N-alkyl amino acid polyether amides are a series of chemically defined surfactants with an amino acid as an alkyl tail linked to the polyether hydrophile through amide bonds [ 255 ]. These have been developed to reconcile the protein stabilization properties of the amino acids (detailed under amino acid-based stabilizers) with that of the non-ester surfactant families. Due to the presence of the amide bond, rather than an ester bond, N-alkyl amino acid polyether amides are resistant to the described HCP-mediated hydrolysis, while they might be still associated with the problem of oxidation as in the case of PSs. N-alkyl amino acid polyether amides also offer the advantage of low batch to batch variability due to their chemically defined structure [ 44 ]. One paramount example within this context is N-myristoyl phenylalanine Jeffamine M1000 diamide (FM1000; ), which, when used in a concentration of 1 mg/mL, could significantly slow down the thermal-induced aggregation of 10 mg/mL abatacept. Dynamic light scattering (DLS) measurements and size exclusion chromatograms demonstrated the reduction of thermal-induced protein particle formation. Interestingly, compared to PS20, PS80, and P188, FM1000 resulted in 3-fold more reduction of protein particle formation [ 255 ]. Following these interesting findings, the efficiency of FM1000 for the stabilization of liquid biotherapeutics under an induced mechanical stress was also investigated [ 256 ]. To this end, the efficiency of FM1000 to inhibit protein particle formation in an immunoglobulin G (IgG) formulation at a concentration of 20 mg/mL following exposure to 24-h-long agitation was compared to that of PS20, PS80, and P188. Interestingly, the rate of protein particle formation was highest in the case of P188, followed by PS80 and then FM1000 and PS20. Compared to conventional surfactants, FM1000 could better facilitate the stabilization of air/water and air/silicon interfaces in IgG containing formulation [ 256 ]. Furthermore, the effect of FM1000 upon the agitation stress of abatacept in IV bags was explored. The protein stability was highest in the case of FM1000, followed by P188, PS80, and PS20. The surface tension measurements revealed that under the same conditions, the CMC of FM1000 was 10 times lower than that of PS80 (0.55 versus 5.5 PPM) [ 257 ]. While the use of these amino acid-based surfactants for protein stabilization has been successfully patented [ 257 ], their industrial translation necessitates more in-depth investigations in terms of potential pharmacological and toxicological side effects. It should also be highlighted, that the current available stability studies were published by the manufacturer of FM1000; thus, it is unclear whether “negative” examples related to protein stabilization of FM1000 would have been disclosed.
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Non-ester sugar-based surfactants such as dodecyl glucoside and dodecyl maltoside ( ) have been shown to reduce the surface tension of water in a matter comparable with SEs [ 191 ]. EPA (Environmental Protection Agency) has reported dodecyl maltoside to be a non-mutagenic, non-toxic, and non-irritating substance [ 252 ]. Dodecyl maltoside was/is further patented for use in nasal, parenteral, and hydrogel formulations [ 253 ]. In the case of different alkyl-β-glycosides, degradation following oral administration and the metabolism of the lipid and carbohydrate moieties through the relevant metabolic pathways have been shown [ 190 ]. Being safe for human consumption, n-dodecyl-β-D-maltoside (DMM) has been exploited as a non-ionic alkyl saccharide surfactant for the stabilization of a therapeutically active peptide (D-alanine Peptide T-amide (DAPTA)) during lyophilization and the subsequent reconstitution of the freeze-dried cake [ 253 ]. The surfactant could successfully extend the product shelf-life independent of the lyophilization-thawing process and could further increase the stability and reduce the aggregation and immunogenicity of the therapeutic peptide [ 253 ]. In a later study, DMM was used as a surfactant to stabilize interferon-β in intranasal and subcutaneous formulations for the treatment of multiple sclerosis [ 254 ]. Within this context, DMM could successfully prevent protein particle formation in solution and reduce the immunogenicity of the final formulation [ 254 ]. Despite these promising results, the use of this surfactant in the formulation of parenteral protein therapeutics in the market remains underexploited.
In terms of stability, little has been reported about POE ethers in general. One might argue that as non-ester surfactants, POE ethers might be superior to PSs in terms of susceptibility to hydrolysis. On the other hand, since Brij ® surfactants contain POE and alkyl chains, the likelihood that they might undergo a similar oxidative degradation to PSs cannot be completely ruled out. The literature holds only a few studies on Brij ® degradation in pharmaceutical formulations [ 78 , 251 ]. The most relevant publications within this context report a metal-mediated oxidation in case of Brij ® 30 [ 78 ] and a photocatalytic degradation of Brij ® 35 in the presence of inorganic minerals [ 251 ]. The ether moieties available in the structure of Brij ® molecules are susceptible to auto-oxidation in contact with air through the formation of hydroperoxides. The oxidation process begins with the formation of a free radical at the α-carbon atom of the ether moiety. According to Currie et al. (2004), the presence of silver ions leads to the removal of hydrogen, which usually is caused by oxygen in non-metal-mediated oxidation processes. Particularly in this metal-induced oxidation of Brij ® surfactants, the susceptibility of long alkyl chains to oxidation is greater than that of short alkyl chains. This has been attributed to the high hydrogen fed capacity to the oxidative process by virtue of the several repetitive methylene units in the saturated carbon chain [ 78 ]. The photocatalytic oxidation of POE moiety, on the other hand, is promoted by UV light incidence and the presence of a photocatalyst. The increase of oxygen concentration influences the rate of oxidation, but only up to a defined concentration where the system reaches a plateau [ 251 ]. With all this said, more profound research is needed to address the stability of the Brij ® surfactants in protein biological formulations and the influence of the degradation products on the stability of the protein cargo. Based on the available data at least, Brij ® surfactants seem to have superior stability to PS surfactants, and they seem to be valid candidates for further investigations within the context of stabilizing parenteral biotherapeutic formulations. However, concerns regarding the potential immunological, hypersensitivity and even anaphylactic reactions originating from the POE moiety of these surfactants must be addressed.
