Development and Validation of an HPLC Gel Filtration Method for Determination of Monomers, Dimers and Aggregates
Challenge: The goal of this work was to investigate the improvement of resolution between all multimer, dimer and monomers in a monoclonal antibody (mAb). The method was initially developed by the sponsor but failed to demonstrate adequate level of sensitivity and selectivity when used during stability studies. In addition the resolution between the analytes (monomer, dimer and multimer) was very poor.
For use as therapeutic agents, protein-based pharmaceutical products must contain acceptably low aggregate levels for the extent of their shelf lives and in-use periods. This requirement involves the need for sensitive, convenient and reliable analytical methods for detection and quantification of these large therapeutically active molecules.
Solution: Size-exclusion chromatography (SEC) has been employed as a sensitive, convenient and reliable analytical technique and as an industry standard for detection and quantification of trace protein aggregates. In SEC, a humanized mAb (proteins and their aggregate complexes) is separated based on differing extent of permeation into the pores of the stationary phase. Large molecules in a mixture elute rapidly, while smaller molecules with greater access to pores elute more slowly. Although SEC offers high throughput efficiency, its analytical accuracy is limited by interactions of the protein with the sieving matrix. Thus, SEC may not always detect aggregates that are loosely associated or very large. Other methods such as semidimentation velocity and electrophoresis have also been evaluated for the ability to quantify trace soluble aggregates of the mAb molecule. Semidimentation, velocity and electrophoresis methods do not rely on a sieving mechanism and may detect aggregates undetectable by SEC.
Diteba has developed and validated a new selective, sensitive and reproducible High Performance Liquid Chromatography (HPLC) method using the latest concepts and technological achievements in gel filtration chromatography. The newly developed SEC method has demonstrated a significant improvement in resolution of all chromatographic peaks (multimer, dimer, monomer and other small molecular fragments) as well as improved sensitivity and robustness. The method was successful implemented by Diteba's sponsor to monitor the drug products' lot-to-lot variability and the products' stability.
Diteba generated a comparison of the soluble aggregates measured by each of these techniques: SEC, Capillary Electrophoresis (CE) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-Page). This work was accomplished for both unstressed and stressed (temperature and acidified) samples and subsequently revealed a lack of agreement between the methods. First, total aggregate percentages differed notably between the methods. This trend appears to depend strongly on the type of sample analyzed. The total aggregate difference between the methods is comparatively small for the stressed samples but much larger for the unstressed samples. Moreover, two methods (SEC and Electrophoresis) detect a different number of protein peaks.
Another source of error was the absorption of aggregates in the solid phase of the column which may occur with covalently or non-covalently-linked aggregates. Under various conditions in this study, SEC has been shown to induce protein losses and to alter antibody association states. Injection of pure dimer resulted in production of monomer and polymer. Each of these disruptive processes clearly modifies the aggregate population during measurement. Furthermore, certain antibody oligometric states show preferential absorption to column gel media. Monomer-dimer unstressed samples demonstrated higher recovery (94%) than stressed samples (90%) with higher aggregate levels. These results highlighted the importance of performing carefully mass balance determinations when drawing conclusions from experiments with SEC. It is still too early to predict the potential value of semidimentation velocity and electrophoresis methods in characterization of therapeutic protein products. The technique appears promising for protein systems difficult to analyze by SEC. Diteba theoretically and experimentally evaluated all the SEC parameters to achieve optimization of the separation technique. Highlights of the HPLC parameters that were investigated include:
The selection of HPLC columns available for SEC separation is quite limited. TOSOH Biosciences offers varieties of TSK-GEL SW, PW, Alpha and Super AW types of columns. The main criterion in choosing between those columns is the molecular weight of the sample and its solubility. Generally TSK-GEL SW-type columns are suitable for separation and quality control of monodisperse biopolymers such as recombinant humanized mAb's. The principle feature of Gel Filtration Chromatography (GFC), a subgroup of SEC, is its gentle non-interaction with the sample, enabling high retention of biomolecular enzymatic activity while separating multimers that are not easily distinguished by other chromatographic methods. However, SEC has limited separation capacity requiring that the molecular weights of the biomolecules differ by at least two fold. In many cases, it has been reported that the chromatographic peaks which did not exhibit baseline separation were resolved by constructing lines at the point of peak intersection orthogonal to the absorbance baseline. Then, fractional concentrations can be calculated by dividing individual peak areas by the sum of peak areas.
Sample load and Injection Volume:
Generally in SEC, sample load on the column is limited due to the absence of the stationary phase that participates in the retention process. High sample loads distort peak shapes and cause an overall decrease in efficiency. Sample volume depends very much on the type of column. On TSK-GEL G3000SW columns a 5-10 μL injection volume ensures optimal results at the 5mg/ml sample concentration.
Proper selection of the mobile phase is necessary to maximize the molecular sieving mechanism and to minimize secondary effects such as ionic and hydrophobic interaction between the sample and the column parking material.
For TSK-GEL G3000SW columns, a mobile phase with a buffer concentration between 0.1 and 0.5 M is recommended. A neutral salt, such as sodium sulphate may be added to the buffer to increase buffer ionic strength. Also, the ionic species of the buffer has an effect on the separation. 0.1 M sodium phosphate buffer together with 0.1 M sodium sulphate has proven to be best choice. A water soluble organic solvent, such as acetonitrile, acetone, methanol or ethanol can be added to the mobile phase, up to a concentration of 20% to prevent columns from fouling by suppressing interactions of possible hydrophobic impurities of the sample.
Column temperature has significant influence on the resolution between multimer and dimer chromatographic peaks. At low column temperatures of 15oC and 20oC, resolution between multimer and dimer peaks are drastically improved while the resolution between dimmer and monomer slightly decreases.
Result: As a result, Diteba developed and validated a new SEC method which was highly efficient and specific in comparison with the method developed previously. Diteba developed CE and SDS-PAGE methods and recommended these for validation and use in conjunction with the SEC method for stability monitoring and release testing of the sponsor's mAb drug product.