1. Cryopreservation Technologies
44 Innovations in Pharmaceutical Technology Issue 54
By Rolf Ehrhardt and
Maria Thompson at
BioCision
In order to maintain their peak functionality, therapeutic cells rely
on being effectively frozen and thawed. This calls for increasingly
optimised and standardised approaches to cryopreservation,
as improvements in consistency and reproducibility will have a
positive impact on patient outcomes
The Big Freeze
Keywords
Cellular therapy
Freezing devices
Cryoprotectants
Cell thawing
The goal of cell cryopreservation is to suppress
the natural processes of change and degradation
inherent within any biological system,and to preserve
viability,structure and function in as perfect a state
as possible.For therapeutic cells,maintaining their
peak achievable functionality is not merely a desirable
outcome,but an obligation subject to the legal and
ethical oversight of institutions such
as the FDA and EMA.However,the
sheer variety of cell types and their
diverse biological characteristics
defies a single method of culturing,
preserving and preparing cells for
clinical use.Nonetheless,standardising
how cells are handled during these
processes is important.
Cryopreservation
While an important part of the process,cryopreservation
has not always been given the same consideration as
other processing steps,like cell characterisation,potency
and safety.Given that pharmaceutical companies must
define their target patient population early on in drug
development,functional activity and toxicity assays
are understandably the focal point of due diligence
when dealing with cellular products.Cryopreservation
of products is often subject to somewhat less stringent
treatment,which can make it a weak link in the cellular
therapy manufacturing workflow.
Therapeutic cells respond to the stress of
cryopreservation in different ways,depending
Image:©roganjosh–morguefile.com

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45Innovations in Pharmaceutical Technology Issue 54
When cells are cooled below 0°C,extracellular ice
crystals begin to form and relative solute concentration
of the freezing media rises.Water flows out of the cell,
beginning the process of cell dehydration.The cooling
rate must be slow enough to allow sufficient egress
of water to minimise the chance of intracellular ice
crystal formation,while at the same time fast enough
to avoid serious dehydration effects.It is for this reason
that cryoprotectants are used:to lower the freezing
point of the extracellular medium,limiting both ice
crystal formation and the osmotic stress that results in
dehydration.Researchers should only use freezing media
that is both fully defined and validated to be optimal for
their cell type.While it is possible that optimal cooling
rates may vary by cell type,the current recommended
freezing procedure for many therapeutic cells is that
they are cooled gradually to approximately -80°C at a
controlled rate of -1°C per minute (2).This cooling rate is
proven to work well for peripheral blood mononuclear
cells,primary cells,stem cells and cell lines.
Freezing Devices
Active control of the freezing process is particularly
important in a regulated environment,where protocols
must comply with GMP standards to deliver a functional
product that is produced in controlled and predictable
conditions.Retention of good functionality requires a
reproducible,validated methodology.
Until recently,this generally meant cryopreservation
needed to be performed using large,expensive,
programmable controlled rate freezers.Such freezers
offer a precisely controlled cooling rate and are
well-validated, but their cost, large footprint and
maintenance issues are a prohibitive drawback,
especially to small or mid-sized companies.In the last
few years,passive freezing devices have gained in
popularity,largely due to their ability to offer equivalent
cooling rates and cell viability results,without the
exorbitant costs and laboratory space required for
controlled rate freezers.Passive cell freezing containers
– particularly those that lower contamination risks
and operating costs by being alcohol-free – are
easily transferrable from the development phase to
the manufacturing suite,and are increasingly being
validated for use in GMP environments (3).
Maintaining the Cold Chain
Once cells and other biological products are safely
frozen,there will inevitably come a time when they
must be transported – either within a facility or to an
end-user.Therapeutic cells,like any other biological
material,are subject to the risk of temperature
fluctuation during transport and,while regulatory
on variables such as cell type,length of storage,
cryoprotectants used,or freezing and thawing
conditions.Optimising cryopreservation to meet the
needs of cellular therapy can be a complicated process.
For example,therapeutic cell viability is often assessed
immediately post-thaw,without taking delayed-
onset cell death into account.The same is true of cell
function.The assays themselves may be well-defined,
but checking functional activity immediately post-
thaw may not give the best indication of what will
occur in the patient.Some cell types require a post-
thaw recovery period of several hours or even days
before functionality is restored; others may be more
dramatically affected by cryogenic storage conditions,
and prone to changes in phenotype or sub-population
frequency (1).
