Pardeshi et al.

Future Journal of
Future Journal of Pharmaceutical Sciences (2023) 9:99
https://doi.org/10.1186/s43094-023-00551-8 Pharmaceutical Sciences

REVIEW Open Access

Process development and quality

attributes for the freeze‑drying process
in pharmaceuticals, biopharmaceuticals
and nanomedicine delivery: a state‑of‑the‑art
review
Sagar R. Pardeshi1, Nilesh S. Deshmukh2, Darshan R. Telange3, Sopan N. Nangare4, Yogesh Y. Sonar5,
Sameer H. Lakade6, Minal T. Harde7, Chandrakantsing V. Pardeshi8, Amol Gholap1, Prashant K. Deshmukh9 and
Mahesh P. More9*   

Abstract
Background Process intensification is a major hurdle in pharmaceutical process scale-up. Solvent removal strategies
have limited the effectiveness of the overall stability of pharmaceutical formulations. The main aim of present review
article is to focus on the use of the freeze-drying process in pharmaceuticals, biopharmaceuticals and nanoderived
therapeutics and their translation into commercial viable products. Unwavering efforts of scientists in the process
intensification of lyophilization promote unique features of products for commercialization. Regulatory agen-
cies are promoting the utilization of a quality-by-design approach to improve product characteristics. Among 300
FDA-approved pharmaceutical industries, 50% of products are freeze-dried. The freeze-drying process is costlier
and requires more time than other drying methodologies. Unstable pharmaceutical dispersions and solutions can be
preferably stabilized by using the freeze-drying method.
Main text This review highlights the utilization of critical quality attributes and process parameters for the freeze-
drying process, which helps to improve the integrity and stability of the formulation. The quality-by-design approach
possibly cuts the cost of the process and saves money, time, and laborious work. The present review focuses prelimi-
narily on the applications of freeze-drying in the development of biopharmaceuticals, including vaccines, proteins
and peptides, and injectable products. In addition, a separate section demonstrating the potential of freeze-drying
in nanoderived therapeutics has been illustrated briefly. The present clinical scenario of freeze-dried pharmaceuticals
and biopharmaceuticals has also been described in later sections of the review.
Conclusions This review underscores the value of integrating Quality by Design into the development of lyophi-
lization processes for pharmaceutical and biopharmaceutical products. By identifying critical process parameters,
delineating a design space, and leveraging advanced monitoring techniques, manufacturers can effectively address
the intricacies of lyophilization. This approach empowers them to produce stable, superior quality products with con-
fidence and consistency.

*Correspondence:
Mahesh P. More
maheshpharma99@gmail.com
Full list of author information is available at the end of the article

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 2 of 31

Keywords Freeze-drying, Quality-by-design, Pharmaceuticals, Biopharmaceuticals, Drug Nanomedicine

Graphical abstract

Background as glucagon at pH 3, and some are stable at extremely

Most of the time, formulation scientists select lyophili- high pH ranges [3]. However, apart from the administra-
zation or freeze-drying technology as the first preferable tion route, pharmaceuticals can be stabilized during the
method during formulation development [1]. Depend- freeze-drying process. Because of its distinct advantages,
ing on the route of administration, the formulator must freeze-drying is an extensive manufacturing method
consider different formulation attributes, such as volume used for improving the stability of medicaments, such
for intravenous, intramuscular, or subcutaneous admin- as nanoparticles (NPs). The methodology used is rela-
istration through reconstituted dispersion [2]. For intra- tively simple, easy to scale up and specifically applicable
muscular or subcutaneous administration, the quantity for small volume products [4]. Freeze-drying involves a
of formulation required is 2 or 10 mL, and the methodol- dehydration process at a lower temperature that involves
ogy is also distinct because the isotonicity and pH of the transitioning from a solid into a gas phase without con-
dispersion did not promote irritation at the site of intra- verting into a liquid phase [5]. It is made up of three
venous injection. Very few therapeutics are stable in a steps: freezing, primary drying, and secondary drying.
liquid state and are miscible in unusual pH ranges, such Water is eventually removed from the ice by the process
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 3 of 31

of sublimation at reduced temperature and pressure stable biopharmaceutical drug products like proteins that
underneath the triple point of water [6]. Considering the are unstable in an aqueous system. Lyophilized forms
process tactics, freeze-dried products are relatively easy have been used for approximately 50% of newly approved
to handle, package and transport [7]. The highly con- biological products, and this figure is likely to increase
centrated liquid suspensions are converted to powdered further [16].
form, where the powder characteristics are similar to
other solid powder formulations and can be blended into
pills, tablets, pellets or easily filled it into capsules [8]. Main text
Furthermore, biopharmaceuticals products have sev- Basics of freeze‑drying
eral benefits over conventional therapeutics and are Water can be categorized into three forms: solid as ice,
transiently useful to treat numerous ailments, such as liquid or gas (in the form of vapours) [1]. Freeze-dry-
hepatitis B virus, arthritis, psoriasis, and multiple types ing employs the sublimation effect, in which solid ice is
of cancer [9]. Other factors that led to the development converted directly into a vapour phase without passing
of biopharmaceuticals and biosimilar products are target- through the liquid phase [2]. Looking at the water phase
based selectivity towards diseases due to the presence diagram (Fig. 1a), at low temperature and variable pres-
of anchored substrates such as monoclonal antibod- sure, water behaves as solid ice. Water is available as a
ies (mAbs), proteins, peptides, and amino acids. The liquid at higher temperatures and pressures and as a gas
engineered particles promote an increase in the blood in the form of vapour at even higher temperatures and
circulation time and reduced protein binding. The reduc- pressures [17]. The sublimation process is slightly oppo-
tion in the frequency of dosage helps to improve patient site, while during the lyophilization process, sublima-
compliance. tion occurs and vapour is sublimed into the condenser.
Proteins and peptide-based formulations are multifac- The process of sublimation occurs below the triple point
eted and challenging compounds that necessitate unique of water. The freeze-drying process optimization is set
industrial operations and handling conditions through- to complete below the triple point of water, which can
out their production, which is attributed to the rising be facilitated at controlled temperature and pressure.
cost of these biosimilar formulations [1, 10]. Biological During lyophilization, a significant amount of energy is
macromolecules have a high molecular weight and are required to convert ice into vapour under very-low-tem-
unsuitable for parenteral administration due to aggrega- perature conditions. To convert 1 gm of ice into water
tion and unstable formulation characteristics. The major- vapour, 2800 J of energy is needed, which is six times
ity of biopharmaceutical formulations are available as more than the energy required for the simple evaporation
dilute or concentrated solutions, suspensions, and dis- process required to convert liquid into gas [17, 18].
persions. Protein-based drug delivery systems are devel-
oped for administration through different routes, such Effect of pressure
as pulmonary [10], oral, intranasal [11], and transder- The most important parameter in freeze-drying process
mal routes [12], that have been extensively researched. is pressure. The pressure in the system is kept low to
Protein-derived formulations have low stability in bio- facilitate effective sample temperature during the freeze-
logical environments and low physicochemical stability drying process [19]. In a pure solvent (aqueous) without
[13]. Protein stability is a major concern that restricts an admixture, the temperature of the sample will be zero
the development of biopharmaceuticals. Protein stability degrees Celsius (0 °C) if the pressure is set at 6.11 mbar,
can be addressed during the preformulation and product which is near to the triple point of water. Currently,
development stages. The particle engineering approach freeze-drying does not use pure water for freezing and
has been utilized to improve the in vivo effectiveness of mostly involves an aqueous system with the required
biosimilar formulations [14, 15]. amount of solid content, excipients, cryoprotectants, and
The mAb-based formulations are typically developed as so on. The composition reduces the melting tempera-
aqueous dispersions for parenteral administration. Liquid ture of the mixture, and therefore, freeze-drying works
dispersions are unstable and prone to degradation with at lower pressure, typically at 1 mbar or below. A pres-
relative environmental factors. Additionally, the mobility sure of 1 mbar corresponds to a temperature of approxi-
of particles creates a more unstable formulation. How- mately reaching to -20 °C [5, 6]. Even at low pressure, the
ever, convertible solid components avoid degradation and temperature of the material drastically decreases at lower
possess limited protein mobility. Conversion of liquid for- level. The minimum pressure achievable in freeze-drying
mulations into stable solid components was accelerated is approximately 0.03 mbar, which corresponds to a tem-
by use of the freeze-drying process. Freeze-drying is the perature of approximately -50 to -60 °C. As a result, pres-
preferred method mostly in the manufacturing process of sure is critical and typically used in the range of 1 mbar
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 4 of 31

Fig. 1 a Water’s triple point at 0.01 °C and 0.00603 atm is depicted in a phase diagram. Freeze-drying is performed under the triple point to convert
ice to vapour without entering the liquid phase. [Reproduced from Assegehegn et al. [17] with kind permission of the copyright holder, Elsevier,
Amsterdam] b State diagram showing Tm, onset of ice melting; Tm′, onset of ice melting in a maximally freeze-concentrated solute matrix; Tg, glass
transition temperature; Tg′, glass transition temperature of the maximally freeze-concentrated solute matrix; Tgw, glass transition of water, and Tc,
collapsed temperature. Tc approximate to Tg′ and increases in the presence of cells. The shaded area shows a temperature range for maximum ice
formation for the solute concentration Cg′. The glass line indicates the glass transition temperature at various solute concentrations [Reproduced
from Santivarangkna et al. [31] with kind permission of the copyright holder, Wiley]

down to 0.03 mbar based on the sample temperature [17, faster. However, the actual process parameter defines that
20]. at lower pressure, the sample temperature is also reduced
and affects the overall lyophilization process. The tem-
Effect of temperature perature of the samples does not go below -50 to -60 °C.
The temperature of the sample is also essential since If the pressure falls below the desired level, the tem-
the temperature gradient between the condenser of perature also falls and deteriorates the freezing process.
the freeze dryer and the sample temperature is driven During the lyophilization process, the pressure should
by sublimation [21, 22]. The sample location in the freeze be maintained at a desirable set point where the sample
dryer varies based on manufacturer specifications in the remains in the frozen state and helps to accelerate the
general case; sample is placed at top of the freeze dryer. process as presented in Fig. 2 [20, 24, 25].
In other cases, chamber is provided for sample placement
or attached as a manifold. The manufacturer designs the Effect of heat transfer
sample compartment based on type of sample, volume The sublimation process consumes much energy, as
of sample and composition. During freeze-drying, the discussed earlier, and is considered to be an important
vapours formed at the top move down to the condenser viewpoint in the freeze-drying process. Two types of
and are collected in the receiver. A small temperature freezing processes are represented: shelf freeze dry-
difference is required between the sample temperature ing and manifold applications [26]. In the first type,
and condenser temperature, which helps to accelerate shelves are created for vials, while stress is applied
the process [18, 23, 24]. The small temperature difference during the freeze-drying process. The shelves are used
and applied vacuum help to move the vapour downwards with or without heat transfer mechanisms, preferably
to the condenser and condensate. In general, the faster conduction or radiation [27]. Conduction utilizes the
the temperature difference is, the faster the drying pro- direct transfer of heat energy from shelves, while the
cess, but the temperature difference should not be more radiation mechanism transfers heat from the top in the
than 15 – 20 °C. downwards direction. At the same time, convection
A very common misunderstanding in freeze-drying is was initiated, which exchanges heat energy from out-
that at lower pressure, the freeze-drying process becomes side to inside shelves. The overall process balances heat
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 5 of 31

