Automated multi-attribute method sample preparation using high-throughput buffer exchange tips

Rationale

The multi-attribute method (MAM) has become a valuable mass spectrometry (MS)-based tool that can identify and quantify the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules in recent years. As we expand the use of the MAM at various stages of drug development, it is critical to enhance the sample preparation throughput without additional chemical modifications and variability.

Methods

In this study, a fully automated MAM sample preparation protocol is presented, where rapid desalting in less than 15 minutes is achieved using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handler. The peptide samples were analyzed using an electrospray ionization (ESI) orbitrap mass spectrometer coupled to an ultra-high-performance liquid chromatography (UHPLC) system with a dual column switching system.

Results

No significant change was observed in product attributes and their quantities compared with manual, low-artifact sample preparation. The sample recovery using the buffer exchange tips was greatly enhanced over the manual spin cartridges while still demonstrating excellent reproducibility for a wide variety of starting sample concentrations. Unlike a plate desalting system, the individual columns provide flexibility in the number of samples prepared at a time and sample locations within plates.

Conclusions

This automated protocol enables the preparation of up to 96 samples with less “at-bench” time than the manual preparation of a smaller batch of samples, thereby greatly facilitating support of process development and the use of the MAM in quality control.

     

Derivatives of Evipal,Alurate, and phanodorn

Zusammenfassung Zur Kennzeichnung von Evipan (1-5-Dimethyl-5-₁-cyclohexenylbarbitursäure), Numal (5-Allyl-5-Isopropylbarbitursäure) und Phanodorm (5-Cyclohexenyl-5-aethylbarbitursäure) wurden Benzylderivate hergestellt und analysiert. Die kristalloptischen Kennzeichen wurden für sechzehn dieser neuen Derivate bestimmt. Die meisten Derivate des Evipans wurden als Öle erhalten und verblieben — mit Ausnahme desp-Nitrobenzylderivates — in diesem Zustande. Die optischen Eigenschaften der Derivate der drei genannten Barbitursäureabkömmlinge unterscheiden sich genügend voneinander, um die Identifizierung der Säuren mit Hilfe der kristalloptischen Daten zu gestatten.     

Kinetics of desorption of ions from quartz and mica surfaces

As part of an investigation concerning the fate of²²⁶Ra during uranium ore milling and long-term taillings storage we have investigated the kinetics of the desorption of alkaline earth, lead and sulfate ions from quartz and mica surfaces into water and dilute nitric acid using a radio-tracer technique. The retention times are sufficiently long to permit the growth of the surface deposits hypothesized in the HILEMAN-SNODGRASS model. The kinetics of desorption of most of these ions is found to agree with the model for rate-limiting desorption proposed by CEREFOLINI. In the case of the desorption of Ba²⁺ from mica surfaces the data suggest some degree of integration of the Ba²⁺ into the mica.     

The formation of hetero-epitaxial deposits of strontium sulfate on mica surfaces

As a continuation of an investigation concerning the fate of²²⁶Ra during uranium ore milling and long-term tailings storage we have grown hetero-epitaxial deposits of strontium sulfate on mica surfaces. The deposits formed readily on the substrate from supersaturated aqueous solutions of the parent ions and appear to be strongly adherent.