RESUMEN
In the past decade, recombinant adeno-associated virus (rAAV) has gained increased attention as a prominent gene therapy technology to treat monogenetic diseases. One of the challenges in rAAV production is the enrichment of full rAAV particles containing the gene of interest (GOI) payload. By adjusting the mobile phase properties of anion-exchange chromatography (AEX), it was demonstrated that empty and full separation of rAAV was improved in monolith based preparative AEX chromatography. When compared to the baseline method using NaCl, the use of tetraethylammonium acetate (TEA-Ac) in the AEX mobile phase resulted in enhanced resolution from 0.75 to 1.23 between "Empty" and "Full" peaks by salt linear gradient elution, as well as increased the percentage of full rAAV particles from 20% to 36% and genome recovery from 59% to 62%. Furthermore, a dual wash plus step elution AEX method was developed. Wherein, the first wash step harnesses TEA-Ac to separate empty and full capsids, which is followed by a second wash step that ensures no TEA-Ac salt is carried over into AEX eluate. The resulting optimized AEX purification method has the potential to be adapted for manufacturing and purification processes involving various rAAV production platforms that experience empty and full rAAV separation challenges.
Asunto(s)
Dependovirus , Vectores Genéticos , Cromatografía por Intercambio Iónico/métodos , Dependovirus/genética , Cápside/química , Clonación MolecularRESUMEN
Extensive testing of hydrolysates of commercially available organosilanes has identified a number of bifunctional organosiloxane compounds that show potential as therapeutics for treatment of diseases characterized by amyloid deposition such as Alzheimer's disease (AD). All of these compounds protect from and/or reverse the metal-induced aggregation of amyloid Abeta(1-42) peptide in dynamic light scattering (DLS) assays in trifluoroethanol (TFE) solutions, protect from and/or reverse the metal-induced loss of alpha-helical structure in TFE solutions of amyloid Abeta(1-42) as measured by circular dichroism (CD), and are able to cross blood-brain barrier models at rates above background using Caco-2 and MDCK cell permeation assays. Based on these studies, we conclude that members of this class of bifunctional organosiloxanes are promising candidates for testing in treatment and/or prevention of AD and other diseases characterized by amyloid deposition.
Asunto(s)
Enfermedades Neurodegenerativas/prevención & control , Siloxanos/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Dicroismo Circular/métodos , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Fragmentos de Péptidos/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Trifluoroetanol/farmacología , Trifluoroetanol/uso terapéuticoRESUMEN
Protein self-association or aggregation is a property of significant concern for biopharmaceutical products due to the potential ability of aggregates to cause adverse toxicological and immunological effects. Thus, during the development of a protein biopharmaceutical, it is important to detect and quantify the level and nature of aggregate species as early as possible in order to make well-informed decisions and to mitigate and control potential risks. Although a deeper understanding of the mechanism of aggregation (i.e., protein-protein interactions) is desirable, such detailed assessment is not always necessary from a biopharmaceutical process development point of view. In fact, the scope of characterization efforts is often focused on achieving a well-controlled process, which generates a product that reliably meets established acceptance criteria for safety and efficacy. In this brief note, we evaluated the utility of size-exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation in their simplest forms, to effectively reveal and confirm the presence of concentration-dependent reversible self-association (RSA) in a monoclonal antibody in the early stages of formulation development. Using these techniques, we also initiated preliminary work aimed at reducing the occurrence of this RSA behavior by varying the pH of the formulation buffer.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Agregado de Proteínas , Dispersión Dinámica de Luz/métodos , Tamaño de la Partícula , Ultracentrifugación/métodosRESUMEN
There is significant scope for more meaningful evaluation of higher-order structure in defining the quality of biopharmaceutical products [Bush L. 2010. Biopharm Int 23(4):14]. We have used isothermal titration calorimetry (ITC) to characterize the Ca(2+) -binding isotherm of a recombinant human factor IX Fc fusion protein (rFIXFc) and the parent recombinant human factor IX molecule (rFIX). Circular dichroism, intrinsic fluorescence, and Fourier transform infrared spectroscopies detected characteristic spectral changes that appear qualitatively consistent with the previously characterized behavior of the factor IX molecule. Sedimentation velocity and dynamic light scattering measurements were recorded in the presence and absence of Ca(2+) over the protein concentration range 1-10 mg/mL. ITC of Ca(2+) binding to rFIXFc reveals a distinctive exothermic-binding isotherm, which is interpreted as consistent with two high-affinity and approximately 14 lower-affinity Ca(2+) sites reported in the literature for human factor IX (Schmidt AE, Bajaj SP. 2003. Trends Cardiovasc Med 13(1):39-45). Analysis of accelerated degradation samples showed significant alterations in Ca(2+) binding, which correlates with significant loss of biopotency and fragmentation by gel chip capillary electrophoresis. Collectively, these data establish a close correspondence in the Ca(2+) -binding characteristics of rFIXFc and its parent rFIX molecule. The utility of ITC to provide a highly pertinent and selective biophysical signature of structure-function for a therapeutic factor protein is discussed.
Asunto(s)
Calcio/metabolismo , Factor IX/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Calorimetría , Cromatografía en Gel , Dicroismo Circular , Electroforesis Capilar , Factor IX/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Dispersión de Radiación , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , UltracentrifugaciónRESUMEN
The orientation of the substrate camphor in the active site of reduced CO-bound cytochrome P450cam (CYP101) as a function of reduced putidaredoxin (Pdxr) addition has been examined by NMR using perdeuterated CYP101 and perdeuterated Pdx as well as isotopically labeled d-camphor. This permits the 1H resonances of CYP101-bound camphor to be observed without interference from the signals of CYP101 or Pdx and confirms assignments of the methyl signals of camphor in the bound form. The Cys4Fe2S2 ferredoxin Pdx is the physiological redox partner and effector of CYP101. The addition of Pdx to the reduced CYP101-camphor-CO complex results in a conformational selection that is slow on the chemical shift time scale with spectral effects observed primarily at the 8-CH3 group of the camphor. The camphor signals are ring current shifted by the heme, and for the 9- and 10-CH3 resonances, these shifts are reasonably well predicted by ring current calculations from the crystal structure of CO-bound CYP101. However, in the absence of Pdx, the 8-CH3 resonance of CYP101-bound camphor is observed at considerably higher field than predicted. Dynamic simulations using ring current shift restraints generated a structure with low chemical shift violations in which the hydrogen bond between the camphor carbonyl oxygen and the OH of Tyr96 is lost, and an expansion of the active site takes place that permits reorientation of the camphor within the active site.