Quality and consistency in reagents is critical to successful drug discovery and development. When targeting a particular protein of interest, in vitro experiments should be performed with proteins of biological properties similar to those for in vivo tests. It is important that molecularity, purity, shape, and degree of heterogeneity remain the same when any alterations are made to the model protein or the formulation buffer. Multi-angle light scattering (MALS) combined with size-exclusion chromatography (SEC-MALS) is a very useful technique to monitor the solution properties of the protein as changes to reagents are made.
Wyatt Technology
Quality and consistency in reagents is critical to successful drug discovery and development. When targeting a particular protein of interest, in vitro experiments should be performed with proteins of biological properties similar to those for in vivo tests. It is important that molecularity, purity, shape, and degree of heterogeneity remain the same when any alterations are made to the model protein or the formulation buffer. Multi-angle light scattering (MALS) combined with size-exclusion chromatography (SEC-MALS) is a very useful technique to monitor the solution properties of the protein as changes to reagents are made.
As an example, crystallization studies typically have a higher rate of success when the proteins involved are simplified, for example, truncated or expressed in bacteria to minimize post-translational modifications. However, these forms may not be amenable to traditional characterization such as plate-based assays and certain biophysical techniques, where native protein is preferred. Affinity tags or changes to buffer excipients may also be required. All of these differences increase the likelihood of altering solution properties.
The example in this note shows how light scattering data obtained with a DAWN® MALS detector and analyzed by ASTRA® software (both from Wyatt Technology, Santa Barbara, California, USA) were used to elucidate the solution properties of protein expressed from two different constructs. Construct A is the shorter of the two, designed for crystallization efforts.
Figure 1: Two protein constructs: A (45 kDa) and B (46 kDa). SDS-PAGE shows similar purity.
Presented with data from Figure 1, combined with knowledge that the enzyme showed activity and was the predominant species in mass spectrometry, one might not question the suitability of this reagent. SEC-MALS data (Figure 2), however, show that this enzyme is in fact quite heterogeneous.
Figure 2: Standard chromatographic trace (UV 208nM) with MALS data (linear traces within the chromatograms represent the mass distribution across the peak). The enzyme from construct B is much more homogeneous, and is in its biologically active form of a dimer (89 KDa).
Construct B addressed this; it is the full-length enzyme. MALS data show that the protein is in its biologically active form (dimer)and is highly homogeneous. This information gave the project team confidence to move forward with this construct for crystallography, NMR, and high-throughput screening (HTS) efforts.
This note graciously submitted by Mark Tardie and George Karam; Pfizer Central Research, Groton, Connecticut, USA.
Wyatt Technology
6330 Hollister Avenue, Santa Barbara, California 93117, USA
Tel. +1 (805) 681 9009
Website: www.wyatt.comE-mail: info@wyatt.com
GC–TOF-MS Finds 250 Volatile Compounds in E-Cigarette Liquids
November 1st 2024A study has used gas chromatography coupled to a time-of-flight mass spectrometer to build an electron ionization mass spectra database of more than 250 chemicals classified as either volatile or semi-volatile compounds. An additional, confirmatory layer of liquid chromatography–mass spectrometry analysis was subsequently performed.
AI and GenAI Applications to Help Optimize Purification and Yield of Antibodies From Plasma
October 31st 2024Deriving antibodies from plasma products involves several steps, typically starting from the collection of plasma and ending with the purification of the desired antibodies. These are: plasma collection; plasma pooling; fractionation; antibody purification; concentration and formulation; quality control; and packaging and storage. This process results in a purified antibody product that can be used for therapeutic purposes, diagnostic tests, or research. Each step is critical to ensure the safety, efficacy, and quality of the final product. Applications of AI/GenAI in many of these steps can significantly help in the optimization of purification and yield of the desired antibodies. Some specific use-cases are: selecting and optimizing plasma units for optimized plasma pooling; GenAI solution for enterprise search on internal knowledge portal; analysing and optimizing production batch profitability, inventory, yields; monitoring production batch key performance indicators for outlier identification; monitoring production equipment to predict maintenance events; and reducing quality control laboratory testing turnaround time.
Multivariate Design of Experiments for Gas Chromatographic Analysis
November 1st 2024Recent advances in green chemistry have made multivariate experimental design popular in sample preparation development. This approach helps reduce the number of measurements and data for evaluation and can be useful for method development in gas chromatography.