Even though the FDA approval of POE ethers as surfactants for parenteral formulations is yet to be obtained, the last decade has witnessed a more detailed investigation of the role of Brij ® as a surfactant in injectable biotherapeutic formulations [ 249 ]. For instance, Agarkhed et al. (2018) explored the effect of PS, Brij ® 35, and several other POE-based surfactants on the mechanical, thermal, and photostability of a mAb injectable formulation [ 250 ]. According to the findings of the mechanical and isothermal stability, as well as the sub-visible particle counting, while all tested non-ionic surfactants demonstrated a similar protein protecting effect, their performance was inferior to that of PS80. On the other hand, formulations comprising non-PS surfactants, including Brij ® 35, could better protect the mAb against photo-oxidation, as was evident from the decrease in methionine and tryptophan oxidation [ 250 ]. In one of the latest studies on the subject, Yue et al. (2020) proposed Brij ® 58 as a good alternative surfactant to PS20 and PS80 for the stabilization of protein injectable formulations [ 249 ]. Through visual inspection and dynamic light scattering, the authors confirmed superior mAb protection ability of Brij ® 58 under forced degradation conditions. Furthermore, ultra-performance liquid chromatography- charged aerosol detector investigations revealed better inherent stability of the surfactant in protein formulations when compared to PS20 and PS80. After 19 days of shaking and incubation at 40 °C for 1 month, no significant reduction of the Brij ® 58 concentration was observed, while the concentrations of PS20/PS80 underwent an approximate decrease of 30%. On top of that, the required concentration of Brij ® 58 as a surfactant was comparably low (0.075 mg/mL versus 0.2 mg/mL in case of PS20/ PS80). In terms of toxicological investigations, the estimated LD 50 in mice was equal to 343.4 mg/kg body weight, which was lower than that reported for PS80 (5000 mg/kg body weight). The authors argued that this would still leave a large safety window for Brij ® 58 in protein formulations, as the required concentration of the surfactant is quite low. Indeed, no side effects were observed in acute toxicological studies when administrated in a concentration of 20 mg/mL in mice [ 249 ]. While these findings highlighted the potential of Brij ® 58 to be used as an efficient PS alternative in the formulation of injectable protein biotherapeutics, the authors also emphasized that the safety of the surfactant in human subjects when incorporated in parenteral formulations is yet to be investigated [ 249 ].
Since POE ethers have shown to provide good stability in topical formulations, their stabilizing effect in protein-based liquid formulations has been compared to the PS surfactants. Bam et al. (1995) investigated how well Brij ® 92 could stabilize the rhGH (recombinant human growth hormone) as a model protein in comparison with PS80 as a reference surfactant [ 246 ]. The surfactant activity was measured through the partitioning behavior of a spin-label with electron paramagnetic resonance to determine the binding stoichiometry [ 247 ]. The obtained results indicated that Brij ® 92 tends to behave in a similar way as PS80 and could effectively stabilize the rhGH protein [ 246 ]. Similarly, through light-scattering experiments, Krause et al. (2002) confirmed that when used in a concentration of 0.001 mg/mL, Brij ® 58 could effectively inhibit the protein particle formation of chemically denatured proteins (glucosidase, citrate synthase, and rhodanese) [ 248 ].
POE ethers (commercially available as Brij ® ) comprise the POE (PEG) chain as a hydrophilic moiety, and the ethoxylated fatty alcohol constituting the lipophilic portion of the surfactant ( ). By using different types of fatty alcohols (lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol), different glycol ethers can be synthesized. According to the FDA Inactive Ingredient Database, Brij ® (cetomacrogol) is a polyether-based surfactant approved as an inactive ingredient solely for topical formulations. This is reflected in the review publications compiled by Jiao (2008) [ 243 ] and Sahoo et al. (2014) [ 244 ] focusing on the use of POE alkyl ethers (Brij ® 35, 78, 98) in pharmaceutical formulations, which mainly comprises a discussion of their role in the development of topical and ocular formulations. In addition, the stabilizing effect of POE ethers in oral protein formulations has also been investigated by Mesiha et al. (1981). The authors demonstrated that the incorporation of Brij ® 58 as a surfactant enhanced the stability of orally administered insulin in male white rabbits [ 245 ].
To sum up, it seems that the use of Poloxamers ® , including P188 and P407, as protein stabilizers in injectable formulations has both advantages and disadvantages depending on the physicochemical traits of the protein, formulation ingredients, and the type of packaging or delivery system in which the formulation is contained. Being FDA-approved excipients already in use for the formulation of protein injectables, these are not associated with safety and toxicological concerns, as is the case for the PS surfactants. However, unlike PSs, Poloxamers ® are not susceptible to HCP-mediated degradation. Nevertheless, it cannot be denied that similar to PSs, the use of Poloxamers ® in injectable protein formulations is still fraught with concerns regarding the instability of the surfactant and the subsequent impact on the protein integrity, the formation of visible and sub-visible particles, and the potential biological interactions. More in-depth research is yet to be conducted to thoroughly address such concerns and to optimize the protein formulations and primary packaging to fully benefit from the use of Poloxamers ® as PS alternatives in such formulations. Moreover, concerns regarding the potential immunological, hypersensitivity, and even anaphylactic reactions originating from the PEG (POE) moiety of these polymers must be addressed.
