IN-DEPTH FOCUS | FORMULATION, DEVELOPMENT & DELIVERY

The importance of disaccharide
excipients in biologics
Many biologic formulations use a variety of high-purity injectable grade disaccharide excipients toensure functionality and stability of the final product. In this article, Sunil Kumar Nataraj, Bastiaan
Dickhoff and Thontesh GC explore the use of these sugars in the stabilisation of biologics and provide
recommendations to manufacturers for enhancing supply chain security.
Abstract“The typeand quality of
excipients plays
a vital role in thedevelopment andoptimisation oflyophilisation
process
parameters”

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According to the US Food and Drug Administration(FDA), biologics include allergenics, blood andblood products, cellular and gene therapy products,
tissue and tissue products, vaccines andproducts for xenotransplantation.1 Biologics also
include monoclonal antibodies (mAbs) from thelist of original Biologic License Application (BLA)
applicants and biosimilars. Excipients play a vitalrole in the functionality of biologics. In addition,
excipients help to prevent the biologic drug fromloss of activity and to maintain stability. For these
purposes, biologic formulations use a variety ofhigh-purity injectable grade disaccharide excipients,
including sucrose and trehalose, among others.
The main manufacturing method for biologics
is lyophilisation, but other techniques
are also applied.

The essential role excipients play in biologic
drugs, alongside the gaps in supply chain security
that have been exposed by drug shortages
related to the COVID-19 pandemic, is alarming.9
In addition, recent quality issues that haveresulted in the closure of plants, heightened tradetensions and the growing numbers of biologics that
require low-temperature storage and shipment,
place an even greater strain on (bio)pharmaceuticalsupply chain systems.9
This review explores the current knowledgeof stabiliser excipients and offers generalrecommendations on excipient and formulation
parameters to be applied in biologics. In addition,
we provide recommendations for enhancing supply
chain security of the high-purity disaccharide
excipients that are extensively used in thelyophilisation of biologics. The latter is of particular
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European Pharmaceutical Review | Volume 26, Issue 01

importance given the current market situationof original BLA and biosimilar applicants relying
on a single supply source for their excipients. It is
recommended that such companies work withreliable partners who supply excipients in alignment
with International Pharmaceutical Excipients
Council (IPEC) guidelines.

Disaccharide excipients in lyophilisation

The type and quality of excipients plays a vitalrole in the development and optimisation oflyophilisation process parameters. The use of
biologic drug-specific excipients contributes
significantly to the drug solution stability(bulk phase). It also aids in the subsequent
optimisation of lyophilisation cycles, with fasterdrying conditions to achieve the desired lyophilised
cake appearance, along with other critical quality
attributes (CQA) such as optimal pH, moisture
content and reconstitution time.
An ideal excipient for lyophilisation should bechemically stable, inert, safe, multifunctional andeconomical.2 Excipients should provide a stableproduct with an acceptable lyophilised cakestructure, low reconstitution time, specific pH
and low residual water content. An improperly
formed lyophilised cake, such as a ‘meltback’ ora ‘collapse’, will lead to incomplete reconstitution
and unacceptable product quality. Low residual
water content is needed to ensure stability
and prevent loss of activity of the biologic.
Low reconstitution time is also necessary forthe biologic to be administered quickly to thepatient. In most cases, following lyophilisation
the finished product is reconstituted in water
for injection/saline/five percent w/w dextroseand injected either via the intravenous (IV) or
subcutaneous (SC) injection route.
Various excipients are used in lyophilisation toachieve several objectives. Table 1 summarises themost important applications of excipients that areused in the lyophilisation process.
Lyophilisation involves freezing the product(ice formation), primary drying (sublimation) andsecondary drying (desorption), which exposes
the biologic drug to freezing and drying stresses.
An annealing step is often introduced duringfreezing to ensure efficient primary drying.
High‑purity disaccharide excipients, such as sucrose
and trehalose, protect the biologic drugs from thefreeze-related (cryoprotectant) and drying-related(lyoprotectant) stresses. There are two theories forthe stabilisation of biologics by disaccharides: thevitrification theory (kinetic stability) and the water
replacement theory (thermodynamic stability).3
The vitrification theory refers to immobilisation of
biologics in a matrix of an amorphous disaccharide,
while the water replacement theory refers to theformation of hydrogen bonds between the hydroxyl

