Implications of AAV affinity column reuse and vector stability on product quality attributes.

In this work, the implications of AAV9 capsid design and column reuse on AAV9 vector product quality were assessed with POROS CaptureSelect (PCS) AAVX and AAV9 resins using sf9 insect cell-derived model AAV9 vectors with varying viral protein (VP) ratios. Chromatographic experiments with purified drug substance AAV9 model feeds indicated consistent vector elution profiles, independent of adeno-associated virus (AAV) VP ratio, or cycle number. In contrast, the presence of process impurities in the clarified lysate feeds resulted in clear changes in the elution patterns. This included increased aggregate content in the vector eluates over multiple cycles as well as clear differences in the performance of these affinity resin systems. The AAV9-serotype specific PCS AAV9 column, with lower vector elution pH, resulted in higher aggregate content over multiple cycles as compared to the serotype-independent PCS AAVX column. Further, the results with vectors of varying VP ratio indicated that while one vector type eluate displayed higher aggregation in both affinity columns over column reuse, the eluate with the other vector type did not exhibit changes in the aggregation profile. Interestingly, vector aggregates in the affinity eluates also contained double-stranded DNA impurities and histone proteins, with similar trends to the aggregate levels. This behavior upon column reuse indicates that these host cell impurities are likely carried over to subsequent runs due to incomplete clean-in-place (CIP). These results indicate that feed impurities, affinity resin characteristics, elution pH, column CIP, and vector stability can impact the reusability of AAV affinity columns and product quality.

Toward MRI and Optical Detection of Zwitterionic Neurotransmitters: Near-Infrared Luminescent and Magnetic Properties of Macrocyclic Lanthanide(III) Complexes Appended with a Crown Ether and a Benzophenone Chromophore.

Thanks to their versatile magnetic and luminescence features, lanthanide complexes have gained a central position in biomedical imaging as magnetic resonance imaging (MRI) contrast agents and optical imaging probes. In addition, appropriate chemical design allows modification of the magnetic relaxation properties of GdIII complexes and the optical properties of visible- or near-infrared (NIR)-emitting lanthanide chelates upon interaction with various biomarkers, which makes them ideal candidates for the creation of responsive agents. In this Forum Article, we demonstrate such design principles as well as the difficulties encountered in the context of neurotransmitter (NT) detection. Lanthanide(III) complexes of a macrocyclic ligand incorporating a benzophenone chromophore and a monoazacrown ether (LnL3) have been synthesized as responsive probes to monitor amino acid NTs either in MRI (Ln = Gd) or in NIR optical detection (Ln = Nd or Yb). The parameters characterizing the water exchange and rotational dynamics of the gadolinium(III) complex were assessed by 17O NMR and 1H NMRD. In the presence of zwitterionic NTs, the inner-sphere water molecule is replaced by the carboxylate function of the NTs in the gadolinium(III) complex, leading to a decrease of the longitudinal relaxivity from 6.7 to 2-2.5 mM-1 s-1 (300 MHz and 37 °C). The apparent affinity constants range from Ka = 35 for γ-aminobutyric acid (GABA) to 80 M-1 for glycine and glutamate, and there is no selectivity with respect to hydrogen carbonate (Ka = 232; pH 7.4). The gadolinium(III) complex interacts with human serum albumin (HSA), resulting in a 60% increase in the relaxivity (20 MHz, 37 °C) in the absence of NTs. The HSA-bound complex, however, was revealed to be less responsive to NTs because of displacement of the GdIII-bound water by HSA, which was confirmed by the hydration number calculated from luminescence lifetimes of the HSA-bound europium(III) complex. The creation of an imaging agent suitable for NIR detection of NTs for an enhanced sensitivity in biological systems using the benzophenone (BP) moiety as the sensitizer of lanthanide luminescence was also attempted. Upon excitation at 300 nm of the BP chromophore in aqueous solutions of NdL3 and YbL3, characteristic NIR emissions of NdIII and YbIII were observed because of 4F3/2 → 4IJ (J = 9/2-13/2) and 2F5/2 → 2F7/2 transitions, respectively, indicating that this chromophore is a suitable antenna. Despite these promising results, luminescence titrations of NdIII and YbIII complexes with NTs were not conclusive because of chemical conversion of the ligand triggered by light, preventing quantitative analysis. The observed photochemical reaction of the ligand is strongly dependent on the nature of the lanthanide chelated; it is considerably slowed down in the presence of NdIII and EuIII.

Using remote substituents to control solution structure and anion binding in lanthanide complexes.

A study of the anion-binding properties of three structurally related lanthanide complexes, which all contain chemically identical anion-binding motifs, has revealed dramatic differences in their anion affinity. These arise as a consequence of changes in the substitution pattern on the periphery of the molecule, at a substantial distance from the binding pocket. Herein, we explore these remote substituent effects and explain the observed behaviour through discussion of the way in which remote substituents can influence and control the global structure of a molecule through their demands upon conformational space. Peripheral modifications to a binuclear lanthanide motif derived from α,α'-bis(DO3 Ayl)-m-xylene are shown to result in dramatic changes to the binding constant for isophthalate. In this system, the parent compound displays considerable conformational flexibility, yet can be assumed to bind to isophthalate through a well-defined conformer. Addition of steric bulk remote from the binding site restricts conformational mobility, giving rise to an increase in binding constant on entropic grounds as long as the ideal binding conformation is not excluded from the available range of conformers.

Aryl-phosphonate lanthanide complexes and their fluorinated derivatives: investigation of their unusual relaxometric behavior and potential application as dual frequency 1H/19F†MRI probes.

A series of low molecular weight lanthanide complexes were developed that have high (1)H longitudinal relaxivities (r1) and the potential to be used as dual frequency (1)H and (19)F MR probes. Their behavior was investigated in more detail through relaxometry, pH-potentiometry, luminescence, and multinuclear NMR spectroscopy. Fitting of the (1)H NMRD and (17)O NMR profiles demonstrated a very short water residence lifetime (<10 ns) and an appreciable second sphere effect. At lower field strengths (20 MHz), two of the complexes displayed a peak in r1 (21.7 and 16.3 mM(-1) s(-1)) caused by an agglomeration, that can be disrupted through the addition of phosphate anions. NMR spectroscopy revealed that at least two species are present in solution interconverting through an intramolecular binding process. Two complexes provided a suitable signal in (19)F NMR spectroscopy and through the selection of optimized imaging parameters, phantom images were obtained in a MRI scanner at concentrations as low as 1 mM. The developed probes could be visualized through both (1)H and (19)F MRI, showing their capability to function as dual frequency MRI contrast agents.

An aryl-phosphonate appended macrocyclic platform for lanthanide based bimodal imaging agents.

Four ligand systems have been prepared whose characteristics are well suited to the design of bimodal MRI and luminescence probes. The lanthanide complexes display high relaxivities and luminescence quantum yields. These properties are retained at higher magnetic fields and in a range of competitive environments including model extracellular medium and cultured cells.

Synthesis and spectroscopic studies on azo-dye derivatives of polymetallic lanthanide complexes: using diazotization to link metal complexes together.

Heteronuclear tetrametallic lanthanide complexes have been synthesized from stable complexes by diazotization and azo-compound formation. Luminescence spectroscopy has been used to show that the complexes used as building blocks are stable under the reaction conditions.