RESUMEN
A chromatographic system was adapted to allow monitoring of eluent of preparative column via absorbance and with the chromatographic analysis of the target macromolecule on the same chromatographic system. The proposed approach was tested on important macromolecules, such as monoclonal antibodies, monoclonal antibody aggregates and plasmid DNA (pDNA). A frontal analysis was made on the preparative column, while a chromatographic on-line analysis was performed by sequentially injecting the preparative column outlet on a convection-based analytical column, operating on the same chromatographic system. Cation and/or anion exchangers were used as the chromatographic media (along with a protein A), depending on the sample to be purified. The method was found to be robust and reproducible. To adjust the limit of detection, an algorithm varying the number of injections was used, enabling accurate monitoring of an early breakthrough for concentrations below 1% of the feed concentration. The accuracy varies according to the applied flow rate, but it is typically in the range of few percent, or even below. Due to its simplicity and flexibility, the proposed method can be easily adapted to a pharmaceutical environment.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Algoritmos , Anticuerpos Monoclonales/aislamiento & purificación , ADN/análisis , Sistemas en Línea , Concentración Osmolar , Plásmidos/genética , ARN/análisis , Proteína Estafilocócica A/análisis , Factores de TiempoRESUMEN
It is important to understand how physiological state of the host influence propagation of bacteriophages (phages), due to the potential higher phage production needs in the future. In our study, we tried to elucidate the effect of bacterial growth rate on adsorption constant (δ), latent period (L), burst size (b), and bacteriophage population growth rate (λ). As a model system, a well-studied phage T4 and Escherichia coli K-12 as a host was used. Bacteria were grown in a continuous culture operating at dilution rates in the range between 0.06 and 0.98 hr-1 . It was found that the burst size increases linearly from 8 PFU·cell-1 to 89 PFU·cell-1 with increase in bacteria growth rate. On the other hand, adsorption constant and latent period were both decreasing from 2.6â10-9 ml·min-1 and 80 min to reach limiting values of 0.5 × 10-9 ml·min-1 and 27 min at higher growth rates, respectively. Both trends were mathematically described with Michaelis-Menten based type of equation and reasons for such form are discussed. By applying selected equations, a mathematical equation for prediction of bacteriophage population growth rate as a function of dilution rate was derived, reaching values around 8 hr-1 at highest dilution rate. Interestingly, almost identical description can be obtained using much simpler Monod type equation and possible reasons for this finding are discussed.