PROTEIN CRYSTALS - Reshaping traditional biotherapeutic formulations

Abstract

While protein therapeutics have enjoyed considerable commercial success throughout the past 3 decades, there still remain formulation and delivery challenges. Due to poor bioavailability and unfavorable pharmacokinetics, frequent administration of large doses is often necessary for clinical benefit. Highly concentrated solutions usually have high viscosity resulting in poor syringeability and injectability. Out of necessity, these products are formulated as low concentration solutions that have to be administered as large volume IV infusions. IV infusions are more expensive, time-consuming, and have to be administered by trained medical professionals. Protein crystals have shown potential to address these issues and can benefit both pharmaceutical developers and patients (Table 1).

Full text

PROTEIN
CRYSTALS
INTRODUCTION
Throughout the past decade, protein-based therapeutics
have emerged as the key driver of growth in the
pharmaceutical industry. R&D pipelines have filled with more
and more biologics and, in recent years, monoclonal
antibodies have become the fastest growing segment of
biological drugs around the world. Despite the success of this
segment, there are specific challenges to overcome when
developing these types of therapeutics. Unlike small molecules,
protein-based therapeutics are almost exclusively administered
by parenteral routes.1Because of the size, biochemical
complexity, and low bioavailability of these macromolecules,
high doses must also be administered. At high concentrations,
protein-protein interactions significantly increase solution
viscosities and may result in the formation of aggregates. This
in turn decreases manufacturability and complicates drug
delivery.2,3 Moreover, aggregates are of special concern
because they have been shown to be associated with altered
biological activity and increased immunogenicity.4-7 Currently,
most protein-based biologics are administered via larger
volume, lower concentration formulations via intravenous (IV)
infusion. However, these procedures are less patient-friendly,
more costly, require trained medical professionals, and often
involve a visit to a clinic.
OVERCOMING CHALLENGES WITH CRYSTAL
FORMULATIONS
From a cost and patient compliance perspective,
subcutaneous injection (SCI) would be the preferred route of
administration. To keep patient discomfort to a minimum, SCI
volumes generally do not exceed 2 milliliters. The high dose
necessary for clinical benefit raises concerns about the
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Reshaping Traditional Biotherapeutic Formulations
By: Don Paul Kovarcik, MBA, and William Wittbold, MS
FIGURE 1
The viscosity of soluble and crystalline suspensions of
Infliximab. Infliximab is a monoclonal antibody
against tumor necrosis factor alpha (TNF-α). It is
marketed under the trade names Remicade®(Janssen
Biotech), RemsimaTM (Celltrion), and InflectraTM
(Hospira) for the treatment of Crohn’s disease,
psoriasis, and other autoimmune diseases. Viscosity
was measured using a Cannon-Fenske viscometer
according to the manufacturer’s instructions. It should
also be noted that small gauge needles can
accommodate viscosities up to about 20 cPs.

