Keeping pace with the increasing demand for high quality drug candidates in pharmaceutical research: Development of a new two-step preparative tandem high performance chromatographic system for the purification of antibodies.

In the area of biological drug development, high throughput (HT) technologies are key to identifying the most promising therapeutic candidate in a time-efficient and market-competitive manner. While efficient cloning and expression methods exist, HT downstream processing mainly relies on liquid handling workstations applying miniaturized chromatography columns or resin-based 96-well plates to shorten process development time. However, there is still a lack of generic, preparative chromatographic methods devoid of aggregates and endotoxins with sufficient throughput. The only truly generic antibody purification strategy including an efficient dimer removal consists of Protein A capture followed by size exclusion chromatography (SEC) as a polishing step. Other polishing methods, including IEX, HIC, and CHT, require an antibody-specific fine tuning. However, standard preparative SEC setups tend to be rather time-consuming, and so limit throughput. In this work, we devised a unique chromatography setup enabling an unattended two-step purification of IgGs on the milligram scale directly from 35 mL clarified cell supernatants, processing up to 48 samples in 44.0 h. By introducing a silica-based SEC column, preparative SEC could be accelerated. By further developing an HT two-step preparative Protein A/alternating column regeneration SEC system using Agilent 1260 Infinity LC components, mAbs can be purified generically by two chromatographic steps in 55 min. In this way, by using a 2-position/10-port valve and two quaternary pumps, two SEC columns can be run in parallel, excluding the cleaning and equilibrating phase from the actual cycle time. By further applying a third pump, the Protein A step can be run independently, resulting in a time-optimized process nesting. By introducing a CETAC ASX-520 autosampler, 48 samples can be run automatically without any user intervention over two working days. The developed system is highly reproducible for all tested human IgG1 antibodies, easily generating milligram scale material sufficient for full characterization of the antibodies and for their use in in vitro and in vivo activity assessments.

Development of nonfibrotic left ventricular hypertrophy in an ANG II-induced chronic ovine hypertension model.

Hypertension is a major risk factor for many cardiovascular diseases and leads to subsequent concomitant pathologies such as left ventricular hypertrophy (LVH). Translational approaches using large animals get more important as they allow the use of standard clinical procedures in an experimental setting. Therefore, the aim of this study was to establish a minimally invasive ovine hypertension model using chronic angiotensin II (ANG II) treatment and to characterize its effects on cardiac remodeling after 8 weeks. Sheep were implanted with osmotic minipumps filled with either vehicle control (n = 7) or ANG II (n = 9) for 8 weeks. Mean arterial blood pressure in the ANG II-treated group increased from 87.4 ± 5.3 to 111.8 ± 6.9 mmHg (P = 0.00013). Cardiovascular magnetic resonance imaging showed an increase in left ventricular mass from 112 ± 12.6 g to 131 ± 18.7 g after 7 weeks (P = 0.0017). This was confirmed by postmortem measurement of left ventricular wall thickness which was higher in ANG II-treated animals compared to the control group (18 ± 4 mm vs. 13 ± 2 mm, respectively, P = 0.002). However, ANG II-treated sheep did not reveal any signs of fibrosis or inflammatory infiltrates as defined by picrosirius red and H&E staining on myocardial full thickness paraffin sections of both atria and ventricles. Measurements of plasma high-sensitivity C-reactive protein and urinary 8-iso-prostaglandin F2α were inconspicuous in all animals. Furthermore, multielectrode surface mapping of the heart did not show any differences in epicardial conduction velocity and heterogeneity. These data demonstrate that chronic ANG II treatment using osmotic minipumps presents a reliable, minimally invasive approach to establish hypertension and nonfibrotic LVH in sheep.

Frequency and age-related course of mitral valve dysfunction in the Marfan syndrome.

Mitral valve (MV) prolapse (MVP) has a high prevalence of 2% to 3% in the general population and thus constitutes the most common cause of severe nonischemic MV regurgitation (MVR). MVP is also common in persons with the Marfan syndrome. However, to date, a large-scale population-based cohort study using modern echocardiographic techniques has not been performed, and the frequency of MVP and the relation of MV dysfunction and age have not been investigated. Therefore, we conducted a population-based cohort study of 204 patients (108 males and 96 females, aged 31.2 ± 16.4 years) with classic Marfan syndrome. We performed echocardiographic follow-up of 174 patients for a mean of 4.4 ± 4.3 years. On the initial or subsequent echocardiographic scan, MVP was present in 82 patients (40%), severe MVR in 25 (12%), and MV endocarditis in 5 patients (2.5%). At 30 years of age, the Weibull cumulative distribution was 42.6% (95% confidence interval [CI] 36% to 50%) for MVP, 56.5% (95% CI 49.3% to 64%) for MVR of any degree, 6.7% (95% CI 3.9% to 11.3%) for severe MVR, and 0.92% (95% CI 0.21% to 3.91%) for MV endocarditis. The cumulative hazard for severe MVR and MV endocarditis was estimated to increase with age. MVP was associated with dural ectasia (p = 0.01), ectopia lentis (p = 0.02), and skeletal involvement (p <0.001). Severe MVR was related to tricuspid valve prolapse (p = 0.002) and to the sporadic form of the Marfan syndrome (p = 0.006). In conclusion, MVP was comparatively frequent in patients with the Marfan syndrome and carries an increased risk of progression to severe MVR and endocarditis, especially in older adults.