Application Review

No. 005/2021

Formulation of peptides & proteins - which technique suit your needs

better?
1. Abstract

The stability of protein/peptide based drug formulations is known to be greater in the solid state
than in the liquid state. A solid formulation offers the advantages of higher stability at ambient
storage temperature and thereby often removes the necessity for cold chain storage and
transportation. Many protein/peptide based drugs are prepared as solid dosage forms by freeze-
drying or spray drying.

Freeze drying or lyophylisation is a very well-established dehydration process for preparing

biopharmaceuticals. It is typically performed with the product directly filled in the final container
and gives a high quality dry cake with a high porosity that enables quick reconstitution of the liquid
form when needed. Freeze-drying formulations are best suited when no further processing is
required after drying and are therefore commonly delivered parenterally by subcutaneous or
intramuscular route after being reconstituted.

Lately, research on powder based methods for protein delivery has increased. Even though very
well-known in the industry, lyophilization is not a direct particle formation method and would
require additional procedures to break the cake into smaller particles. Spray drying on the other
hand is an elegant one-step process to produce a powder with unique particle characteristics from
a biopharmaceutical formulation. Even though high temperatures are applied during the process,
most of the energy is used to evaporate the liquid and the actives will not be subjected to
excessive temperatures. When appropriate formulation strategies are used to minimize the effect
of heat and drying on the biomolecules, spray drying is an effective, well-established research
and production tool to obtain powders of proteins and peptides formulation opening the door to a
wider scope of deliver methods.

2. General Method Overview – Means of production

Proteins play an important role in most of the fundamental processes in living cells where most
of the information is encoded in nucleic acids and controlled by proteins. Proteins are all made
up of building blocks called amino acids and they have a highly organized three dimensional
struction in solution which is tightly related to their biological function.
Peptide are smaller version of proteins. The three-dimensional structure is less well defined but
there biological importance is not to be minimized1,2.

Proteins and peptides are known to be responsible for a wide range of life-essential functions and
processes in the body including signal transduction, heart rate regulation, food intake and growth.
They also find applications of the highest importance in the fields of biomedicine, biotechnology,
bioengineering drug discoverey, drug delivery, cosmetic or nutrition. Molecules such as insulin or
aspartame are famous example of applications for peptides and proteins. 3–5

In drug development, proteins and peptides are often considered as an attractive therapeutic
agent against various diseases. In biomedical or biochemical research, these biomolecules can
be used as references in quantitative studies, growth factors and cytokines for cell culture, active
enzymes for biochemical assays, carrier proteins for movement of molecules, inhibitors for
blocking pathways or antigens for use with antibodies.

Proteins and peptides manufacturing (Figure 1) can be achieved through chemical synthesis,
using recombinant microorganisms or cell free expression systems and via extraction from their
natural environment.3,5 The used strategy will be largely determined by the size and the chemical
features of the required molecules.

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 Synthesis

Solid
Solution
phase
phase

Biotechnology

Recombinant DNA sequence of

culture protein of interest

Ribosome
Protein

Extraction
Extraction

Figure 1: Peptides and proteins can be manufactured through chemical synthesis, using biotechnology techniques or after
extraction from their natural environment.

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3. General Method Overview – Workflow

In peptide and protein production, there is no one-size-fits-all solution and the best-performing
methodology should be identified for each case. Chemical and biotechnology technologies are
commonly used to maximize yields and product purity and can as well be combine when required
to overcome synthesis challenges.2,6

Peptides can be produced chemically using mainly solution-phase synthesis (SPS) or solid-phase
synthesis (SPPS). A combination of both methods using various ligation strategies is also
possible.5,7–9

Synthesis Purification Isolation Formulation

Analytics

Figure 2: Peptide production using the chemical route

Peptides are created by a step by step addition of amino acids to the peptide chain through a
serie of protection and coupling steps, until the required sequence is completed.8,10 The
composition of the sequence of amino acid is determining the peptide properites, it is therefore
very important to maximize yield when adding an amino acid to the chain.8,9

