an impact on the toxicity of the material to be handled,
and must be taken into account
when it comes to determining the
necessary containment.

Denk and Flckiger Antibody drug conjugates

2. Suitable containment
for achieving the required
limit value of 50 ng/m3
What do we mean when we talk
about a limit value of 50 ng/m3?
Consider a sweetener tablet weighing approx. 100 mg. In order to dilute these 100 mg to a concentration
of 50 ng/m3, it would take a volume
of 2 million m3 (from 100 mg/m3 to
100 ng/m3 = a factor of 1 million;
from 100 ng/m3 to 50 ng/m3 a further factor of 2; so 2 million m3 in
total).
The Empire State Building in New
York, with its height of 450 m, has
one of the highest volumes of any
building in the world at approximately 1 million m3. This volume
would make it possible to dilute
100 mg to a concentration of
100 ng/m3. To then achieve a concentration of 50 ng/m3 would take another factor of 2, i. e. two Empire
State Buildings. Placed one on top
of the other, these two Empire States
Buildings would give a total height of
900 m. A number of key measures
need to be in place to ensure that
an OEL of 50 ng/m3 can be achieved.

3. Measures to ensure
compliance with the
exposure limit
The first step in the process involved
in manufacturing ADCs is to produce
the toxin, or payload, a highly active
substance that has a defined low exposure limit. The pure toxin is
handled again when the payload is
added to the conjugation process,
which is why this is another step
that calls for high containment. Following sterile filtration, the further
critical stages are the filling of the
product into vials, freeze-drying and
the crimping of the vials.
What technologies exist for meeting the requirements of extremely
low OELs?
In the last decade, a whole host of
technologies have been developed
with a view to transferring highly active or highly hazardous products

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Suitable airlock system for loading

the highly active substance into the
isolator and for removing material
and waste
High-performance filter technology
Hygienic design for cleaning on
product changeover
Air-tight system, in particular airtight gloves (glove testing)

4. Interfaces between the

inside and the outside of
the isolator
Design errors are most commonly
made in the systems used to place
the material in the isolator and remove it again, in the filter technology
and in the gloves.
A system is needed to transfer the
highly active pharmaceutical substance into the isolator, and to remove empty containers or product
residues from the isolator. These
transfer systems are also critical, as
they involve a connection between
the interior and exterior of the isolator. There are various systems available for carrying out these transfers:
. Transfer airlock with interlocked
doors between the pre-chamber
and the main chamber of the isolator

Figure 1: ADC Aseptic Fill & Finish isolator with the option of lyophilisation (source of all
images: SKAN AG).

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Endless liner technologies

Rapid transfer ports (RTPs), also
referred to as alpha-beta ports
These systems all have weak points
of varying significance that could
cause a possible containment breach,
and often prevent exposure levels of
< 50 ng/m3 from being achieved.
Containment of < 50 ng/m3 typically
calls for a combination of two barrier
systems e. g. an RTP between the
exterior and the airlock (pre-chamber) on the isolator, as well as a
locked door between the airlock
and the isolator interior. The pressure in the airlock is higher than in
the main chamber of the isolator.
Another example of a double-barrier
system is an airlock with an endless
liner system between the material
airlock and the main chamber, and
another endless liner to transfer material from the pre-chamber to the
exterior of the isolator.
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5. Filter technology
The filter technology used together
with the isolator is another critical
area.
Given that personal protection
isolators are operated in negative
pressure, suitable filter technologies
are also required at the air inlet and
outlet points of the isolator.
Possible filter systems are:
. Bag-in/bag-out filter
. Push-push filter cartridge
. FiPa filter cartridge
All of these filter systems are suitable
for preventing the highly active substance from escaping from the isolator. Most filter systems, however,
present a risk in terms of Good Manufacturing Practice (GMP) with
bag-in/bag-out filters and pushpush filters, for example, there is
the possibility of recontamination
from the old, used filter to the new
one as the filters are changed. Particles of old product can then become detached from the new filter
and enter the area containing the
product, and this is critical because
this recontamination is often not discovered. The FiPa filter cartridge pre-

