Letter pubs.acs.org/journal/abseba
Amino-Acid-Incorporating Nonionic Surfactants for Stabilization of Protein Pharmaceuticals Joshua S. Katz,*,† Yujing Tan,‡ Krishna Kuppannan,‡ Yang Song,§,⊥ David J. Brennan,⊥ Timothy Young,⊥ Lu Yao,† and Susan Jordan† †
Formulation Science, Core R&D, The Dow Chemical Company, 400 Arcola Road, Collegeville, Pennsylvania 19426, United States Analytical Sciences, Core R&D, The Dow Chemical Company, 1897 Building, Midland, Michigan 48674, United States § Department of Chemistry, University of Illinois at Urbana−Champaign, 405 North Matthews Avenue, Urbana, Illinois 61801, United States ⊥ Formulation Science, Core R&D, The Dow Chemical Company, 1712 Building, Midland, Michigan 48674, United States ‡
S Supporting Information *
ABSTRACT: With the rapid development of protein-based pharmaceutical products over the past decade, one of the biggest challenges in product development is maintaining the structural stability of proteins during purification, processing, and storage. In this work, the design of a new class of surfactants, polyethermodified N-acyl amino acids, is presented. One surfactant from this series, containing a phenylalanine moiety, demonstrated remarkable stabilization against aggregation of several model protein drugs. Dynamic light scattering, size exclusion chromatography, and circular dichroism all show the rate of thermally accelerated protein aggregation slowed. IgG aggregation was reduced by 3-fold compared to polysorbate controls. Testing of Orencia, a prescription biologic drug for rheumatoid arthritis, demonstrated a 36% improvement in monomer retention upon heat-aging. KEYWORDS: biopharmaceutical, aggregation, surfactant, stabilization
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The vast majority of products formulated with surfactants contain either a polysorbate (e.g., Tween20 or Tween80) or poloxamer (Pluronic) as they both have well-established safety profiles.9 Although polysorbates and poloxamers have been utilized because of their safety in injectable formulations, there has been little research into how they as surfactants might be optimized to effect protein stabilization. To that end, a series of surfactants that are built on common, inert building blocks and that could improve on the incumbent technologies for protein stabilization has been designed. These novel surfactants comprise a fully saturated 14 carbon alkyl chain, commonly found in natural lipids, an amino acid that could fine-tune the functionality of the surfactant, and a polyether nonionic hydrophilic headgroup. The synthesis of these materials was a straightforward two-step amidation reaction, first between myristoyl chloride and an amino acid and then between the acylated amino acid and a monoamine functional Jeffamine Mseries polyether (Scheme 1, for full synthetic details, see the Supporting Information). The use of amide rather than ester bonds was chosen as ester hydrolysis has been implicated in
iotech drugs, pharmaceuticals derived from proteins or other biologically derived macromolecules, are rapidly emerging as a dominant class of pharmaceuticals.1 Nearly onequarter of all pharmaceutical sales was for biotech products in 2014 with 46% of the top 100 products expected to be biotech by 2020.2 Because of the relatively fragile nature of protein materials, development of biologic actives that are both therapeutically beneficial and sufficiently stable to withstand processing, distribution, and administration remains a significant challenge.3 The pharmaceutical industry employs a variety of process techniques and formulation ingredients to improve the shelf-stability of biologic formulations, but due to regulatory limitations, the current toolbox is fairly small.4 Dry powders of proteins are often more stable so many first generation product offerings are dried, either by lyophilization, or more recently, spray drying.5 However, dry products lead to challenges in reconstitution and insoluble species that cause immunogenicity. Therefore, liquid formulations, where feasible, are the preferred delivery form. To improve liquid formulation stability, formulators employ various excipients designed to stabilize the biologic, including buffers, salts, sugars, and surfactants.4 Surfactants stabilize proteins by blocking the proteins’ access to hydrophobic surfaces (vial walls or air interfaces) on which they can denature and then aggregate and/or by blocking protein− protein interactions which could also lead to denaturation.6−8 © XXXX American Chemical Society
Received: May 3, 2016 Accepted: June 23, 2016
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DOI: 10.1021/acsbiomaterials.6b00245 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Letter
ACS Biomaterials Science & Engineering Scheme 1. Synthesis of N-Acylamino Acid Surfactants