Table 1
Examples of excipients used in lyophilisation of biologics categorised
according to their functionality
Category

Function

ExamplesStabiliser

Protects against chemical andphysical instability during
processing or application

Sucrose, trehalose, mannitol,
amino acids and others

Bulking
agent

Provides appropriate structure
to the lyophilised cakeMannitol, sucrose, trehaloseand others

Surfactants

Prevents protein aggregation

Polysorbate 20 and 80 and others

Buffers

Maintains the pH of the solution
near the value at which theprotein has greatest stability

Citrate, phosphate buffers
and others

Tc modifier

Allows primary drying at higher
temperatures to enable lower
primary drying time

Dextran (1Kda), Ficoll®
(non‑ionic copolymer ofsucrose and epichlorohydrin)group of the disaccharide and the polar groupsof the biologic drug.
Key parameters to be studied during freeze
drying for an appropriate stability and shelf life ofa biologic formulation are: 1) the glass transition
temperature (Tg) of disaccharide excipients and Tgof the combination of the in situ formed amorphous(glassy) disaccharide-protein (biologic drug);2) the Tg of the maximally freeze concentrated
solution (Tg’); and 3) the collapse temperature(Tc), beyond which the lyophilised cake collapses.
Any temperature increase beyond Tc would resultin a meltback (collapse).4
Disaccharides with a high Tg are said to bebeneficial for maintaining biologic drug stability.3
The biologic drug is expected to increase the Tgof the formulation; therefore, disaccharides with ahigh Tg are known to complement the formulation.6
However, all of this depends on the type of biologic
drug studied.
Primary drying should be carried out below theTc otherwise there will be a loss of lyophilisate
structure and its biological activity. However, many

“Excipient
suppliers
should have astrengthened andsecured supply
chain system, inwhich they havegood control
over end-to-end
processes, fromsourcing theirraw materials todelivery of their
excipients to the
manufacturersof finished drug
products”

Table 2
Tg’, Tc and Tg of typical excipients used in lyophilisation
ExcipientTg’ (°C)

Tc (°C)Tg (°C)

Sucrose-32 to -35-31

60 to 65a

Trehalose-27 to -29a

-29a

107 to 111a

Mannitol-28 to -35b–*–*

Dextran-10c-9 to -10c

-151.8c

Ficoll®

-19b-19 to -20b

–*

a,b

b

a = Bjelošević M et al3b = Baheti A et al4
c = Mahmood Y et al16
* Value not found in the literature references11

IN-DEPTH FOCUS | FORMULATION, DEVELOPMENT & DELIVERY

Sunil Kumar
Nataraj
Sunil is a pharmaceuticalleader with on-the groundexperience of providing
problem-solving solutions
across the US, Europe,
Japan and India. Suniljoined the Technology &Innovation department
at DFE Pharma in 2017as product application
specialist in Germany.
Sunil has pharmaceuticalproduct development
expertise in oral solids
focusing on ODT's andcontinuous twin screw
granulation supporting
continuous manufacturing.
Sunil focuses on enhanced
customer R&D interaction
catering to current
pharma market trends
and ever changing pharma
regulatory landscapes.

excipients have a low Tc threshold. To achieve
efficient primary drying with reduced drying
time, a Tc modifier is often used which helps dry
at higher temperatures during primary drying.
Furthermore, to reduce the primary drying time,
the ratio of stabiliser to bulking agent must be
optimised (with a starting ratio of 1:2 of stabiliser
to bulking agent).3
A summary of stabilisers, Tc modifiers, along withcritical temperatures are shown in Table 2.
Aside from temperature control during thelyophilisation process, the avoidance ofthe Maillard reaction via the use of certain
reducing disaccharides plays an importantrole in the formulation of stable biologic drugs.
The degradation rate due to the Maillard reaction
generally increases with increasing moisture
content, with little or no degradation below
a water activity of 0.25 and with a maximum
degradation around 0.75. Because of this,non‑reducing disaccharides, such as sucrose andtrehalose, are mainly used in biologic drugs.3