After synthesis, the crude product contains the desired peptide in solution together with some
impurities (deletion peptides, truncated peptides, incompletely deprotected peptides, modified
peptides, byproducts,…) which need to be removed.8,9 Purification strategies usually comprise a
combination of separation methods that take advantage of the physiochemical characteristics of
peptides such as their size, charge and hydrophobicity. Commonly used purification techniques
are size-exclusion chromatography, ion exchange chromatography (IEC), partition
chromatography and high-performance liquid chromatography (HPLC).8,9,11 Reverse-phase and
ion-exchange chromatography are preferred, giving their high resolution, robustness and
reproducibility output.12 After analytics, fractions containing a sufficiently pure peptide will be
isolated and formulated.8

Conventional approach for peptide isolation would be to remove the solvent using rotary
evaporation, parallel evaporation or freeze-drying.8,9 Although the synthesis step is the most
important part of the workflow, purification and isolation issues can become a cause for many
bottlenecks and should be evaluated carefully.

4. Key Applications

The size of peptide is one of the key criteria for determining the most appropriate manufacturing
technology. While chemical synthesis is known to be faster, more flexible and less expensive for
the manufacturing of up to a multi-100 kg of peptides with a maximum of 50 amino acids,
recombinant technologies is an attractive option for the production of large amounts of long and
complex peptides composed of natural amino acids.10,14 In the recombinant approach, the natural
machinery of the cells is used to produce heterologeous peptides and proteins. 14
The first protein produced with recombinant technology was recombinant insulin. This was a
milestone for modern biotechnology and the starting point for the development of many other
recombinant peptides and proteins.14
Peptides application are as varied as peptides themselves and they seem to be responsible for
several benefit on human heath including anti-aging, anti-inflammatory, anti-microbial activities.
These molecules are produced by almost all living beings, however in limited quantities.
Researchers have therefore tried to synthesize peptides and proteins to study their properties
and use them in various areas. Thanks to there unique characteristics and functionalities,

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 peptides and proteins find many applications in diagnostics, drug discovery, biomedicine, drug
delivery, health care, cosmetic, nutrition or other fields. Some of the most popular peptides include
insulin to treat diabetes, collagen for anti-aging and skin health, creatine as food supplement to
build up muscles and enhance athletic performances or aspartame as a zero-calorie sweetener
for beverages.15

5. Formulation

Drug formulation is the process of designing and producing drugs that will be sold to patients.
Several substances are combined with the active ingredient. The ingredient, once elaborated,
may still not be directly incorporated into a formulation as the ingredient may be unstable or
display undesirable qualities. In order to facilitate storage before incorporation in a final
formulation, the ingredient needs to be preformulated either in a liquid or in a solid form. There is
not general preferred technique for selectig a drying method for a biopharmaceutical. The choice
usually depends on the final use of the compound and of company preferences. Drying methods
should however be tailored to the individual biopharmaceutical, considering parameters such as
the coumpound characteristics and future use but also the financial aspects and the production
capacity and requirements. Spray drying and freeze-drying are both commonly used for solid
formulation of peptides and proteins, with their advantages and disadvantages. The following
section will review these technologies.16

Table 1: Comparison of the main characteristics of freeze drying and spray drying.16,17

Spray Drying Freeze Drying

Drying time Short - Seconds Long - Days / weeks
yield 50-80% on lab scale units 100% in vial
> 0°C - primary drying
Product temperature 50°C or above
20-40°C - secondary drying
Ice crystal formation (solid-liquid
Shear, atomization, air-liquid
interfacial stress), change of ionic
Stresses interfacial, mecanical, thermal,
strength and pH, phase separation,
dehydration
dehydration, crystallisation