Recently, the stabilizing properties of PSs and P188 in mAb formulations under different stress and storage conditions have been compared [ 241 ]. These studies have indicated that both PSs and P188 could effectively protect mAbs from interfacial stress in liquid vial formulations. However, unlike formulations containing PSs, clear protein–polydimethylsiloxane (PDMS) particles were observed in vials of protein formulations stabilized using P188 after long-term storage at 2–8 °C. Moreover, the use of P188 as surfactant in vials with a bromobutyl teflonized and siliconized stopper led to the formation of molecule-specific visible and sub-visible protein–PDMS particles in liquid mAb formulations, when stored for an extended period of time [ 241 ]. The protein–PDMS particles found in this study have been attributed to the siliconized stopper used in the primary packaging, resulting from a shift of silicone oil traces from the non-product contacting side to the product contacting side during the bulk storage and processing of the stoppers [ 241 ]. Hence, while P188 seems to be a useful alternative to PSs for stabilizing liquid mAb formulations, a change of chemical composition of the PDMS stoppers is necessary to avoid the formation of protein–PDMS particles during storage, even at low temperatures. Even in cases where non-PDMS stoppers have been used, Fourier-transform infrared microscopy has revealed the formation of fluoropolymer particles in the solution. On the other hand, a related study examining the impact of excipients on the functionality of prefilled syringes discovered that when compared to P407 and P188-containing formulations, those containing PS80 showed a significant increase in glide force. While this was mostly observed at higher temperatures (40 °C), which indicates a low risk of syringe failure during storage at room temperature, an increase in protein concentration seems to accelerate this process and result in an increase of glide force at 25 °C. In comparison, the performance of P188 in terms of maintaining syringe functionality and stability, particularly in the case of HCLF, was shown to be superior [ 242 ].
Although the chemical degradation of P188 and P407 in the solid state has been examined and published in greater depth, their chemical stability in solution has not been thoroughly investigated [ 240 ]. In one of the few studies in the subject, Wang et al. (2019) examined the degradation of P188 in solution as a function of the buffer type, pH, temperature, trace metals, and peroxides [ 240 ]. The results obtained using direct injection gas chromatography-mass spectroscopy were indicative of significant degradation of the polymer in 10 mM histidine buffer. Within this context, the thermal oxidation of the PPO block initiates the oxidation of P188 triggered by the secondary radicals in the polymer chain, which leads to secondary hydrogen peroxide. As a result, the oxidation of histidine could be catalyzed by both secondary free radicals and secondary hydrogen peroxide produced during the P188 oxidation process [ 240 ]. The authors concluded that, relative to PS surfactants, P188 has a lower stability liability in liquid formulations [ 240 ].
Since Poloxamers ® , as efficient stabilizers of injectable protein biotherapeutics, have hitherto successfully found their way to the market, it is worth first detailing the stability and potential degradation of these surfactants [ 160 ]. P188 has been shown to degrade in the solid state at high temperatures due to auto-oxidation and chain cleavage, resulting in aldehydes, organic acids, and smaller molecular weight polymers [ 238 ]. The solid-state degradation of the P188, trackable based on the pH shifts, aldehyde formation, and changes in molar mass of the polymer has been shown to initiate at around 40 °C [ 238 ]. P407, on the other hand, has been shown to be stable, and the thermal degradation only initiated after 21 days at 80 °C, starting from the PPO center block, resulting in chain scissions in both PPO and POE polymer blocks [ 238 , 239 ]. In this study, P188 comprised less antioxidant compared to the P407, which the authors mentioned as a potential reason behind the higher sensitivity of the former to solid-state degradation. Based on the proposed mechanism, the solid-state degradation of P407 is associated with the generation of free radicals [ 239 ]. It should be, however, noted that these degradation reactions are triggered at higher temperatures, which are at any rate deteriorating to the stability of the protein and are hence avoided during the production, storage, and administration phases of protein biotherapeutics.
Triblock copolymers of polyethylene oxide-polypropylene oxide-polyethylene oxide (POE-PPO-POE), commercially available as Poloxamers ® and Pluronics ® , possess surfactant properties resulting from the hydrophobic PPO block in the middle and the hydrophilic POE blocks on the sides ( ) [ 231 ]. Different Poloxamers ® vary mainly in the molecular mass of the PPO core and the percentage of POE content [ 232 ]. The name of the copolymers commonly starts with the letter “P” (for Poloxamer ® ) followed by three digits, the first two of which, when multiplied by 100, give the approximate molecular mass of the PPO core. Multiplication of the last digit by 10 provides information of the percentage POE content [ 160 , 233 ]. The two most well-known members of this family are Poloxamer ® 188 (P188; PPO molecular mass of 5400 g/mol and 80% POE content) and Poloxamer ® 407 (P407; PPO molecular mass of 4000 g/mol and 70% POE content). According to the FDA Inactive Ingredient Database, P188 is approved for use in a variety of formulations as an emulsifying, solubilizing, defoaming, and dispersing agent. In fact, P188 is one of the well-considered PS alternative surfactants, as it is already incorporated in commercial biotherapeutic formulations such as Gazyva ® (0.2 mg/mL) [ 234 , 235 ], Orencia ® (8 mg/mL) [ 236 ], Norditropin ® (3 mg/mL) [ 237 ], and Hemlibra ® (0.5 mg/mL) [ 234 ].
The benefit of the non-ester surfactants mainly lies in their lack of sensitivity to enzymes currently detected in protein formulations. The most important surfactant class of these include block polyethylene-propylene glycol and polyoxyethylene fatty ethers, as well as non-ester sugar-based surfactants such as dodecyl glucoside and dodecyl maltoside, and N-alkyl amino acid polyether amides such as N-myristoyl phenylalanine Jeffamine M1000 diamide (FM1000) ( ).
