Opalescence of an IgG1 Monoclonal Antibody Formulation is Mediated by Ionic Strength and Excipients - Can increase in ionic strength result in higher viscosity? - BioPharm International

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Opalescence of an IgG1 Monoclonal Antibody Formulation is Mediated by Ionic Strength and Excipients
Can increase in ionic strength result in higher viscosity?


BioPharm International
Volume 22, Issue 4

METHODS

Opalescence Determination

Opalescence of the samples was assessed according to the European Pharmacopoeia (EP) 5.0 (2.2.1). Opalescence reference suspensions were made using hexamethylenetetramine and hydrazine sulfate. Samples were evaluated at a volume of 11 mL in 20 mL glass vials by comparing them to the reference suspensions in diffused daylight against a black background.

Turbidity Measurements

The opalescence of MAb1 was measured by subtracting the optical density (OD) at 350 nm from the OD at 550 nm using an HP spectrophotometer as previously described.12

Size Exclusion High Performance Liquid Chromatography

Size exclusion high performance liquid chromatography (SEC–HPLC) was performed using a TSKgel 3000SWXL column from Tosoh Corporation (Tokyo, Japan) with a mobile phase consisting of 25 mM phosphate, 0.3 M NaCl, pH 7.0. The flow rate was 0.5 mL/min. The temperature of the column was maintained at 25 C. The samples were detected at 230 nm.

Dynamic Light Scattering (DLS)

The particle sizes of samples tested were measured by dynamic light scattering (DLS) using a Zetasizer Nano System from Malvern Instruments (Malvern, UK). A total of 10 measurements for each sample were taken and the results were reported as the Zave, the average hydrodynamic size.

Viscosity Measurements

Viscosities were measured using a LVDV-III Ultra Programmable Rheometer by Brookfield Engineering (Middleboro, MA). Sample volumes of 0.5 mL were tested for each measurement.

Determination of the Osmotic Second Virial Coefficient Values

The osmotic second virial coefficient was obtained using a Dawn Heleos multi-angle light scattering (MALS) instrument from Wyatt Technology Corporation (Santa Barbara, CA). The formulations containing buffer and 150 mM NaCl (no PS-80) were diluted and injected into the instrument as follows: 1:2.5 (8.20 mg/mL), 1:5 (4.10 mg/mL), 1:10 (2.05 mg/mL), and 1:20 (1.03 mg/mL). The Rayleigh equation was used by the computer software to generate data on the Zimm plot. The software also calculated the molecular weight and particle size of the sample.




The Rayleigh equation is described as follows (Malvern Instruments):13

(Equation 1)

in which C is the particle concentration (g cm-3 ), Rθ is the the Rayleigh ratio—the ratio of scattered light to incident light of the sample (cm-1), M is the sample molecular weight (g mol-1 ), A2 is the 2nd virial coefficient (cm3 mol g-2), P(θ) is the angular dependence of the sample scattering intensity, and K is the optical constant, which is calculated using Equation 1.1:




(Equation 1.1)


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