some destabilization pathways caused by polysorbate surfactants.10 As the new surfactants have a polyether hydrophile similar to the polysorbates and poloxamers, improvement in oxidative stability is not expected and study of this phenomenon was not included as part of this study.11 NMR analysis confirmed the presence of a single species with structure consistent with the product. For ease of reference, surfactants were named xMy where x is the one letter amino acid code and y is the approximate molecular weight of the Mseries Jeffamine. OM1000 was a control surfactant with no amino acid. Critical micelle concentration (CMC) was determined for each surfactant by tensiometry (Table S1). The surfactants had CMC’s in the range of 120−140 mg/L (ppm) with the exception of AM1000 (74 ppm) which had limited solubility in water (the cause of which we were unable to determine), which could alter the measured results, and GM2000 which, because of the more hydrophobic headgroup (25 mol % propylene oxide content) had a two-order of magnitude lower CMC of only 5 ppm. The CMC’s for GM1000, FM1000, and OM1000 are modestly higher than those reported for Polysorbate 20 and 80 (72 and 16 ppm, respectively) but an order of magnitude less than Poloxamer 188 whose CMC is approximately 1000 ppm.12,13 As environment and the presence of amphiphilic species can shift CMC, these numbers should not be considered absolutes, but rather orders of magnitude.14 Many biologic drugs on the market and in development are built on an immunoglobulin platform and as a result, IgG was chosen as a model protein to assess surfactant stabilization. Initial screens of aggregation were completed using a highthroughput dynamic light scattering instrument with temperature control.15 None of the surfactants screened altered the aggregation onset temperature, but it was observed that the FM1000 sample grew in size at a different rate as temperature increased above aggregation onset (Figure S1). Consequently, future studies focused on the FM1000 surfactant as the primary test material and for clarity is the only test surfactant reported in the data to follow. The altered rate of growth behavior following onset of aggregation led to a probe of the rate of aggregation growth at constant temperature just below the aggregation onset (Figure 1a and Figure S2). Holding just below aggregation temperature allows acceleration of the aggregation process that would otherwise occur at ambient temperature, enabling more rapid testing. When held isothermally at 65 °C, the aggregate size grew at a rate approximately three times slower than polysorbate controls over a 16 h period. All samples performed better than a surfactant free control that rapidly aggregated to sizes above the
Figure 1. (a) 1 mg/mL IgG (aggregate) size as a function of time when held isothermally at 65 °C as measured by DLS. Note that the vast majority of control data is off the scale of this chart. (b) 0.4 mg/ mL IgG β-sheet signal evolution with 0.1 mg/mL surfactant during an isothermal hold at 65 °C, measured by CD.
scale of the plot shown. N-dodecyl-β-D-maltoside, a surfactant that has been explored elsewhere for protein stabilization, was also included in this study and did not perform qualitatively differently than either polysorbate control (Figure S2).16 Protein aggregates often have large amounts of β-sheet structure as hydrophobic regions come together, and as a result, circular dichroism (CD) can be used to track aggregation through monitoring of the β-sheet signal.17,18 Figure 1b shows the evolution of the β-sheet signal (218 nm) for IgG samples formulated alone or with Polysorbate 20 or FM1000. Approximately 30% less β-sheet structure is formed for the FM1000 sample, further confirming that FM1000 helps to maintain the native and nonaggregated state of IgG. Following the initial promise of improved stabilization demonstrated with IgG, the next step was to demonstrate stabilization using an actual biologic drug. After analyzing several different available drugs, abatacept (brand name, Orencia), a fusion protein of the CH2 and CH3 Fc domains of human IgG1 and extracellular soluble domains of human CLTA-4, was chosen.19 The primary reason for choosing abatacept is that it is commercially available in two forms: a dry lyophilized powder and a prefilled syringe. Because only the prefilled syringe contains a stabilizing surfactant (Poloxamer B
DOI: 10.1021/acsbiomaterials.6b00245 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Letter
ACS Biomaterials Science & Engineering 188), the surfactant-free dry form could be obtained and reformulated with either the incumbent surfactant or an experimental surfactant. There are other differences in the formulation ingredients (see Table S2), but following dilution to experimental conditions those ingredients are not expected to have an appreciable effect on formulation stability compared to the surfactant, nor are there any other differences across the experimental groups aside from the surfactant added. Abatacept has two different thermal transitions, one at about 55 °C corresponding to the CTLA-4 and IgG CH2 domains unfolding and one around 80 °C corresponding to the IgG CH3 domain.19 The first study was a staged isothermal DLS with holds at 50, 60, and 73 °C (Figure S3). When held below the first transition temperature, the hydrodynamic radius (Rh) of abatacept increases in size by about 20% as measured by the DLS if formulated with Polysorbate 20, Poloxamer 188, or FM1000. The surfactant free control and sample formulated with Polysorbate 80 grew to much larger sizes. While the FM1000 did not reduce the apparent aggregate size, the rate of growth is slower for the FM1000 sample (Figure 2a). At 60 °C, the FM1000 sample did lead to an additional increase in size (which steadied) that was not observed with the Poloxamer 188 or Polysorbate 20 samples. All samples rapidly grew in size to larger aggregates at 73 °C. A direct comparison was also made
