Purpose: The Poloxamer 188 is a widely used pharmaceutical excipient, and in recent years has also been used as a critical ingredient in cell culture bioprocessing. During cell culture, poloxamer 188 acts as a shear protectant by reducing the detrimental effect of localized shear forces on cell viability. However, it has been shown recently that lot-to-lot variability of poloxamer 188 due to the presence or absence of hydrophobic impurities may result in significantly variable performance during bioprocessing. While consequences are clear, the underlying physico-chemical mechanism of the behavior is not yet fully understood. In this work, classical colloidal methods contribute to the understanding of poloxamer 188 behavior in solution.
Methods: In the presented work, solutions of Kolliphor® P188 Bio were formulated in a wide array of formulations in absence and presence of hydrophobic impurities. Colloidal measurements were taken using a series of techniques to elucidate measurable effects using standard techniques. Previously sited techniques include reversed-phase high performance liquid chromatography (RP-HPLC) where hydrophobic impurities were linked to poor cell culture results. Static surface tension measurements as a function of concentration, both total surfactant as well as the ratio of hydrophobic impurity concentration are shown. In addition, photon correlation spectroscopy (Malvern) is utilized in order to characterize micelle sizes of poloxamer in the presence and absence of hydrophobic impurities. Finally, differential scanning calorimetry (DSC, TA Instruments) was utilized to study micelle formation and self-assembly behavior of poloxamer in the presence and absence of hydrophobic impurities.
Results: Recent publications have also suggested that the use of static surface tension measurements can predict subtle changes in hydrophobic impurities, and specifically that the highest purity poloxamer would yield the highest measured static surface tension. This was not found to be the case, as the effect of impurities on surface tension resulted in a sometimes increased result and other times as a lowered result; based both on concentration of total polymer as well as surfactant ratio with impurities. Furthermore, photon correlation spectroscopy showed that the formation of mixed micelles of poloxamer 188 with hydrophobic impurities did not occur, but rather separate micelles were present in solution at all concentrations. This was further corroborated by DSC measurements highlighting self-assembly behavior of poloxamer 188 was not significantly affected by impurities. Consequently, this lends to the hypothesis that hydrophobic impurities function in a “displacement mechanism”, where poloxamer 188 is no longer able to adequately provide shear protection through the mechanism shown in Figure 1, rather than interact with poloxamer 188 causing the ineffective protection.
Conclusion: Trace levels of hydrophobic impurities in poloxamer 188 may have a significant and adverse impact on the performance as shear protectant when used in bioprocessing – the use of Kolliphor® P188 Bio mitigates this risk by reducing the presence of many hydrophobic impurities present in poloxamer 188 grades which are not dedicated for the use in biologics. Through a series of physical chemistry experiments, it is shown that the presence of hydrophobic impurities does not affect the physico-chemical behavior of poloxamer 188, lending evidence to the hypothesis that the impurities rather operate independently and function by displacing the shear protection pseudo-coating of poloxamer 188.