A high yielding IFNAR1 ECD mammalian expression process for use in autoimmune disease drug development.

Interferon-alpha receptor 1 (IFNAR1) is a target of interest for recombinant biotherapeutics that block the JAK/STAT pathway. This pathway is believed to play a role in many diseases including Hepatitis B and C, Herpes Simplex, Multiple Sclerosis, and other autoimmune disorders. By using IFNAR1 as a target to block Type I IFN from binding to the JAK/STAT pathway and prevent activation of this target, autoimmune disease progression can be modulated. Current IFNAR1 extracellular domain (ECD) expression and purification protocols are labor intensive with low product yield and limited scalability. In this work, we evaluate three different expression systems (baculovirus, human embryonic kidney 293 (HEK293×), and Chinese hamster ovary (CHO)) to improve expression of IFNAR1 ECD. We demonstrate the benefits of utilizing mammalian CHO cell transient transfection to increase expression titer, as well as an improved two-step purification process performed using immobilized metal affinity chromatography (IMAC) as the capture step and Ceramic Hydroxyapatite (CHT) Type II for HMW impurity removal in flow through mode. This process showed an 20-fold increase in productivity compared to the baseline process as measured by grams purified per liter of cell culture fluid. Lastly, the improved process showed good scalability, enabling efficient purification of 3.6 g of product from a 30 L scale bioreactor.

The Use of a GroEL-BLI Biosensor to Rapidly Assess Preaggregate Populations for Antibody Solutions Exhibiting Different Stability Profiles.

An automated method using biotinylated GroEL-streptavidin biosensors with biolayer interferometry (GroEL-BLI) was evaluated to detect the formation of transiently formed, preaggregate species in various pharmaceutically relevant monoclonal antibody (mAb) samples. The relative aggregation propensity of various IgG1 and IgG4 mAbs was rank ordered using the GroEL-BLI biosensor method, and the least stable IgG4 mAb was subjected to different stresses including elevated temperatures, acidic pH, and addition of guanidine HCl. The GroEL-BLI biosensor detects mAb preaggregate formation mostly before, or sometimes concomitantly with, observing soluble aggregates and subvisible particles using size-exclusion chromatography and microflow imaging, respectively. A relatively unstable bispecific antibody (Bis-3) was shown to bind the GroEL biosensor even at low temperatures (25°C). During thermal stress (50°C, 1 h), increased Bis-3 binding to GroEL-biosensors was observed prior to aggregation by size-exclusion chromatography or microflow imaging. Transmission electron microscopy analysis of Bis-3 preaggregate GroEL complexes revealed, in some cases, potential hydrophobic interaction sites between the Fc domain of the Bis-3 and GroEL protein. The automated BLI method not only enables detection of transiently formed preaggregate species that initiate protein aggregation pathways but also permits rapid mAb formulation stability assessments at low volumes and low protein concentrations.