Drug Development & Delivery June 2016 Vol 16 No 5
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exponential relationship between protein
concentration and viscosity.8High
viscosity levels reduce syringeability
(loading) and injectability (delivery to
patient). It has been demonstrated that
highly concentrated crystalline
suspensions do not result in a similar
increase in viscosity (Figure 1).
The viscosity of a suspension (η) is
primarily determined by viscosity of the
formulation vehicle (η0) and the
suspension viscosity is dependent on the
crystal volume fraction (Φ) (Figure 2).
Because protein crystals are highly
organized, tightly packed structures
(Figure 3), their volume fraction is
considerably less than an equivalent
number of protein molecules in solution.
The advantages of protein crystals aren’t
just limited to lower viscosity.
Proteins are complex
macromolecules that require a specific
three-dimensional structure in order to be
biologically active. The interactions that
drive and stabilize higher order
secondary, tertiary, and quaternary
structures are inherently weak and
mainly driven by hydrophobic
interactions but also stabilized by
hydrogen bonds, salt bridges, and
disulfide bonds. As a result, proteins are
susceptible to physical or conformational
degradation. A number of external
factors can cause physical degradation,
including higher temperatures, pH,
mechanical agitation, and high shear
forces to name a few. Crystals present a
uniquely stable form of proteins and help
protect against many forms of
degradation. Moreover, studies have
shown that the crystallization process
does not affect the biological activity of
a protein (Figure 4).
Crystal formulations have long been
used for long-acting versions of small
molecule drugs. Crystal formulations of
therapeutic proteins can also be used to
develop products with extended-release
properties. Crystal Infliximab
administered monthly has the same effect
as the soluble form administered weekly
in a TNF-αmouse model (Figure 5).
Due to improved handling,
increased stability and the possibility of
controlled release, crystal formulations of
small molecule therapeutics have been
on the market for decades.9However, to
date, insulin is the only biologic
available in a crystal formulation. What
then are the key issues that are
preventing the widespread development
of these advantageous formulations?
CHALLENGES OF DEVELOPING
A CRYSTAL SUSPENSION
FORMULATION
There are two main challenges to
developing a crystal suspension
formulation. The first is to the find a
robust crystallization condition that will
produce crystals within a short period of
time – sufficiently short for GMP
manufacturing, preferably less than 24
hours. The second challenge is the
development of a drug product
formulation that is suitable for injection
while maintaining stability in the crystal
structure. Finding conditions in which a
protein crystallizes is the initial
challenge, and oftentimes those
crystallization conditions have properties
that are not suitable for introduction into
patients (ie, non-GRAS excipients, not
isotonic, etc). The second, and often
more difficult challenge is then to
reformulate the crystal suspension into
excipients suitable for injection that also
maintain crystal integrity and molecular
function upon dissolution.
GMP MANUFACTURING OF
THERAPEUTIC CRYSTAL
FORMULATIONS
Proteins have been crystallized for
structural studies in biochemistry
laboratories for over 50 years. However,
the requirements of and methods for
producing protein crystals for therapeutic
purposes are significantly different. Most
crystallographers want a single large
size crystal (> mm) for structure studies,
FIGURE 2
Einstein equation for suspensions.
FIGURE 3
Crystal size as a function of the number of protein molecules.

whereas formulation scientists want very
high concentrations of uniform crystals (>
200 mg/mL) that are several orders of
magnitude smaller, typically 5 to 30 µm.
Protein aggregation is a concern during
crystallization - if precipitant amounts are
too high, then the individual protein
molecules assemble too rapidly and not
in order resulting in aggregation. A lot of
time and effort is invested in finding the
optimal balance to regulate crystal
assembly and growth without
aggregation.
In addition, the ideal crystallization
conditions change as the project moves
from vapor diffusion screening to micro-
batch screening. Conditions that work at
the 3-µl level usually don’t translate well
to the 15-µl level. Scaling the volume of
the crystallization reaction affects how
the crystals form. In early development
(volumes < 3 μL), evaporation is the
primary driver for crystallization. As
water evaporates from the small drop,
the concentration of excipients increases
until crystals form (if conditions are
right). In larger volume reactions (> 15
μL and up), there is insufficient surface
area for evaporation to be the main
driving force behind crystallization. By
this point, however, optimization efforts
likely have determined the conditions that
don’t rely on evaporation to produce
crystals with the desired properties. To
scale further, it is necessary to move into
tank systems (50 mL and up). Tank
systems, because of their significantly
larger volumes, introduce additional
variables that can affect crystal yields
and quality; these variables include
mixing rate, impeller design, order of
excipient addition, and temperature.
Following the discovery of the
optimal crystallization conditions, the
next step is formulation development.
This can be as challenging as
developing the optimal crystallization
process. Even when a robust process to
make small (10 mL) batches of uniform
crystals has been developed, the
excipients are typically not generally
regarded as safe (GRAS). Often, the
protein crystals need to be reformulated
into GRAS excipients suitable for
subcutaneous injection that are also in
the desired pH, osmolality range, and
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FIGURE 4
Cultured L-929 mouse fibroblast cells were detached, diluted to 2x105 cells
per mL and added to 96-well plates (100 μL per well). TNF-αneutralization
assays were performed by incubating mouse fibroblast cells overnight in
the presence of 100 pg/mL TNF-αand various concentrations of Infliximab
or dissolved crystals of Infliximab. The number of viable cells was
determined by using a commercially available cell proliferation assay kit.
FIGURE 5
Approximately 100 μL of Infliximab (20 mg/mL) was subcutaneously
administered to C57BL/6NTac-TgN(TNF- α) mice at a dose of 8 mg/kg in
both soluble (weekly) and crystallized (monthly) forms. Non-specific IgG
was used a control.