Fig. 2 The figure illustrates (1) The comparison of lyophilization and sublimation process. (2) The impact of reduced pressure on the freeze-drying.
(3) The influence of temperature on freeze-drying. (4) The effect of heat transfer or exchange on freeze-drying process

transfers across all the shelves and regulates the drying There are some systems available without heatable
process. The balancing mechanism improves the dry- shelves. In this case, the process takes a much longer time
ing characteristics and accelerates the rate of drying. to dry because the energy available will be delivered by
Recent reports highlight the importance of heat trans- convection only. In the second type, one end of the mani-
fer during the freeze-drying process: an increase in the fold is attached to the valve, and the other end is attached
heat transfer process by 1 °C can shorten the freezing to the freeze dryer [28]. The second type utilizes heat
time of the sample by 13%. In short, a 5 °C increase in from the environment, which initiates the exchange of
temperature can fruitfully reduce drying time to half heat energy directly from the external environment. The
[23, 24]. sample vessel is encapsulated completely, while a larger
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 6 of 31

area is exposed to the external environment. The higher Implementation of quality by design (QbD) in freeze‑dried
exposure surface can accelerate the freezing process. product development
At ambient temperature conditions where laboratories Quality by design (QbD) is a method of choice in formu-
are not completely air-conditioned it utilizes manifold lation development to obtain robust and quality prod-
devices for freeze-drying applications. The rate of freez- ucts followed by continuous improvement [35]. QbD
ing using a manifold device can also be improved on is an organized approach preferably select predefined
warm days [24, 29]. parameters with major objective to screen the product
Freezing is very important consideration in the case of or process. The QbD considered fundamental consid-
crystalline and amorphous samples. It is often misunder- eration and select process control tactics as quality con-
stood that once the sample reaches the freezing point, trol parameters. Additionally QbD promotes quality risk
it becomes completely frozen, which is not correct. The management workflow to eliminate the associated errors
sample is cooled to zero degrees, is not yet frozen and [36]. QbD is particularly helpful in designing robust pro-
goes to the level called supercooling, so it cools below cesses with well-understood operational limits and their
the freezing point and forms ice crystals. The entire sam- significance. Implementation of QbD is complex and
ple crystallizes, and the sample achieves a frozen state at challenging work in the pharmaceutical industry. The use
0 °C. However, at this stage, the sample is not completely of QbD in formulation development minimizes the trial
frozen, as it contains a portion of the solution and a cost of validation batches. Therefore, implementation of
portion of ice [24, 29, 30]. To achieve complete solidifi- QbD principles, developing a better understanding, and
cation of the sample, we need to achieve a temperature possibly designing simple templates for the development
that is below the freezing temperature, also known as the of complex formulations is the current need.
eutectic temperature or collapse temperature, as shown From an operational standpoint, freeze-drying is an
in Fig. 1b [31]. Similarly, for amorphous samples, freez- expensive and time-consuming process. As a result, the
ing temperature should only have a solid and rubbery process must be brief, repeatable and robust. To design
state that can achieve at lower temperature. For achiev- a freeze-drying method, one must first define the essen-
ing a glassy state, there is a need to go below the glass tial formulation parameters and control them in the
transition temperature or collapse temperature, which process design. The temperature of the heating fluid,
is typically even lower than the eutectic temperature of pressure in the drying chamber, and timeframe of both
crystalline samples. Therefore, amorphous samples typi- drying stages throughout primary and secondary drying
cally need to be freeze-dried at lower temperatures or are some of the freeze-drying process parameters that
pressures than crystalline samples [32]. If the sample have a major impact on the quality of finished products
goes above the critical solution temperature, one can see [21]. If such variables are not controlled, they can cause
a collapse in the sample. In general, the solid sample is product denaturation, melting, or collapse of the dried
surrounded by ice, and it turns into a gas (vapour) and cake. Therefore, systematic development of the freeze-
left the solid at the bottom. In the case where the sample dried product by the implementation of QbD is required
goes above the collapse temperature, part of the sample to obtain a quality product. According to ICH Q8 (R2),
starts melting, some of the solvents become liquid again different effective QbD tools can be used for the develop-
and start to redissolve. After melting, the solvent starts ment of robust quality formulations (Fig. 3).
to sublimed and convert it into spongy hard material
instead of conversion into fluffy cake. The hard spongy Quality target product profile (QTPP)
material may create a problem in the final application QTPP is an outline of expected product characteristics
[30, 32, 33]. To avoid collapse of material and hardening and is used to design quality into the product and pro-
of sample, few commercial manufacturers provide auto- cess. QTPP embodies the overall objectives of the qual-
matic monitoring of collapse temperature measurement. ity of the drug product development program. Strategies,
Where it ensures and avoids conversion into hard spongy prior knowledge, and experience of a process or avail-
material. The recent advancements in the design of freeze ability of equipment and facilities could influence the
dryers allow the measurement of sample temperature choice of QTPP. QTPP is a knowledge-based system, so
and ensure that it will remain below the collapsed tem- it should be utilized in the identification of drug product
perature. A temperature sensor placed on each central characteristics of freeze-dried products. Some probable
vial at the bottom of each shelf makes the freeze-drying QTPP elements of freeze-dried products are represented
process highly efficient and reproducible [24, 34]. in Fig. 4.
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 7 of 31

Fig. 3 Quality by design tools

Fig. 4 Quality target product profile of freeze-dried products

Critical material attributes (CMAs), critical material fall within a specific range. The freeze-dried product’s
parameters (CPPs), and critical quality attributes (CQAs) CQAs are usually those factors that impact product
The desired quality of the material for use in freeze-dry- quality, safety, efficacy, purity, strength, drug release,
ing must be determined by taking into account its use stability, and so on. CQAs are normally linked with the
as well as knowledge and understanding of its physico- active substance, additives, intermediates (in-process
chemical, biological, and microbiological characteristics, steps) and drug products in freeze-drying. The possible
which can impact product characteristics. As a result, CMAs, CPPs and CQAs for the freeze-dried product are
before beginning the development cycle, CMAs should depicted in Table 1.
be identified. A CPP is one whose variability has an effect
on a CQA and should thus be checked or controlled to Risk assessment of the freeze‑drying process
ensure that the system achieves the best quality. The Risk assessment is a valued science-based method used
freeze-drying process consists of a series of unit opera- for quality risk management by identifying the poten-
tions and need to rectify each steps/stages. tial impact of CMAs and CPPs on drug product CQAs
The CQAs have physicochemical, biological, or (3). Different models have been used previously for risk
microbiological attributes or characteristics that must assessment, such as risk ranking, fishbone diagrams, and
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 8 of 31

Table 1 Probable CMA’s, CPP’s and CQA’s of freeze-dried product development

Critical material attributes (CMA’s) Critical process parameters (CPP’s) Critical quality attributes (CQA’s)

Buffer Shelf temperature Cake appearance

Surfactant Freezing ramp rate Reconstitution time
Fill volume Freezing temperature pH
Total solid content Hold time for freezing Residual moisture content
Ionic strength Chamber pressure at primary drying Potency
Configuration of vial Ramp rate to primary drying hold Concentration of solution
Stopper Primary drying temperature Particulate matter
− Hold time for primary drying Content uniformity
Ramp rate to secondary drying hold Drug impurity
Secondary drying temperature Sterility
Hold time for secondary drying −

failure mode effects analysis (FMEA) [37, 38]. The risk CMAs and CPPs can be defined by knowledge obtained
assessment of the freeze-dried products is carried out through risk assessment and the design of experiments
by linking input and process variables to CQA. The rela- for their effects on CQA [41]. The effect of each param-
tive risk that each variable presents was ranked as high, eter on freeze-dried products was estimated, and operat-
medium, or low based on their criticality. Attributes ing ranges for each parameter were selected as the design
that could have a high or medium impact on the CQA of space to obtain a robust formulation. ANOVA, response
freeze-dried products will be selected for further inves- surface diagrams (RSDs), contour plots, interaction
tigation. No further action is required for low-impact plots, main effects and other statistical tools were used
attributes [39]. to establish the design space. It was considered that the
process within the design space will result in a product
meeting defined quality.
Design of experiment (DOE) and design space
In DOE, inputs (variables) and output (response) form a Control strategy for the preparation of freeze‑dried
complicated matrix in which the input variables may be products
interlinked or independent of each other. It is challenging The control strategy is a planned set of controls that com-
to understand all potential variations and their impact prises process monitoring, practical controls, lot release
on the quality of a drug product. DOE tools help scien- testing, in-process controls, comparability testing, char-
tifically collate most variations in a minimum number of acterization testing and stability testing. Control strategy
experiments. Some possible input variables are the con- development requires an organized process involving a
centration of a solution, amount of excipients, freezing team of experts, linkage of the development of a prod-
rate, annealing temperature, secondary drying tempera- uct to the manufacturing process and process equipment
ture, etc. [40]. The ultimate goal of the DOE in the freeze- controls. An example of a control strategy for the freeze-
dried product is to optimize critical parameters identified dried product is depicted in Table 2, which is built upon
in risk assessment and to achieve the desired quality
product. Two types of designs can be used in DOE, i.e.
screening design and optimization design. The purpose Table 2 List of critical process parameters and control limits for
of using a screening design is to test pairs of combina- freeze drying [Reproduced from Radhakrishnan et al. [42] with
tions in only a few experimental runs, thereby saving kind permission of the copyright holder, Springer, Cham]
time and resources. Some commonly used screening Freeze-drying steps Parameter Control limits
designs are fractional factorial design, Plackett‒Burman
design and Taguchi design. The critical variables identi- Freezing Freezing temperature Not more than − 40 °C
of shelf
fied in the screening design will be further developed by
Primary drying Chamber pressure Below 150 microns
optimization design. Some commonly used optimization
Primary drying Shelf temperature Not more than + 35 °C
designs are full factorial design, Box‒Behnken design and
Primary drying Hold time Not less than 18 h
central composite design.
Secondary drying Shelf temperature Between 35 and 65 °C
The linkage between input, process and output was
Secondary drying Hold time Not less than 5 h
described in the design space. The operating ranges of
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 9 of 31

the outcome of extensive product and process under- chemical stability to the nanoparticles as presented in
standing studies [42]. Fig. 5 [45]. It reduces the mechanical stress during freez-
ing and drying steps and improves the stability of nano-
Selection of lyoprotectants and their impact on stability particles [46]. Bulking agents (mannitol, glycine, sucrose),
Lyophilization is an industrial process that converts buffers (Tris HCl, histidine, and phosphate), structure
an aqueous nanoparticle suspension into an amor- modifiers, tonicity adjusters (salts), stabilizers (glucose,
phous-crystalline solid by freezing, primary drying and dextran, sucrose, trehalose, lactose, mannitol and ala-
secondary drying [43]. These processes generate mechan- nine) and collapse inhibitors (glucose, dextran, maltose,
ical stress on the nanoparticles, causing aggregation and maltotriose, maltopentose and maltoheptose) are the
reducing nanoparticle stability [44]. Lyoprotectants are most commonly used inert excipients as lyoprotectants
inert excipients introduced exclusively into the aqueous and cryoprotectants in the freeze-drying process [45].
suspension before lyophilization to provide physical and Moreover, lyoprotectants also act as cryoprotectants and

