Purpose:
Monoclonal antibodies (mAbs) are currently the dominant class of protein therapeutics. mAbs may undergo various degradation processes during product expression and purification from host cells, formulation and storage, which may alter the safety and efficacy of the drug product. Consequently, a number of critical quality attributes (CQAs) must be monitored throughout drug development and production to ensure biotherapeutic substances are suitable for clinical use. Aggregation is a common degradation process for therapeutic proteins that can result from partial unfolding or other types of conformational changes in protein structure to form dimers, and other higher order structures. Protein aggregation is considered a CQA as aggregates may reduce product efficacy and have been found to result in immunogenic effects. Hence, it is a regulatory requirement to monitor the aggregation profile of therapeutic proteins.
Size exclusion chromatography (SEC) is the most commonly applied method for protein aggregate analysis. Even though SEC columns are typically run at conditions that generate relatively low back pressure, the columns show a tendency toward shorter lifetimes. This is related to the inherent mechanical fragility of wide pore particles. However column fouling due to contamination is also commonly regarded as a potential cause of short lifetimes. Short column lifetime implies higher cost for biopharmaceutical laboratories that need to analyse large numbers of samples on a daily basis. In this work we evaluated the lifetime of commercially available columns used for mAb aggregate analysis.
Thermo ScientificTM VanquishTM Flex quaternary UHPLC system was utilized to monitor protein aggregation of the commercial drug substance bevacizumab. Detection was performed using the Thermo Scientific™ Vanquish™ Diode array detector with a Thermo ScientificTM LightPipeTM 10 mm standard flow cell. MAbPac SEC-1, 4.0 x 300 mm column and the MAbPac SEC-1, 7.8 x 300 mm column were used.
Protein Standards were also used to monitor column performance. The following molecules (with a range of sizes) were used to verify size exclusion: Thyroglobulin, BSA, Ribonuclease A, Myoglobin & (the small molecule) Cytidine.
Peak efficiency, retention time, and resolution between monomer and dimer, as stability indicating parameters were evaluated. Several vials containing fractions of the same mAb drug products solution were kept in the cooled autosampler. Injections were made from a single vial, and then moved to a new one, when the sample volume in the initial vial was estimated to be low. Peak width of the monomer was observed to be dependent on the aging of the sample. It was also observed that sample aging was accelerated in the vial from where the sample was withdrawn. This phenomenon was investigated and the most obvious potential causes, such as sample depletion or solvent evaporation were ruled out. The behaviour could not be explained by the time this abstract was submitted. Concerning the column stability, the performances of all columns tested were preserved at least for 1000 injections and sometime almost 2000 injections, contradicting the expectations of fragility for this class of columns.
The MAbPac SEC-1 column coupled to the Vanquish Flex Quaternary UHPLC system is a robust platform for aggregate analysis of mAbs. Consistent retention time, excellent peak symmetry and exceptional column efficiency were observed over the course of 2000 injections on two different column dimension. Data were measured with one specific UHPLC instrument without the use of guard columns. Column lifetime with other instrumentation was not assessed.
Amy Farrell
– The National Institute for Bioprocessing Research and TrainingMauro De Pra
– Thermo FisherAlexander Ley
– Thermo Fisher ScientificKevin McCowen
– Vertical Marketing, Thermo Fisher Scientific, San DiegoKrisztina Radi
– Thermo FisherFrank Steiner
– Thermo Fisher ScientificJonathan Bones
– The National Institute for Bioprocessing Research and TrainingKevin McCowen
– Vertical Marketing, Thermo Fisher Scientific, San Diego245 Views