Polysorbates: Part(icle) of the Problem?

"Primary engines of value creation”—this is the role that leading market commentators see biologic drugs playing in the evolving biopharmaceuticals market (1). Demand for protein-based, large-molecule therapies has been growing steadily for decades, but few could have predicted the boom which followed in the wake of COVID-19. Significantly accelerated by the pandemic, sales of biologic drugs are predicted to exceed those of traditional small-molecule medications by 2027 (1). Runaway demand, exciting scientific breakthroughs, and robust interest from governments and the public: the outlook is bright for biologics. To fulfill this growing potential, producers will need to make drug stability a priority, and this could mean reconsidering the use of a ubiquitous processing ingredient.

Polysorbates (PS 20 and PS 80) are the surfactants most used in biopharmaceutical formulations to protect proteins from denaturation, aggregation, and surface adsorption; these surfactants are present in as many as 90% of marketed therapeutic antibodies (2). Despite their popularity, polysorbates are prone to a number of issues, with serious implications for drug safety, stability, and efficacy. As pressures mount, could it be time for the industry to adopt a new favorite excipient solution for biologics? This article will explore the findings of two recent studies comparing the performance of polysorbates with hydroxypropyl -cyclodextrin (HPβCD)—an alternative biologic stabilizer.

Reliable, multifunctional, and safe, is HPβCD the answer to the question: how do you solve a problem like polysorbates?

Setting the scene

To fully understand the “problem” with polysorbates, the industry must examine the essential role ultrafiltration/diafiltration (UF/DF) and protein stabilization during the final formulation stage play in biologic drug manufacturing, and why processing aids are crucial to their success. As Figure 1 shows, UF/DF comes relatively late in downstream biopharma processing, helping achieve the desired concentration and formulation composition of biologics, such as monoclonal antibodies (mAbs) and recombinant proteins. Though an indispensable processing step, the shear stress and continuous adsorption/desorption interactions with the filtration membrane during UF/DF subject active ingredients to harsh conditions. This typically results in protein aggregation and the generation of visible and subvisible particles, jeopardizing the safety and efficacy of the drug. It is here that surface active molecules, such as polysorbates, enter the frame. During high-stress processes, polysorbates act as a chaperone, allowing aggregation-prone hydrophobic sites on the surface of the biologic to pass more easily through membranes to preserve their folded structure and reduce interface exposure (3).

With decades of evidence pointing to their effectiveness in preventing protein adsorption and aggregation, as well as their demonstrated safety, polysorbate 80 (PS 80) and polysorbate 20 (PS 20) are considered the surfactants of choice in more than 90% of approved mAb products (2). Due to their amphiphilic (both hydrophilic and hydrophobic) properties, mAb proteins have a high propensity for surface-mediated unfolding and adsorbing into interfaces (3). Polysorbates compete with proteins at interface points, reducing the interfacial exposure of the proteins. Despite their widespread use, there are several serious questions surrounding the effectiveness of polysorbates in biologic processing. Polysorbate degradation via oxidation and hydrolysis can produce peroxides and fatty acids that could negatively impact the quality and stability of a medication’s therapeutic protein. Polysorbates can also be nonspecifically absorbed onto UF/DF filter membranes, leading to the formation of micelles bigger than the membrane’s molecular weight cut-off, which in turn causes inconsistencies and membrane blockage. As the biopharma industry moves to make efficiency its central priority, researchers begin to wonder, could an alternative excipient help flip the script?

Polysorbates vs. HPβCD in UF/DF

UF/DF is an integral part of downstream biotherapeutic processing, though it’s not without its challenges. A typical UF/DF process involves several passes through a pump and membrane cassette, often taking several hours to complete (4). During this process, active proteins are exposed to a range of physical stresses, including interfacial interaction, which, as discussed previously, can lead to the formation of higher order aggregates and particulates (4). Therefore, to survive the UF/DF process intact, proteins need an accompanying surfactant to smooth their multiple passes through the membrane cassette and protect them from interfacial stresses (4). For the majority of biologic processing steps, polysorbates are the solution of choice; however, their usefulness runs out by the UF/DF stage. Not only do polysorbate molecules tend to bind nonspecifically with filter membranes, but their propensity to form micelles bigger than the membrane’s molecular weight cut-off can cause filters to become blocked during the UF/DF process (4). Researchers at Roquette postulated that HPβCD could offer biopharma producers an alternative excipient solution, better suited to the requirements of the UF/DF process.

