Therapeutic protein immunogenicity

Do cold-chain breaks contribute to this problem?

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:

A role for protein misfolding in immunogenicity of biopharmaceuticals

"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:

Interferon immunogenicity: technical evaluation of interferon-alpha 2a.

(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.

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