Fig. 5 The use of lyoprotectant for stabilization of aqueous nanoparticle dispersion with inhibition of the particles aggregation due to reduced
mechanical stress on the particles surface for the same
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 10 of 31

vice versa [44]. The details of commonly used excipients endothelial and pericyte cell lines as a part of the retinal
as lyoprotectants in nanoformulations are tabulated in vascular system due to their biodegradability and bio-
Table 3. compatibility characteristics. Lyophilization of FA-NPA
Romeo and colleagues prepared and assessed the effect and FA-NPB polymeric nanosuspensions using HP-β-CD
of lyophilized ferulic acid-loaded polymeric nanoparti- at 5% w/v resulted in particle sizes in the range of ~ 150–
cles for ocular drug delivery. Ferulic acid (FA) is a well- 200 nm, PDI values of ~  < 0.2 and zeta potential values
known antioxidant that has low bioavailability due to of > − 20 mV. It was indicated that lyoprotectant could
low aqueous solubility. Biodegradable polymers, such inhibit particle aggregation following rehydration and
as poly(lactic acid) (PLA) and poly(lactic-co-glycolic provide long-term stability. The presence of HP-β-CD
acid) (PLGA), were used to prepare ferulic acid-loaded on the surface of nanoparticles promotes easy absorp-
and unloaded polymeric nanoparticles (FA-NPA and tion and reconstitution in aqueous media. The strong
FA-NPB) using the nanoprecipitation method. The hydrogen bonding between the nanoparticles and cryo-
nanoparticles were lyophilized using hydroxypropyl-β- protectant reduces the collapse temperature of the final
cyclodextrin (HP-β-CD) lyoprotectant and evaluated for formulation. The strong hydrogen bonding shifts the
stability studies. Final reports showed that unloaded NPA glass transition temperature to a slightly lower value. This
and NPB carriers at a concentration range of 0.25–1 mg/ causes a reduction in the primary drying step in the lyo-
mL and 0.25–2.5 mg/mL produced no cytotoxicity in philization process. The developed amorphous powder

Table 3 Different types of excipients commonly used as lyoprotectants in freeze-drying of nanoformulations

Excipients as lyoprotectants Molecular Optimum concentration of Glass transition Functions References
weight (g/mol lyoprotectants (% w/w or temperature (Tg,
or kDa) mM) ℃)

Bulking agents
Glycine 75.07 10 − 70 to  − 90 ℃ Provide bulk to the formula- [43, 47, 48]
Hydroxyethyl starch 670 kDa 12.5 44 ℃ tion, particularly if the product [49, 50]
concentration to freeze-dry
Lactose 342.3 1–2 101 ℃ seems to be very low [51, 52]
Mannitol 182.18 6 13 ℃ [49, 53]
Sucrose 342.3 2.5 − 46 ℃ [54, 55]
Trehalose 378.33 20 106 ℃ [47]
Buffers
Citrate 192.12 NA 105 ℃ Adjust and maintain pH [56]
Histidine 155.15 3.5 mM − 32 ℃ changes during freezing [57, 58]
Phosphate 94.97 20 mM NA [59]
Tris HCl 121.14 NA − 81 ℃ [60]
Stabilizers
Alanine 89.09 1–5 − 10 to 35 ℃ Offer protection from freezing [61, 62]
Dextran 40–70 kDa 2 68.2℃ and drying stresses dur- [63, 64]
ing the freeze-drying process
glycerol 92.09 NA NA
Polyethylene glycol 400 380–420 g/mol 25–35 − 63 ℃
Polyvinyl pyrrolidone NA 7.5 NA
Sodium chloride 58.44 5M NA
Tonicity adjusters
Glycine 75.07 10 − 70 to  − 90 ℃ Produce an isotonic solution [43, 47, 48]
Glycerol 92.09 NA NA while controlling osmotic
pressure
Mannitol 182.18 6 13 ℃ [49, 53]
Sucrose 342.3 2.5 − 46 ℃ [54, 55]
Sodium chloride 58.44 5M NA
Collapse temperature modifiers
Hydroxypropyl-β-cyclodextrin 1387 5–10 418 ℃ Raise the product’s collapse [65]
temperature to attain optimal
drying temperatures
NA indicates non-availability of said references
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 11 of 31

reduces the aggregation potential and enhances the sta- of particle size ~ 80–100 nm and PDI ~ 0.19–0.24. The
bility of both nanoparticles. Hence, HP-β-CD is consid- reduction in particle size ascribed the amorphization of
ered an efficient lyoprotectant and assists in enhancing lyophilized formulation, while 0.5% w/v concentration
the long-term stability of polymeric nanoparticles tar- of cryoprotectant can effectively stabilize the particles as
geted for ocular delivery [66]. shown in Fig. 6a–d.
In another study, Telange and coworkers studied the The low dispersibility of nanoparticles following rehy-
effect of five lyoprotectants on the particle size and poly- dration and hydrolysis of chitosan-induced severe aggre-
dispersity index (PDI) value of mangiferin phospholipid gation leads to increase its particle size and PDI value
complex-loaded phytosomal soft nanoparticles. These by ~ 1900 nm and 0.9, respectively. Histidine buffer (pH
nanoparticles, after lyophilization, showed a smaller 6.5) lowered the particle size (< 200 nm), and PDI (~ 0.32)
particle size and PDI. In this study, glucose, fructose value may be due to H-bonding and cation-π interac-
(monosaccharides) and trehalose, maltose, and sucrose tions between chitosan and histidine. The presence of
(disaccharides) were used as lyoprotectants at 0.5%, 1%, lyoprotectant and histidine buffer (pH 6.5) at ~ 0.5% w/v
2% and 5% w/w, respectively. The findings of the study and ~ 3.5 mM could be useful for improving the stabil-
revealed that glucose and fructose at 2% and 5% w/w ity (Fig. 6e) and gene delivery of chitosan/DNA nano-
concentrations reduced the particle size and PDI values particles [57]. Furthermore, the SEM study also revealed
to ~ 936.39 nm and ~ 0.68 of soft nanoparticles compared aggregation of nanoparticles (Fig. 6f-a) in the absence of
to their lower concentrations. This significant effect of excipients, whereas distinct nanosized spherical particles
both monosaccharides showed that the formation of are formed (Fig. 6f-b) in the presence of lyoprotectants.
weak interactions with soft nanoparticles reduced aggre- Chow and coworkers fabricated lyophilized curcumin-
gation. Likewise, trehalose and maltose at 2% and 5% w/w loaded polyethylene glycol (PEG)-PLA nanoparticles
reduced the particle size and PDI value to ~ 931.50 nm using flash nanoprecipitation technology and studied its
and 0.62, respectively, compared to their lower concen- effects on the long-term stability of curcumin nanoparti-
trations. It was suggested that the solid form of these cles. Curcumin is a highly lipophilic compound. Its nano-
excipients during lyophilization improved the alliance particles association with a copolymer stabilizer exhibited
with nanoparticles and reduced their particle size and low stability that may be due to weak binding interaction
PDI more significantly. Compared to 5% w/w, sucrose at with the hydrophobic cores. The prepared nanosuspen-
2% w/w reduced the particle size and PDI by ~ 906.69 nm sion was lyophilized using kleptose lyoprotectant. The
and 0.50, respectively, due to its amorphous nature and study demonstrated that variable factors, i.e. mixing rate,
easy interaction with nanoparticles, which improved the the molecular weight of PEG-PLA, and the drug-to-sta-
stability of the soft nanoparticles. The study confirms that bilizer ratio, influenced the curcumin particle size and its
sucrose enhances redispersibility and long-term stabil- distribution to a great extent. The mixing rate and molec-
ity at a low concentration acts as a superior cryoprotect- ular weight of PEG-PLA at ~ 4500 and 2–5 k generated
ant and enhanced the nanoparticle’s redispersibility and particle sizes of ~ 70–75 nm, indicating that mass trans-
long-term stability via the formation of hydrogen bond- portation from water to the organic phase followed by
ing interactions with nanoparticles surfaces [67]. supersaturation and nucleation reduced the particle size
Veilleux and colleagues prepared lyophilized highly of curcumin nanoparticles. However, after preparation
concentrated chitosan/deoxyribonucleic acid (DNA) the curcumin nanoparticle suspension showed a cloudy
nanoparticle formulations for gene delivery at clini- nature with the appearance of recrystallized curcumin.
cally relevant dosages. Chitosan (degree of deacetyla- The recrystallization leads to high molecular mobility
tion ~ 92–93.7%) and plasmid EGFPLuc were used to of PEG-PLA. The stability issue was addressed by use of
prepare chitosan/DNA nanoparticles. These nanoparti- polyvinyl pyrrolidone (PVP) as a costabilizer. Compared
cles were freeze-thawed to select the optimal concentra- to PVP K-30, polyvinyl alcohol (PVA), D-α-tocopheryl
tion of lyoprotectants from mannitol, sucrose, dextran polyethylene glycol 1000 succinate (TPGS), Pluronic
(5 kDa) and trehalose. The histidine buffer is used during F68, and leucine at 0.5% w/v offer excellent stability. The
dispersion and further lyophilized to get the free flowing enhanced stability may be due to hydrogen-bonded inter-
powder. The chitosan/DNA nanoparticles shows parti- action between PVP and the PEG block copolymer. The
cle size > 100 nm in the presence of mannitol (0.1–0.3% interaction also prevents drug leakage during long-term
w/v) with polydispersity index value > 0.1. The particle storage. Lyophilization of the curcumin nanosuspen-
size increases may be due to the crystallization of man- sion with lactose, trehalose, mannitol and glucose at a
nitol causing particle aggregation. In comparison with 10% w/v concentration produced a higher glass transi-
mannitol, other cryoprotectants like sucrose, trehalose tion temperature of approximately − 28 °C and − 43 °C
and dextran (0.5% w/v) demonstrate effective reduction and particle size was enlarged to approximately 275 to
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 12 of 31

Fig. 6 Evaluation of potential lyoprotectants for CS/DNA polyplexes using freeze–thaw (FT). Prior to (fresh) and after a freeze–thaw cycle, Z-average
diameter and PDI, or intensity mean diameter, of polyplexes formulated with up to 3% w/v mannitol (a), sucrose (b), dextran 5 kDa (c) or trehalose
(d) (1FT). Histidine concentration optimization for lyophilization of CS/DNA polyplexes (e). SEM images of CS/DNA polyplexes formulated
without or with excipients (f) [Reproduced from Veilleux et al. [57] with kind permission of the copyright holder, Elsevier, Amsterdam]

