Polysorbate-80 (PS-80) is believed to protect proteins against interfacial stresses at the air-liquid and glass-liquid interfaces, as well as between protein molecules. For surfactants, the CMC is the point at which surfactant molecules begin to form micelles in solution. Parenteral formulations containing surfactants often include them at concentrations far greater than the CMC and little work has investigated formulations with surfactants near or at the CMC level. A previous agitation study performed on mAb-1 showed that a surfactant such as PS-80 is necessary in the formulation to mitigate visible and sub-visible particulate formation. Agitating the protein with no PS-80 present in the formulation resulted in a significant amount of visible particulate formation, giving the sample a “snow globe-like” appearance. However, no particles were observed in a formulation that contained half the PS-80 amount in the target. While the level of PS-80 in this formulation exceeded the CMC value, this led to the questions of what would occur upon agitation of mAb-1 as the PS-80 level approached or was lower than the CMC. This investigation aimed to determine if visible particulate formation after agitating mAb-1 could be correlated to the CMC value of the PS-80 lot used in this experiment.
First, the CMC value for the specific PS-80 lot was determined via a surface tension versus PS-80 concentration curve. Surface tension was determined using a Kruss tensiometer. Next, seven different formulations were prepared at varying PS-80 levels above and below the CMC value of the specific PS-80 lot used in this experiment. These formulations ranged between 0 mg/mL PS-80 and 0.2 mg/mL PS-80 (target PS-80 amount in drug product formulation). With four formulations below and three formulations above the calculated CMC value (0.00674 mg/mL PS-80). After material formulation, three vials of each formulation were filled in the intended container closure system. One vial from each formulation was then placed on a shaker at 300 rpm for 24 hours at uncontrolled room temperature (approximately 25 °C) and another was kept outside on the lab bench to serve as a room temperature control. The third vial was immediately frozen at -80°C to serve as a T=0 control sample. Samples were inspected for visual appearance noting sample color, clarity, and visible particulates. Sample turbidity was measured using a HACH 2100. Sub-visible particulates were assessed using HIAC. Sample quality was determined using UV-Vis spectroscopy and size exclusion HPLC. Surface tension measurements were made for each formulation.
The CMC of the utilized PS-80 lot was determined to be 0.00674 mg/mL PS-80. After the 24 hour agitation hold, the only sample that showed a large amount of visible particulates was the formulation containing 0 mg/mL PS-80. The agitated samples containing 0 mg/mL PS-80 and 0.003 mg/mL PS-80 were visually observed as more turbid than other samples. The results obtained from HIAC and HACH 2100 turbidity testing showed that those two samples had more sub-visible particulate matter, and were more turbid than the other samples. As expected, samples with PS-80 concentrations below the CMC showed a decrease in surface tension as the CMC was approached; the samples with PS-80 concentrations at or immediately above the CMC remained constant. Surface tension measurements ranged from 68.412 mN/m (for 0 mg/mL PS-80) to 46.330 mN/m (for 0.2 mg/mL PS-80). No significant differences were seen between formulations in protein concentration or % high molecular mass species as determined by size exclusion HPLC. All samples were within ±1 mg/mL of the target concentration and no unexpected degradation was observed by size exclusion HPLC.
Results from the study demonstrate that PS-80 is necessary in the mAb-1 formulation to mitigate visible and sub-visible particulate formation under agitation stress. This effect occurs even at PS-80 concentrations lower than the CMC value of the specific PS-80 lot used. However, agitated samples with the lowest PS-80 concentration of 0.003 mg/mL showed higher sub-visible particulate counts and turbidity levels than all other agitated samples. One formulation sample with moderate PS-80 concentration below the CMC (0.005 mg/mL) resulted in sub-visible particle levels that were unchanged. At moderate PS-80 concentrations lower than the CMC, liquid interfaces are not saturated with surfactant molecules, but may still provide some protection for the protein from shear stresses at interfacial surfaces.