break loose energy (BLE - how much
force is needed to expel the material
from a syringe).
Downstream purification of the
desired crystal size can be a challenge.
Even with the tightest controls, in each
batch, there will be distribution of
various crystal size populations.
Centrifugation can be used to purify;
however, it is not a preferred method.
Centrifuge bottles can shed particles thus
contaminating the crystals. It’s possible to
pre-clean and irradiate the bottles prior
to centrifugation, but this adds additional
steps to the process. In addition,
operators have to manually handle and
pour to/from the bottles, introducing risk
of spills, errors, and contamination. A
better option would be an automated,
closed system such as tangential flow
filtration (TFF). It reduces the chance for
human error, is gentler, and reduces the
risk of contamination when compared to
centrifugation.
The next steps in the process are fill
finish manufacturing, release testing, and
visual inspection. There are two major
areas of concern when filling crystalline
therapeutics: suspension uniformity and
fill weight accuracy. In addition to the
typical release assays for a protein-
based biologic, it’s also important to
perform extensive dissolution and
biophysical characterization studies of
the API pre- and post-crystallization to
show protein isn’t affected by the
crystallization process or the crystals
themselves. The final step is manual
visual inspection. Manual visual
inspection is the standard in both the US
and Europe and heavily relies on the
experience, training, and skill of the
operator. Specialized training is needed
to identify the potential defects in
opaque crystalline suspensions that
resemble a milky fluid (Figure 6).
THE SOLUTION – ALTHEA’S
CRYSTALOMICS® FORMULATION
TECHNOLOGY
To help clients develop crystalline
formulations, Althea offers access to a
proprietary Crystalomics®Formulation
Technology. Althea’s unique portfolio of
intellectual property encompasses
crystallization, cross-linking, and
complexation of proteins for therapeutic
use. It includes patents, proprietary
knowledge, and expertise to develop
ideal crystallization conditions, stable
crystalline formulations, and scale-up for
GMP manufacturing of crystalline
suspension drug products. The
technology allows companies to produce
highly concentrated formulations with
low viscosity, enabling low-volume doses
and increased stability. The resulting
crystalline suspensions are easier to
administer and offer the chance to
extend the patent life of high-value
biologic drugs. A typical crystallization
workflow conducted at Althea is shown
in Figure 7.
Althea has been successful
developing crystallization conditions and
stable crystal suspension formulations for
over 100 molecules, including
antibodies, hormones, enzymes, and
peptides from human, animal, and
microbial sources.
SUMMARY
While protein therapeutics have
enjoyed considerable commercial
success throughout the past 3 decades,
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FIGURE 6
Syringe filled with a crystalline suspension of recombinant human growth
hormone.
FIGURE 7
A workflow diagram of a typical crystallization project.

