Temperature stress destroys immunogenicity, does
it also create it?
The FDA now recognizes that therapeutic
protein immunogenicity is a major problem. As a result, the
pharmaceutical industry spends much time and effort optimizing therapeutic
protein structure and manufacturing processes in an effort to reduce the
severity of this problem. However two simple facts remain: 1)
proteins are inherently delicate molecules that can easily degrade or
aggregate in response to freezing or storage at high temperatures; 2)
degraded and/or aggregated proteins have an increased chance of being
immunogenic.
It is well known that
vaccines are temperature sensitive, and that vaccines can easily be
inactivated by exposure to temperatures outside of the standard 2 oC
to 8 oC refrigerated storage range. It is also well known that,
even in the US, the cold chain is fragile and prone to frequent disruption.
As a result, the CDC recommends/requires that the temperatures of vaccines
be recorded at least twice a day. Additionally, for freeze-tolerant
vaccines such as smallpox, vaccine vial monitors (VVM), have been developed
that warn users if the vaccine has been inactivated due to excess heat.
It is also well known that many vaccines are inactivated by freezing.
Temperature stress can also
create immunogenicity. For example, a recent article suggests that
heat denaturation is a simple method to improve the immunotherapeutic
potential of allergens
1.
By contrast, for protein based biotherapeutics,
relatively little work has been done in exploring the links between cold
chain breaks and subsequent development of unwanted antibodies that may
neutralize the biotherapeutic, or worse shut down a naturally produced
growth factor or cytokine. A recent article by
van Regenmortel gives a good introduction to this area. Other recent work in this field
includes work by
Hermerling, Schellekens,
and others
1,
2,
3,
4. In a recent 2007 article, Maas, Hermeling, Brouma, et. al.
conclude:
"Storage Increases the Level of Misfolded Protein in Biopharmaceuticals -
Most protein pharmaceuticals can be stored for prolonged periods of time
without losing their bioactivity. However, since proteins have the intrinsic
propensity to loose their unique native structure, some fraction of proteins
may gradually loose their structure and degrade. We examined the effect of
storage on the level of protein with amyloid-like structure in a number of
biopharmaceuticals, i.e. insulin, human albumin and somatropin, proteins
previously known to be able to form aggregates or amyloid under certain
conditions. Figure 2 shows that the level of protein with amyloid-like
properties increases when these biopharmaceuticals were examined closer to
their expiration date. These findings show that protein misfolding is
time-dependent in biopharmaceuticals, when measured by elevated markers for
amyloid structure."
"...in
general, biopharmaceuticals can have amyloid-like properties and that these
properties can be induced upon storage or conditions of stress. These data
indicate that misfolded proteins with amyloid-like properties can be
responsible for enhanced immunogenicity of biopharmaceuticals and breaking
of tolerance. Based on our results, we propose a unifying mechanism by which
individual immunogenic factors, such as oxidation or formulation changes,
via the formation of misfolded protein with common amyloid-like properties,
ultimately lead to an (enhanced) immune response...
...Our
findings indicate that various biopharmaceuticals have a tendency to misfold,
which may result in the generation of immunogenic proteins with amyloid-like
properties in various drug products that are on the market."
Other relevant work includes
Hochuli, cited below:
(Hochuli E., Department of Biotech Production and Process Development,
F. Hoffmann-La Roche Ltd., Basel, Switzerland, USA., J Interferon Cytokine
Res. 1997 Jul;17 Suppl 1:S15-21.)
Observations from some studies with interferon-alpha 2a (IFN-alpha 2a)
have shown the presence of neutralizing antibodies in a proportion of
patients. As a result, an investigation into the production of antibodies to
IFN-alpha 2a was undertaken. A number of technical aspects of its production
and storage were investigated, including the possibility of an incorrect
structure, which could affect the immunogenicity of the IFN-alpha 2a
molecule. These investigations demonstrated the presence, in vials of
IFN-alpha 2a, of both interferon-interferon (IFN-IFN) aggregates and
aggregates of interferon with human serum albumin (HSA), the excipient of
the galenical form of IFN-alpha 2a (IFN-HSA) aggregates. The amount of
aggregates is temperature dependent, there being very little increase in
aggregate content over time when vials are stored at 4 degrees C. The
relative immunogenicity of IFN-alpha 2a increased when the vials were stored
at ambient temperature but not when stored at 4 degrees C. These findings
demonstrate that the immunogenicity of IFN-alpha 2a is likely to be related
to the storage temperature. Storage of IFN-alpha 2a vials at 2-8 degrees C
is now recommended. A new formulation has been introduced that does not
contain HSA as an excipient, removing the possibility of IFN-HSA
aggregation.
As Hochuli suggests, in
some cases formulation
optimization may be sufficient to eliminate such problems. However
for clinically important biotherapeutics where such optimization fails, the LifeTrack offers another
option. Here IP licensing is
available.
