Companies:  ALV.  Brookhaven Instruments   Malvern. Wyatt  

Light scattering is one of the few absolute methods available for the determination of molecular mass and structure and is applicable over a broader range of Mw than any other methods. When size-exclusion chromtography (SEC) is used with on-line multi-angle laser light scattering (MALLS) detection, the weight, number and z-average values for obth mass and size may be obtained for most samples. Included in such measurements is the ability to caclulate both differential and accumulative distributions of the Mw and mean square radii. (Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004). 

Liquid chromatography and in particular high performance size exclusive chromatography, HPSEC, is a useful tool for the characterization of polymers. Typically, samples are prepared and injected into a chromatgoraphy where they are pumped thorugh columns that seaprate the molecules based on their hydrodynamic size, smaller molecuels tend to remain longer in the interstices of the columns and thus elute at later times than large molecules. Historically, the chromatograph with its separating columns and concentraiton sensitive detector was claibrated by using nearly monodisperse polymeric standards spanning a broad range of molecular weight, MW. The MWs present in the unkown sample were thus derived form a measurement of the time required for each separated fraction of sample to pass through the chromatograph relative to the corresponding times for the narrow calibration standards. With the advent of in-line light scattering detectors, the need to calibrate was no longer required, since a light scattering detector combined with a concentraiton detector permitted the determiantion of MWs and sizes, and their distributions, on an absoltue basis. (Shortt US Patent No: 5,528366). 

Dynamic Light scattering (DLS):

products: 

Wyatt Technology DyanPro Plate Readr: can perform high-throughput screening with dyanmic light scattering (HTS-DLS), It contains 96, 384 or 1536 well plates and performs temeprature scans of all samples simultaneously for a temperature range of 4-85C. A biomolecule’s staility is not an entirely intrinsci property, as it is influenced by the concetnraiton at which the protein is formulated and buffer composition. Protein stability must be measrued as a function of specific ion type, ionic strengh, pH and excipient profile for an optimal and successful formulation. (Wyatt Technology “The diffusion interaction parameter (Kd) as an indicator of colloidal and thermal stability” 

High throughput light scattering applications are routinely applied in the pharmaceutical industry including dynamic light scattering (DLS) and multi-angle light scattering (MALS). DLS is based on the measurement of intensity fluctuations of scatterid light due to Brownian motion. Autocorrelation curves can then be calculated form the intesity flucutations and fitted to dervie the translational diffusion coefficient D. In the prsence of interactions the moelcuels diffuse according to an effective diffusion coefficient D whcih deviates form the diffusion coefficient of the moelcuels D0. This effective diffusion coefficient D exhibits a linear relationship as a function of mAb concetnration, D=D1(1+KDc), with the slope KD equal to the diffusion itneraction parameter. A positive slope indicates repulsive intermoelcuels interactions, whereas a negative slope indicates attractive intermoelcular interaction.  (Lorenzen Chapter 14, “Mesauring self-assocaition of antibody lead candidates with dynamic light scattering” in “Therapeutic Antibodies Methods and Protocols, Methods in Molecular Biology, springer Protocols, pp. 1-347, 2022)

Various studies suggest that undesriable solution behaviors such as elevated viscosity are caused by short live transient “clusters” that result form attractive prtoein-protein interactions (PPI), The protein interaction parameter (Kd) calculated form low-protein-concetnraiton dyynamic light scattering (DLS) mreasurements is often used to relate PPI to solution viscosity at high protein concetnration. For example, the potein interaction parameter (Kd) calculated from low protein concenration DLS measruements were related to solution viscosity at high protein concentration. These empirical correlations were used to conclude that negative KD values, and therefore attractive PPI, at least qualitatively, predict the large increases in viscosity observed at high mAb concetnration. (Woldeyes, “Molecular-scale understanding of protein interactions and solution viscosities” dissertation, 2018). 

DLS is based on the fluctuations of the scatterd light intensity. It is robust and amendable to high throughput methodeoloy (about 100 samples/day per instrucement). Colloidal stability can be measrued by measuring the concetnration depednence of the prtoein collective diffusion coefficeint via DLS with protein-protein interactions being parameterized via the interaction parameter KD and comparing diffusion coefficient values measrued usying Taylor disperson analysis. (Sikka, sirat “Studying Protein-Protein interactions using dynamic light scatteringa nd Taylor Disperson Analysis” Dissertation. 

A negative KD value (protein-protien interation parameter obtainable from DLS) can be used to udnerstand the effects of NaCL and ArgHCL on protein-protein interactions. A negative KD value indicates an attractive prtein-protein interaction, while a positive KD value indicates a repulsive protein-protein interaction. (Alsabih “Towards an improved predictor for the colloidal stability of unfolded prtoeins through probing aggregation behaviour in solutions containing chemical denaturants” Thesis. 

With UV absorbance (multi-angle light scattering (MALS) +UV):

Ion Exchange:

Bailey (US Patent Application 16/579,220, published as US 2020/0018771 and US 13/850,664, published as US 10481164; see also US 17/495,963 published as US 2022/0026442) disclose a method for determing a stop collection point during IEX which includes monitoring the eluate with a laser light scattering detector and a UV absorbance detector and calculating a fraction LS/UV ratio for each fraction until he peak max LS/UV is determined, calculating a normalized LS/UV ratio for all subsequent fractions, wherein an increase in the normalized LS/UV ratio indicates an increase in the amount of impurities in the eluate pool and terminating the eltuion when the normalized LS/UV ratio reaches a predetermined value. In one embodiment, the normalized LS/UV ratio is calculated by dividing the fraction LS/UV ratio by the peak max LS/UV ratio. When a purificaiton process is operated in bind and elute or gradeint elution mode, the protein product is eluted first and aggregated species are present in the tail of the elution peak. Real time monitoring of the change in the ratio allows for a real time determiantion of the stop collection point based on the properties of the desired product at the time of elution. A monomeric protein peak, for instance, will have a constant ratio of LS/UV. Any deviation in that ratio signifies the product stream is contaminated with non-monomer impurities. The ration of the light scattering (LS) to absorbance concentration (for example, UV) is determiend real time. In one embodiment, eluate from a protein A column was diverted using a slipstream port to a AKTA UV-900 by pump and then passed to a Heleous II (Wyatt Technology) light scattering detector. The signals were processed in real time using a software program that cacluated the LS/UV ration of the elution preaks, “fraction LS/UV ratio” using the signals form the light scattering detector and UV absorbance. The purest fraction was the peak max. The stop collection point for the elution was set at a predetermiend normalized LS/UV ratio in the elution pool. An increase in high molecular eight cotaminants in the elution was reflected in an increase in the normalized LS/UV ratio. 

With Size-exclusion (LS +SEC):

The SEC-MALLS technique does not rely on relative Mw standrds for column calibration and yields absolute Mw estimates direclty from the angular dependence of scatterd light intesity as a funciton of concentration, as formulated by light scattering theory. Oliva, Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins” Current drug discovery technologies, 1(3), pp. 229-242 (2004).

Harman “Charcterization and analysis of thermal denaturation of antibodies by size exlusion high-performance liquid chromatography with quadruple detection” (Analytical Biochemistry 325 (2004) 227-239) discloses that SEX coupled with online light scattering and UV visible spectroscopy provides a very powerful tool for studying protein size, shape and aggregation. 

Kalonia (US 2007/0178013) discloses appatuses and methods for simltaneous measurment of protein concentraiton and scatered light intensity.