By Dr. Megha Agrawal, Dr. William Cho, Dr. Shyamasri Biswas, Dr.

Kim Van Vliet, Contributing Editors

Lyophilization
Pushing the Limits
for Pharmaceutical
Biotechnology Products

yophilization method corresponds

to a vacuum technology based
dehydration technique that is frequently used in the Pharmaceutical and
Diagnostic Industries. It enables liquid
or slurry frozen biotechnology products
to be dried under vacuum. Lyophilization
method is pushing the limits for many
pharmaceutical biotechnology processes. The principal advantage of the lyophilization technology over conventional
crystallization, ltration, or precipitation
based drying methods is the preservation
of chemical and biological potency, homogeneity in the nal product, and ease
of dispensing/metering before the nal
packaging of the products. Lyophilization technology has recently received a
great deal of attention. This is proven by
the fact that prestigious Purdue University has created a new lyophilization consortium, LyoHUB (https://pharmahub.
org/groups/lyo) that is aimed to advance
lyophilization technology to make food,
pharmaceuticals and biotech products safer and more affordable to the society. As
the lyophilization technology becomes a
logical, time-effective, and cost-sensitive
option, the current pharmaceutical industry is looking for advancements in the lyophilization manufacturing services to bet-

6ACUUM4ECHNOLOGY#OATINGsMarch 2016

ter manage the growing number of drugs

that are currently being developed.
Important vacuum processes are involved to initiate and complete the lyophilization steps. A lyophilizing unit consists of a vacuum chamber that contains
product shelves capable of cooling and
heating containers and their contents (Figure 1). A vacuum pump, a refrigeration
unit, and associated controls are connected to the vacuum chamber [2]. To remove
solvent in a reasonable time, vacuum is

applied during the drying process. The

required vacuum level is typically in the
range of 50 to 100 bar. A two-stage rotary
vacuum pump is usually used to achieve
the required vacuum level. For advanced
lyophilization, large chambers with multiple pumps are used [2]. The process involves chemicals that are generally placed
in containers such as glass vials that are
placed on the shelves within the vacuum
chamber. Subsequently, the cooling elements within the shelves freeze the prod-

Figure 1. The Lyophilization unit containing various components. [Source: Nireesha et al 2013]
www.vtcmag.com

27

uct. The vacuum pump then evacuates the

chamber after the product is frozen and
the product is then heated. It is followed
by the transfer of heat by thermal conduction from the shelf, through the vial, that
ultimately reaches into the product [2].
Protein-based therapeutics is of importance in the biopharmaceutical industry
and considered effective clinical method
to treat a wide spectrum of diseases. Lyophilization generally results in improved
protein stability profile that is extremely
important for effective development of
therapeutics. In this regard, a lyophilized
silk fibroin hydrogel matrix (lyogel) for
the sustained release of pharmaceutically relevant monoclonal antibodies was
described [3]. Researchers demonstrated
that lyophilization of antibody-loaded silk
hydrogels significantly improved longterm antibody release properties over the
parent hydrogel material [3]. They proposed that silk lyogels can be prepared
in two forms: low density -sheet networks (LDBN) and high density -sheet
networks (HDBN). The transition from
LDBN to HDBN occured between 20
mg(cm3)1 and 42 mg(cm3)1 -sheet
structure in silk lyogels. The researchers
observed that in water rich environment,
enhanced silk-antibody interactions were
rapidly reversed, and also observed a release behavior comparable to that of the
hydrogel (Figure 2 top). In the HDBN
form, significant hydration resistance was
observed. Lower water content within
the HDBN lyogels resulted in the lowering of the competition of water with the
silk-antibody interactions (Figure 2 bottom). The decreased kinetics of water-silk
interactions replacing antibody-silk interactions resulted in the sustained release of
untrapped antibody [3].
Researchers developed lyophilization
into a promising approach in order to
achieve long-term stable vaccine formulations [4]. Lyophilization of cells, e.g.
red blood cells has also been investigated. Further, advances in gene therapy
have brought biomedical researchers and
biotechnologists together to employ lyophilization as an effective method for the
preservation of nucleic acid based pharmaceuticals. Researchers have reported
that lyophilization has the capability to

28

Figure 2. A schematic illustration of water uptake and antibody release behavior for low (~20
mg (cm3)1) and high (>35 mg (cm3)1 -sheet density silk lyogels. Lyophilization based removal
of water leads to increased silk-antibody interactions [Source: Guziewicz et al 2011]

both stabilize the nucleic acid against

degradation and to preserve the structures [4]. Other recent developments have
shown expansion of the traditional field
of lyophilization that includes lyophilized
biological reference standards. Such biological reference standards are usually
processed from a single large-batch of
well-characterized biological material and
compared to the traditional lyophilization,
the lyophilization of biological materials
are performed in ampoules instead of
vials, guarantees long-term stability and
ease of distribution [4].
Researchers have also investigated
pharmaceutical applications of lyophilisates in the solid state. For example,
several applications of solid lyophilisates
have been investigated. Researchers utilized the unique properties of the highly
porous structures of lypohilisates. They
showed that orally disintegrating tablets
(ODTs) can be manufactured by lyophilization based technologies such as the
ZydisTM (R.P. Scherer, UK), LyocTM (Farmalyoc, France) or QuicksolvTM (Janssen Pharmaceutical, USA) technology
[4]. The advantage of ODTs prepared
by lyophilization is the unique porosity
that guarantees rapid dissolution in the
saliva. Further, research on such preparation method indicated that it could have
high potential for oral vaccine ODTs.
Also, published reports showed wafers as