Efficiency optimized

The focus of this study into HPβCD’s potential as an alternative to polysorbates was two-fold: assess its permeability through commonly used filtration membrane types and capacity to reduce particle formation during the UF/DF of human plasma immunoglobulin G (IgG) and adalimumab solutions (4). In the first instance, varying concentrations of HPβCD were observed in the retentate and permeate of solutions passed through a regenerated cellulose-type membrane following centrifugation (4). This indicated that HPβCD may be unsuitable for regenerated cellulose membranes, as it tends to be retained during ultrafiltration (4). With another commonly used polyethersulfone (PES) membrane, however, the results were a complete contrast. In all four variations tested, concentrations of HPβCD remained constant, providing convincing evidence that unlike polysorbates, it is able to pass freely through PES membranes (4). What’s more, these results remained largely consistent when the excipient was tested via an alternative tangential flow filtration (TFF) setup, adding credence to HPβCD’s suitability for UF/DF processes (4).

Regarding the goal of mitigating particle formation, results were similarly promising. Using the same TFF setup, researchers conducted UF/DF experiments using the proteins human plasma IgG and adalimumab in the presence of different concentrations of HPβCD. In the diafiltration setup, both the retentate and permeate were recirculated for four hours to mimic the duration of a typical commercial drug production process. The results at the end of the recirculation process suggested that HPβCD effectively modulated the rate of turbidity (measure of clarity in liquids) increase in the human plasma IgG solution (4). In fact, the turbidity of the sample was reduced by more than two times in the presence of just 10 mM of HPβCD, while dynamic light scattering (DLS) data also showed that it had lowered the relative intensity of larger particles (> 0.1 m) compared to the control sample (4). The addition of a larger, 25 mM concentration of HPβCD reduced the rate of turbidity increase in the adalimumab solution yet further, meaning that the final turbidity was more than four times lower in the sample containing HPβCD than in the control group.

Researchers were able to observe HPβCD’s effectiveness in reducing UF/DF-induced particle formation in both the human plasma IgG and adalimumab solutions too. Looking at the human plasma IgG specifically, a 10% protein loss was observed in the control, whereas all samples containing HPβCD achieved almost complete protein recovery at the end of the four-hour recirculation (4). Not only then is HPβCD suitable for UF/DF where polysorbates are not, but its presence could actually boost efficiencies in biologic production lines—no small point in a time of rising demand for the industry.

The potential benefits HPβCD can bring to the biologic manufacturing process are not exclusive to the UF/DF process. Indeed, this multifunctional excipient exhibits continuous functionality into the final stages of formulation, particularly protein stabilization.

Polysorbates vs. HPβCD: Protein stabilization

The presence of protein aggregates and visible/sub-visible particles within a biologic formulation can compromise product quality and long-term stability, as well as lead to an unwanted immune response in the patient. As such, numerous health regulators around the world require biopharmaceutical companies to closely monitor visible and subvisible particle formation within formulations by comprehensive characterization, as outlined in United States Pharmacopeia (USP) <787>, USP <788>, and USP <789> (5). With scrutiny mounting on the issue of particle formation, recent research has sought to investigate the potential of alternative surfactants that could offer a more stable proposition than traditional polysorbates, with the prime candidate being HPβCD.

A sturdier solution

Due to its regulatory status as an approved excipient for oral and parenteral administration, HPβCD has been considered as a functional biologic surfactant for more than a decade (6). There is robust evidence to support HPβCD’s effectiveness in preventing thermal or agitation- and lyophilization-induced protein aggregation, in addition to its capacity to reduce interfacial stress-induced particle formation during UF/DF (5). This stabilizing effect has been attributed to the excipient’s ability to reduce protein–protein hydrophobic interactions and displace proteins from interfaces thanks to its weak surface activity (5). Unique properties such as those listed above formed the foundation of a recent investigation into HPβCD as a potential replacement for conventional polysorbates in biologic formulations.