Table 3
Some marketed formulations
containing trehalose7
Trade name

Monoclonal antibody

Blenrep®

Belantamab

Fasenra®

Benralizumab

Avastin®

Bevacizumab

Table 4

Adcetris®

Brentuximab Vedotin

Some marketed formulations containing
sucrose as a stabiliser

Padcev®EJFV

Enfortumab Vedotin

Uplizna®

inebilizumab

Trade name

Monoclonal
antibody/vaccine

Gazyva®

Obinutuzumab

Benlysta®

Belimumab

Ocrevus®

Ocrelizumab

Entyvio®

Vedolizumab

Phesgo®

Pertuzumab

Inflectra®

Infliximab-dyyb

Lucentis®

Ranibizumab

Kadcyla®

Imfinzi®

Durvalumab

Ado-trastuzumabemtansine

Rituximab® &Hyaluronidase

Keytruda®

Pembrolizumab

Rituxan Hycela

Nucala®

Mepolizumab

Cosentyx®

Secukinumab

Nulojix®

Belatacept

Monjuvi®

Tafasitamab

Myalept®

Metreleptin

Tepezza®

Teprotumumab

MMR® II

Measles, mumps andrubella virus vaccine

Mvasi®

Bastiaan Dickhoff
Bastiaan leads the
Product Application
Specialists team at DFE
Pharma and his expertiselies in understanding
powder behaviour inbatch and continuous
processes coupled withmultivariate analysis andquality by design. Prior toworking at DFE Pharma,
he worked at RSSLand GlaxoSmithKline.
Bastiaan holds a PhD
from the Departmentof Pharmaceutical
Technology andBiopharmacy at
Groningen University,
the Netherlands, where
he specialised in therelationship between
the role of lactose(excipient) and APIs forinhalation purposes. He
has published various
papers on the relationshipbetween lactose and APIs.

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Sometimes lactose monohydrate is used; forinstance, in the stabilisation of glucarpidase.5
The primary drying phase is the most time- andenergy-consuming stage in the lyophilisation
process and significantly contributes to the total
cost of lyophilisation.3 Secondary drying involves theremoval of the last traces of water by desorption.
In this process, product temperature would be lessthan or about 50°C and drying time should not begreater than six hours to prevent loss of activityof the protein (biologic). In most cases, target
moisture content of the finished product (cake) is
less than one percent w/w. This can be assessed by
Karl Fischer water content analysis.3
Another important factor contributing to thestabilisation of biologics is the ratio of stabiliser tobiologic drug used in the formulation. For effective
stabilisation, a general recommendation is that themolar ratio should be at least 360:1 for stabiliser
to biologic drug to avoid denaturation.3
The use of stabiliser excipients with high Tg’,
Tc and Tg is beneficial for reduction of primary
drying. For this reason, trehalose is increasingly
being used in mAb formulations as the first
choice excipient5 (Table 3). Selection of the rightstabiliser excipient depends on the type andconcentration of the biologic drug together with thephysicochemical compatibility with other excipientsin the formulation. It is important that the stabiliser
excipient is in an amorphous state throughout

Herceptin®
Ontruzant®
Herzuma®

Trastuzumab

Kanjinti®
Herceptin®
Hylecta®

Trastuzumab andHyaluronidase

Astrazenaca-Oxford
vaccine (AZD1222)13
Moderna vaccine(mRNA-1273)14

COVID-19 vaccine

Pfizer-BioNTech
vaccine (mRNABNT162b2)8,15
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European Pharmaceutical Review | Volume 26, Issue 01

the shelf life of the product (lyophilised cake).
There are few reported cases of cake instability thatare associated with crystallisation of mannitol whenused as a stabiliser. In contrast, both sucrose andtrehalose are not known to crystallise during thelyophilisation process and therefore are often more
effective in the stabilisation of the biologic drug
than mannitol.3

Current usage of disaccharide excipients

The disaccharide excipients sucrose and trehalose
have been used in several commercialised biologic
drug products, as described in Tables 3 and 4.
To support pharmaceutical companies in the
manufacture of biologic drugs (including vaccines),
DFE Pharma has extended its portfolio with highpurity low endotoxin-grade trehalose and sucrose
under the BioHale® brand.12