Although the FDA and EMA have confirmed the low oral toxicity and non-mutagenicity of Kolliphor ® EL, anaphylactic reactions associated with parenteral formulations, in which the surfactant has been used as a delivery vehicle or an excipient, have been reported [ 219 , 220 , 221 , 222 , 223 , 224 , 225 ]. This is not a side effect specific to Kolliphor ® EL, but a drawback shared by all PEG (POE) containing excipients. Similar to the PS structures, the PEG moiety can induce mild to strong immunogenic and hypersensitivity reactions. Between January 1977 and April 2016, 37 case reports of immediate-type hypersensitivity to PEG emerged, of which 28 (76%) described hypersensitivity reactions that met anaphylaxis criteria [ 226 ]. This shared drawback of all PEG containing excipients is not taken seriously, as certain proteins or their aggregates can also trigger immunogenic reactions of their own, or else induce the secretion of various immunoglobulins [ 227 , 228 , 229 ]. Combined, these might lead to immunogenic reactions ranging from milder site-of-injection swelling and pain to hypersensitivity reactions and anaphylactic reactions in extreme cases [ 152 , 230 ]. Hence, more detailed clinical studies should be dedicated to uncovering the full extent and the reasons behind the immunological concerns raised in the case of PEG-based surfactants to thoroughly address the issue of the suitability of the surfactant for the stabilization of injectable protein formulations.
Similar to polyoxyl 15 hydroxy stearate, polyoxyl 35 castor oil (also known as Cremophor ® EL and Kolliphor ® EL) is a non-ionic surfactant belonging to the PEG fatty ester family ( a). It is made of reacting castor oil and ethylene oxide in a molar ratio of 1:35, and has an HLB value in the range of 12 to 14 [ 217 , 218 ]. The surfactant has been approved by the FDA for use in pharmaceutical formulations through intramuscular, intravenous, intravesical, subcutaneous, ophthalmic, sublingual, and topical administration routes, and it has been mainly used for the parenteral delivery of small molecular drugs [ 161 ]. In one study, the ability of Kolliphor ® EL to stabilize amylase over two months in the presence of hydrogen peroxide has been compared to that of Kolliphor ® HS 15 and PS80. Fluorescence measurements revealed the ability of Kolliphor ® EL in the preservation of amylase and the reduction of the oxidative degradation to be superior to that of PS80. In addition, similar to PS80 and Kolliphor ® HS 15, Kolliphor ® EL could efficiently protect the BSA exposed to mechanical stress for a period of 7 days [ 204 ].
Few studies have shown the ability of Kolliphor ® HS 15 to stabilize proteins, e.g., amylase and bovine serum albumin (BSA) [ 204 ]. However, as the ester bond available in the structure of Kolliphor ® HS 15 is chemically and enzymatically cleavable, the surfactant is expected to be, at least to some extent, associated with similar drawbacks as PS surfactants. Furthermore, the presence of the POE moiety renders the molecule susceptible to oxidative degradation [ 211 , 212 ]. Here again, the rate of surfactant degradation along with the impact of degradation products on the protein stability as well as quality of the final product has to be investigated in detail, in order to provide a comparative overview of the surfactant’s efficiency in comparison to PSs for the stabilization of protein therapeutics. As in the case of any other excipients used for parenteral biotherapeutic formulations, the analytical characterization of the surfactant to detect impurities, as well as the plausible side products from the auto-degradation process, is important for quality control. Several techniques have been hitherto developed to quantitatively examine Kolliphor ® HS 15 composition. Examples include the use of ultraperformance liquid chromatography coupled with a nano quantity analyte detector to quantify the lipophilic moieties as well as hydrophilic free PEGs in the formulation [ 213 ], or else application of gel permeation chromatography to quantify Kolliphor ® HS 15 structural components [ 214 ]. Another most outstanding method is the use of liquid chromatography tandem mass spectrometry with an ion suppression effect, through which the identification of twelve oligomers by electrospray ionization has been possible. Based on these analyses, the lipophilic and hydrophilic components in Kolliphor ® HS 15 have been quantified to be around 63.3% and 36.7%, respectively [ 215 , 216 ]. Notwithstanding, except for the FFAs, little has been practically uncovered about the degradation products of this surfactant and how these will affect protein stability. Moreover, the potential interactions of the surfactant with the primary packaging have not been fully investigated either. Hence, a thorough investigation of the applicability of Kolliphor ® HS 15 for the stabilization of protein biopharmaceutics, the stability of the surfactant and the effect of potential degradation products on the quality of the formulation, and the potential interactions of the surfactant with the primary packaging inspires further research.
The toxicological profile of Kolliphor ® HS 15 has been hitherto investigated, having shown a superior safety and toxicity profile in comparison with PS80 [ 209 ]. In animal models, Kolliphor ® HS 15 shows no acute toxicity in rats (intravenous LD 50 > 1 g/kg body weight, oral LD 50 > 20 g/kg body weight), mice (oral LD 50 > 20 g/kg body weight), dogs (intravenous LD 50 > 3 g/kg body weight), and rabbits (intravenous LD 50 > 1 g/kg body weight) [ 192 , 210 ]. Parenteral administration of 100 mg/kg body weight of Kolliphor ® HS 15 has been shown to induce pseudo-allergic reactions associated with histamine release, though no histopathological changes or signs of specific toxicity have been observed [ 210 ]. These allergic reactions, however, have not been limited to Kolliphor ® HS 15, and have been observed in the case of Kolliphor ® EL, another PEG fatty acid, and PS80. Overall, the results of these investigations conclude the safety and low toxicity of Kolliphor ® HS 15 as a non-ionic surfactant in comparison with other commercial non-ionic PEG-based surfactants [ 199 ].
