(T1530-07-38) Poloxamer 188 Variant for the Formulation of Biologics

Purpose: Today, only three surfactants are commonly used as stabilizer in parenteral formulations of biologics: Polysorbate 80, Polysorbate 20 and Poloxamer 188. Polysorbates have a long history of use and are effective. But they are also complex mixtures, are prone to chemical and enzymatic degradation and known to form reactive impurities. [1,2]. Poloxamer 188 is a triblock copolymer with a more defined polyether structure, it is effective at slightly higher concentrations, more stable and not susceptible to degradation by residual host cell lipases. However, in the presence of silicon oil, protein-PDMS particles were detected in vials after long-term storage for some protein formulations [3]. It was observed that certain Poloxamer 188 lots that contained more hydrophobic species, were more effective stabilizers, than other typical poloxamer 188 lots. [4,5]. It is the aim of our study to investigate the performance of a more hydrophobic poloxamer 188 variant in the formulation of biologics.

Methods: Kolliphor® P 188 and Kolliphor® P 188 Bio are commercial products of BASF. More hydrophobic poloxamer 188 variants were synthesized by BASF and analyzed for their molecular weight at peak maximum by GPC and for their “late eluter content” by HPLC [4] Formulations of sterilely filtered 1 mg/mL mAb4 solutions in 20 mM histidine buffer at pH 6 with or without poloxamer were prepared in quadruplicate and filled into 2R glass vials (Schott AG, Mainz, Germany) to a volume of 0.8 mL and closed with FluoroTec® injection stoppers (West Pharmaceutical Services, Inc., Exton, USA). To induce antibody aggregation, the formulations were stressed on a horizontal shaker (IKA Werke, Staufen, Germany) for 15 hours at 300 rpm in a tilted position. Subvisible particles were characterized and quantified in duplicate by means of flow-imaging microscopy (FIM) on an MFI 5200 instrument from ProteinSimple (San Jose, USA). Statistical analysis was conducted by two-way ANOVA followed by a Dunnett’s multiple comparison test using GraphPad Prism version 8.0.2. Statistical significance is indicated by * p < 0.05, ** p < 0.01 and *** p < 0.001. Quantification of surfactant or antibody adsorption on siliconized surfaces was performed using quartz-crystal microbalance (QCM) on a QSense Omega QCM-D instrument (Biolin Scientific, Gothenburg, Sweden). A gold-coated sensor (QSX 301) was siliconized in-house and mounted into the instrument. To analyze adsorption, a sequential flush was conducted. It began with either neat buffer (Blank) or a 0.01 mg/mL poloxamer solution, followed by a buffer rinsing step. This was followed by an mAb4 solution at a concentration of 1 mg/mL, and finally another buffer rinsing step. The amount of adsorbent was quantified from the rinsing step.

Results: Table 1. Characterization of poloxamer variants by titration according to Ph. Eur., late eluter content (HPLC) and relative molecular weight at peak maximum (M_p) (GPC). The late eluter content is categorized as low ( < 10%), medium (10% - 20%), or high ( > 20%). The relative M_p values are categorized as low ( < 9600 g/mol) medium (9600 g/mol – 11000 g/mol), or high ( > 11000 g/mol). The average molecular weight (M_w) of PLX (8) is out of the compendial poloxamer 188 specification (7680 g/mol - 9510 g/mol). Both the HPLC and GPC method classify the poloxamer hydrophobicity in a similar order. Figure 1. Subvisible particles in mAb4 formulations with or without (mAb4 RT) 0.001% (w/v) poloxamer were quantified by FIM. Formulations without P188 were stored at room temperature (RT) for 15 h and analyzed accordingly. Statistical analysis with P188 (2) as control (lowest Mw, low early eluter, low M_p) were conducted for formulations containing P188. The results show that both P188 (4) and PLX (8) have significantly lower average particle counts, even for larger particles (≥ 25 µm), indicating the positive impact of poloxamer hydrophobicity on subvisible particle formation. Figure 2. mAb4 adsorption to surfactant pre-covered PDMS sensors was carried out using QCM. Pre-rinsing steps were conducted with or without (Blank, neat buffer) 0.001% (w/v) poloxamer. Upon examining the second rinsing step with neat buffer, it was observed that PDMS surfaces pre-covered with more hydrophobic poloxamers (P188 (4) and PLX (8)) exhibited lower adherence of mAb4.

Conclusion: The eight poloxamer variants tested herein vary in their hydrophobicity, which is shown by HPLC and GPC analysis. The comparison of the M_p is only meaningful if the amount of EO of the poloxamer variants is the same. We could show that the hydrophobicity of the poloxamer variants has an impact on the subvisible particle formation, significantly reducing the formation of subvisible particles in the mAb4 formulation. Furthermore, we could show that a pre-adsorption of a more hydrophobic poloxamer variant reduces antibody adhesion to siliconized surfaces. This could prevent the formation of protein-PDMS visible particles. Our studies indicate the benefits of a more hydrophobic P188 variant as surfactant in therapeutic protein formulations.

References: [1] Wuchner, K. et al.: Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates. Journal of Pharmaceutical Sciences, 2022
[2] Yuk, I. H. et al.: Formulation mitigations for particle formation induced by enzymatic hydrolysis of polysorbate 20 in protein-based drug products: insights from a full-factorial longitudinal study, AAPS Open, 2022
[3] Grapentin, C. et al.: Protein-Polydimethylsiloxane Particles in Liquid Vial Monoclonal Antibody Formulations Containing Poloxamer 188. Journal of Pharmaceutical Sciences, 2020

Acknowledgements: The authors thank Marion Bendel, Yvonne Matheis, Peter Stengel and Werner Wacker (all BASF SE) for their contributions to this work.

Table 1. Characterization of poloxamer variants by titration according to Ph. Eur., late eluter content (HPLC) and relative molecular weight at peak maximum (Mp) (GPC). The late eluter content is categorized as low ( < 10%), medium (10% - 20%), or high (> 20%). The relative Mp values are categorized as low ( < 9600 g/mol) medium (9600 g/mol – 11000 g/mol), or high (> 11000 g/mol). The average molecular weight (Mw) of PLX (8) is out of the compendial poloxamer 188 specification (7680 g/mol - 9510 g/mol). Both the HPLC and GPC method classify the poloxamer hydrophobicity in a similar order.


Figure 1. Subvisible particles in mAb4 formulations with or without (mAb4 RT) 0.001% (w/v) poloxamer were quantified by FIM. Formulations without P188 were stored at room temperature (RT) for 15 h and analyzed accordingly. Statistical analysis with P188 (2) as control (lowest Mw, low early eluter, low Mp) were conducted for formulations containing P188. The results show that both P188 (4) and PLX (8) have significantly lower average particle counts, even for larger particles (≥ 25 µm), indicating the positive impact of poloxamer hydrophobicity on subvisible particle formation.


Figure 2. mAb4 adsorption to surfactant pre-covered PDMS sensors was carried out using QCM. Pre-rinsing steps were conducted with or without (Blank, neat buffer) 0.001% (w/v) poloxamer. Upon examining the second rinsing step with neat buffer, it was observed that PDMS surfaces pre-covered with more hydrophobic poloxamers (P188 (4) and PLX (8)) exhibited lower adherence of mAb4.