Denk and Flckiger Antibody drug conjugates

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safely into or out of a manufacturing

process. When it comes to safely remaining below an OEL of 100 ng/m3,
isolator technology is used in most
cases. Originally developed for the
nuclear industry, rigid isolators
have also been in use for many years
in pharmaceutical manufacturing for
highly active substances. Such an isolator comprises a contained space to
which access is obtained by means of
gloves integrated in a glass panel (see
Fig. 1). The interior of the isolator is
operated in negative or positive pressure, depending on the requirements.
For straightforward personal protection, the isolator is operated in negative pressure in order to prevent the
hazardous substance from escaping.
In aseptic manufacture, product protection has top priority and therefore
requires the isolator to be operated
in positive pressure.
Prior to the sterile filtration of the
ADC, personal protection usually has
priority, which is why straightforward personal protection isolators
operated in negative pressure are
often used up until this stage. Isolators also vary in ways that can make
them suitable for substances with
OELs below 50 ng/m3.
The following requirements apply
to an OEL < 50 ng/m3:

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age inspection should be carried out

on a regular basis, and the document
entitled How risky are pinholes in
gloves? A rational appeal for the integrity of gloves for isolators from
the Parenteral Drug Association
(PDA) provides further information
(see Bibliography).

vents this from happening (see

Fig. 2). FiPa filter technology was developed as a filter isolator, and the
FiPa is designed as a closed system
that is opened into the isolator interior. The closed FiPa is attached to
the isolator, with the opening on the
FiPa towards the interior of the isolator being accessed from outside.
The dust-laden air from the isolator
can enter the FiPa, and the dust is
deposited on the filter. During the
product change, the opening in the
FiPa into the isolator interior is
sealed again from the outside. The
isolator can now be cleaned. Following the cleaning process the FiPa is
removed with no risk to the operator or the product to be manufactured next.1)

6. Hygienic design and

glove testing
The concept of hygienic design refers
to the optimal cleanability of the interior surfaces of the isolator or of
devices built into the isolator. As
mentioned earlier, cleanability is extremely important as product residues on these surfaces present,
from a GMP perspective, a risk of
cross-contamination between two
different substances manufactured
in sequence. Particularly important
in the design of an isolator are the
seals on the glass panels and doors.
Static seals are the most basic option,
but they are also seen as the most
critical. Static seals have the disadvantage that they wear out over
time and can allow dust to be deposited and penetrate critical areas. This
process of wear can also affect the
airtightness of the isolator. During
or following cleaning in particular,
opening the glass panel can cause
the product residues deposited in
the seals to become detached and
escape from the isolator or into its
interior. Inflatable seals are a better
option in terms of hygienic design,
and allow for highly accurate sealing
1) See Bibliography for details of contamination-free filter change.

7. Weighing the toxin

and transferring it to the
conjugation reactor

Figure 2: FiPa open towards the interior of

the isolator.

of the glass panel. The seal and its

functionality can also be tested using
validated measuring equipment.
Other important points are the design of the seals for gloves, as well as
glove testing. When it comes to attaching the gloves, there are various
possibilities for ensuring that the
necessary level of containment is
achieved. Most of these options
make use of a double O-ring groove,
which is needed to ensure a closed
changeover in the event of a damaged glove. From a containment perspective these O-ring attachments
are a weak point, as containment
cannot be achieved if the gloves are
fixed incorrectly. It is also important
to prevent the highly active substance from accessing and becoming
deposited around the O-rings, as this
area is also difficult to clean. An ideal
solution is an additional seal on the
O-ring and on the sleeve of the glove,
in order to prevent the substance
from reaching the O-ring.
Glove testing is the final stage in
the safety check of this area (see
Fig. 3), and involves analysing the
gloves for small tears or pinholes.
The gloves are used to work inside
the isolator, and are therefore exposed to risk of damage. This dam-

Denk and Flckiger Antibody drug conjugates

If the toxin/payload is in powder

form, it is vital after dispensing to
ensure that no further transfer step
involving the handling of powder is
required, i. e. the transfer of the dispensed material into the conjugation
reactor. The transfer systems currently available are not completely
suitable for this step, and it is more
advisable to dissolve or suspend the
powdered substance in a suitable
liquid within the isolator. Once the
highly active substance is in the
liquid phase, it should be transferred
to the conjugation container using
either a peristaltic pump or an AT
connector (see Fig. 4). Both are single-use systems and can ensure safe
and adequately contained transfer.
With the peristaltic pump, it should
be ensured that the product hoses
connecting the conjugate container
are cleaned before being removed.
Here it is important that the hose
connectors are designed in such a
way that spillage is prevented when
the hoses are disconnected. The AT
Connect system makes this possible
thanks to a special connecting and
disconnecting process, and was developed to transfer sterile liquids
from a container into an isolator for

Figure 3: Wireless glove tester.

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sterile filling into vials or syringes.