vtcmag@vtcmag.com

promising systems for drug delivery via

the buccal mucosa [4].
In other work on lyophilization of pharmaceutical biotechnology products, the
bacteria Lactobacillus rhamnosus GG
were suspended and lyophilized in various types of aqueous two-phase emulsions: PVP/dextran, PEG4000/dextran
and PEG8000/dextran [5]. In another
related work, researchers investigated
the viability of lyophilized Lactobacillus
rhamnosus IMC 501 and Lactobacillus
paracasei IMC 502 using different protective agents (i.e., glycerine, mannitol,
sorbitol, inulin, dextrin, Crystalean)
and compared with semi skimmed milk
(SSM) control [6].
In a recent work, researchers demonstrated a silk biomaterial platform with
highly tunable mechanical and degradation properties for engineering and regeneration of soft tissues such as, skin,
adipose, and neural tissue, with elasticity
properties in the kilopascal range (Figure
3). They prepared lyophilized silk sponges
under different process conditions and the
effect of silk molecular weight, concentration and crystallinity on 3D scaffold formation, structural integrity, morphology,
mechanical and degradation properties,
and cell interactions in vitro and in vivo
were studied. In their interesting findings,
they reported that lyophilized silk sponges
supported the adhesion of mesenchymal

March 2016 Vacuum Technology & Coating

sors for process development, monitoring

and control that are aimed at developing
standards for lyophilization equipment
performance, testing and validation. We
also anticipate that further advances in the
technical design of the vacuum chamber
for the lyophilization process will make it
possible for large chamber and multi-stage
vacuum based commercial production of
high-quality products for future applications in the pharmaceutical industry.
References for Further Reading
1. General Principles of Freeze Drying. American Lyophilizer, INC. 2016.

Figure 3. Lyophilized Silk Sponges: A Versatile Biomaterial Platform for Soft Tissue Engineering [Source: Rnjak-Kovacina et al 2015]

stem cells throughout 3D scaffolds, cell

proliferation in vitro, and cell infiltration
and scaffold remodeling when implanted
subcutaneously in vivo [7].
Concluding Remarks
Vacuum lyophilization method has
proven to be effective for a number of applications in the pharmaceutical and diagnostic industries. Many biological products that are of clinical significance can
be developed into better functional pharmaceutical biotechnology products by
the application of lyophilization. For example, development of stable lyophilized
protein drug products has been shown by
the researchers. However, more research
is required to optimize the parameters that
are considered critical on the basis of their
relationship to lyophilization cycle development and protein product stability. New
processes are being developed to make
lyophilization a superior technology that
include the development of new sensors
to have them less bulky and better distributed within the system to eventually gain
better control of the processing. Biotechnologists and vacuum technologists are
working together to develop advanced
process analytical technologies and sen-

Vacuum Technology & Coating March 2016

2. GR.Nireesha, L.Divya, C.Sowmya, N.Venkateshan, M. Niranjan Babu and V.Lavakumar, Lyophilization/ Freeze Drying- An Review. International Journal of Novel Trends
in Pharmaceutical Sciences (IJNTPS),
2013, 3 (4): 87-98.
3. Nicholas Guziewicz, Annie Best, Bernardo Perez-Ramirez, and David L. Kaplan,
Lyophilized Silk Fibroin Hydrogels for
the Sustained Local Delivery of Therapeutic Monoclonal Antibodies. Biomaterials,
2011, 32 (10): 26422650.
4. Tushar R. Jadhav and R. S. Moon, Review
on Lyophilization technique. World Journal
of Pharmacy and Pharmaceutical Sciences
(WJPPS), 2015, 4 (5): 1906-1928.
5. Katarzyna Leja, Radosaw Dembczyski,
Wojciech Biaas, Tomasz Jankowski, Production of dry Lactobacillus rhamnosus
GG preparations by spray drying and lyophilization in aqueous two-phase systems.
Acta Sci. Pol., Technol. Aliment., 2009,
8(4): 39-49.
6. Michele Savini, Cinzia Cecchini, Maria
Cristina Verdenelli, Stefania Silvi, Carla
Orpianesi, and Alberto Cresci, Pilot-scale
Production and Viability Analysis of
Freeze-Dried Probiotic Bacteria Using Different Protective Agents. Nutrients, 2010,
2(3): 330-339.
7. Jelena Rnjak-Kovacina, Lindsay S. Wray,
Kelly A. Burke, Tess Torregrosa, Julianne
M. Golinski, Wenwen Huang, and David
L. Kaplan, Lyophilized Silk Sponges: A
Versatile Biomaterial Platform for Soft Tissue Engineering. ACS Biomater. Sci. Eng.,
2015, 1 (4): 260270.

www.vtcmag.com

29