Control of particle characteristics Yes No

Capital cost Moderate Very high

Operating cost Moderate High
Production Continuous Batch

Spray-drying
Spray drying is currently the preferred solution for drying many small molecules and for
amorphous solid dispersion. Thanks to its particle engineering possibilities and low cost, it is
gaining popularity for drying formulations of biopharmaceuticals. Dehydration via spray drying
happens in a matter of seconds, making it a high throughput, continuous, easily scalabe and
economic technology. Investment and operational costs of spray drying are significantly lower (4-
7 times) than those of freeze drying even though hot gas is generated and consumes a high
amount of energy.16,18 The ability to control particle characteristics of the dried powder by
changing process parameters make spray drying an ideal option for applications such as
pulmonary and nasal delivery where particle characteristics are important. It also enable a good
processability of the powder that can readily be tableted or filled intocapsules after being mixed
with excipients if needed.16

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 Spray drying is accomplished by dissolving, emulsifying
or dispersing the core substance in a solvent or in a
solution of carrier material. The material is then
atomized and sprayed into the drying chamber where a
hot stream of drying gas will help evaporate the solvent
to produce dry solid particles that will further be
separated from the gas stream and collected (Figure
3).16,19–22
The process parameters, the properties of the feed and
the equipment design are variable that can be adjusted
to modify the characteristics of the final product.
Spray drying can be considered a high throughput
process since it is drying very quickly compared to other
drying techniques. It provides the advantage of weight
and volume reduction. The transformation of a liquid
Figure 3: Functional principle of conventional
spray dryer
product into a dry powder is done in a single step, which
makes the method advantageous in terms of costs,
scale-up and process simplification. The powder can be
fully engineered and processed into tablets/capsules without milling or other secondary
processing. Moreover, even though spray drying exposes biomolecules to shear, air-liquid,
interfacial and dehydration stresses, most temperature sensitive substances like enzymes,
proteins, antibiotics, etc. can be spray dried without major loss of activity 16,19–22.
Due to a loss of product on the wall of the drying chamber and into the exhaust air, yields in
laboratory scale experiments are far from optimal and are reported to be in the range of 20-70
%16,19–21. This requires working with larger amounts of biological substances, which can be
expensive and challenging at the early stages of the development. The introduction nof high-
efficiency cyclones however led to increased yields that can reach over 80%. At industrial scale,
yields increase with larger scale setups since the lost fraction is a smaller part of the production
volume. Insufficient forces of liquid atomization and the ineffectiveness of the cyclone to
effectively separate fine particles with a diameter below 2 μm, makes the production and the
recovery of sub-micron particles tedious. This phenomenon has to be considered in the
development of drug delivery systems such as intravenous administrated pharmaceuticals 16,19–21.
Laboratory scale spray drying also fail to produce particles with a size range above 50 μm –
similar to those produced at large scale. This needs to be taken into account during lab scale
screening since it could lead to some issue later during scale up when dissolution profile of
particles and powders are important parameters.

Freeze-drying
Freeze-drying or lyophilization is an effective way of drying a material. It is using the physical
principle of sublimation, which involve the direct transition between the solid and the gaseous
phase, bypassing the liquid phase. The frozen sample is dried under vacuum without being
allowed to thaw. This process is suitable for a wide range of applications such as the preservation
of delicate material against degradation or decomposition, the preservation of product
characteristics and initial shape, the conservation of products that require fast rehydration or the
conditioning of product for further use.23
The crucial parameters in freeze drying are pressure and temperature. A typical freeze drying
process involves two stages – freezing and primary drying. For some samples a secondary drying
might be required in order to remove solvent molecules tightly attached to the sample and reduce
moisture further (Figure 4). Each process step has distinctive requirements in terms of pressure
and temperature depending on sample characteristics.23

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 Figure 4: Main steps of a freeze-drying process – Freezing, primary drying and secondary drying.