Another FDA-approved surfactant belonging to the PEG fatty esters is polyoxyl 15 hydroxy stearate, commercially labelled as Kolliphor ® HS 15 and Solutol ® HS 15. This surfactant has been approved by the FDA for the improvement of solubility of low water-soluble drugs in parenteral and oral formulations [ 198 ]. Kolliphor ® HS 15 is a non-ionic surfactant synthesized using 1 mol 12-hydroxystearic acid and 15 mol ethylene oxide ( a) [ 199 ]. Its chemical structure is based on 12-hydroxystearic acid (lipophilic moiety) polyethoxylated at both the carboxyl and the hydroxyl groups with PEG (hydrophilic moiety). As a mixture, Kolliphor ® HS 15 also contains free PEG as a side product of the epoxidation reaction [ 199 ]. It possesses a hydrophilic–lipophilic balance (HLB) value of about 16 and a critical micelle concentration (CMC) ranging from 0.06 and 0.1 nM when dissolved in water at 21 °C [ 198 , 200 ]. Due to these properties, Kolliphor ® HS 15 has efficient solubilization properties, having been thus used mainly for the solubilization of poorly soluble vitamins and hydrophobic drugs, such as clotrimazole, carbamazepine, ketoconazole, danazol, piroxicam, fenofibrate, and cinnarizine [ 198 , 201 , 202 , 203 ]. Kolliphor ® HS 15 can also be used to protect macromolecules and some protein formulations [ 204 ]. As an FDA-approved non-ionic surfactant, Kolliphor ® HS 15 has been used in oral, parenteral, and ophthalmic formulations for several years [ 205 ]. It has also been investigated for the development of various types of injectable nanoparticle formulations, including but not limited to self-emulsifying drug delivery systems, solid self-emulsifying drug delivery systems, lipid nanocapsules, solid lipid nanoparticles, and nanostructured lipid carriers, as well as other liquid and solid dispersion formulations [ 198 ]. Within the context of the marketed injectable drugs, it has been mostly used as a component of non-biotherapeutic formulations. These include injectable formulations of ibuprofen eugenol ester [ 206 ], propofol (2,6-diisopropylphenol) [ 207 ], and a mixture of α-tocopherol, ascorbic acid, and β-carotene [ 208 ].
However, as these are associated with relatively safe toxicological profiles, further investigations regarding their potential application for protein stabilization are meaningful. PEG stearates have been non-lethal in animal tests at a concentration of 10 g/kg body weight, leading to minimal ocular irritation in rabbits and resulting in merely low-level skin irritation in rabbits when tested at 100% concentration, and induced no irritation in humans [ 194 ]. While low molecular weight PEG-2 stearate and PEG-9 stearate can influence the skin barrier function at concentrations of 5% w/v, higher molecular weight PEG-40 stearate does not exert the same effect [ 195 ]. Feeding animal models with PEG-8, −40, and −100 stearates accounted for no impact upon the mortality rate, growth, and histopathological and hematological abnormalities [ 196 ]. Similarly, PEG fatty acid esters have been shown to induce no genotoxic effects [ 193 , 196 , 197 ], while carcinogenic and chromosomal aberration data are yet to be provided.
Structurally speaking, PEG stearates, similar to PS surfactants, are prone to hydrolysis of the ester bond present in their chemical structure, as well as the oxidation of the POE moiety. However, little is known about the rate of degradation, and the potential influence thereof upon the quality of the final product. Furthermore, no information regarding the efficiency of the surfactant in stabilizing different proteins and the required concentration range is in hand. Adjunct to the factors related to the stability of these surfactants, the potential interactions of the PEG stearates with the components of the primary packaging and the subsequent influence upon the final product quality is yet to be investigated.
According to the FDA Inactive Ingredients Database [ 161 ], four different PEG stearates are approved for use in pharmaceutical products. Within this context, PEG 40 stearate is used in formulations for ophthalmic, oral, and topical application. The use of PEG 8 and PEG 100 stearates has been approved in topical and oral formulations, whereas PEG 2 stearate is merely applicable in topical formulations. PEG stearates are common excipients in cosmetic products, mainly due to their viscosity and solubility enhancement properties, along with their low toxicity, humectant, and emulsifying properties [ 194 ].
Polyethylene glycol (PEG) stearates, also referred as macrogol stearates, polyoxylstearates, POE stearates, or ethoxylated stearates, are non-ionic surfactants that contain a POE (also known as PEG) segment that endows their hydrophilic character and a saturated fatty acid as the hydrophobic moiety (mainly octadecanoic acid) linked with an ester bond ( a) [ 192 ]. The average length or the molecular weight of the polymer chain is often indicated in the name of the compound, e.g., PEG 8 stearate, and the ethylene oxide monomer range can be between 2 to 150 units [ 192 , 193 ]. The longer the polymer chain in the compound, the more water-soluble it is [ 192 ].
Similar to SEs, monoesters of sugars other than sucrose can serve as potential candidates for protein stabilization. These have been introduced since a few decades as non-toxic and biodegradable emulsifiers for use in the food industry [ 190 ]. Garofalakis et al. (2000) investigated various sugar monoesters of xylose, galactose, and lactose esterified with different hydrophobic chain lengths. All sugar monoesters could reduce the surface tension of the water in a manner comparable with commercial SEs (C8-C16 alkyl chain). The authors further observed a decreased critical aggregation concentration of the surfactants with increasing carbon chain length [ 191 ]. This early study provides a view of the unexploited potential of some sugar monoesters, from which pharmaceutical formulations could benefit. These will be, however, potentially associated with similar advantages and drawbacks detailed in the case of SEs.