512 nm. The ratio of particle size after lyophilization to produces reconstitution solution at similar concentration
initial particle size (i.e. S ­f/Si) was approximately 3.05: using vial inversion method. The trehalose (5% w/v) and
5.67, respectively, and the higher ratio contributes to pre- sucrose (10% w/v) show a significant reduction in particle
cipitation that appears after redispersion. This indicates size ratio to ~ 0.99: ~ 1.05. Trehalose (5% w/v) considered
that these lyophiloprotectants failed to preserve the cur- as a suitable lyoprotectant based on reconstitution time
cumin nanoparticles. In contrast, kleptose (HP-β-CD), a and avoids aggregation during lyophilization process.
collapse temperature modifier at a 1.25% w/v concentra- Lyophilized gemcitabine core shell nanoparticles pos-
tion, produced a low S ­ f/Si ratio of ~ 1.22 and resulted in a sess high encapsulation efficiency (~ 40.5% w/w), small
transparent solution following redispersion. The presence particle size (243 nm) and PDI (0.16). The presence of
of kleptose and its amorphous characteristics enhances albumin on the core shell nanoparticles improves update

than GEMCITE® marketed formulation (~ 51%). The

the stability of the curcumin nanoparticles and avoids efficiency to 91% MCF-7 cell line comparatively higher
aggregation. The presence of the lyoprotectant kleptose
promotes the long-term stability of curcumin nanoparti- IC50 value was calculated on third day of study and was
cles at the lowest concentration in support with stabilizer found to be 16 µM. The process of lyophilization signifi-
and costabilizer through its amorphous nature [68]. cantly improves the stability, encapsulation and cytotox-
Chitkara and colleagues developed a lyophilized core– icity potential of gemcitabine [69].
shell nanoparticle preparation using gemcitabine and
assessed its stability, in vitro and in vivo cytotoxicity stud- Lyophilized drug product development
ies on MCF-7 cell line and dimethyl benz [α] anthracene Development of an oral drug delivery system
(DMBA)-induced breast cancer models. Hydrophilic Since its inception, the oral route is considered as the
bovine serum albumin and hydrophobic PLGA were used best route for the delivery of many drugs or active mol-
as core and shell materials, respectively. The core–shell ecules [70]. From the component of patient conformity,
formulation loaded with gemcitabine was lyophilized the oral route is much more justifiable [71]. It can be used
using different lyoprotectants, such as inulin, lactose, for systematic as well as local drug delivery applications.
mannitol, sorbitol, sucrose, and trehalose, at 5 and 10% Principally, it provides an abundance of merits, includ-
w/v concentrations. The findings indicated that trehalose ing non-invasiveness, ease of use and simplicity for self-
at 5% w/v increased the reconstitution of the formulation administration [71]. The limitations of oral drug delivery
in the water compared to other lyoprotectants. Sucrose system include the process factors, drug release, content
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 13 of 31

analysis and stability. The formulation scientist focuses shows a low disintegration time (less than 1.5 min) and
on imparting better drug release characteristics to oral enhanced dissolution rates of ODT. Moreover, it pro-
formulations. The process factors include uneven particle vides 100% drug release within 6 min. Hence, the use of
size, low flowability, less surface interaction or formation the lyophilization method for the development of gum-
of complex due to ionic species available. There is con- based ODTs will open a new path for oral drug delivery
stant need to alleviate the processing factors in designing applications [79]. A similar line of work reported by Bae
solid dosage forms, and freeze-drying technique may be et al. explored hepato-peptide-loaded ODT formulations
helpful for such improvements. developed for the treatment of neurogenic bladder dys-
Presently, the involvement of freeze-drying is function prepared using lyophilization method. It shows
immensely important, with an ongoing quest on the an extremely low disintegration time (30 s) and rapid
development for the oral drug delivery systems [72]. The release of peptides (almost 97%) within 15 min. Addition-
earlier reports suggest that lyophilization is known as ally, it shows good stability in different stability zones for
the desiccation process wherein favourable component 30 days. The use of sorbitol avoids structural collapse or
prosperities are ascribed to the freeze-drying that helps shrinkage of the lyophilized tablet during the lyophiliza-
in the preparation of stable pharmaceutical products tion process [80]. In another study, deferasirox-based
[73]. The multiple parameters affect the product stability directly compressed and lyophilized fast disintegrating
in solid state such as flow characteristics, moisture con- tablets were prepared. Notably, the lyophilized tablet
tent and processability with additional emphasis on pro- shows a low disintegration time (5 s) and a faster dissolu-
cess-related and formulation-related variables [74]. For tion rate in comparison with the compressed form of the
introducing new products for the oral administration, it tablet. The lyophilization process embeds high porous
is interesting to know the detailed process and princi- structure in the tablet matrix, which was absent in the
ples of freeze-drying [73]. In recent decades, numerous case of compared with the directly compressed tablet.
types of lyophilized products have been developed for Accordingly, it resulted in enhancement of bioavailability
the delivery of drug molecules via the oral route [75]. In as well as rapid onset of action. Overall, it could be a bet-
2020, Al-Amodi and coauthors developed febuxostat- ter dosage form to improve patient compliance compared
loaded self-nanoemulsifying delivery systems (FSNEDS) to the currently engaged dosage forms for the treatment
followed by the preparation of lyophilized tablets for of geriatric, paediatric populations [81]. Ahmed and cow-
transmucosal delivery applications. The lyophilization of orkers report rosuvastatin-loaded pullulan-based tablets
FSNEDS provides a porous structure and fluffy matrix prepared using lyophilization process. The drug-loaded
that help to reduce the disintegration time of tablets as chitosomes help to improved bioavailability as well as
well as maximum drug release. As a result, it increased anti-hyperlipidaemic and antioxidant activity. The tab-
the oral bioavailability of febuxostat up to 146.4%. The let rapidly disintegrates within 1.48 min because of the
lyophilization process helps to enhance the bioavail- distinct porous surface (Fig. 7a) and sponge-like inner
ability and pharmacokinetic parameter of poorly soluble structure of the freeze-dried tablet (Fig. 7c), whereas the
drug candidate [76]. Salama and coauthors designed an marketed directly compressed tablet formulation exhibits
oral disintegrating tablet (ODT) using the atorvastatin a smooth surface (Fig. 7b) and compact inner structure
calcium-based dry emulsion technique followed by lyo- (Fig. 7d), as confirmed by scanning electron microscopy.
philization. In brief, gelatin has been used as a binder for In the future, the lyophilization technique can be used as
lyophilized ODT and as a matrix former, whereas sugar a suitable alternative for the development of fast dissolv-
alcohol plays the role of cement for the porous structure ing tablets along with improvement of oral bioavailabil-
of the lyophilized matrix. In addition, it provides excel- ity and pharmacodynamics as well as pharmacokinetic
lent disintegration and a good release profile for ODT activity [82]. Hosny and coauthors reported the prepa-
that results in the enhancement of oral bioavailability ration of sildenafil citrate-loaded self-nanoemulsifying
[77]. Gulsun and coauthors reported terbutaline sulphate system-based oral lyophilized flash tablets. A mixture
ODTs by freeze-drying method and comparative evalua- of clove oil/oleic acid (10%) was selected as the oil base,
tion of directly compressed tablets. The lyophilized ODT whereas Tween 20 (60%) and propylene glycol (30%)
demonstrates a low disintegration time (11 s) and quicker were selected as the surfactant and cosurfactant that
dissolution profile in compared to the directly com- forms nanoemulsion (65 nm). The flash tablet was pre-
pressed tablet (11 min). Overall, it will help to improve pared using fumed silica, hydroxyl propyl methylcellulose
patient compliance and can be a suitable substitute for (HPMC) and sodium starch glycolate (SSG) followed by
conventional oral dosage forms [78]. Huanbutta and col- freezing of the mixture. Freeze-drying was carried out
leagues developed the tamarind seed gum-based ODT at − 45 °C for 24 h, which resulted in the formulation of
of diclofenac sodium using the freeze-drying method. It patch-like structures. The porous powder formed was
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 14 of 31

Fig. 7 Scanning electron microscope (SEM) images for the surface (a) and inner structure (c) of the chitosomes freeze-dried tablets and marketed
drug product (b and d), respectively [Reproduced from Ahmed et al. [82] with kind permission of the copyright holder, Elsevier, Amsterdam]

compressed into tablets by adding a suitable amount of distribution of the nanosuspension. The highly porous
diluents and disintegrating agent. The disintegration structure of the lyophilized nanosuspension matrix
time was decreased to 3 s, while flash tablet releases 68% resulted in rapid disintegration as well as dissolution
of sildenafil within the first 1 h. The study suggests that enhancement [85]. Sharma and colleagues report the
the bioavailability of sildenafil was improved after lyo- curcumin-loaded lipid nanoparticles for oral delivery
philization process in comparison with marketed tablet. prepared using lyophilization method. The lyophiliza-
In summary, it can be used as an outstanding approach tion of the formulation was accelerated in the presence
for enhancing the bioavailability of water-insoluble drugs of 10% w/w trehalose. Mainly, trehalose has been used
[83]. as a good cryoprotectant due to its high glass transition
Powar and Hajare developed a lyophilized nanosus- temperature, lack of chemical reactivity, poor hygrosco-
pension of ethinyl estradiol using a quality-by-design picity, etc. Trehalose imparts good physical and chemi-
approach. An ethinyl estradiol containing nanosuspen- cal stability to formulations [86]. Li and coauthors
sion was prepared by using a high-pressure homog- reported the preparation of curcumin-HP-β-CD inclu-
enizer followed by freeze-drying with mannitol act as sion complexes using a cosolvency method followed
cryoprotectant. Significantly, it provides a porous drug- by lyophilization. As a result, HP-β-CD improves the
loaded lyophilized nanosuspension that enhances the oral bioavailability of curcumin by 2.77-fold. Therefore,
stability and relative bioavailability by twofold [84]. the preparation of inclusion complexes at an indus-
Ibrahim and coauthors developed silymarin-containing trial scale can be feasible using the simple cosolvency
lyophilized nanosuspension-based tablets for immedi- and lyophilization method. In addition, this method
ate release applications. The use of mannitol provides can be more suitable for solubility enhancement and
the formation of a lyophilized cake with a porous struc- improving the oral bioavailability of poorly soluble
ture, which results in effortless conversion into suspen- candidates [87]. In another study, cosolvency-lyophi-
sion and preserves the actual nanosized particle size lization method-based encapsulation of thiostrepton
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 15 of 31

