RESUMEN
We investigated the discoloration of a highly concentrated monoclonal antibody (mAbZ) in sodium acetate (NaAc) and histidine/lysine (His/Lys) buffer after exposure to visible light. The color change of the mAbZ formulation was significantly more intense in NaAc buffer and developed a characteristic absorbance with a λmax of ca. 450 nm. We characterized this photo-chemically generated chromophore by comparison with visible light photo-degradation of a concentrated solution of a model compound for protein Trp residues, N-acetyl-l-tryptophan amide (NATA). The photo-degradation of NATA generated a chromophoric product with a λmax of ca. 450 nm and UV-vis spectroscopic properties identical to those of the product generated from mAbZ. This product was isolated and analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and 1H, 13C, and 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. MS/MS analysis reveals a product characterized by the loss of 33 Da from NATA, referred to as NATA-33. Together, the NMR data suggest that this product may be N-(2,4-dihydrocyclopenta[b]indol-2-yl)acetamide (structure P3a) or a tautomer (P3b-d).
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Luz/efectos adversos , Proteolisis/efectos de la radiación , Triptófano/análogos & derivados , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/efectos de la radiación , Tampones (Química) , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masas en Tándem , Triptófano/metabolismo , Triptófano/efectos de la radiaciónRESUMEN
Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.
Asunto(s)
Anticuerpos Monoclonales/química , Ácidos Linoleicos Conjugados/efectos de la radiación , Ácidos Oléicos/efectos de la radiación , Polisorbatos/efectos de la radiación , Composición de Medicamentos , Estabilidad de Medicamentos , Isomerismo , Ácidos Linoleicos Conjugados/química , Ácidos Oléicos/química , Oxidación-Reducción , Fotólisis , Polisorbatos/química , Estabilidad ProteicaRESUMEN
Polysorbates are used ubiquitously in protein therapeutic drugs to help minimize adsorption to surfaces and aggregation. It has been recognized that polysorbate can itself degrade and in turn result in loss of efficacy of therapeutic proteins. We studied the 2 main pathways of polysorbate 80 (PS80) degradation, enzymatic ester hydrolysis, and oxidation. Degraded polysorbates were quantified through mass spectrometry to identify the loss of individual components. Next Langmuir trough adsorption isotherms were used to characterize changes in the surface activity of the degraded polysorbates. PS80 degraded via hydrolysis results in slower surface adsorption rates, whereas the oxidized PS80 show increased surface activity. However, the critical micelle concentration remained unchanged. A monoclonal antibody was formulated with stock and degraded polysorbates to probe their ability to prevent aggregation. Hydrolyzed polysorbate resulted in a large increase in particle formation during shaking stress. Oxidized PS80 was still protective against aggregation for the monoclonal antibody. Monomer loss as measured by SEC was comparable in formulations without PS80 to those with esterase hydrolyzed PS80. Monomer loss for oxidized PS80 was similar to that of nondegraded PS80. Hydrolysis of PS80 resulted in free fatty acids which formed insoluble particles during mechanical agitation which stimulated protein aggregation.
Asunto(s)
Anticuerpos Monoclonales/química , Polisorbatos/química , Tensoactivos/química , Composición de Medicamentos , Estabilidad de Medicamentos , Hidrólisis , Modelos Químicos , Oxidación-Reducción , Agregado de Proteínas , Estabilidad Proteica , Proteolisis , Estrés MecánicoRESUMEN
Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG2 mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process. A correlation between the level of free thiol in the HCCF (as a result of antibody reduction) and aggregation during the low pH step was established, wherein higher levels of free thiol in the starting sample resulted in increased levels of aggregates during low pH treatment. The elevated levels of free thiol were not reduced over the course of purification, resulting in carry-over of high free thiol content into the formulated drug substance. When the drug substance with high free thiols was monitored for product degradation at room temperature and 2-8°C, faster rates of aggregation were observed compared to the drug substance generated from HCCF that was purified immediately after harvest. Further, when antibody reduction mitigations (e.g., chilling, aeration, and addition of cystine) were applied, HCCF could be held for an extended period of time while providing the same product quality/stability as material that had been purified immediately after harvest. Biotechnol. Bioeng. 2017;114: 1264-1274. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Medios de Cultivo/química , Disulfuros/química , Contaminación de Medicamentos/prevención & control , Animales , Células CHO , Cricetulus , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Oxidación-ReducciónRESUMEN
Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.
Asunto(s)
Abatacept/farmacología , Diseño de Fármacos , Inmunosupresores/farmacología , Abatacept/química , Animales , Antígeno B7-1/inmunología , Antígeno B7-2 , Estabilidad de Medicamentos , Humanos , Inmunosupresores/química , Unión Proteica/inmunologíaRESUMEN
In recent years, there has been an increasing concern over the association between computer use and reports of discomfort, aches and pains in students. It is suggested that the physical set-up and individual styles of using interactive media has an influence over this discomfort. As children grow up, they will interact and use computers throughout most of their life. Healthy computing may be vital to preventing/reducing the incidence of discomfort/pain associated with interactive media. This research paper will describe a study, which has collected health and comfort data on the incidence and prevalence of self-reported computer-related musculoskeletal discomfort/pain among 6th and 7th grade students in three middle schools in New England. General base line data from this three-year study is reported.