The same principle can also be applied to the closed transfer of a liquid
from an isolator to a container, with
the passive adapter of the AT Connect being attached to the conjugation container. The active part of the
AT Connect adapter is located on the
isolator, and the passive part is connected from the conjugate container
to the active part of the isolator and
locked in place. Once it has been
locked, the active part can be opened
and connected to the liquid container in the isolator to allow for
safe, closed transfer.

8. Aseptic fill and finish

Once the ADC has been sterile-filtered, the fill and finish process can
take place. The following aspects are
important parts of the manufacturing process:
. ADC transfer to the vial-filling area
. Vial-filling
. Freeze-drying (lyophilisation)
. Crimping
. Washing the outer surface of the
vial
. Inspection
. Packaging
The entire manufacturing process
from vial-filling to the freeze-dried
pharmaceutical product takes place
under cleanroom class ISO 5 conditions. Given that the ADC product is
a highly hazardous substance, it is
recommended that these manufacturing steps be handled using isola-

tor technology. Isolator technologies

are widely used in aseptic manufacture, and have the benefit that critical aseptic areas require no direct
access by operating personnel (see
Fig. 1).
Requirements regarding aseptic
manufacture in isolators are described in the PDA paper entitled
"Development and Quantification of
H2O2 Decontamination Cycles" (see
"Bibliography").
In accordance with GMP guidelines on aseptic manufacture, isolators are operated in positive pressure
in order to protect the product. Personal protection, however, calls for
operation in negative pressure, in order to prevent the active substance
from escaping. How can these GMP
and personal protection requirements be reconciled?
This can be facilitated through the
integration of additional safety systems. These safety systems are essentially similar to those used in the personal protection isolators described
in this article:
. Special seals on the glass panels
and regular inspection of these
seals
. FiPa technology to prevent the
highly hazardous substance from
reaching the ventilation channels
. Absolute hygienic design
It is also necessary to prevent the
substance from being spread through
the isolator should a vial happen to
break, and this is achieved by means
of various pressure cascades.

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The more critical the area, the

lower its pressure in comparison
with the other areas. Targeted air
flows to the FiPa filters reduce the
spread of the substance particularly during vial-filling and the unloading of the freeze-dryer. These
measures facilitate containment to
< 10 ng/m3, as verified in accordance
with the Good Practice Guide from
the International Society for Pharmaceutical Engineering (ISPE) entitled
Assessing the particulate containment performance of pharmaceutical equipment (see Bibliography).

9. Packaging
Following the freeze-drying and vial
crimping processes, the dried powder is contained in sealed vials. These
vials are washed before they leave the
isolator in order to prevent contamination by traces of product that
came into contact with the exterior
surface of the vials during filling, or
due to the breakage of one of the
vials.
The risk of a vial being broken during inspection and packaging remains,
however. The inspection and packaging of the vials must, for this reason,
also be protected by means of suitable
isolator technology (see Fig. 5).

10. Conclusion
ADCs are a new generation of highly
active and hence extremely challenging substances in the pharmaceutical

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Figure 4: AT Connect for safe transfer of the solution to the conjugation container.

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While the quantities manufactured

in the initial phase of development
are low, there is a significant risk of
coming into contact with the product if suitable protective measures
are not taken. Isolator technologies
are suitable for handling ADCs safely,
but require innovative solutions and
safety precautions.
Figure 5: Packaging machine with
integrated isolator technology.

industry that call for state-of-the-art

containment for their manufacture.

Bibliography
Angela Gessler, Alexander Staerk, Volker Sigwarth, Dr Claude Moirandat: How risky
are pinholes in gloves? A rational appeal

for the integrity of gloves for isolators,

PDA paper, Vol. 64, No. 3, May-June 2011.
Frank Lehmann, Jrg Lmkemann: Safe
change filter systems for containments in
the pharmaceutical industry, Pharm.
Ind. 73, No. 9, 1683 1694 (2011).
Patrick Vanhecke, Volker Sigwarth, Dr Claude
Moirandat: A potent and safe H2O2 fumigation approach, PDA Paper, Vol. 66,
No. 4, July/August 2012.
Volker Sigwarth, Dr Claude Moirandat: Development and quantification of H2O2
decontamination cycles, PDA Paper,
Vol. 54, No. 4, July/August 2000.
ISPE Good Practice Guide Assessing the
particulate containment performance of
pharmaceutical equipment 2nd edition
2012. Available from www.ispe.org.
ISPE Containment Manual. Available from
www.ispe-dach.org.

Editor-in-chief: Claudius Arndt, Managing editor: Jens Renke. Publisher: ECV Editio Cantor Verlag fr Medizin und Naturwissenschaften GmbH, Baendelstockweg 20,
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