Method Development Challenges for Analysis of Aggregation in Protein Mixtures
Many of the challenges facing researchers in the proteomics and biopharmaceutical fields are related to the need to obtain as much information as possible on very limited samples. UPLC technology with BEH columns can address these challenges.
These can be addressed through:
1. Column Selection
In order to achieve an acceptable protein separations mode for a targeted protein molecule, several factors should be investigated that have direct impact on the detection process such as column chemistry, UPLC, BEH, C4, C8, C18, phenyl, OST and T3 reverse phase stationary phases, SEC, gradient elution time and slope, composition of mobile phase, ion pairing reagents, pH, consideration, temperature of column and sample compartments, detection methods and appropriate wavelengths or mass fragments.
Ion exchange and Size Exclusion Chromatography (SEC) are commonly used for the analysis or characterization of proteins. The complete analysis and characterization of these species is most efficient when orthogonal analytical techniques are applied. Each technique should be based on different physical properties of the protein and can include chromatographic methods such as size exclusion, ion exchange and reversed-phase chromatography.
Among the available analytical tools, reverse phase separation is a popular analytical tool for protein and peptide separations because it often provides better resolution over traditional ion exchange and gel filtration methods. The power and utility of RP-UPLC is typified in the separation of cytochrome c isoforms. The different phase chemistries and separation modes have provided unique selectivity increasing the resolution options.
The selected column should provide sensitive, stable, efficient, reproducible separations of proteins and their impurites. The low-bleed feature, and microbore and capillary dimensions of the columns listed above make them ideal for proteomics and other LC-MS applications. The selectivity and efficiency offered by the chosen column can provide maximum power for resolving complex mixtures of protein and peptide maps. Exceptional pH stability can allow for the full use of mobile phase pH to adjust and to separate complex protein or peptide mixtures.
2. Mobile Phase
When used at traditional concentrations, typically above 0.05% (v/v), TFA (trifluoroacetic acid) can effectively mask silanol interactions. Under those conditions, most modern RP-separations provide good peak shapes. However, TFA also reduces the sensitivity of mass spectrometry analyses. When TFA is absent or used at very low concentrations for the purpose of maximizing mass spectrometry sensitivity, the inertness or lack of silanol activity of the column can become increasingly relevant to attaining the best chromatographic performance. It can be noted that the differences in column inertness are obvious under low or no TFA conditions. Results can demonstrate that under the same conditions, UPLC BHE C18 columns provide superior resolution for most of the peak groups in the product but it can also lack sensitivity in comparison to a BEH C4 column with a wider pore size 300A. This clearly illustrates that choosing the best column is a critical element for developing efficient and sensitive protein separation methods.
There are several reasons why it is advantageous to use mobile phases with basic pH values (>pH 7). Among them may be altered ionic character of the protein, different selectivity of the separation, stability of the protein and the need to clean the column between injections. To be certain that the residual protein and other contaminants are removed from the column, a caustic wash of 0.1N NaOH is commonly employed. Normally, silica-based materials cannot withstand this treatment. However, UPLC columns have demonstrated high stability to caustic washing agents or run in high pH mobile phases without damage.
A protein-based molecule tends to aggregate which might be missing in RP-separation. Therefore, a different methodology may be required to monitor these changes in your investigated samples. Protein aggregation is the most common and troubling appearance of protein instability in almost all phases of protein drug development. The presence of any insoluble aggregates in a protein biopharmaceutical is generally unacceptable for product release. Protein aggregation occurs readily in almost all biopharmaceutical processes, especially during fermentation, refolding, purification, formulation and storage. Recognition of different causes of protein aggregation in these processes is the basis for selecting different techniques or methods for aggregation inhibition.
Aggregation has recently attracted a lot of attention from the FDA due to a number of incidents in which stability issues were caused by container leachables or extractables or changes in excipients. Since aggregation can induce immunogenic reactions with potentially severe consequences, the FDA is requiring manufacturers and analytical laboratories to pay more attention during release testing of protein API's and finished products. Aggregation is typically the primary degradation pathway for many protein-based products which are often formulated at high concentrations as high dosage volumes. The mechanisms of aggregation of proteins in general are not very well understood and it is a subject of case-to-case studies depending upon size and structure of the particular product. Identifying the specific mechanism of protein aggregation is essential in order to be able to change or adjust the manufacturing process in a way that minimizes aggregation.
Firstly, traditional and simple instrumental methods have been attempted in monitoring protein aggregation such as the turbidimetric method. This measures the optical density of the sample based on light scattering in the near UV or visible region where proteins have negligible absorption.
Size exclusion chromatography (SEC) is the gold standard for measuring aggregation. As we have observed, the conditions for reverse phase chromatography can interfere with the interactions required for aggregation. Novel high resolution SEC technology implemented by us in combination with UPLC-UV/MS detection has demonstrated excellent performance in improvement for the separation of molecule aggregates.
We can now add IEX and SEC to the range of techniques available for protein analysis that benefit from UPLC technology. Combining the low dwell volume of the UPLC system and the higher pressure capabilities of newer ion exchange and size exclusion materials, improves resolution, sensitivity and speed which cannot be achieved by conventional methods for bio-separations. In the detection of biomolecule impurities, specifically the determination of charged variants and aggregates, this has been demonstrated. The application of these techniques gives improved detection of impurities and often leads to faster analysis.
These types of methodologies can also be used for forced degradation studies. Forced degradation studies involving exposure of the drug substance to harsher conditions than the product should be expected and determining at what point and how it degrades is important. Stability testing of biological drugs should be more comprehensive than that of small molecule compounds. Therefore, holding time, shipping, freeze/thaw cycles, temperature, exposure to oxygen and light, physical stress and excipient and container compatibility parameters must be investigated. It is strongly advised that some characterization work be performed early in the development of a drug to identify the degradation pathways so that potential problems associated with stability, storage conditions and formulation design can be avoided during clinical trials.