(hydrophobic antibiotic) was stabilized via steric micel- Freeze‑dried biopharmaceuticals

lar aggregates. Esparaza and coauthors reported that Biopharmaceuticals are organic entities that are popu-
phosphate-buffered saline (PBS) salts improved cake larly recognized as biological drugs that are derived from
porosity, accelerated the reconstitution time of lyophi- a biological organism by recombinant DNA technology
lized cakes and allowed efficient thiostrepton encapsu- or biotechnology and intended for the treatment and
lation [88]. Routray and colleagues prepared lyophilized prevention of diseases [14]. At present, biopharmaceu-
cinnacalcet hydrochloride-loaded solid lipid nanoparti- ticals have been highly valued as therapeutic agents that
cles using mannitol (100% w/w) as a matrix former. The include peptides, enzymes, monoclonal antibodies, oli-
particles with a lower size (less than 200 nm) give a high gonucleotides, nucleic acid derivatives, DNA prepara-
surface area and offer faster dissolution. In addition, tions, antibody‒drug conjugates, vaccines, immunosera,
lyophilization of solid lipid nanoparticles provides good hormones, therapeutic proteins, etc. [2, 92]. These bio-
redispersibility and an enhanced dissolution rate. As a therapeutic agents possess better specificity and potency
result, lyophilized solid lipid nanoparticles improve the than small molecules but offer many challenges, such as
pharmacokinetic parameters and oral bioavailability of stabilization of their highly labile, delicate, unstable, sen-
cinnacalcet hydrochloride compared to the pure drug sitive structures. In intent to provide stable biopharma-
and optimized solid lipid nanoparticles [89]. ceutical formulations, low-temperature lyophilization
Textural effect of freeze-dried formulation was assessed or cryodesiccation is the most popular processing tech-
using image processing and artificial intelligence. Fol- nique in practice. The technique improves stability along
lowing freeze-dried carrot was taken as case study to with thermolabile, and sensitive biopharmaceuticals to
understand the use of AI in freeze-drying process. The manufacture and serve stabilized biotherapeutic prod-
digital camera images of carrot and freeze-dried car- ucts for the betterment of mankind as presented in Fig. 8
rot was extracted and converted to colour channel L, a, [15]. Freeze-drying or lyophilization is recognized as a
b, R, G, B, X, Y and Z creates 181 channels for assess- gentle process to concentrate or dry biologically active
ment. Machine learning algorithms find 20 effective tex- substances.
tural parameters for each model. The Trees, Rules, Meta, Cryodesiccated biotherapeutic products provide
Lazy and functional groups classify the models and pro- several benefits, such as ease of handling and storage,
vide highest accuracy for the determination of difference reduction in transportation costs, and improved stabil-
between carrot and freeze-dried carrot [90]. ity without damaging physical structure with retained
Residual moisture (RM) content is an important tool potency. Freeze-dried biopharmaceuticals may be recon-
in assessment of freeze-dried product. Machine learn- stituted very quickly and easily, which is particularly fea-
ing and NIR spectroscopy were used to evaluate the level sible for vaccines and antibodies and mostly supportive
of RM as quantitative value. The linear regression and in the case of a medical emergency where quick admin-
neural network model were used to predict the RM with istration of medication is a need. The extensive demand
creation of architectural categorization. The learning step and usage of freeze-dried biopharmaceuticals have made
uses root-mean-square error for calculating RM with it a potential candidate likely to take over the dominant
supportive parity and absolute error plot use to visualize position in the global market. Biopharmaceuticals are
the effect. The effect of sucrose concentration (3, 6 9%) on one of the fastest-growing sectors in the pharmaceuti-
lyophilization was observed with comparative evaluation cal industry and are evidenced by the current scenario
of trehalose on arginine mixture. The 6% sucrose model of several lyophilized biopharmaceuticals available in
provides consistent effect for prediction of RM [91]. the global pharma market. Examples of some block-
The lyophilization process offers many merits, such as buster freeze-dried biopharmaceuticals are trastuzumab
rapid disintegration time, flow characteristics, improved (Genentech), etanercept (Amgen), rituximab (Biogen
dissolution rate, faster drug release, high stability and Idec), and infliximab (Janssen Biotech) [93]. Designing a
bioavailability. Moreover, the physicochemical character- protocol for the development of stable liquid prototype
istics of drugs and excipients can be modified by the use formulations is mostly the first step for the development
of lyophilization process. Additionally, the finished dos- of novel biopharmaceuticals; moreover, this approach
age form characteristics can be changed by use of modi- has been carefully studied and consistently practiced
fied process tactics such as use of lyophilization process. for the development of biopharmaceuticals in the biop-
Owing to this, lyophilization holds new positions in the harmaceutical industry [94, 95]. Figure 9 illustrates the
field of oral drug delivery systems. The improving appli- routine process associated with the manufacturing of
cability of lyophilization process in designing oral drug biopharmaceuticals.
delivery is pertinent and appealing with the introduction The production of typical biopharmaceuticals promi-
of the latest new dosage forms. nently involves upstream and downstream processes.
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 16 of 31

Fig. 8 The challenges of biotherapeutic agents and implementation of freeze-drying process to address this issue of the same

Upstream process (step 2) signifies the method, which purification process of the biological product from the
involves the microbes or mammalian cells to cultivate, substrate to a desired final product. Each step involved
harvest, and produce the desired substance or other bio- in the purification is adequate to remove various types
molecules associated with necessary steps such as choice of impurities [96]. Downstream processing generally
of a cell line, selection of culture media, regulation of encompasses steps such as extraction or isolation, puri-
growth parameters and process parameter optimization fication and polishing (removal of particular contami-
to execute optimal conditions for the growth of cell cul- nants and unwanted target biomolecules that may have
ture in a well-controlled condition. The major objective been generated during the process of isolation and puri-
of the upstream process is the transformation of starting fication). The primary objective behind carrying out the
material into the required metabolic product. The down- downstream process is to produce the purest product
stream process (step 3) elaborates the separation and with maximum yield in a short period at an efficient cost.
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 17 of 31

products, are available as lyophilized formulations. Lyo-

philized marketed biopharmaceuticals have several
advantages over liquid formulations, including enhanced
long-term storage/shelf life and superior stability dur-
ing transportation and storage. Moreover, the improved
stability of lyophilized biopharmaceuticals often avoids
the emergence of stability concerns during development
or at the commercial stage. These elements make lyophi-
lized formulations an outstanding commercial candidate
[99, 100]. Since unstable therapeutic proteins, such as
interferon or haemophilia factors, are lyophilized, which
retains therapeutic potency after lyophilization and allow
to store at ambient temperature. The use of lyophilization
improves ease of storage, handling, shipping, transporta-
tion, administration and distribution of the biopharma-
ceutical product, which makes the methodology more
convenient for underdeveloped countries or regions
where the cold chain has yet to be established [101]. Lyo-
philization of biopharmaceuticals is a well-established
process and is commonly used in the pharmaceutical
industry for stabilizing high-cost, sensitive, and labile
bioproducts. A few important lyophilized bioproducts
are discussed below, such as vaccines and proteins.

Vaccines
The development of the freeze-dried vaccine started in
the nineteenth century. In 1909, the first freeze-dried
smallpox vaccine formulation was reported. Thereaf-
ter, a multidose dried powder formulation of Bacillus
Calmette–Guérin (BCG) and the smallpox vaccine was
developed for immunization. Later, in 1940, the "Dry
Vax" smallpox vaccine was introduced in several coun-
tries for mass immunization. In addition, in 1960, a lyo-
philized rabies vaccine was successfully launched that
extended the shelf life of the vaccine and made a vital
impact on providing postexposure protection in rabies
outbreaks [102]. There are several freeze-dried vaccines
available in the global pharma market that possess the
Fig. 9 Steps involved in the manufacturing of biopharmaceuticals potential to trigger the required immune response. Some
blockbuster marketed freeze-dried biotherapeutic vac-
cines are listed in Table 4.
Step 4 represents the obtained purified biological prod- Generally, cryodesiccated live attenuated viruses fre-
uct that can be preserved as frozen liquid, crystallized quently need a complex formulation that involves vari-
solid and ready-to-fill formulated liquid. Additionally, the ous excipients [104]. Such lyophilized vaccines consist
consequential process may result in a fill-finish operation of multiple antigens, a bulking agent to produce prod-
that elaborates the development of freeze-dried biothera- uct appearance up to the mark, a buffer to set a pH at
peutics [97, 98]. a required value, tonicity modifiers, and stabilizers to
Many biopharmaceuticals are not satisfactorily pro- impart the required protection to the viruses against
viding stability over 2-year period, drastically decreas- chemical and physical degradation throughout manufac-
ing shelf life in a liquid state. Currently, more than 50% turing and storage [104, 108]. The freeze-drying process
of marketed biopharmaceuticals, such as monoclonal mainly accomplishes the generation and extension of the
antibodies, vaccines, therapeutic proteins and plasma stability of the vaccine, yet freezing and the low-temper-
ature dehydration operation themselves are very stressful
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 18 of 31

Table 4 Examples of freeze-dried vaccine, manufacturer, and administration route of globally available viral and bacterial vaccines
[103–107]
Vaccine Abbreviation Manufacturer Recommended Trade name (Brand) Route of administration
storage

Haemophilus influenzae type b vaccine HiB GSK 2–8 °C HIBERIX Intramuscular

Measles, mumps, and rubella vaccine MMR MERCK 2–8 °C M-M-R II Subcutaneous
Meningococcal polysaccharide vaccine MPSV4 Sanofi Pasteur 2–8 °C Menomune-A/C/Y/W-135 Subcutaneous
(quadrivalent)
Measles and rubella virus vaccine MR Serum 2–8 °C MR-Vac Subcutaneous
Yellow Fever Vaccine YF Sanofi Pasteur 2–8 °C Yf-Vax Subcutaneous
`Bacillus Calmette–Guerin BCG Serum 2–8 °C TUBERVAC Intradermal
(Bacterial Vaccine)
Rabies Vaccine – Serum 2–8 °C RABIVAX-S Intramuscular
Measles Vaccine MCV Serum 2–8 °C M-Vac Subcutaneous
Measles, Mumps and Rubella Vaccine MMR Serum 2–8 °C TRESIVAC Subcutaneous
Rubella Vaccine - Serum 2–8 °C R-VAC Subcutaneous
Rotavirus Vaccine RV Serum 2–8 °C ROTASIIL Oral
Influenza Vaccine (Trivalent) IIV3 Serum 2–8 °C NASOVAC-S Intranasal
Haemophilus b Conjugate Vaccine HiB Sanofi Pasteur 2–8 °C ActHIB Intramuscular
Bacillus Calmette–Guerin strain BCG Serum 2–8 °C SII-ONCO-BCG Intravesical
Instillation
measles and rubella vaccine MR Merck 2–8 °C M-R-VAX II Subcutaneous
Measles, Mumps, Rubella and Varicella MMRV Merck 2–8 °C ProQuad Subcutaneous
Meningococcal,Hib (Groups CY-Hib) HibMenCY-TT GSK 2–8 °C MENHIBRIX Intramuscular
Rotavirus (Monovalent) RV1 GSK 2–8 °C ROTARIX Oral
Herpes Zoster RZV Merck 2–8 °C ZOSTAVAX Subcutaneous
Varicella VAR Merck 2–8 °C VARIVAX Subcutaneous
Rabies – Novartis 2–8 °C RabAvert Intramuscular
Rabies – Sanofi Pasteur 2–8 °C Imovax Intramuscular