Accordingly, the potential of SE surfactants for the stabilization of injectable protein biotherapeutic formulations, particularly at a mechanistic level and with regard to the minimum required concentrations as well as the long-term stability of the surfactant under different conditions, has to be investigated. Within the context of the latter, given the presence of the ester bond, SEs will not be devoid of the same drawback that complicates the use of PS surfactants in protein formulations. The chemical and enzymatic hydrolysis of the SE surfactants can raise the same issues, e.g., precipitation of the FFAs, creation of the visible or sub-visible particles, and endangering protein stability, leading to potential immunogenic reactions. However, the rate of surfactant degradation, the size of the potential FFA particles, and the minimum required concentration of the SEs for protein stabilization are factors that need to be investigated to enable a decent comparison of the SEs with the PSs. On the other hand, SEs offer obvious advantages when compared to their conventionally used counterparts. For example, the chemical structure of SEs renders them less susceptible to oxidative degradation when compared to the PSs, which can significantly improve the quality of the final product over long-term storage. Additionally, the lack of POE moiety decreases the risk of immunogenic, hypersensitivity and anaphylactic reactions. In all, while SEs seem to have potential to serve as effective PS substitutes, comparative studies are required to state conclusively as to whether SEs could perform equal or superior to PSs when used for the stabilization of protein biotherapeutics.
As commonly used surfactants in the food industry, some information regarding the biocompatibility and safety of the SEs is already in hand. In 1992, the Scientific Committee for Food stated an acceptable daily intake (ADI) of 0–20 mg/kg body weight/day for SEs of fatty acids and sucro-glycerides derived from palm oil, lard, and tallow fatty acids. In 2004, the European Food Safety Authority (EFSA) re-examined the safety of these food additives based on new research and granted a group ADI of 40 mg/kg body weight/day for SEs of all fatty acids [ 176 ]. Additionally, the absorption, distribution, and metabolism of the SEs has been extensively investigated. Following oral intake, higher fatty acid esters of sucrose, such as octa- and hepta-esters (e.g., Olestra [ 179 ]), are excreted unmetabolized, while lower esters are partly hydrolyzed and absorbed as sucrose and individual fatty acids [ 180 , 181 , 182 ]. However, one study reported that intravenous administration of sucrose monopalmitate in a relatively high dose of 0.5 g/kg could lead to hemolytic reactions in rats [ 183 , 184 ]. Interestingly, no hemolysis has been reported in case of the intravenous injection of palm oil SEs and lard and tallow SEs in rats and mice, respectively [ 184 ]. These findings point out the safety of SEs, at least in animal models. Nonetheless, more profound toxicological studies are required to ensure the full safety of these surfactants following intravenous application in human subjects. At the moment, only a few articles have described the use of SEs in pharmaceutical products tested on humans, focusing mostly on transdermal delivery systems, where SEs have been utilized as emulsifiers, or else for the control of drug release and the enhancement of cutaneous absorption [ 185 , 186 , 187 ]. The only case where SEs have been used for the formulation of parenteral injectables has been the exploitation of sucrose laurate as a POE-free Cremophor ® EL substitute in parenteral formulations for the enhancement of the solubility of the poorly water-soluble drugs, such as cyclosporin A, docetaxel, paclitaxel, and etoposide [ 188 , 189 ].
Sucrose fatty acid esters or sucrose esters (SEs) are non-ionic surfactants with a sucrose backbone as the hydrophilic group, and a maximum of eight fatty acids per molecule as the hydrophobic moiety ( a). The fatty acids are most commonly lauric, myristic, palmitic, stearic, erucic, and oleic acid [ 162 ]. SEs are FDA-approved excipients used mostly in the food and cosmetic sectors and, to a lesser extent, in the pharmaceutical sector [ 163 , 164 ]. According to the FDA Inactive Ingredients Database [ 161 ], two members of the SE class, namely sucrose stearate and sucrose palmitate, are approved for use in oral and topical formulations of small molecules [ 164 ] for drug solubility enhancement [ 165 , 166 ], controlled drug release [ 167 ], and drug absorption and penetration enhancement [ 168 , 169 , 170 ]. SEs are also used as emulsifiers for the formulation of microemulsions for transdermal drug delivery or in food and cosmetic application [ 171 , 172 ], and for the preparation of different micro- and nanoparticulate systems [ 173 , 174 , 175 ]. The most important SEs used within this context include sucrose stearate, sucrose palmitate, sucrose erucate, sucrose laurate, sucrose myristate, and sucrose oleate [ 176 ]. These are biodegradable and biocompatible surfactants, as they comprise a cleavable ester bond in their chemical structure. Following oral intake, most of these hydrolyze into FFAs and saccharide constituents, rendering them gastrointestinal digestible [ 177 , 178 ]. On top of that, SEs are odorless, flavorless, non-toxic, and non-irritant to the skin. For such reasons, they have been granted the GRAS (generally recognized as safe) status by the FDA [ 178 ].
The main surfactants in this category include the sucrose fatty acid esters and sugar monoesters, polyethylene glycol (PEG) stearates, and PEG fatty esters, ( a). Similar to PS surfactants, these are associated with the main advantage of biodegradability by virtue of the cleavable ester bond in their structure, and have thus been approved by the FDA for use in the food, cosmetic, and pharmaceutical industries (see FDA Inactive Ingredient Database [ 161 ]). Yet again, the ester bond renders these prone to enzyme-mediated hydrolysis when used in the formulation of protein therapeutics.
As surfactants are the most common class of excipients with established protein stabilization abilities, it comes as no surprise that the first group of PS alternatives are proposed from this class. Here, we classified PS alternative surfactants into two main categories. The first includes the surfactants that, like PSs, comprise an ester bond in their structure. This means that, similar to the case of PSs, these are associated with the risk of enzyme-mediated cleavage of the ester bond ( b). Unlike PS surfactants, however, some of these might be less prone to auto- and photo-oxidation and are hence expected to offer advantages in this regard (see the upcoming sections for more information) [ 160 ]. The second group involves PS alternatives, which, given the lack of ester bond in their structure, are resistant to enzymatic hydrolysis and in cases, potentially, auto- and photo-oxidation. In this section, we will debate each group of these surfactants separately and discuss selected examples.
