ABSTRACT
Several companies were authorized to treat COVID-19 patients with monoclonal antibodies within 1-2 years of the start of the pandemic. These products were discovered, developed, manufactured, clinically tested, and approved under emergency-use authorization at unprecedented speed. Pandemic urgency led to novel development approaches that reduced the time to clinical trials by 75% or more without creating unacceptable patient or product-safety risks. Hundreds of thousands of patients now benefit from these therapeutics that have reduced the rates of hospitalization and death. The chemistry, manufacturing, and control development strategies set a new precedent of speed, safety, and demonstrated clinical benefit and will likely have a lasting impact on the development of future monoclonal antibody therapies for not only infectious diseases but also for oncology, inflammation, and rare diseases.
Subject(s)
COVID-19 , Humans , Antibodies, Monoclonal/therapeutic use , ImmunotherapyABSTRACT
N-linked glycosylation is a primary source of heterogeneity associated with recombinant monoclonal antibodies and plays a key role in a myriad of drug properties associated with biological function. The glycosylation profile of recombinant monoclonal antibodies is influenced by an array of cell culture inputs which must be carefully controlled in order to engineer the desired glycan distribution. A platform reduced intact mass method applied to monoclonal antibodies has been validated as a quantitative method to monitor the relative mannose-5 level as a surrogate for overall high mannose content in cell culture as a control strategy to ensure product quality and process consistency. The method was shown to be linear, accurate, specific, and precise for an IgG1 and IgG4 mAb allowing relative quantitation of mannose-5 in the range 0.8-11.0% and 1.0-6.2%, respectively. The method can be applied at several stages of the production process from cell culture harvest to drug substance/drug product and is amenable to routine GMP batch testing in a quality control laboratory. Testing upstream during cell culture rather than for product release allows for an earlier assessment of product quality as the glycosylation profile remains unchanged during downstream purification.
Subject(s)
Antibodies, Monoclonal/metabolism , Recombinant Proteins/metabolism , Glycosylation , Immunoglobulin G/metabolism , Mannose/metabolism , Models, BiologicalABSTRACT
We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.
Subject(s)
Drug Design , Erythropoiesis , Polymers , Polymers/chemistry , Polymers/chemical synthesis , Proteins/chemistry , Proteins/chemical synthesis , Amino Acid Sequence , Animals , Cell Line , Circular Dichroism , Drug Stability , Electrophoresis, Polyacrylamide Gel , Erythropoietin/chemistry , Erythropoietin/pharmacology , Hematocrit , Humans , Isoelectric Point , Mice , Molecular Sequence Data , Molecular Structure , Molecular Weight , Polymers/pharmacokinetics , Polymers/pharmacology , Protein Folding , Proteins/pharmacokinetics , Proteins/pharmacology , Rats , Receptors, Erythropoietin/drug effects , Receptors, Erythropoietin/metabolism , Recombinant Proteins , Spectrometry, Mass, Electrospray IonizationABSTRACT
Radiolabeled monoclonal antibodies are widely used in the detection and treatment of cancer. However, several problems still prevent full clinical exploitation of these reagents. Low tumor/background ratios in radioimmunoscintigraphy and high background radioactivity in therapy are the foremost among these. The strategy of pretargeting which separates the tumor-targeting step from radiolocalization step may overcome these limitations. One pretargeting approach, based on the streptavidin-biotin system, has been demonstrated to successfully treat cancer in preclinical models (Proc. Natl. Acad. Sci. 97 (2000) 1802). In this report we describe the synthesis of several electrophilic chelates, designed for use in vivo. In this new pretargeting approach, we have used protein engineering to prepare an antibody that can bind selectively and irreversibly to certain of these metal chelates. This improves upon approaches based on the immunogenic protein streptavidin and the endogenous ligand biotin.