processes for product development [109, 110]. To mini- share the lyophilized biopharmaceutical market [113],
mize the negative impact produced by the procedure, which covers various types of antibodies (e.g. mono-
the well-designed optimized formulation and steps play clonal, fused, domain), small therapeutic proteins (e.g.
a crucial role. Optimization of the formulation suggests enzymes, hormones, cytokines), and complex biologi-
a method for establishing the best combination of excipi- cals (e.g. pegylated proteins, antibody‒drug conjugate).
ents by studying various permutations and combinations The large structure, delicate nature and different physi-
that will have a specific role concerned either with the cal and chemical properties of therapeutic proteins usu-
process or with the protection of the key component dur- ally result in poor stability in aqueous solution and thus
ing and subsequent lyophilization [25, 111, 112]. Several create a challenge to make formulations stable [114,
optimization protocols and stabilizing agents play a vital 115]. Lyophilization is the best-known method for the
role in the stabilization of the freeze-dried live, attenu- stabilization of unstable liquid protein biopharmaceu-
ated vaccine manufacturing process. ticals into a solid-state dosage form. The lyophilization
process relies greatly on the protein moiety and excipi-
Proteins ents used in the formulation. Admitting protein formu-
Over the past two decades, outstanding growth has been lations developed in assorted forms to continue stability.
marked in the development of therapeutic proteins, while Certainly, the nature of proteins is affected by different
recombinant DNA and hybridoma techniques play a degradation pathways during the freeze-drying process,
crucial role in the advancement of such biopharmaceu- which may be due to stress encountered by the respective
ticals. In 1982, the first approved recombinant protein method that affects the quality of therapeutic proteins
was insulin, and sequential biotherapeutic growth hor- [116]. Due to chemical degradation, protein products
mone and blood-clotting factor VIII were also reported. come across the destruction of covalent bonds and
Presently, almost 30% of therapeutic protein products undergo hydrolysis and oxidation reactions [117, 118].
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 19 of 31

The list of commonly experienced chemical instabilities storage conditions is recommended for long-term stabil-
includes disulphide exchange, elimination, etc. Particu- ity purposes [131].
larly physical instabilities include denaturation, aggrega- Regular excipients with distinct properties that impart
tion (association of native monomers, surface-induced more than one property are selected during preparation.
aggregation, etc.), precipitation (haziness or cloudiness), Excipients such as buffers help to balance the variation in
and surface adsorption (adsorption of native or partially pH, surfactants can manage stress-induced aggregation
unfolded protein) [119–121]. All the physicochemical and are used as bulking agents, oxidation is prevented
stress factors revamp the final product as well if an indig- using antioxidants, low molecular weight carbohydrates
enous structure is not preserved, which then leads to an are used to maintain physiological osmolality, etc. Many
emulated ambiguous finish product. However, due to the others are used to improve stability along with cryopro-
defined stability of proteins, freeze-drying results in pro- tective action [132, 133]. The stability assessment of dried
tein unfolding and subsequent destabilization. However, formulations is also investigated using product residue
one of the most compelling properties of proteins is the content as an alternative measure for understanding
three-dimensional conformation of biological activity. physical factors [134]. At the global level, the Interna-
This involves complexity and is oftentimes associated tional Pharmaceutical Excipient Council (IPEC) provides
with weak forces [122–124]. The freeze-drying process goods manufacturing practices and good distribution
imparts beneficial properties and promotes stability. The measures. The IPEC regulates the number, concentration
stability of a protein is an imperative characteristic and and types of excipients used during the process of manu-
is subjected to assorted designs of other stress elements, facturing. The excipient concentration in protein-based
such as pH, temperature, light and organic solvents. The formulations was slightly higher than in other formula-
aggregation and degradation of protein due to the stress tions. The choice of excipients in protein-based formula-
effect were set to appear in the final formulation, while tions is dependent on the type of dosage form, route of
efficacy was analysed after delivery [125, 126]. Moreo- administration, content and clinical acceptance [135].
ver, all these stresses often correlate with degradation Various types of stress conditions are applied during
and make them a favourable barometer of the instability the drying process and lead to changes in the integrity
and quality of a finished product. The instability of pro- of the formulation. The conformation changes in the
teins has also been analysed via intermolecular bonds, polymer structure during drying lead to destabilization
morphology and hydrophobicity, but the process is very of particulate matter. Destabilization can be avoided by
complex [126, 127]. If the process is not controlled, it studying process factors, or the use of single or combi-
leads to a difference in integrity, which appears to curtail nations of stabilizers may be considered a suitable alter-
therapeutic competence and activate immunogenic reac- native to alleviate stress [136]. The stabilizer can protect
tions in patients and hence necessitates amalgamation the protein through a distinct mechanism out of these
of the distinct analytical technique. As proteins undergo two major mechanisms of vitrification and water replace-
numerous instabilities, the delivery system selected must ment. Vitrification involves inactive forms of protein
magnify the storage stability and perpetuate in vivo effi- matrix composed of stabilizer, which counters the struc-
cacy. Hence, for the design and expansion of therapeutic tural changes. Water replacement centred on the con-
proteins, stability is of major concern and is given defi- struction of hydrogen bonds between the hydroxyl group
nite relevance in the preformulation studies [128]. of the stabilizer and the polar group of the protein. The
The freeze-drying process prevails over the long-term change in hydrogen-bonded interactions helps to main-
stability argument and is beneficial for shipping when tain the structure of the protein and thereby stabilize it
correlated to liquid protein therapeutic products. The in the environment [117, 137]. Vitrification theory mainly
factors elaborated above are based on the type of excipi- exhibits kinetic stabilization, although water replacement
ent composition used during preparation, and pertinent serves a thermodynamic part. Kinetic stabilization is
operational process parameters are important consid- firmly associated with T ­ g, which is necessary for conveni-
erations when dealing with the stability of protein-based ent storage and transport conditions [138].
products [129]. Depletion of water content and molecu- In the solid state, a glass matrix containing excipients
lar potency during lyophilization may shorten as the and proteins is analysed for thermomechanical analy-
physicochemical properties of proteins may be altered sis. The Tg value suggests a reduction in the rate of deg-
during storage [130]. Following regulatory guidelines, radation of proteins in the presence of excipients. The
the list of excipient use for parenteral application must protective action of the excipient is dependent on the
be of supreme quality, sterile and stable during the prod- concentration of the excipient used for encapsulation
uct life cycle. The right choice of excipient and most ideal [139, 140].
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 20 of 31

There are distinctive illustrations of the stabilization of by far the most efficient way to maintain the therapeutic
proteins in aqueous environments. The molecular states efficiency of protein medicines [101].
of proteins in solid forms explore the functional proper- Lyophilization is thought to be a pleasant way to con-
ties of formulations. In biopharmaceuticals, notably, the centrate or dry pharmacologically important chemicals.
secondary and tertiary structures of the protein can be For more than 40 years, enzymologists have used it as a
identified through methods that build on structural char- normal practice, and pharmaceutical companies have
acterization in solid forms of protein in the interim of followed suit. This is because enzyme/protein/peptide-
formulation development. Hence, significant formulation based medications are much more durable in solid form
protects the solid states of the native structure of a pro- (as opposed to solution). As a result, freeze-dried parti-
tein. The adaptation of a proportionate path can direct cles have benefits in terms of preservation and transpor-
the solid state of the protein and eliminate inadequate tation. They also extend the shelf life of the product at the
formulations for stability studies [141, 142]. The develop- endless mode. Sublimation and desorption remove water
ment and formulation of stable biotherapeutics require from such a frozen sample during freeze-drying (lyophili-
more intellectual effort than the conventional method of zation). It is a three-step procedure that includes chilling,
small molecule formulation. A great deal of research con- initial drying, and secondary drying. Although cryopro-
cerning the delicate, labile, complex, large, and unstable tectants can preserve proteins against denaturation even
structure of biopharmaceuticals evidenced the develop- during the early phases of their development, lyophilo-
ment of freeze-dried biopharmaceuticals. Lyophilization, protectants are required to avoid protein deactivation
a quickly growing technique, has a high potential to during the drying [114, 145, 148–150]. By turning the
provide stability and retain the therapeutic efficacy of mixture of volatile particles into solid powder as well as
sensitive biopharmaceutical products. The pace of freeze- cake by the sublimation process, lyophilization or freeze-
dried biopharmaceutical development has opened the drying is the preferred method for improving the long-
doors to accepting new opportunities and challenges term durability of small-molecule [39, 151–154]. The
to stabilize and make better biotherapeutics. The profi- majority of the literature on pharmaceutical lyophiliza-
ciency to govern and operate during the lyophilization tion focuses on the drying of medicinal proteins. Further-
process will benefit the establishment of competent biop- more, research into the utilization of lyophilization for a
harmaceutical products with ameliorating stability. variety of additional medicinal purposes has begun [144].
In another study, the researcher reported lyophilization
Freeze‑dried injectables of a parenteral rhEGF (recombinant human epidermal
Freeze-drying grew from a research laboratory to a well- growth factor) formulation. Excipients were screened
established technology for the protection of biophar- using both unannealed and annealed drying proce-
maceutical molecules. The technology was established dures. Excipients and formulations were chosen based on
in the twentieth century [143, 144]. Fundamental prin- freeze-dry microscopy, which was also utilized to define
ciples in formulation development, as well as process freeze-drying parameters. The chemical stability, protein
design, are being introduced throughout this era. Even structure, and bioactivity of excipients were assessed uti-
though we are now in the twenty-first century, lyophiliza- lizing a comprehensive range of analytical procedures to
tion remains the preferred method for a diverse variety assess their effect on freeze-drying recovery and dried
of pharmaceutical applications, and it is still undergoing product stability at 50 °C. The inclusion of sucrose or
continuous improvement and extension [145]. Several trehalose increased the durability of rhEGF after freeze-
difficulties have arisen, and even well-accepted ortho- drying. The maximum stability was attained by adding
doxy has been called into question [144]. The growing dextran, sucrose, trehalose, or raffinose to the dry sample
use of complex structures of bioactive compounds (e.g. after it had been stored at 50 °C. Mostly during freeze-
recombinant proteins, peptides, polynucleotides), as well drying as well as delivery processes, the chosen compo-
as supramolecular drug carriers (e.g. liposomes) in phar- sition blend of sucrose and dextran may avoid protein
maceuticals, highlights the importance of preparations degradation. In dried formulations, the degradation rate
that enable products to be stored and distributed while measured by RP-HPLC may be reduced by 100 times at
maintaining their standard functionality [114, 146, 147]. 37 °C and 70 times at 50 °C when compared to the liquid
Even at chilled temperatures (2–8 °C), several protein formulation. These findings suggest that the freeze-dried
medicines have a significant proclivity for physical and product is a suitable alternative for rhEGF stabilization
chemical deterioration in aqueous systems and are there- [146].
fore not stable enough just to withstand preservation Nanomaterials composed of human serum albu-
for the 1–3 years required for commercial goods. When min (HSA) were freeze-dried in the presence of several
compared to alternative formulations, freeze-drying is cryoprotective compounds, and their physicochemical
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 21 of 31