The resultant study compared the chemical stability of HPβCD against that of PS 20 and PS 80 under various stress conditions. This included heat and chemical stressors, as well as its capacity to stabilize proteins within a formulation. When subjected to heat stress, HPβCD showed little change in product recovery (90.7–100.7% recovery depending on the HPβCD grade), while PS 20 and PS 80 both underwent significant degradation, with only 11.5% and 7.3% undegraded product remaining, respectively (5). Similar results were recorded when the surfactants were subject to autoclave, light, and oxidative stresses, with the HPβCDs remaining almost completely stable, while both polysorbates displayed some level of degradation (95.5%–98.8% usable product remaining for PS 20 and 85.5%–97.4% for PS 80) (5).On closer analysis, researchers found the HPβCDs’ chemical structure remained consistently stable, compared to the significant hydrolytic degradation and oxidation seen with the polysorbate molecules (5).

Finally, attention turned to HPβCD’s performance as a functional surfactant in a test formulation. Researchers evaluated the stability of various model mAbs when subjected to light stresses. Compared with the more traditional PS 80 preparation, the mAb formulated in the presence of HPβCD showed a notable decrease in protein aggregation, a superior monomer, and total protein recovery while reducing both protein aggregation and subvisible particle formation (5). Evidently, HPβCD excipients hold immense potential as biologic stabilizers.

Problem solved?

At the end of this compare-and-contrast exercise, it is important to acknowledge the real value polysorbates bring to biologics processing. In a wide array of applications, they act as effective buffers and mediating agents, and their years of evidence as safe and nontoxic excipients cannot be discounted—but neither can the clear potential of HPβCD. In the studies discussed above, it can be seen what a significant impact stabilizing cyclodextrins could have on protein stability, mitigating particle formation, and streamlining vital processes. In some instances, such as UF/DF, substituting traditional polysorbates for HPβCD appears to be the logical choice, but this will not be so clear cut in every scenario. The future of biologics processing then could be one where these two surfactant groups are used interchangeably or even in tandem, to produce the very best outcomes for patients. That is, after all, the ultimate problem every drug producer is seeking to solve.

References

1. GlobalData. Biologic Sales to Pass Innovative Small Molecules in Next Five Years, Says GlobalData. globaldata.com, May 10, 2022.
2. Bollenbach, L.; Buske, J.; Mader, K.; Garidel, P. Poloxamer 188 as Surfactant in Biological Formulations—An Alternative for Polysorbate 20/80? Int. J. Pharm. 2022, 620, 121706.
3. Grabarek, A. D.; Bozic, U.; Rousel, J.; et al. What Makes Polysorbate Functional? Impact of Polysorbate 80 Grade and Quality on IgG Stability During Mechanical Stress. J. Pharm. Sci. 2020, 109 (1), 871–880. DOI: 10.1016/j.xphs.2019.10.015
4. Hong, S.; Peng, T.; Gokhale, R. Mitigating Particle Formation During Ultrafiltration/Diafiltration of Biologics with KLEPTOSE HPβCD (Hydroxypropyl Beta-Cyclodextrin). Whitepaper, 2023.
5. Zhang, H.; Hong, S.; Tan, S. S. K.; et al. Polysorbates versus Hydroxypropyl Beta-Cyclodextrin (HPβCD): Comparative Study on Excipient Stability and Stabilization Benefits on Monoclonal Antibodies. Molecules 2022, 27, 6497. DOI: 10.3390/molecules27196497
6. Serno, T.; Carpenter, J. F.; Randolph, T. W.; Winter, G. Inhibition of Agitation-Induced Aggregation of an IgG-Antibody by Hydroxypropyl-Beta-Cyclodextrin. J. Pharm. Sci. 2010, 99, 1193–1206. DOI: 10.1002/jps.21931

About the author

Tao Peng, PhD, is biopharma research manager at Roquette.

Article details

BioPharm International®
Vol. 37, No. 5
May 2024
Pages: 29–33

Citation

When referring to this article, please cite it as Peng, T. Polysorbates: Part(icle) of the Problem? BioPharm International 2024, 37 (5) 29–33.