between the Poloxamer 188 and the FM1000, looking at the total percentage of abatacept that remained in monomeric form following heat aging at 52 and 65 °C (Figure 2b). At both temperatures, the FM1000-containing formulation contained a higher concentration of abatacept monomer, 36% (52 °C) and 290% (65 °C) more monomer than the Poloxamer 188 control after 22 h. How the effect of the FM1000s and Poloxamer 188’s different CMC’s may contribute to these results will be the focus of future studies. Tracking the β-sheet signal over time isothermally at 65 °C (Figure S4) and during a temperature ramp (Figure S5) indicates that addition of FM1000 leads to improved structure maintenance than control formulations. Since thermal transitions do not shift as a function of formulation, the mechanism of stabilization is likely not through direct interaction between the surfactant and protein at the site of primary denaturation. Together, these data indicate that the FM1000 markedly improves the thermal stability of abatacept in solution compared to controls, including the incumbent technology employed by the drug manufacturer. In a final experiment, stabilization of infliximab (brand name, Remicade), a monoclonal antibody drug formulated with Polysorbate 80, was probed by isothermal DLS (Figure S6). In this experiment, the polysorbates clearly outperform the FM1000 surfactant in maintaining monomeric structure for longer periods of time before rapid aggregation occurs. FM1000 performed worse than the control without added surfactant. (A trace amount of surfactant present in the initial formulation provided by the drug formulator is present in all formulations equally). Interesting to note is that the shape of the aggregation curve is very different compared to abatacept or IgG (Figures 1a, Figures S3 and S6), suggesting a different mechanism of aggregation. These data indicate that the FM1000 surfactant is likely not a “cure-all” solution to aggregation stabilization of proteins and also suggests that more work is warranted to better understand how surfactant and protein structures drive a protein’s thermally induced aggregation behavior and stability. In this work, a novel series of surfactants based on inert, biologically relevant building blocks has been developed. One particular surfactant (FM1000) was found to be especially promising for stabilization of IgG and an IgG-derived pharmaceutical. Because the FM1000 surfactant did not provide any stabilization to the infliximab, future work should focus on better understanding the structure function relationships for both protein and surfactant structures. One potential mechanism could include phenylalanine interacting with specific domains on the IgG and abatacept, leading to improved stabilization compared to other surfactants. This study focused specifically on the use of surfactants to prevent thermally induced aggregation, monitored by various techniques. Further work to determine the effect of this series of surfactants on agitation-induced aggregation due to changes at the air/water/ surface interfaces would be interesting. Additional work will focus on the underlying mechanisms that enable the observed improved stability and further expansion of the surfactant structures built off this motif.
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Figure 2. (a) Initial rate of abatacept (10 mg/mL, 1 mg/mL surfactant) size growth after heating to 50 °C measured by DLS. (b) Percent abatacept (0.4 mg/mL, 0.1 mg/mL surfactant) monomer retention following heat aging at 52 °C (solid lines, diamonds) or 65 °C (dashed lines, circles) as measured by SEC. Standard deviations are less than 2% for two samples per time point and formulation.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00245. C
DOI: 10.1021/acsbiomaterials.6b00245 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
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Beta-1b and Reduces Its Immunogenicity. Journal of Neuroimmune Pharmacology 2011, 6, 158−162. (17) Fink, A. L. Protein Aggregation: Folding Aggregates, Inclusion Bodies and Amyloid. Folding Des. 1998, 3, R9−R23. (18) Greenfield, N. J. Analysis of the Kinetics of Folding of Proteins and Peptides Using Circular Dichroism. Nat. Protoc. 2007, 1, 2891− 2899. (19) Fast, J. L.; Cordes, A. A.; Carpenter, J. F.; Randolph, T. W. Physical Instability of a Therapeutic Fc Fusion Protein: Domain Contributions to Conformational and Colloidal Stability. Biochemistry 2009, 48, 11724−11736.
Complete materials and methods along with supporting tables and figures (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENTS The authors thank Florin Dan, Yongfu Li, Cassie Fhaner, and Maggie Covington for their assistance with the analytical techniques. We thank Robert Schmitt for his guidance and insight. This work was funded by The Dow Chemical Company.
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