The scientist must determine if cryopreserved cells
maintain the same potency and function at the point
of infusion into a patient as they did before they
were frozen.Freezing and recovery media should be
carefully chosen to match the cell type in question,and
researchers need to ensure that the components of the
media are fully defined.Perhaps the most important
consideration for cryopreserving cells,particularly
in a Good Manufacturing Practice (GMP) regulated
environment,is to confirm that the cryopreservation
methodology itself – and the technology supporting
it – is fully optimised and standardised.
Given the natural proclivity for cells to respond and
adapt to their environment,there is an ongoing need
for cryopreservation techniques to adapt in a way
that minimises variability and subjectivity at every
possible point when the process is transferred from
development to manufacturing.The complete process
includes freezing,cold chain management during
transportation and thawing.Luckily,necessity informs
invention,and as cellular therapy gains momentum,
we are beginning to see a paradigm shift towards
more optimised,standardised and translatable
cryopreservation technologies.
Cell Freezing
This is the starting point of any cryopreservation
workflow,and while pharma R&D teams may not always
have the time and resources to devote to optimising
freezing rates and media for each cell type,these factors
will have an important influence on ultimate cell survival.
Fortunately,a great deal of scientific effort has been
devoted over the years to understanding the effects of
various freezing parameters on cell survival and recovery.
Generally speaking,freezing protocols are based on
avoiding the physiological effects of dehydration and
ice crystal formation.

3. 46 Innovations in Pharmaceutical Technology Issue 54
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containers,and are capable of maintaining temperatures
below -50°C for over 24 hours.Liquid nitrogen dry
vapour shippers can now be used in place of dry ice
shipping,and can provide cooling for 10 days or more
while fragile biospecimens are shipped.Other liquid
nitrogen-based solutions exist that are more suitable
for short-distance transport.
The International Society for Biological and
Environmental Repositories recognised a hand-portable
liquid nitrogen-based carrier as their‘Outstanding
New Product’of 2015.The CryoPod™ Carrier holds
samples at below -150°C for up to four hours,while
providing automatic temperature alarms,monitoring
and logging,so that users will immediately be alerted if
the temperature has been compromised at any point in
the journey.Such products point towards a shift to more
stringent,standardised temperature-control solutions.
Re-Thinking CellThawing
In the field of regenerative medicine,the final step in
the cryopreservation workflow involves thawing the
cryopreserved cells so they can be administered to the
patient.Controlling the rate of thawing is as important
as freezing,and optimising both is crucial to cell survival
and function.Only recently,however,has cell thawing
been given the same serious consideration that cell
freezing has been subject to (7).
During thawing,many of the internal processes of cell
freezing are reversed.As cells are warmed,extracellular
ice melts and water can rush into the cell,causing it to
swell or even burst.If thawing is done too slowly,or heat
application is uneven,tiny ice crystals formed during the
freezing process can grow and become large enough
to cause damage.The shock of transit back to a viable
state can also cause oxidative stress,triggering apoptotic
pathways and delayed onset cell death.
Water Bath Technology
By far the most common method of thawing cells
is through the use of a heated water bath.When
agencies promote guidelines for maintaining optimal
temperatures during clinical studies and manufacturing
processes,temperature control during transport is still
poorly upheld.A recent US study found that the top
six globally administered vaccines (including those for
polio,diphtheria and measles) lose up to 36% of their
effective shelf-life due to failures in meeting cold chain
requirements,as measured by vaccine vial monitoring
(4).And this is not an isolated report; another study
found that most,if not all,vaccines are exposed to
freeze damage risk at some point throughout the
shipping process (5).
Temperature fluctuation problems are not only limited
to long-distance shipping;they can occur during cross-
campus or cross-town transport,during transfer to
and from liquid nitrogen storage,or even simply when
cryostorage boxes or liquid nitrogen canes are shifted
around to access specific samples.But while transport
of temperature-sensitive biologicals may not be
standardised,scientists are beginning to recognise that
even short-term increases in temperature can expose cells
to the dangers of thermal cycling and ice recrystallisation,
lowering cell viability,recovery and,in the case of stem
cells,even the ability to be pluripotent (6).The good news
is,in recent years,several transportation and storage
solutions have come to market that are aimed at more
stringently controlling the cold chain,whether it be during
long-distance transport or movement within a medical,
research or biopharma manufacturing facility.