The vast majority of the water should be removed by the end of the primary drying phase. The
residual moisture content of the product may now be 5 – 10% due to water bound to the matrix.
At this stage, ice should not be present anymore. The secondary drying step removes the
adsorbed water molecules by desorption. In order to achieve ideal conditions for desorption, the
lowest possible pressure as well as a further increase of the shelf temperature is required. Again,
product stability must be considered when choosing the shelf temperature. Secondary drying is
usually performed for shorter time periods. At the end of secondary drying, the product moisture
content should be in the range of 1 – 5%.22,23
Freeze drying is usually the favorite choice for the preservation of a wide range of pharmaceutical,
mainly when stability in the liquid state is not adequate, storage requirements are too rigorous or
when the product is required in solid form. It is well suited for formulations that do not require
further processing after drying since they can be filled directly in vials, which can be sealed in the
drying after the cycle, in order to avoid potential contaminations. Freeze drying strengths lie in the
low process temperatures, high yields, great product uniformity and often, high quality in term of
activity, water content and/or stability. The accurate control of the process enables the production
of a product of the highest quality since it minimizes risk of intrinsic products properties such as
collapse, eutectic melt or glass transition temperatures being exceeded.22,23
Even though freeze-drying shows non-negligible benefits for sensitive products, industries are
investigating alternative methods such as spray drying, due to the high cost, long process time
and limited volumes associated with lyophilization at production scale. 16,22

6. Key parameters to consider

The main consideration in drying of protein formulations is long-term stability, which is mainly
associated to water content. Small molecules can usually be formulated without excipient and
the moisture content usually reach a low enough value so that the formulation remains stable for
long periods of time. The situation for proteins is however reported to be more complicated since
they are labile molecules. The stability of proteins is highly related to the water content of the
formulation and to the conformational structure of the molecule. Proteins need water to avoid
denaturation and therefore, formulation should be optimized and the drying process should be
properly control to avoid stability issues.24
Even though a protein can exhibit instability during the drying process, the protein may refold
completely and display good pharmaceutical stability after reconstitution if no irreversible reaction
such as aggregation occurs during storage or during reconstitution.24
Before looking into process parameters, it is important to fully understand the specificity of the
molecule in the given formulation. A properly designed formulation should take into account any
issues with associated the drying process such as the drying stress and corrects these issues
before starting with the design of the process. Knowing how the protein or peptide has been
produced and purified will also help to put in place the most adequate formulation and drying
process by understanding the molecule degradation pathway.24

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Spray-drying
The results of the spray drying method highly depend on material properties, equipment design
and correlation of the process parameters. Those factors have an influence on the quality of the
final product in term of morphology, residual moisture and particle size. Optimization of the
process is usually achieved by a “trial and error” approach, however, understanding the basic
guidelines of spray drying can help the user with intelligent use of the equipment. 25,26 General
guidelines for optimizing the parameters and informations related to its impact on product are
summarized in Figure 5.18

To minimize thermal degradation of the protein and peptides, the key focus should be on the
temperature and the residence time. It is commonly said that the oulet temperature is the maximal
temperature experienced by the product or the temperature at which the molecule spends the
longest time.16,18 It is therefore a key process parameter to prevent thermal degradation. If the
outlet temperature is lower than the degradation temperature of the pharmaceutical, the material
will experience less thermal degradation. A too low outlet temperature can however result in
shorter shelf life due to an increased residual water content. It is therefore important to optimize
the outlet temperature of the produc to obtain an adequately dry powder, without inducing
degradation. Generally, spray drying of biopharmaceuticals is carried out with outlet temperature
below 100°C, with very low feeding rates.
The residence time is
important with respect to
the complete drying of the
droplets and to control the
particle temperature so
that thermal degradation
of heat sensitive materials
can be minimized. Fine
particles containing a high
amount of surface
moisture will experience
an easy evaporation and
therefore require a short
residence time. Fine to
semi-coarse spray that
need to be dried to a low
moisture content require a
medium residence time
while coarser spray that
need to achieve lower
residual moisture content
require a long residence
Figure 5: Influence of process parameters on the product characteristics
time.26 The time required
to evaporate a droplet of pure water is reported to be of 0.03 s for a 10 um droplet and 3 s for a
100 um droplet. This drying times is assumed to be considerably increased for a spray dired
particle that forms a crust.27 The typical residence time for a laboratory scale spray dryer is of 0.2-
0.35s s.27 the residence time is given by the drying chamber volume and the drying gas flow rate.
The drying chamber volume being fixed by design and not adjustable, only the drying gas flow
rate can be modified. In order to maximize capacity however, it is always preferred to run
commercial spray dryers at maximum drying gas flow rate.16