features were studied following reconstitution. The vari- prelyophilization composition and (2) the headspace
able concentrations of cryoprotectants like sucrose, pressure in the final lyophilized container was reduced.
trehalose and mannitol were used during the process. The physical properties of cakes formed from prelyophi-
The cryoprotectants avoids aggregation of PEGylated lization products containing a range of TBA concentra-
nanoparticles via freeze-drying approach. Particle devel- tions were studied. Under stress conditions, the stability
opment was detected throughout all freeze-dried compo- of antibodies containing TBA in liquid and lyophilized
sitions in the presence of cryoprotectants. Clustering of forms was assessed. At a TBA content of 5% w/v, recon-
HSA nanomaterials during the freeze-drying technique stitution time was reduced by more than half (> 50%). A
was inhibited in the presence of sucrose, mannitol, and headspace pressure was less than 50 Torr in the lyophi-
trehalose. Even though all of the additives were found to lized vial resulted in a 50-per cent reduction in reconsti-
be appropriate stabilizers for freeze-drying HSA nano- tution time [156]. The list of commonly used additives in
materials, sucrose and trehalose outperformed in terms freeze-dried formulations has been described in detail by
of long-term storage stability [150]. Mehmood et al. [157].
A group of researchers developed a parenteral dosage
form for EO9-encoded experimental cytotoxic medica- Lyophilization of nanoderived therapeutics
tion. The solubility and stability of EO9 in water were sig- Nanoderived therapeutic agents have a better com-
nificantly improved using freeze-drying. Quality control pliance structure than the conventional therapeutics
of the lyophilized formulation revealed that the fabrica- available [158]. The multiple number of nanoderived
tion process did not affect the integrity of EO9. When- therapeutics or in a simple term referred to as nanomedi-
ever stored at + 4 °C in a dark setting, the preparation is cine [159]. Nanoderived therapeutics have the capability
stable for one year, according to accelerated stability test to work as theranostics and can have dual functionality
data [155]. as diagnostics and therapeutics for specific disease types.
Ayen and colleagues developed doxorubicin-loaded Designing nanomedicine while understanding their phys-
(PEG)3–PLA nanopolymersomes. Nanopolymersomes icochemical characteristics and maintaining their integ-
were made using the nanoprecipitation method and rity throughout the formulation cycle is a challenging
lyophilized in the presence of several lyophiloprotect- task for formulation scientists. The major step in com-
ants before being assessed for physicochemical quali- mercialization is the formulation cycle, which is con-
ties. Finally, the findings reveal that doxorubicin-loaded sidered a benchmark. The interaction of nanocarriers at
micropolymersomes can be lyophilized using inulin (5% the biointerface is also an important component in for-
w/v) without losing their physicochemical features and mulation development, and recent studies have defined
can be preserved at 2–8 °C for more than a year [145]. computational strategies to comply with the selectivity
Butreddy et al. (2020) provided a broad overview of the of polymers. Most of the nanoderived formulations are
lyophilization process and examined various critical con- available as dispersions, or during formulation develop-
cerns and product development features, such as formu- ment, liquid suspensions are prepared [160].
lation, process optimization, container closing systems, The dispersion systems are extremely unstable and
scale-up principles and medicinal product quality [151]. deteriorate the encapsulated actives [161]. The dis-
Mockus and coworkers show that the effect of QbD persed phase particles aggregate due to charged species,
method could be used in the process parameter develop- hydrolysis, uneven particle distribution of particles, and
ment of a small molecule freeze-dried injectable medi- denaturation in the presence of solvent, which may pos-
cation. The model ingredient was sodium ethacrynate. sibly result in premature drug leakage. The instability
The main breakdown products of sodium ethacrynate may be harmful, as it leads to toxic effects after inges-
are dimers formed by Diels–Alder condensation in the tion. The alternative approach to improve the stability
freeze-dried solid-state and hydrolysis of the unsaturated of dispersed nanomedicine is conversion into a dry solid
ketone in an aqueous solution [39]. component by removing, extracting or evaporating sol-
In another study, Franzé and coauthors examine the vents by various particle engineering approaches, such
key production parameters that can ensure an output as spray drying, freeze drying, spray freeze drying, tray
with desirable properties while also increasing the pro- drying, and vacuum drying. Freeze drying is one of the
duction process effectiveness [153]. most promising methods to improve the integrity of par-
Prolonged reconstitution durations for lyophilized ticulate matter and stabilize particles in the solid state.
biomaterials with protein-rich concentrations can be The removal of solvent in the sublimation state (in the
inconvenient for the end user. Luoma and coauthors vapour state) and via desorption is also called the lyo-
outlined two strategies for reducing the reconstitution philization process and includes primary and secondary
period: (1) tert-butyl alcohol (TBA) was included in the drying, respectively. The method has been extensively
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 22 of 31

used in pharmaceuticals and the food industry. Lyophi- was used as cryoprotectant, which retains the spherical-
lization improves the stability of nanoderived therapeutic shaped prepared nanoparticles effective for storage with
agents and improves the shelf life of encapsulated actives improved release characteristics [165].
[162]. Major limitations of nanodervied therapeutic are Biological actives such as messenger RNA (mRNA)
aggregation of colloidal mass, and formation complex require deep freezing temperature to stabilize the formu-
may diminish the therapeutic potential of loaded actives. lations like vaccine. Maintaining deep freezing tempera-
The presence of organic solvent may interfere in the drug ture is challenging task as it creates complications during
content analysis as traces of solvent decrease the total storage, distribution and increase cost of accessibility in
concentration of actives upon storage like in the case low-income countries. The lipid-encapsulated mRNA
of liposome presence of chloroform decrease the active nanoparticles (mRLNPs) were prepared using spin freez-
concentration of bioactives upon storage. Other drying ing technique to provide continuous manufacturing
methods like surface drying, spray drying, tray drying, air modalities. The high ionizable weight ratios between
drying, etc., drastically modify the morphological char- lipid and mRNA prevent premature release of bioactives.
acteristics of nanomedicine, which hamper the release The phosphates and Tris buffer were used during lyophi-
mechanism. Additionally surface-anchored material may lization to maintain the stability. The transfection prop-
degraded in case of alternative processing steps used for erties of mRLNP were increased after lyophilization and
targeted nanomedicine and decrease the stability and maintained the integrity of formulation up to 12 weeks
performance characteristics. The lyophilized method [166].
has considerable importance in the development of
nanomedicine, which preserves and protects the actives Patents and clinical status
encapsulated inside polymeric matrices. Along with mor- Cryogenic modifications are relatively important in the
phological characteristics was retained at controlled tem- development of drug delivery systems. It improvises the
perature processing. storage, transportation and handling of pharmaceuticals.
Targetability and cryoprotectant ability of hyaluronic Freeze drying was adopted to enhance the product char-
acid (HA) were analysed on PLGA nanoparticles and acteristics. For the past few years, freeze-drying tech-
liposome formulation. The HA increases interaction at niques were used in addition to conventional techniques
the polymeric and lipid surfaces and improves redisper- for tableting. Freeze-drying methodologies are robust
sion characteristics after lyophilization process. Classical and dissolve unique features of materials. The technique
saccharides require 10–20% of lyoportectant, while HA helps to explore the material characteristics in solid‒solid
concentration was lesser. High hydrophilic HA having as well as solute-solid interactions. The freeze-dried for-
variable molecular weight (4.8 kDa and 14.8 kDa) pro- mulations patented by the world-renowned manufac-
vides strong interaction with phospholipids. The high turer are highlighted in Table 5.
molecular weight HA shows least particle size less than Clinical investigation of freeze-dried formulations was
300 nm and less variability (less than 20%) before and better than other conventional dosage forms. Bavarian
after lyophilization. The HA considered as promising tar- Nordic scheduled a clinical phase II study to identify the
geting as well as cryopreservation characteristics [163]. bioequivalence between liquid frozen and freeze-dried
Targeting to ocular inflammation is a difficult task for formulations containing a smallpox vaccine. The trial was
the formulation scientist as multiple layers of barrier conducted for immunogenicity and safety against two
avoid the transportation of therapeutically active agent. comparative formulations. A similar comparative evalua-
The Licochalcon A (Lico A)-loaded PLGA NPs trans- tion updated in Table 6 provides the safety and efficacy of
ported across ocular barrier using cell penetrating pep- freeze-dried formulations against conventional formula-
tides B6 and Tet-1 prepared using lyophilization process. tions containing biologicals.
To mitigate physical stress, different grades of lyoprotect-
ants were used such as disaccharides, alcohol and sugar Conclusion
alcohol. The prepared nanoparticles converted to semi- Desiccation, lyophilization, freeze-drying and sev-
solid state at physiological condition due to the presence eral other terms have been devised for advance dry-
of temperature-sensitive polymer poloxamer 407. The ing techniques use for pharmaceutical product. Till
functionalized PLGA NPs support biocompatibility in date no alternative has proposed for lyophilization
Caco-2 cell studies, while RAW 264.7 cells show strong process and leading methodology adopted for biop-
anti-inflammatory response [164]. harmaceutical and nanoderived therapeutics. The
Isradipine-based PLGA nanoparticles were prepared QbD-based application is increasing, and regulatory
using the QbD approach and provide improved kinetics agencies are recommending the use of QTTP, CPP,
and stability to designed drug delivery system. Mannitol CQA and CMA for improving process characteristics
Table 5 Freeze-dried patented inventions (https://​paten​ts.​google.​com/)
Therapeutics/encapsulant Carriers/particulate system Title Inventor/assignee/applicant Patent application number

