laboratory-scale-production-and-purification-of-a-therapeutic-antibody

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

University of Sydney

Improving the physical stability of spray-dried proteins is essential for enabling pulmonary delivery of biotherapeutics as a noninvasive alternative to injections. Recently, a novel combination of two amino acids - l-arginine (l-Arg) and l-glutamic acid (l-Glu), has been reported to have synergistic protein-stabilizing effects on various protein solutions. Using spray-dried bovine serum albumin (BSA) reconstituted in solution as a model protein, we investigated the synergistic effect of these amino acids on the physical stability of proteins. Five BSA solutions were prepared: (1) BSA with no amino acids (control); (2) with 50 mM l-Arg; (3) with 200 mM l-Arg, (4) with 50 mM l-Glu and (5) with 25:25 mM of Arg:Glu. All solutions were spray-dried and accelerated studies at high temperatures were performed. Following accelerated studies, monomer BSA loss was measured using SE-HPLC. We found that l-Arg significantly improved the physical stability of spray-dried BSA even at low concentrations, however, when combined with l-Glu, was ineffective at reducing monomer BSA loss. Our findings demonstrate the limitations in using Arg-Glu for the stabilization of spray-dried BSA. Furthermore, we found that a low concentration of l-Glu enhanced monomer BSA loss. These findings may have significant implications on the design of future biotherapeutic formulations.

Lack of a synergistic effect of arginine-glutamic acid on the physical stability of spray-dried bovine serum albumin.

Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium oxide (D2O), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of D2O on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that D2O enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T (ThT), however, the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with D2O can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.

The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin.

It has been over four decades since the development of monoclonal antibodies (mAbs) using a hybridoma cell line was first reported. Since then more than thirty therapeutic antibodies have been marketed, mostly as oncology, autoimmune and inflammatory therapeutics. While antibodies are very efficient, their cost-effectiveness has always been discussed owing to their high costs, accumulating to more than one billion dollars from preclinical development through to market approval. Because of this, therapeutic antibodies are inaccessible to some patients in both developed and developing countries. The growing interest in biosimilar antibodies as affordable versions of therapeutic antibodies may provide alternative treatment options as well potentially decreasing costs. As certain markets begin to capitalize on this opportunity, regulatory authorities continue to refine the requirements for demonstrating quality, efficacy and safety of biosimilar compared to originator products. In addition to biosimilars, innovations in antibody engineering are providing the opportunity to design biobetter antibodies with improved properties to maximize efficacy. Enhancing effector function, antibody drug conjugates (ADC) or targeting multiple disease pathways via multi-specific antibodies are being explored. The manufacturing process of antibodies is also moving forward with advancements relating to host cell production and purification processes. Studies into the physical and chemical degradation pathways of antibodies are contributing to the design of more stable proteins guided by computational tools. Moreover, the delivery and pharmacokinetics of antibody-based therapeutics are improving as optimized formulations are pursued through the implementation of recent innovations in the field.

Mouhamad Reslan

Faculty of Pharmacy

University of Sydney

Laboratory Scale Production and Purification of a Therapeutic Antibody

Mouhamad Reslan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Faculty of Pharmacy

University of Sydney

Laboratory Scale Production and Purification of a Therapeutic Antibody

Faculty of Pharmacy