This category of potential alternative excipients includes disaccharides and their chemical derivatives, sugar alcohols, cyclodextrins, and semisynthetic polysaccharides. These are natural or in cases semi-synthetic compounds associated with an acceptable safety profile and have been hitherto exploited for protein stabilization, mainly during lyophilization. The chemical structures of these compounds are presented in .
Disaccharides
Disaccharides such as sucrose and trehalose are well-known for their ability for protein cryopreservation and protein stabilization in the lyophilized state [258]. What might be less widely known is their ability to stabilize proteins in solution. As osmolytes, sucrose and trehalose exert their protein stabilizing effect indirectly by altering the solvent properties and the consequent protein–solvent interactions [259]. By surrounding the protein molecules, they also separate them from one another, thereby reducing the protein–protein interactions [259]. Sucrose ( a) has been shown to inhibit the protein particle formation of recombinant interleukin-1 receptor antagonist (IL-1ra), which tends to form a soluble dimer during storage at 30 °C under minimal stressful conditions [260]. It is proposed that proteins with an increased surface area tend to be thermodynamically less stable in the presence of sucrose compared to those with a more compact structure. This could lead to a shift of equilibrium from the aggregated to the unaggregated species over time [260].
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Trehalose ( a), according to the literature, acts superior to sucrose in terms of protein stabilization, mainly through its specific solvent structure and dynamics [261]. Sun et al. (1998) demonstrated the superior ability of trehalose/glucose over sucrose/glucose mixture to preserve the stability and the biological activity of a dehydrated dehydrogenase enzyme, after long-term storage at 44 °C (ca. 80 days vs. 5 days), 60 °C (>70 h vs. ca. 50 h), and 75 °C (ca. 30 h vs. <10 h) [262]. It has also provided protection and stabilization of the enzymes like restriction during the vacuum-drying process [263]. Accordingly, trehalose is a well-established excipient for the cryo- and lyoprotection of different proteins.
Research has highlighted the effective concentration of trehalose to maintain the protein stability during parenteral biotherapeutics (bevacizumab, ranibizumab, trastuzumab, recombinant antihemophilic factor) to oscillate between 10–100 mg/mL, but it is usually used in combination with PS surfactants, as it cannot provide adequate protein stability in the absence of the latter [264]. Since the pharmaceutical industry currently endeavors towards finding a substitution for polyethoxylated surfactants in biotherapeutic formulations, unusual approaches have been taken to provide trehalose better protein stabilization by modifying the molecule’s chemical structure [265,266,267,268,269,270]. One of these strategies involves the modification of trehalose with fatty acids to provide the molecule surfactant properties (similar to sucrose fatty acid esters). Schiefelbein et al. (2010) synthesized 6-O-monolauroyl- α, α-trehalose, and investigated its ability to stabilize human growth hormone (hGH) subjected to agitation stress. At a concentration of 1 mg/mL, the trehalose-based surfactant could stabilize the protein as effectively as PS20 and PS80 [265]. As a trehalose fatty acid ester, 6-O-monolauroyl-α, α-trehalose is prone to hydrolysis. Thus, a similar synthetic approach was proposed by Messina et al. (2017), who produced 4 trehalose regioisomers with an ether moiety. The compound was shown to prevent protein particle formation (>97%) against thermal and mechanical-induced stresses [266]. In a recent study, synthetic linear and 4-arm star glycopolymers (mannose, galactose, arabinose, lactose, and trehalose) were investigated in terms of maintaining the conformational stability and reducing the protein particle formation tendency of mAb1 as a model protein [267]. The stress conditions used in this study involved storage at 25 °C and 40 °C for up to 7 weeks. According to the static light scattering and differential scanning calorimetry results, the conformational and colloidal stability of the mAb1 were affected by the sugar monomer concentration as well as the polymer structure (linear or star). Unlike their low molecular weight sugar counterpart, which increased mAb 1 formulation stability even at low concentrations, glycopolymers had the opposite effect, leading to the destabilization of the Ab [267]. This was most pronounced in the case of trehalose-based glycopolymers at higher concentrations and during long-term storage both at 25 °C and 40 °C for up to 7 weeks. These results demonstrate that unlike low molecular weight sugar excipients, their corresponding glycopolymers cannot act as efficient protein stabilizers [267]. In another study, Mancini et al. (2012) synthetized a trehalose sidechain glycopolymer further conjugated to a thiolated hen egg white lysozyme. This approach led to superior stability of the lysozyme against lyophilization, and high temperatures compared to POE and trehalose excipients [268]. Nonetheless, it also required the modification of the protein, which can potentially affect the protein’s activity. In a different study, trehalose glycopolymers, along with four different monomer modifications, were synthetized, and their efficiency in terms of preserving the activity of horseradish peroxidase and glucose oxidase at higher temperatures and after several lyophilization cycles was investigated [269]. In comparison with trehalose monomers, the polymerized glycopolymer made of four trehalose monomers better preserved the protein activity and conformational stability. As an additional benefit, the glycopolymer was shown to be non-toxic for different cell lines when examined up to a concentration of 8 mg/mL [269]. In the most recent study, the authors continued their research on the same trehalose glycopolymer by chemically conjugating it to insulin. This prevented insulin from undergoing thermal and agitation-induced protein particle formation in solution. When tested in mice, the conjugate had a significantly more prolonged plasma circulation than the free insulin [270]. Furthermore, from a safety perspective, the glycopolymer has been shown to be biocompatible and non-toxic to mice when administered 1.6 mg/kg body weight [269].
