Determination of molecular masses of proteins in solution: Implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory (original) (raw)
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Journal of Pharmaceutical and Biomedical Analysis, 2001
High-performance size-exclusion chromatography (SEC) based on UV-Vis detection is a relative technique for molecular weight determination whereas procedure based on multi-angle laser light scattering (MALLS) is both rapid and absolute. The two methods using recombinant human growth hormone (rHGH) and b-lactoglobulin samples were compared. A calibration curve for the chromatographic system was generated based on standard proteins and the data were fitted by least squares to a third order polynomial model. The molecular weight from the conventional SEC method for both proteins was higher than the reported values. The molecular weight of rHGH from MALLS was 23.1 9 0.57 and 21.2 9 0.80 kDa using differential refractive index (SEC-MALLS/RI) and UV (SEC-MALLS/ UV-Vis) detectors as mass detectors. Both values agree, within experimental error with the molecular weight sequence of rHGH, 22.1 kDa. In contrast, the molecular weight from LS for b-lactoglobulin was 22.5 9 0.55 kDa by SEC-MALLS/RI and 23.0 91.22 kDa by SEC-MALLS/UV-Vis, respectively, values always higher than those supplied by the manufacturer, 18.4 kDa. The reproducibilty of the SEC-MALLS/UV-Vis method versus the SEC-MALLS/RI method was performed using the concordance correlation coefficient. The method%s reproducibility was accepted by assuming a precision of 98% and a 1% loss in precision.
Current Drug Discovery Technologies, 2004
The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and/or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates. Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution. The purpose of the current review is to describe and discuss the capability of the SEC/LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor/ligands. For this, the "two or three detector" methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes. Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.
Methods in molecular biology (Clifton, N.J.), 2006
Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of molar mass and oligomeric state in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step, while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis.
Journal of Agricultural and Food Chemistry, 1992
Size exclusion chromatography with low-angle laser light scattering detection (SEC/LALLS) has been applied to the separation and characterization of a number of proteins as a function of molecular weight using different SEC columns and elution buffers. The use of a LALLS detector in conjunction with a concentration detector eliminated the need for calibration of the SEC column. Data acquisition and analysis were under computer control. The technique has been demonstrated to determine molecular weights for a variety of proteins, including natural bovine and natural porcine somatotropins. The LALLS detector combined with an SEC column was effective in determining an accurate molecular weight even under conditions when the SEC columns' separation was not strictly size-dependent. Applications of the technique to protein samples that contain dimers and higher order aggregates, i.e., samples which may arise in protein stability and formulation studies, will be discussed.
Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hy-drodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.
Biomolecules, 2022
Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique that determines the absolute molar mass (molecular weight) of macromolecules in solution, such as proteins or polymers, by detecting their light scattering intensity. Because SEC-MALS does not rely on the assumption of the globular state of the analyte and the calibration of standards, the molar mass can be obtained for proteins of any shape, as well as for intrinsically disordered proteins and aggregates. Yet, corrections need to be made for samples that absorb light at the wavelength of the MALS laser, such as iron–sulfur [Fe-S] cluster-containing proteins. We analyze several examples of [2Fe-2S] and [4Fe-4S] cluster-containing proteins, for which various corrections were applied to determine the absolute molar mass of both the apo- and holo-forms. Importantly, the determination of the absolute molar mass of the [2Fe-2S]-containing holo-NEET proteins allowed us to ascertain a change i...
Methods in molecular biology (Clifton, N.J.), 2008
Mass spectrometry (MS) is widely used within structural and functional proteomics for a variety of tasks including protein quality assessment, identification, and characterization. MS is used routinely for the determination of the total mass of proteins, including N-glycosylated proteins, analysis of selenomethionine incorporation, crystal content verification, and analysis of N-glycosylation site occupancy. Protocols for sample preparation, data collection, and analysis are given.A recent development is the fluorescence-based thermal shift (ThermoFluor) assay. It uses an environmentally sensitive dye, Sypro Orange, to monitor the thermal stability of a protein and investigate factors (e.g., buffers, additives, and ligands) affecting this stability. This chapter describes the application of this method using a 96-condition in-house screen. The measurements are performed on a commercially available real-time PCR machine. Multiangle or static light scattering (SLS) is a very powerful ...
Accuracy of Protein Size Estimates Based on Light Scattering Measurements
Open Journal of Biophysics, 2014
There are two types of light scattering measurements: static light scattering (SLS) and dynamic light scattering (DLS). The SLS method is used to estimate the molecular weight (MW) of particles by measuring the time-averaged intensity of light scattered by the particles, whereas the DLS method is used to estimate the diffusion coefficient of particles by observing the time-correlation of scattered light intensity. These techniques have recently been applied to the investigation of the aggregation, denaturation and folding, and complex formation of proteins in solution. However, the accuracy of protein size measurement by light scattering is poorly understood. In the present study, we carried out the size measurements of five globular proteins by SLS and DLS at a detection angle of 90˚ and compared these data to measurements made by size exclusion chromatography (SEC). The difference (%) between the MW estimated from each method and the MW calculated from the amino acid sequence (namely the calibration residual error) was regarded as an index of measurement accuracy. The averaged calibration residual errors were 5.2 and 4.7 for SEC and SLS measurements, respectively. For the DLS measurements, the extrapolation of the apparent hydrodynamic radii to a protein concentration of zero may effectively eliminate the interparticle and hydrodynamic interactions and significantly reduced the averaged calibration residual error to 4.8%. Our results suggested that the size of globular proteins can be estimated using light scattering measurements with an accuracy equivalent to that of SEC.
Light-Scattering Immunoassay of Specific Proteins: A Review
Annals of Clinical Biochemistry: International Journal of Laboratory Medicine, 1983
Immunoprecipitation techniques have been employed in the measurement of specific proteins for many years. The early techniques of gel precipitation are now gradually being replaced by nephelometric and turbidimetric measurement of the antigenantibody complex in free solution. The reason for this is primarily due to an increased awareness of the value of specific protein measurement combined with improvements in immunochemical reagents and advances in instrumentation. Light-scattering measurements of the antigenantibody reaction were performed as early as 1938 by Libby.' However, it was not until specific antisera became available that immunonephelometry became widely used. The work of Ritchie et al. 2 using a continuous-flow system helped to identify the need of the analyst for high-affinity antibodies and a means of enhancing the reaction rate. During the 1970s the basic work of Savory et al. 3 on the kinetics of the antigen-antibody reaction paved the way for discrete nephelometric systems. At the present time a large number of commercial systems are available for immunological assays based on light scattering, including some using centrifugal analysis.s This surge of interest in immunonephelometry has stimulated a number of workers to look again at the technique of turbidimetry with the aid of polymer enhancement and high-affinity antibodies.
Flow injection analysis: Rayleigh light scattering technique for total protein determination
Analytical and bioanalytical chemistry, 2003
A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of total protein concentrations. This method is based on the weak intensity of RLS of bromothymol blue (BB) (3',3"-dibromothymolsulfonephthalein) which can be enhanced by the addition of protein in weakly acidic solution. A common spectrofluorimeter was used as a detector. It was proved that the application of this method to quantify the total proteins in real samples by using bovine serum albumin was possible. The RLS signal was detected at lambda(ex)= lambda(em)=572 nm. The linear range was 7.0-70.0 microg mL(-1), the detection limit was 3.75 microg mL(-1), the reproducibility was 5.5% (n=7), and the sample throughput was 26 h(-1).