Freeze-drying
When developing a freeze drying process, the most critical step is freezing since it establishes
the microstructure of the dried product. The product must be frozen at a temperature that is low
enough to be completely solidified.24 Most liquid products, or formulations, freeze by forming ice
crystals. Size and shape of the ice crystals depend on the cooling speed and define the freeze
drying ability; rapid cooling results in small ice crystals while slower cooling leads to larger ice
crystals. In terms of freeze drying, small ice crystals are more challenging to remove from the
product than large ones. Yet, the freezing temperature of a formulation is defined by its
characteristics and composition.23

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 Formulations can generally freeze in two different ways; eutectic mixtures contain substances
that freeze at lower temperatures than the water surrounding them. When cooling an eutectic
mixture, water is the first to separate from the substances and it freezes to ice. The formulation
may now appear frozen but the remaining substances are actually still liquid. They form
concentrated areas that freeze eventually at temperatures below the freezing point of water. The
temperature where all components of the mixture are properly frozen is called eutectic
temperature. This is the critical temperature of the formulation and the maximum temperature the
formulation can endure during the freeze drying process. Applying vacuum to an incompletely
frozen eutectic mixture, may result in the destruction of the product as unfrozen components
expand when placed under vacuum.23

The other class of mixtures is amorphous and form glassy states when frozen. With decreasing
temperature, the formulation becomes more and more viscous and eventually freezes to a
vitreous solid at the glass transition point. For amorphous products, the critical point in terms of
stability is called collapse temperature. The collapse temperature is typically slightly lower than
the glass transition point. Amorphous products are generally very challenging to freeze dry.23
It is important to understand the physical form of the solute (i.e., whether crystalline or amorphous)
after freezing since it will have an influence not only on the drying characteristics, but also on the
appearance of the final product, and product stability during storage.24

Proteins are molecules with a relatively high glass transition temperature and are therefore rather
easy to freeze and dry. The critical issues are the critical changes that can occure during the
freezing process and that can contribute to the denaturation of the proteins. When freezing, the
final structure in which the protein will be embedded is created. If this structure is not right, the
protein will be locked in a wrong confromation and can potentially lose potency. It is therefore
important to assess the freezing way (slow, quick,…) and its impact on the protein integrity.24

For primary drying, the cycle is usually developed using information from the glass transition
temperature to define shelf temperature and chamber pressure. Phase duration can be
determined using tools such as end point determination (temperature and/or pressure) or based
on scientific knowledge. While the primary drying procedure is developed using information from
the thermal characteristics of the molecule, the secondary drying cycle is developed using
information on the thermal stability of the molecule. The temperature of the shelf and duration
may impact product potency and these parameters should be kept at a reasonable to avoid any
issue. Secondary drying also determines the residual moisture level in the product and therefore
it’s stability and shelf life. The remaining humidity can increase degradation reactions during the
product shelf life and should, therefore, be kept at a low level, typically below 3%. 24

7. Solid protein formulations prepared by spray drying and freeze drying17

Process Protein/Peptides Stabilizers Ref.