there still remain formulation and
delivery challenges. Due to poor
bioavailability and unfavorable
pharmacokinetics, frequent
administration of large doses is often
necessary for clinical benefit. Highly
concentrated solutions usually have high
viscosity resulting in poor syringeability
and injectability. Out of necessity, these
products are formulated as low
concentration solutions that have to be
administered as large volume IV
infusions. IV infusions are more
expensive, time-consuming, and have to
be administered by trained medical
professionals. Protein crystals have
shown potential to address these issues
and can benefit both pharmaceutical
developers and patients (Table 1).u
REFERENCES
1. Vugmeyster et al. Pharmacokinetics and
toxicology of therapeutic proteins:
Advances and challenges. World J Biol
Chem. 2012 Apr 26;3(4):73-92.
2. Child J. Minireview: Protein Interactions.
University of New Hampshire Scholars'
Repository, Fall 2012.
3. Palm et al. The Importance of the
Concentration-Temperature-Viscosity
Relationship for the Development of
Biologics. BioProcess International. Mar.
2015.
http://www.bioprocessintl.com/manufactu
ring/antibody-non-antibody/importance-
concentration-temperature-viscosity-
relationship-development-biologics/.
4. Patel et al. Stability Considerations for
Biopharmaceuticals, Part 1: Overview of
Protein and Peptide Degradation
Pathways. BioProcess International. Jan.
2011.
http://www.bioprocessintl.com/manufactu
ring/formulation/stability-considerations-
for-biopharmaceuticals-part-1-332821/.
5. Particle Sciences, Inc. Protein Structure.
Technical Brief 2009, Vol. 8.
http://www.particlesciences.com/
news/technical-briefs/2009/protein-
structure.html.
6. Watts A. (University of Bath), Biological
Drugs – Practical Considerations for
Handling and Storage. Presentation, May
2013.
7. Kashi R. (Merck Research Laboratory),
Challenges in the Development of Stable
Protein Formulations for Lung Delivery.
Presentation, AAPS Symposium, Sep.
2011.
8. Skalko-Basnet N. Biologics: the role of
delivery systems in improved therapy.
Biologics Target Ther. 2014;8:107-114.
9. Basu et al. Protein crystals for the delivery
of biopharmaceuticals. Expert Opinion
Biological Ther. 2004;4(3):301-317.
BIOGRAPHIES
Don Paul
Kovarcik is
the Technical
Marketing
Specialist at
Ajinomoto
Althea, Inc. He
is responsible
for developing
technical
marketing
pieces for all aspects of Althea’s business,
including drug product (fill finish)
manufacturing, drug substance
manufacturing, Crystalomics®Formulation
Technology, and Corynex®Protein
Expression System. Prior to joining Althea,
he worked in a variety of marketing and
business development roles at Lonza in their
research products and cell therapy contract
manufacturing business units; specifically
focused on the development of pluripotent
stem cell product and service offerings. He
earned his BS in Biochemistry from Virginia
Tech and his MBA from Carnegie Mellon
University.
William
Wittbold is
the Manager for
Crystalomics®
Technology
Transfer at
Ajinomoto
Althea, Inc.
After earning his
BS and MS in
Microbiology
from the University of Massachusetts
Amherst, he worked in positions with
InfiMed Therapeutics, University of
Massachusetts Medical School, Altus
Pharmaceuticals and Wyatt Technology.
With an extensive background in protein
crystallization, biophysical characterization,
and assay development, he guides client
and internal projects from screening through
GMP manufacturing and fill finish. He has
diverse experience working with clients
ranging from startups to large
pharmaceutical companies.
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Benefits of Crystal Formulations
Pharmaceutical Developers
Patients
Ability to formulate highly concentrated
proteins in small injection volumes
Better compliance without time-
consuming IV infusions
Scalable to support both clinical stage and
commercial manufacturing
Self-injection that doesn’t require
trained medical personnel
Maintains biochemical characteristics and
bioactivity of the soluble protein
Improved patient comfort via use of
finer gauge needles
Improvement in syringeability and
injectability
Non-injection routes of administration
possible
Flexibility in dosage form–oral, pulmonary,
topical and subcutaneous injection possible
Fewer treatments via controlled and
extended release formulations
Opportunity to extend patent life of branded
protein-based therapeutics
Same therapeutic benefits as low
concentration formulations
TABLE 1
Summary of the benefits of protein crystal formulations.