Developability considerations should be integrated with lead engineering of antibody drug candidates in interest of their cost effective translations into medicines. To explore feasibility of this imperative, we have performed rational mutagenesis studies on a monoclonal antibody (MAB1) whose development was discontinued owing to manufacturability hurdles. Seven computationally designed variants of MAB1 containing single point (V44K, E59S, E59T and E59Y) and double (V44KE59S, V44KE59T and V44KE59Y) mutations in its light chain were produced in Chinese Hamster Ovary (CHO) cells and purified by using platform processes employed during commercial scale production of monoclonal antibodies. MAB1 and its variants were formulated in the same platform buffer and subjected to a battery of experiments to assess their solution behaviors, and biological activities. Five of the seven (71%) variants of MAB1 demonstrated improved biophysical attributes in multiple experimental testings. Contrary to the commonly expressed reservations about potential biological activity loss upon developability optimizations, the improvements in solution behavior of MAB1 also increased its biological activity up to ~180%. In particular, concentrate-ability and apparent solubility of V44KE59S improved to ~150% and ~160%, respectively. Its diffusion interaction parameter (kD) reduced to 28% and viscosity at ~100 mg/ml decreased to less than half of the corresponding values for MAB1. V44KE59S is also slightly more active and its transfections in CHO cells were more productive. It also degraded slower than MAB1 in three month long 25°C and 40°C formulation stability studies. These results open doors to an exciting realm of structure-based biologic drug design where developability and biological activity can be simultaneously optimized at the molecular engineering stages.

During fill-finish manufacturing, therapeutic proteins may aggregate or form subvisible particles in response to the physical stresses encountered within filling pumps. Understanding and quantitating this risk is important since filling may be the last unit operation before the patient receives their dose. We studied particle formation from lab-scale to manufacturing-scale using sensitive and robust protein formulations. Filling experiments with a ceramic rotary piston pump were integrated with a rinse-stripping method to investigate the relationship between protein adsorption and particle formation. For a sensitive protein, multilayer film formation on the piston surface correlated with high levels of subvisible particles in solution. For a robust protein formulation, adsorption and subvisible particle formation were minimal. These results support an aggregation mechanism that is initiated by adsorption to pump surfaces and propagated by mechanical and/or hydrodynamic disruption of the film. The elemental analysis confirmed that ceramic wear debris remained at trace levels and did not contribute appreciably to protein aggregation.

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During fill-finish manufacturing, protein-pump surface interactions can induce subvisible particle (SVP) formation which poses a risk to drug product quality and patient safety. Despite this risk, there have been no concerted efforts to understand the effects of piston pump design on SVP formation.We've systematically varied the design of the piston-cylinder interface to minimize SVP formation for a therapeutic immunoglobulin. The clearance factor, surface roughness factor, and their combined interaction significantly affected particle concentrations, quantitated by light obscuration and microflow imaging. Optimized pump designs reduced particle levels by 1-2 orders of magnitude compared to the off-the-shelf equipment. At the piston surface, scanning electron microscopy revealed evidence of protein film abrasion, a process which ejects SVPs from the piston-cylinder interface as wear debris. Computational fluid dynamics and quartz crystal microbalance were applied to simulate fluid flow and protein adsorption phenomena in the pump respectively. The risk of protein film abrasion was modeled along a hypothetical Stribeck curve, thereby interconnecting design parameters, lubrication conditions, and SVP formation. Our findings support implementation of a modular pump platform with interchangeable pistons; this approach would enable the pump design to be customized based on each protein's propensity to form SVPs. This flexible approach can benefit pharmaceutical manufacturers and patients alike by accelerating tech transfer and improving process control.