Supply chain security of
disaccharide excipients

The need to improve the pharmaceutical supply
chain was recognised even before the COVID-19
pandemic.9 Several events in recent history have
put pharmaceutical supply chains under pressure
and as such have demanded supply chain
improvements. These events include, among others:
quality issues at manufacturing sites, closure ofplants for environmental reasons, heightened
trade tensions and the rising demand in emerging
markets in Asia-Pacific/Latin America. The growing
numbers of biologics that require low-temperaturestorage shipment has also influenced drug
shortages in recent years.9
The current COVID-19 pandemic has added
further pressures to the security of pharmaceuticalsupply chains. A survey held by Pharmaceutical
Technology (April 2020) on the confidence of thepharma supply chain in maintaining adequate
supply of medicines during the outbreak of
COVID-19, showed that 42.2 percent of the
respondents felt “doubtful”.9 This shows theimportance, for all companies involved, of
establishing proactive strategies for building
and maintaining supply chain security.
Sourcing excipients through a global supply
chain network will likely be essential for supporting
globalised pharma manufacturing. Companies that
supply excipients to pharmaceutical companies
internationally need to have multiple supply points
spread across the globe to supply their materials
within the requested delivery times. In addition,
excipient suppliers should have a strengthened
and secured supply chain system, in which they
have good control over end-to-end processes,
from sourcing their raw materials to delivery of
their excipients to the manufacturers of finisheddrug products.
The IPEC Guide and various IPEC
presentations10,11 on the qualification of excipientsoutlines various topics that excipient suppliers andpharmaceutical companies could and should work
closely together on in order to enhance supply
chain security. For instance, supplier qualification
is an important part of managing and securing
the supply chain for a pharmaceutical company.
In addition, pharmaceutical companies arerecommended to utilise supplier questionnaires,
review pharmacopoeial compliance, audit
their suppliers’ facilities and identify any risks
involved in excipient parameters beyond thosementioned on COA.

Thontesh GC
Thontesh is currently
Manager within the Global
R&D group at Apotex
Pharmaceuticals. He has
14 years of experience insterile formulation design,
process development,
scale-up and processvalidations of injectables,
micelles, pre-filled
systems, controlled drugdelivery systems andophthalmic solutions
along with productlifecycle management.
Thontesh is a subject
matter expert inlyophilisation cycle
designs, development
and optimisation.
This also includes
working with different
types excipients, such as
various sugars, polymers,
buffers and solventsused for lyophilised
product development.

References
1. Vaccines, Blood & Biologics [Internet]. U.S. Foodand Drug Administration. 2021 [cited 21 January2021]. Available from: https://www.fda.gov/vaccinesblood‑biologics.
2. Rayaprolu BM, Strawser JJ, Anyarambhatla G. (2018)
Excipients in parenteral formulations: selection
considerations and effective utilization with small
molecules and biologics. Drug Dev. Ind. Pharm. 44,
1565–1571.
3. Bjelošević M, Pobirk AZ, Planinšek O, Grabnar PA.
(2020) Excipients in freeze-dried biopharmaceuticals:Contributions toward formulation stability andLyophilisation cycle optimisation. Int. J. Pharm
576 119029.
4. Baheti A, Kumar L, Bansalb A. (2010) Excipients usedin lyophilization of small molecules J. Excipients andFood Chem. 1 (1).
5. Mensink MA, Frijlink HW, Maarschalk KVDV,
Hinrichs WLJ. (2017) How disaccharides protect
proteins in the solid state and during drying (review):
Mechanisms of stabilization in relation to stressconditions Eur. J. Pharm. Biopharm, 114 288–295.
6. Bjelošević M, Seljak KB, Trstenjak U, Logar M, Brus
B, Grabnar PA. (2018) Aggressive conditions during
primary drying as a contemporary approach to
optimise freeze-drying cycles of biopharmaceuticals.

dfepharma.com/Corporate/News-and-Events/
News/DFE-Pharma-launches-ultimate-stabilizationaid-for-biologics.
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Available from: https://www.astrazeneca.com/media9. David Alvaro P. Rethinking the Global Pharmaceuticalcentre/press-releases/2020/azd1222hlr.html.
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Pharmasalmanac.com. 2021 [cited 20 January 2021].
Against COVID-19 Achieved Success in First Interim
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