28,29
IgG Trehalose, Sucrose, PEG
30
Lyzozyme PEG, Glycerol, Sucrose, Trehalose, Dextran
Freeze Drying
31
BSA Glucose, Sucrose, Maltose, Trehalose, Maltotriose
32
Anti-IgE antibody Histidine, Arginine, Glycine, Aspartic acid
Trehalose, Sucrose, Leucine, Glycine, Lysine, 17,33,34
IgG
Phenylalanine
Trastuzumab Trehalose, HPβCD, βCD 35

36
Anti-IgE Mab, rhDNase Mannitol, Trehalose, Scrose
Spray Drying 37
Catalase Arginine, Glycine, Histidine
38
Influenza vaccine HEPES buffer, Phosphate buffer
39
Alkaline phosphatase Sodium carboxy methylcellulose
40
EPO Dextran

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8. Tips and Tricks

∙ Proteins and peptides are best stored at -80°C or on ice, however freezing them at -20°C
is not recommended. Prior to storage, purified protein should be sterile filtered. Rapid
freezing using dry ice/ethanol mixture is preferable than slow freezing at -20°C and the
addition of sucrose or glycerol often increases stability during storage, freezing and
thawing cycles. Drying is best for long-term storage. When spray drying or freeze-drying
proteins, care should be taken in choosing the protein solvent, moreover a protectant
such as trehalose should be used to stabilize the molecules and help them retain their
functional activity.13

∙ As much as possible, measure the degradation temperature of your biopharmaceutical,

as well as the glass transition temperature of the biopharmaceutical formulation
(molecule of interest + excipients).18,23

∙ The use of sugars and sugar derivatives can be used to stabilize spray-dried and freeze-
dried biopharmaceutical against dehydration and thermal stresses.16

∙ The formulation design should take into account the issues associated with freeze-drying
and/or spray drying and correct them. It is very difficult to design a freeze-drying cycle
around a formulation that has not been made for freeze-drying (e.g, proteins in phosphate
buffered saline are the worst things to lyophilize).24

∙ The freeze-drying cycle should be developed and optimized for the product. The idea of
“one cycle fit all” is a common misunderstanding that cannot be applied.”24

∙ Freezing methods used before lyophilization can have a significant influence on the
structure of the ice formed, affecting both the water-vapor flow during primary drying and
the final product. Understanding and controlling how a solution freezes can lead to shorter
freeze-drying time and more stable products.23

9. References

[1] Deb, P. K.; Al-Attraqchi, O.; Chandrasekaran, B.; Paradkar, A.; Tekade, R. K. Chapter 16 -
Protein/Peptide Drug Delivery Systems: Practical Considerations in Pharmaceutical
Product Development. In Basic Fundamentals of Drug Delivery; Tekade, R. K., Ed.;
Advances in Pharmaceutical Product Development and Research; Academic Press, 2019;
pp 651–684. https://doi.org/10.1016/B978-0-12-817909-3.00016-9.
[2] Lloyd-Williams, P.; Albericio, F.; Giralt, E. Chemical Approaches to the Synthesis of
Peptides and Proteins; CRC Press, 1997.
[3] Guzman, F et al. Peptide Synthesis: Chemical or Enzymatic. Electron. J. Biotechnol. 2017,
10 (2).
[4] Petrou, C.; Sarigiannis, Y. 1 - Peptide Synthesis: Methods, Trends, and Challenges. In
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[5] Uhlig, T.; Kyprianou, T.; Martinelli, F. G.; Oppici, C. A.; Heiligers, D.; Hills, D.; Calvo, X. R.;
Verhaert, P. The Emergence of Peptides in the Pharmaceutical Business: From Exploration
to Exploitation. EuPA Open Proteomics 2014, 4, 58–69.
https://doi.org/10.1016/j.euprot.2014.05.003.
[6] Funfrock, P. Challenges in Chemical and Recombinant Peptide Production Processes.
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[7] Mueller, L. K.; Baumruck, A. C.; Zhdanova, H.; Tietze, A. A. Challenges and Perspectives
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[8] Ag, B. Bachem Peptide Guide. 24.
[9] Biotage - A Holistic Approach to the Peptide Workflow in Drug Discovery
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