Analytical characterization of coformulated antibodies as combination therapy

Abstract

When two therapeutic agents are combined in a single formulation, i.e., coformulated, the quality and safety of the individual agents must be preserved. Here we describe an approach to evaluate the quality attributes of two individual monoclonal antibodies (mAbs), designated mAb-A and mAb-B, in coformulation. The mAbs were fractionated from heat-stressed coformulated drug product (DP) by hydrophobic interaction chromatography. Each purified mAb fraction was then compared with mAb-A and mAb-B in their individual formulations from the same drug substance sources used to make the coformulated DP lot, which was subjected to the same stress conditions. Product variants were evaluated and compared by using several analytical tests, including high-performance size exclusion chromatography (HPSEC), reducing and nonreducing gel electrophoresis, ion-exchange chromatography, capillary isoelectric focusing, and peptide mapping with mass spectrometry. Intermolecular interactions in coformulated and photostressed DPs were studied by evaluating aggregates fractionated from coformulated DP by HPSEC. Aggregate fractions of coformulated DP contained dimers, but not coaggregates, of mAb-A or mAb-B. Moreover, extensive assays for higher-order structure and biological interactions confirmed that there was no interaction between the two mAb molecules in the coformulation. These results demonstrate that the two coformulated therapeutic mAbs had the same quality attributes as the individually formulated mAb-A and mAb-B, no new quality attributes were formed, and no physicochemical, intermolecular, or biological interactions occurred between the two components. The approach described here can be used to monitor the product quality of other coformulated antibodies. ARTICLE HISTORY

Stability enhancement in a mAb and Fab coformulation

“…Few studies have investigated the individual stability and degradation of therapeutic protein components in coformulations 4,5,35 . Therefore, there is no current consensus or strategy for guiding the development of stable coformulated products.…”

“…Among these products, most antibodies are formulated and administrated as individual therapeutic agents. However, recent clinical trials also report a number of combined administration routes showing promising results as new treatments [2][3][4][5][6] . The combination of nivolumab (Opdivo, anti-PD-1 antibody) and ipilimumab (Yvery, anti-CTLA-4 antibody), for example, inhibits two immuno-oncology checkpoints that shows encouraging response and survival rates in melanoma patients compared to the nivolumab single therapy 7,8 .…”

“…Thus, a formulation must provide the means to stabilize the combined molecules against these degradation pathways, during long-term storage 17 , 20 – 22 . As combination products are only recently emerging, there have been few studies into their stability in the mixture, with the aim of understanding how the multiple protein components interact and degrade during storage or in the final stages of drug product manufacturing 4 , 23 .…”

Advancements in the co-formulation of biologic therapeutics

“…Capillary electrophoresis (CE) and imaged capillary isoelectric focusing (iCIEF), separate the mixed biologics based on their charge heterogeneity [ 67 , 68 ]. These methods can be used along with chromatography to map out the degradation species in solution.…”

“…These methods can be used along with chromatography to map out the degradation species in solution. A recent study of a mixture of two antibodies tested the feasibility and limitations, of combining HPLC and CE in the analysis of co-formulated products [ 67 ]. It was previously suggested that mass spectrometry (MS) could be coupled to CE to characterise complex mixtures of antibodies [ 69 ].…”