13


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).


Figure 2. LenS3


MALS enhanced optics, showing the low angle (LALS), right angle (RALS) and high angle (HALS) light scattering detectors.


ultimately increasing the scattering intensities. The chamber’s non-refractive material prevents the scattered light hitting the wall from bouncing back, leading to stray light and subsequent noise. The conical shape defi nes the forward (10°) and backward (170°) angles of collection of scattered light, while the perpendicular (90°) measurement is made at the centre of the channel through a separate observation window equipped with a spherical lens.


Improved optics The LenS3


4.


MALS detector also features improved optics to provide even more intensity of


scattered light and cleaner signals (Figure 2). Using a green laser (0


compared to the typical red laser (0 proportional to 1/0


Figure 3: Angular dissymmetry plot for direct Rg


=505nm), the scattered intensity increases by a factor of three, =660nm) used in most LS instruments, as scattering is


The optical bench includes mirrors in both the backscattering and forward scattering positions, with a hole where the incident beam can travel through the mirrors so that the incident beam is effectively eliminated. Only the annulus of light at the desired angles 10° and 170° is collected.


to traditional MALS instruments, comes from the combination of the following elements: • The wider angles of measurement:


o True, usable ultra-low angle (LALS at 10°) for accurate and direct MW determination without extrapolation


o Ultra-high angle (HALS at 170°) used in conjunction with the LALS and the 90° angle (RALS) to detect the smallest difference in scattered intensity for Rg measurements of smaller molecules.


• The novel fl ow channel:


o Elongated conical shape to maximise scattering volume and thus signal intensity. o Black, inert polymeric material eliminates stray light to reduce noise. • The advanced optics: o A green laser to increase the intensity of scattering by a factor of three. o Elimination of the incident beam to obtain a cleaner signal at low and high angles.


Table 1. Experimental chromatographic conditions for the applications. Monoclonal Antibody Oligonucleotide Columns


Mobile phase


TSKgel UP-SW3000 (2 µm, 4.6 mm x 30 cm)


100 mmol/L NaH2 SO4 PO4 ,


pH 6.8 + 100 mmol/L Na2


Flow Rate 0.35 mL /min


Temperature 25°C Detection Sample


MALS


TSKgel UP-SW2000 (2 µm 4.6 mm x 30 cm)


0.5 mol/L NaCl, 0.1 mol/L EDTA, pH 7.5 0.1 mol/L Na2 0.03% NaN3


SO4 , in 0.1 mol/L


phosphate buffer 0.3mL/min 30°C


UV@260 nm; MALS


Herceptin biosimilar 20 bases custom oligonucleotide with MW= 6141 Da (1 mg/mL)


1 mL/min 40°C MALS


Polystyrene standards Table 2. Rg of polystyrene standards in toluene measured by LenS3


Sample ID MW [Da]


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.


A5000 F-1 F-2 F-4


F-10 F-20


5,796


10,650 18,554 40,510


Conc.


[mg/mL] 4.81 4.27 3.22 2.79


100,432 1.97 195,787 1.02


Rg by LenS3


[nm] 2.11 3.04 4.34 6.59


10.48 15.78


Rg MALS. 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% Polystyrene


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


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


TSKgel GMHHR-N (5 µm, 7.8 mm x 30 cm)


Toluene


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.


Overall, the gain in performance and sensitivity of the LenS3 MALS detectors, as compared


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.


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


Benefi ts for the characterisation of macromolecules With the higher sensitivity provided by the LenS3


MALS detector smaller quantities of determination.


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


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®


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.


LenS3 (trastuzumab), was analysed by UHPLC-SEC-MALS using the


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