Aripiprazole Carboxymethyl cellulose, hydroxy- Controlled release sterile freeze-dried Otsuka Pharmaceutical Co Ltd CA2543242C
propyl or ethylcellulose, polyvinylpyr- aripiprazole formulation and inject-
rolidone able formulation thereof
Bioactive material comprises: cell, cellulose acetate phthalate (CAP), Dry glassy composition comprising Advanced Bionutrtion Corp US9731020B2
microbe, virus, protein, enzyme, vac- carboxy-methyl-cellulose, pectin, a bioactive material
cine, or a drug sodium alginate, starches, and modi-
fied starches, and cyclodextrins
Sugars and hydrolysed proteins Polysaccharide (cellulose acetate Stabilizing composition for biological Intervet International BV AU2013234931B2
phthalate (CAP), carboxy-methyl- materials
cellulose, pectin), disaccharides
(trehalose, sucrose, lactose), and car-
boxylic acids
Live attenuated virus Sugar and an amino acid stabilizer Dry formulations of vaccines that are Intervet International BV AU2014326855B2
Pardeshi et al. Future Journal of Pharmaceutical Sciences

room temperature stable

Tetrodotoxin Hydroxyethyl starch or hydroxypro- Stable freeze-dried pharmaceutical Wex Medical Ltd CA2529598C
pyl cyclodextrin, sucrose, lactose, formulation of tetrodotoxin
and polyglucose
Docetaxel Polyethylene glycol monomethyl Freeze-dried formulation of nano Simcere Pharmaceuticals Company JP2016528187A, US9683073B2
(2023) 9:99

ether and D, L-lactide polymer micelle of docetaxel and its Ltd

preparation method
mRNA molecule Protamine, Trehalose Dry powder composition comprising Curevac AG US10729654B2
long-chain RNA
Bendamustine, Vendamustine Mannitol Bendamustine pharmaceutical com- Cephalon Inc US20200237726A1, KR20170096221A
positions for lyophilization
Recombinant Clostridium botulinum Aluminum-salt adjuvant, Trehalose Process for preparing an immuno- University of Colorado US8808710B2, DK2829282T3
neurotoxin protein logically active adjuvant-linked dried
vaccine composition
Anti-tissue factor Histidine, sucrose, trehalose, mannitol, Antibody–drug conjugate (ADC) Genmab AS JP2019163319A, US20200246477A1
and glycine lyophilized formulation
Proteasome inhibitors Cyclodextrin, surfactant and bulking Lyophilized preparations of protea- CEPHALON, INC., CEPHALON FRANCE TWI484981B
agent some inhibitors
Medicinal substances (analgesic, anti- Gelatine (molecular weight Process to form an orally disintegrat- Merck Sharp and Dohme BV Intervet US9095516B2
inflammatory) of 2 × 104 g/mol) ing tablet for human use Inc
Anoectochilus roxburghii Xylitol, microcrystalline cellulose, Chewable tablet containing freeze- Minnan Normal University CN104783180A
polyvinylpyrrolidone, and magnesium dried anoectochilus roxburghii
stearate powder and preparation method
of chewable tablet
Metallic nanoparticles Solvents for dispersion Manufacturing method of coated OKUMURA Haruki JPWO2014057564A1,
material using freeze-drying method WO2014057564A1
Virus or viral protein subunit Non-polymeric sugar Method of obtaining thermostable Merck Sharp and Dohme Corp US9782470B2
dried vaccine formulations
Page 23 of 31
 Pardeshi et al. Future Journal of Pharmaceutical Sciences

Table 5 (continued)
(2023) 9:99

Therapeutics/encapsulant Carriers/particulate system Title Inventor/assignee/applicant Patent application number

Swine fever attenuated viruses Mucous membrane immunologic Oral attenuated freeze-dried vaccine Zhang Zhigang CN108969492B
adjuvant and immunopotentiator for swine fever and preparation
method thereof
Page 24 of 31
Table 6 Clinical trial updates for comparative bioequivalence studies for freeze-dried formulations (https://​clini​caltr​ials.​gov/)
Sr. no. Clinical trial phase Title Disease Sponsor company Status Identifier number

1 Phase II A Phase II Trial to Compare a Liquid- Smallpox Bavarian Nordic Completed NCT01668537
frozen and a Freeze-dried Formula- (August 2012–October 2020)
tion of IMVAMUNE (MVA-BN®)
Smallpox Vaccine in Vaccinia-naïve
Healthy Subjects
2 Phase I Bioequivalence Trial of Liquid Anovulation replacement therapy Merck KGaA, Darmstadt, Germany Completed (December 2014–July NCT02317809
Pardeshi et al. Future Journal of Pharmaceutical Sciences

Versus Freeze-Dried Pergoveris® 2018)

in Pituitary Suppressed Healthy
Premenopausal Female Subjects
3 Phase III Freeze-Dried MVA-BN® Lot Consist- Smallpox Bavarian Nordic Completed (October 2018–May NCT03699124
ency Smallpox Trial 2021)
(2023) 9:99

4 Phase I r-hGH Liquid Multidose Versus Recombinant Human growth EMD Serono Completed (December 2009– NCT01034735
Freeze-dried Multidose Bioequiva- hormone supplement October 2013)
lence Trial
5 Phase III A Study in Infants to Test Two Lot to lot consistency of human GlaxoSmithKline Completed (October 2006– NCT00382772
Preparations (Freeze-dried or Liq- rotavirus vaccine November 2016)
uid) of the Rotavirus Vaccine (HRV
Vaccine)
6 – Freeze-Dried Black Raspberries Oral Cancer Ohio State University Comprehen- Completed (July 2012–February NCT01504932
in Preventing Oral Cancer Recur- sive Cancer Center 2020)
rence in High At-Risk Appalachian
Patients Oral Cancer Survivors
7 Phase I A Study to Evaluate the Safety hereditary angioedema CSL Behring Completed (January 2013–April NCT01760343
and Pharmacokinetics of Two For- 2013)
mulations of C1-esterase Inhibitor
8 Phase I Pergoveris FD and Liquid China BE Bioequivalence study for pituitary Merck Healthcare KGaA, Darmstadt, Currently Recruiting (May 2021) NCT04899193
Study suppressed healthy premenopau- Germany
sal Chinese female
Page 25 of 31
Pardeshi et al. Future Journal of Pharmaceutical Sciences (2023) 9:99 Page 26 of 31

during freeze-drying process. ODT tablet characteris- Availability of data and materials
The data that support the findings of this study are available from the cor-
tics were highly improved after adopting the lyophiliza- responding author, upon reasonable request.
tion method with increasing in the process ability, flow
characteristics and overall performance. For creating Declarations
highly porous structure, the freeze drying is considered
as effective process outcome for biopharmaceutical and Ethics approval and consent to participate
This work does not contain any clinical or preclinical assessment and hence
pharmaceutical products. The integrity of biological did not require any ethical approval or participatory consent from volun-
component was well preserved with improved stabil- teers. Studies involving plants must include a statement specifying the
ity at ambient temperature supports ease of handling, local, national or international guidelines and legislation and the required or
appropriate permissions and/or licences for the study. The present review
transportation and long-term storage. Rate of aggre- article does not involve experimental part or did not use any plants or parts
gation in liquid as well as dispersed system is very of plants, and hence, permission or licence is not required for the study. It is
high while freeze drying alter process steps and avoid comprehensive assessment or earlier research reports.
complex formation. Furthermore, considering the Consent for publication
advantages of freeze drying, it is considered to viable The authors declare no conflict of interest.
alternative for pharmaceuticals, biopharmaceuticals
Competing interests
and nanodervied therapeutics (nanomedicine), novel The authors declare that they have no competing interests.
drug delivery systems, etc.
Author details
1
Department of Pharmaceutics, St. John Institute of Pharmacy and Research,
Abbreviations Palghar, MS 401404, India. 2 Department of Quality Assurance Techniques,
NPs Nanoparticles Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erand-
mAbs Antibodies wane, Pune, MS 411038, India. 3 Datta Meghe College of Pharmacy, Datta
QbD Quality by design Meghe Institute of Higher Education and Research (Deemed to Be University),
QTTP Quality target product profile Sawangi (Meghe), Wardha, MS 442001, India. 4 Department of Pharmaceu-
CMAs Critical material attributes tics, H. R. Patel Institute of Pharmaceutical Education and Research, Shirpur,
CPPs Critical material parameters Dist‑Dhule, MS 425405, India. 5 Department of Quality Assurance, R. C. Patel
CQAs Critical quality attributes Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur,
FMEA Failure mode effects analysis Dist‑Dhule, MS 425405, India. 6 Department of Pharmaceutics, RMD Institute
DOE Design of experiment of Pharmaceutical Education and Research, Pune, MS, India. 7 PES’s Modern
RSDs Response surface diagrams College of Pharmacy, Sector No. 21, Yamunanagar, Nigdi, Pune, MS, India.
8
FA Ferulic acid Industrial Pharmacy Laboratory, Department of Pharmaceutics, R.C. Patel
PLA Poly (lactic acid) Institute of Pharmaceutical Education and Research, Shirpur, MS 425 405,
PLGA Poly (lactic-co-glycolic acid) India. 9 Department of Pharmaceutics, Dr. Rajendra Gode College of Pharmacy,
HP-β-CD Hydroxypropyl-β-cyclodextrin Malkapur, Dist., Buldhana, MS 443 101, India.
PDI Polydispersity index
DNA Deoxyribonucleic acid Received: 7 September 2023 Accepted: 26 October 2023
PEG Polyethylene glycol
PVP Polyvinyl pyrrolidone
PVA Polyvinyl alcohol
TPGS D-α-tocopheryl polyethylene glycol 1000 succinate
DMBA Dimethyl benz [α] anthracene
ODT Oral disintegrating tablet References
SEM Scanning electron microscopy 1. Emami F, Shokooh MK, Mostafavi Yazdi SJ (2023) Recent progress in
HPMC Hydroxyl propyl methylcellulose drying technologies for improving the stability and delivery efficiency
SSG Sodium starch glycolate of biopharmaceuticals. J Pharm Investig 53:35–57. https://​doi.​org/​10.​
PBS Phosphate-buffered saline 1007/​s40005-​022-​00610-x
BCG Bacillus Calmette–Guérin 2. Mitragotri S, Burke PA, Langer R (2014) Overcoming the challenges in
rhEGF Recombinant human epidermal growth factor administering biopharmaceuticals: formulation and delivery strategies.
HSA Human serum albumin Nat Rev Drug Discov 13:655–672. https://​doi.​org/​10.​1038/​nrd43​63
TBA Tert-butyl alcohol 3. Bjelošević M, Pobirk AZ, Planinšek O, Grabnar PA (2020) Excipients in
FSNEDS Febuxostat-loaded self-nanoemulsifying delivery systems freeze-dried biopharmaceuticals: contributions toward formulation
stability and lyophilisation cycle optimisation. Int J Pharm 576:119029.
Acknowledgements https://​doi.​org/​10.​1016/j.​ijpha​rm.​2020.​119029
The authors are grateful to Management and Chairman, Dr. Rajendra Gode 4. Patel SM, Jameel F, Sane SU, Kamat M (2015) Lyophilization process
College of Pharmacy, Malkapur, (MS) India, for providing necessary facility. design and development using QbD principles. In: Jameel F (ed)
Quality by design for biopharmaceutical drug product development.
Author contributions Springer, New York, doi:https://​doi.​org/​10.​1007/​978-1-​4939-​2316-8_​14.
SRP and MPM contributed to the design of the study; SHL, DRT and MTH 5. Pardeshi S, More M, Patil P, Pardeshi C, Deshmukh P, Mujumdar A, Naik
collected the data/sample articles; SNN, YYS and AG performed the content J (2021) A meticulous overview on drying-based (spray-, freeze-, and
analysis and analysis the data; NSD, CVP and PKD drafted the paper; all authors spray-freeze) particle engineering approaches for pharmaceutical tech-
have read and approved the final manuscript. nologies. Dry Technol 39:1447–1491. https://​doi.​org/​10.​1080/​07373​937.​
2021.​18933​30
Funding 6. Yoon K, Narsimhan V (2023) Comparison of vial heat transfer
This work did not contain any supportive funding agency. coefficients during the primary and secondary drying stages of
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