A recent AAPS webinar addresses physical and chemical protein degradation and novel analytical methods.
By Mark Crawford
Danny Chou, Ph.D., founder and president of Compassion BioSolution, Tim Menzen, Ph.D., pharmacist and chair-elect of the AAPS Protein Aggregation and Biological Consequences (PABC) focus group, and Sreedhara Alavattam, Ph.D., principal scientist and senior group leader at Genentech, recently presented a webinar entitled Formulation Strategies to Prevent Protein Degradation.
Assessment of product attributes is an evolving process, from the discovery phase to clinical stages to product approval and commercial manufacturing. Developing a commercial process and formulation for a compound must be undertaken in a safe, reliable, and consistent manner. This means conducting appropriate analytical, structural, and biological tests on the components to determine function, safety, stability, microbiological safety, and dosage form integrity.
A key consideration for any product in development is protein degradation and its impact on protein stability and shelf life.
The AAPS PABC focus group sponsored the webinar to discuss why it is important for pharmaceutical and biotechnology scientists to understand the fundamentals of protein formulation and analytical development—including formulation strategies that minimize the negative impacts of protein degradation.
The webinar is divided into three sections:
- Strategies and mechanisms for preventing physical degradation
- Strategies and mechanisms for preventing chemical degradation
- Novel analytical methods, especially for particle analysis (subvisible and visible particles)
PHYSICAL DEGRADATION: Mechanisms and Strategies for Prevention and Management
The physical stability of proteins is largely determined by thermodynamic reactions that occur at the interface. Interfacial stability can often influence protein conformation and stability. Sources of interfacial stress include the air -liquid interface in the head space of vials or syringes, solid-liquid interfaces in bioprocessing containers and tanks, and the liquid-oil interface in delivery devices where silicone oil is used as the lubricant, such as in siliconized prefilled syringes.
Protein aggregation is a form of protein instability. Protein aggregation during manufacturing, storage, and shipping is typically caused by a two-step aggregation model of unfolding, followed by diffusion-controlled aggregation. This process can be affected by temperature, stirring, exposure to the air/liquid or solid-liquid interface, extreme high or low pH, ionic strength, dehydration, and free radical and/or UV exposure.
Aggregation can also be impacted by colloidal stability and stirring. Under ambient conditions, colloidal stability can play a prominent role in aggregation (bovine serum albumin is a good example). Stirring-induced aggregation is directly proportional to the surface area of air/liquid interface—reducing the interfacial area reduces the negative impacts of the stirring. Complete protection against stirring can be accomplished by adding a surfactant.
Any strategy for enhancing the physical stability of proteins in solution requires:
- the development of formulations that enhance conformational stability of native state,
- ligands that preferentially bind the native state,
- minimized protein-protein attractive forces (pH, ionic strength, solutes) that modulate protein surface charge or hydrophobicity,
- minimal exposure to interfaces (reducing interfacial stress), and
- reducing/eliminating the interfacial area (addition of surfactant).
Understanding how these factors impact protein aggregation is essential for success.
CHEMICAL DEGRADATION: Formulation Strategies for Prevention
Three key chemical degradation processes—deamidation, isomerization, and oxidation—can have profound negative impacts on protein stability and shelf life.
Deamidation and isomerization are spontaneous intramolecular reactions. No extrinsic factors are required; the reactions are driven only by the pH of the reaction media. Since water is a reactant, these reactions occur more readily in liquid formulations than in solid state. Deamidation and isomerization are also highly sequence dependent. For example, Asn/Gly and Asn/Ser are more susceptible for deamidation than Gln/Gly and Gln/Ser. Asp/Gly and Asp/Ser are the most susceptible for isomerization.
Both Asn deamidation and Asp isomerization are pH-dependent. Optimizing pH is the best approach for stabilizing proteins against deamidation and isomerization. Deamidation reactions are base-catalyzed and increase between pH 5–8; optimal pH for preventing deamidation is usually pH 3–5. Isomerization reactions are acid-catalyzed and occur usually at pH 4–6; therefore the preferred pH to stabilize against isomerization is pH >7.
Oxidation is a form of degradation that commonly occurs during protein process development (cell culture, purification, formulation) and normal handling. Causes of oxidation include alkyl peroxides from degraded polysorbate (via autoxidation), light exposure during processing and storage, metal ion leachates from stainless steel surfaces, and vaporized hydrogen peroxide. Typical amino acid residues that are “readily” oxidized in proteins include Tyr, Trp, Met, His, Cys, and disulfide (Cys-Cys). In particular, antioxidants such as L -methionine, sodium thiosulfate, catalase, or platinum prevent Met oxidation in rhuMAb HER2, presumably as free radicals or oxygen scavengers.
INDOLE DERIVATIVES HAVE BEEN PROPOSED TO PREVENT TRP OXIDATION IN MAB FORMULATIONS
Metal ion chelators such as ethylene-diaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) have also been suggested as potential excipients in mAb formulations that prevent oxidation via metal ion sequestration.
Various stress models are used in the industry to understand and tackle protein oxidation challenges. These include azodiisobutyramidine dihydrochloride (AAPH) (mimics alkyl peroxides from degraded polysorbate), light (mimics ambient light exposure during processing and storage), and Fenton (mimics metal residue from stainless steel surfaces). The best way to prevent oxidation of Met and Trp is through screening for antioxidants
ANALYTICAL METHODS FOR PROTEIN DEGRADATION
Methods for testing protein degradation must cover all aspects of protein stability and aggregation. Proteins aggregate via different pathways—for example, conformational-dependent aggregation, colloidal stability-dependent aggregation, or heterogeneous nucleation of interface-dependent aggregation.
Choosing a method for sizing and (semi)quantification of particles depends on several factors, including broad particle size range, broad concentration range, heterogeneous composition, and physiochemical properties such as viscosity and refractive index.
As an example, effective methods for measuring protein degradation in nm monomer-oligomer aggregates are high pressure size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, analytical ultracentrifugation (AUC), dynamic light scattering (DLS), laser diffraction, and static light scattering methods.
Other useful analytical methods for protein degradation include AF4, AUC,turbidity, DLS, flow cytometry, electric zone sensing, resonant mass measurement, nanoparticle tracking analysis, flow imaging, light obscuration, visual inspection, and fluorescence microscopy.
Methods for detecting chemical changes are isoelectric focusing (IEF) (gel), capillary IEF, ion-exchange chromatography, capillary electrophoresis, reverse phase chromatography, hydrophobic interaction chromatography, mass spectrometry, and nuclear magnetic resonance (NMR).
Methods for detecting conformational changes are UV (second directive), far-UV circular dichroism (CD), near-UV CD, Fourier-transform infrared spectroscopy, fluorescence, differential scanning calorimetry, differential scanning fluorescence, hydrogen–deuterium exchange, NMR, small angle neutron scattering, and small angle X-ray scattering.
Imaging flow cytometers combine particle sizing/counting/morphology with fluorescence properties.
Staining of particles with specific fluorescent dyes allows for differentiation (for example, silicone oil and protein).
In conclusion, the analysis of protein degradation requires numerous analytical methods that address colloidal, chemical, and conformational changes. Dependent on readout, cost, throughput, and other factors, these methods can be used from discovery to release testing. Multidimensional methods, with standardized, automated, and miniaturized sample preparation, can be especially beneficial. Standardized reference samples and harmonized methods also allow for better cross-lab comparison of results.
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Mark Crawford is a freelance writer specializing in science and technology based in Madison, Wis.