NovelTransport Systems
For a long time,homemade dry ice containers were
the only method for shipping temperature-sensitive
biomaterials.With the emergence of the cellular
therapy industry,it became recognised that this type
of container was too unreliable to entrust products
representing a company’s major investment in time and
money – not to mention what might be a patient’s best
hope for survival.Recently,more robust dry ice-based
transport systems – and even mobile dry ice-based
workstations – have emerged to deliver more reliable
temperature stability than conventional Styrofoam
Temperature fluctuation problems are not only limited
to long-distance shipping; they can occur during cross-
campus or cross-town transport, during transfer to
and from liquid nitrogen storage, or even simply when
cryostorage boxes or liquid nitrogen canes are shifted
around to access specific samples

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47Innovations in Pharmaceutical Technology Issue 54
References
1. McKenna KC, Beatty KM, Vicetti Miguel R and
Bilonick RA, Delayed processing of blood
increases the frequency of activated CD11b+
CD15+
granulocytes which inhibit T cell function,
J Immunol Methods 341: pp68-75, 2009
2. Bissoyi A, Nayak B, Pramanik K and Sarangi SK,
Targeting cryopreservation-induced cell death:
A review, Biopreservation and Biobanking
12(1): pp23-34, 2014
3. Stone M et al, Maximizing PMBC recovery and
viability: A method to optimize and streamline
peripheral blood mononuclear cell isolation,
cryopreservation and thawing, BioProcess
International, April 2015
4. Kartoglu U et al, Use of cool water packs to
prevent freezing during vaccine transportation
at the country level, PDA Journal of
Pharmaceutical Science and Technology 63(1):
pp11-26, 2009
5. Kumru O et al, Vaccine instability in the cold
chain: Mechanisms, analysis, and formulation
strategy, Biologicals 42: pp237-259, 2014
6. Deller RC, Vatish M, Mitchell DA and Gibson MI,
Synthetic polymers enable non-vitreous cellular
cryopreservation by reducing ice crystal growth
during thawing, Nature Communications 5:
p3,244, 2014
7. Thompson M, Kunkel E and Ehrhardt R,
Balancing the Cryopreservation Equation,
Cold Facts, January 2015
examined in the context of a GMP-regulated
environment, this method is fraught with problems.
Due to the fact that water is an efficient carrier of
contaminating organisms, GMP regulations either
prohibit water bath use, or specify that a water bath
be cleaned and refilled after each use.This method
of thawing can also be very subjective.The thawing
endpoint is based on each operator’s observation –
often determined by repeatedly lifting the vial out of
the bath to peer at it – of when the last ice crystal is
ready to melt.
Often,no set times are applied and the researcher’s
attention may be momentarily called away to another
task, adding to the risk of variable endpoints and
potential overcooking of cells.The problem is
compounded when more than one sample is involved.
In a clinical setting, cryobags are often used to freeze
large volumes of cells.When these bags are thawed,
the first bag may take just four minutes. However, the
first bag cools the temperature of the water bath and
then, when the second bag is added, it may take much
longer. Again, the process is inconsistent and will lead
to variable results.
A more standardised approach would greatly benefit
researchers, in-process manufacturing personnel and
clinicians alike. An automated cell thawing system
is now on the market that is designed to detect the
solid-to-liquid phase change within a cryovial.
The system is water free, small enough to fit inside
a tissue culture hood and has already been validated
in several labs that follow GMP regulations (3).The
technology is adaptable to other vessel formats – for
example, bags, bigger vials and multiple vials – giving
it the versatility to be adopted into many different
clinical models.
Better Prospects
Today, we are at a stage where the cellular therapy
industry is just beginning to develop. As with all
new endeavours, there are growing pains to be
overcome before the infrastructure can be put
into place. In addition, the creation of guidelines
for the standardisation of workflow methods is
still ongoing.
Cryopreservation is one of the lynchpins of cellular
therapy, and improvements in consistency and
reproducibility will no doubt benefit patient outcomes
down the line. Optimising cryopreservation will
improve the safety and efficacy of cellular drug
products. It is therefore important that we adopt
tools and procedures that both reduce variability
and streamline the process as we move forward.
Dr Rolf Ehrhardt is the President and Chief
Executive Officer of BioCision, a leading
provider of innovative tools for standardising
sample and biomaterial handling,
cryopreservation and storage procedures.
Previously, he was responsible for the early
clinical development of a novel hepatitis C virus protease
inhibitor at Intermune, was Vice President of Preclinical
Development at Corgentech and founding President of
BioSeek. Rolf earned his medical and doctoral degrees with
distinction from the Technical University of Munich, Germany.
Email: rolf@biocision.com
Dr Maria Thompson is Vice President of
Scientific Affairs at BioCision. She has a
background in molecular genetics, and over
18 years of experience in pharmaceutical and
diagnostics R&D. Maria has held a variety of
roles including Head of Genome Wide Screening
for type 2 diabetes, Six Sigma Black Belt, Principal Consultant and
Head of Scientific Affairs, in addition to running APEX Think, a
private consulting company. She has a BSc in Genetics and a
PhD in Molecular and Cellular Biochemistry from the Royal
London School of Medicine, UK. Email: maria@biocision.com