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.1 Because 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 exponential relationship between protein concentration
and viscosity.8 High 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
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-α mouse model
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.9 However,
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
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-Generally Regarded As Safe
(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, 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
In addition, the ideal crystallization
conditions change as the project moves from vapor diffusion screening to
microbatch 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 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 break loose energy (BLE - how much force
is needed to expel the material from a
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.
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).
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
3. Palm et al. The Importance
of the Concentration-Temperature-Viscosity Relationship for the Development of Biologics.
BioProcess International. Mar. 2015.
4. Patel et al. Stability
Considerations for Biopharmaceuticals, Part 1: Overview of Protein and Peptide
Degradation Pathways. BioProcess International. Jan. 2011.
5. Particle Sciences, Inc.
Protein Structure. Technical Brief 2009, Vol. 8. http://www.particlesciences.com/news/technical-briefs/2009/proteinstructure.
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.
9. Basu et al. Protein
crystals for the delivery of biopharmaceuticals. Expert Opinion
Biological Ther. 2004;4(3):301-317.
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.
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.