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16 Buyers’ Guide 2021


Oligonucleotide The LenS3


MALS was also used to


investigate low MW oligonucleotides at extremely low concentrations. Accurate molecular weight profi ling of an unpurifi ed oligonucleotide sample was successfully achieved. The results in Figure 5 demonstrate excellent sensitivity and reproducibility of retention time and MW.


Polystyrene Figure 3: Angular dissymmetry plot for direct Rg determination.


o Elimination of the incident beam to obtain a cleaner signal at low and high angles.


Molecular weight and Rg determination


Thanks to the presence of a low angle measurement with an excellent signal-to- noise ratio, MW can be measured directly on the LenS3


MALS without the need for


an extrapolation procedure such as the complex Zimm plot. The only underlying assumption is that if the molecules are within the size range that SEC columns can separate, the scattering intensity at 10° equals the intensity at 0° [1]. The molecular weight of the molecules is simply proportional to LALS intensity.


A new method for determining Rg was


developed to take advantage of the detector design. From Rayleigh’s equation for suffi ciently dilute solutions, one can defi ne the angular dependence as the ratio of the intensity of scattered light at a given angle to that at 0° angle. A plot of this ratio for the LALS, RALS and HALS against the angle of observation is used to map the angular dependence pattern and provide a direct Rg


determination from the slope of the obtained curve (Figure 3).


Benefi ts for the characterisation of macromolecules


With the higher sensitivity provided by the LenS3


MALS detector smaller quantities


of material are required to characterise samples accurately, which is critical in applications where the quantity of sample available may be a limiting factor. For protein applications, the presence of aggregates and fragments of an antibody can also be detected down to a much lower


level when compared to traditional MALS designs.


Samples with a low molecular weight or a low refractive index increment (dn/dc) inherently scatter a lower amount of light. A MALS detector such as the LenS3


Analysis of polystyrene standards was conducted in toluene for size determination. Table 2 shows Rg


values below 10 nm for the


fi rst time ever reported by light scattering. The values observed by the LenS3


validated by small-angle X-ray scattering (SAXS) measurements reported in the literature [2].


allows


the MW of those types of molecules to be measured with greater accuracy.


Additionally, the position of the extreme angles and their higher signal-to-noise ratio opens new areas of applications for the size


measurement of macromolecules with an Rg below 10nm.


Examples of applications


Experimental Conditions Experimental conditions for the three application examples are listed in Table 1.


Monoclonal Antibody


A biosimilar of Herceptin® (trastuzumab), was analysed by UHPLC-SEC-MALS using the LenS3


MALS detector. Decreasing


amounts of sample were injected to determine the limit of detection in these conditions. Aggregates, monomer, and monoclonal antibody fragments were readily observed down to 50 ng of loading. Figure 4 (A and B) shows that its monomer was easily detected at only 2ng loading.


Table 2: Rg Conclusions


The novel confi guration of the LenS3 MALS detector features an elongated fl ow chamber made of non-refractive inert material, associated with an improved optical bench and a green laser. This new design increases signal intensity while minimising noise, resulting in exceptional detection sensitivity.


Furthermore, with extreme angles at 10° and 170° in addition to the 90° angle, the instrument can detect angular dependence to an extremely low level to measure the lowest Rg


ever reported by light scattering. The detector also takes advantage of the low angle to measure MW directly and accurately without extrapolation.


This innovative approach represents the fi rst signifi cant advancement in light scattering technologies in four decades. This will benefi t the analysis of all types of macromolecules, from biomolecules to synthetic polymers.


MALS are


of polystyrene standards in toluene measured by LenS3


Sample ID MW [Da]


A5000 F-1 F-2 F-4


F-10 F-20


5,796


10,650 18,554 40,510


100,432 195,787


Conc.


[mg/mL] 4.81 4.27 3.22 2.79 1.97 1.02


Rg by LenS3


[nm] 2.11 3.04 4.34 6.59


10.48 15.78


MALS. Rg by SAXS


[nm] 2.04 2.93 4.32 6.69 N/A 16.2


Difference [%]


3.37% 3.69% 0.46% 1.51% N/A


2.63%


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