The above-debated studies highlight both the benefits and limitations of disaccharides for the stabilization of protein biotherapeutics. Disaccharides offer the advantage of safety and lack of toxicity for parenteral, particularly intravenous, use. Sucrose and trehalose are FDA-approved GRAS excipients commonly used in the injectable formulations. While these are excipients of choice for the protection of the proteins against thermal and lyophilization stresses, their ability to protect the proteins in solution remains to be optimized. In many cases, the use of sucrose and trehalose alone cannot ensure adequate protein stability, as these can only minimize protein–protein interaction to a certain degree and are not as effective against sheer and interface stresses [258]. To overcome this issue, other classes of glycopolymers based on the disaccharides have also been developed. However, a further issue to consider is the excipient stability. Both sucrose and trehalose are susceptible to acid hydrolysis, though sucrose is much more prone to this phenomenon than trehalose [271]. Hydrolysis of disaccharides creates reducing sugars, which must be avoided as they can damage the proteins. Nevertheless, this is usually not an issue unless the pH is lower than 4, which is not the case for protein biotherapeutics [84]. Reducing sugars such as lactose or maltose must be avoided, as they can chemically interact to proteins through the Maillard reaction during storage [84]. Taken together, while disaccharides might not be adequate PS alternatives in their crude form [264], they can provide a base for chemical modification, or else be used in combination with other classes of stabilizers, which can hopefully pave the way for the synthesis of more suitable PS alternatives.
Sugar alcohols
Sugar alcohols such as glycerol, sorbitol, and mannitol ( b) stabilize proteins in solution as well as during lyophilization or storage [272,273,274]. Similar to trehalose and sucrose, these mainly serve as osmolytes, protecting the integrity of the protein by altering the protein–solvent interactions [272], and by replacing the water molecules during lyophilization [273,274]. Glycerol has been shown to shift the conformation of the native protein towards a more compact state [275]. It also inhibits protein unfolding and aggregation through the stabilization of aggregation-prone intermediates. The main reason behind this phenomenon is the amphiphilic nature of glycerol, which arranges at the interface of the hydrophobic protein regions and the polar solvent [275]. To what extent glycerol can improve the protein stability is, however, dictated by the nature of the protein in question.
Mannitol, a hexahydro alcohol, is often used as a bulking agent in freeze-dried and spray-dried formulations [276,277]. In an amorphous state, mannitol also provides protein stabilization during lyophilization, as it provides intimate H-bonding with the protein at molar ratios of 360:1 or even higher. This leads to the shift of the lattice structure of the protein towards an amorphous state, which renders these more stable than their crystalline counterparts [276,277]. However, research has also demonstrated that the protein stabilization effect of mannitol occurs up to a certain concentration of the excipient. In spray-dried recombinant humanized mAb formulation, mannitol has been shown to exert a protective effect up to an excipient: protein ratio of 200:1. At higher concentrations, the crystallization of mannitol exerts a detrimental effect upon the protein stability [278]. Literature holds many other reports describing the detrimental effect of crystallized mannitol upon protein stabilization [279,280,281,282,283]. On the other hand, a recent review details varying techniques to achieve a successful lyophilized injectable formulation without the inconveniences that mannitol conveys. As a detailed discussion about the optimization of freeze-drying cycle parameters in multi-component formulations is out of the scope of the current paper, the reader is referred to a detailed review by Thakral et al. (2022) for further information [284]. The excipient to protein ratio has also been reported as essential to maintain the stability of lyophilized protein formulations during storage [285]. Today, mannitol, along with PSs, is a component of several marketed mAb formulations, including but not limited to Humira®, Simulect®, and Ilaris® (Ilaris liquid formulation contains mannitol, while the lyophilized formulation contains sucrose instead) [15,286].
Sorbitol is another common sugar alcohol used in marketed mAbs parenteral formulations such as Vyepti®, Anthim®, Simponi®, etc [286]. It has been shown to protect the proteins in a manner similar to mannitol but is associated with the drawback of recrystallization over time, which negatively impacts the protein stability during long-term storage [287,288]. As in the case of mannitol, excipient to protein ratio seems to play an important part, with sorbitol-recrystallization-mediated protein instability being less pronounced when lower protein concentrations are used [288]. The combination of sorbitol and sucrose has shown a synergistic protein stabilizing effect [289,290].
Like disaccharides, sugar alcohols possess acceptable safety profiles for intravenously injectable formulations. Glycerol, the backbone of many lipids, is found in vegetable and animal fats and oils within regularly consumed foods. Following ingestion, glycerol is absorbed from the intestine and then follows two degradation pathways; it is transformed to glycogen and carbon dioxide or used as a precursor for the anabolism of fatty acid in the body [291]. Glycerol has been listed as GRAS, included in the FDA Inactive Ingredients Database for pharmaceutical applications, and added to the Canadian list of acceptable non-medicinal ingredients. Given its metabolism in the human body, glycerol is widely employed in parenteral, topical, oral, and ophthalmic formulations, as well as a solvent and sweetener [292]. In general, glycerol as a food additive or excipient has been proven to be non-irritant and non-toxic [291]. Low concentrations of glycerol for oral formulations present scarce side effects [293]. Nevertheless, high oral doses of glycerol can induce hyperglycemia, nausea, headache, and thirst. Intravenous or oral administration of glycerol for the reduction of cerebral edema [294] has been linked to side effects such as hemolysis, hemoglobinuria, and renal failure [295,296]. Of course, the concentration of glycerol used for such purposes is potentially much higher than the amounts necessary to stabilize protein formulations. Another side effect of high dosage use is the mucous membranes and skin irritation [297].
Mannitol and sorbitol are also GRAS, FDA-approved excipients and, as previously mentioned, part of the marketed injectable formulations. They are listed in the FDA Inactive Ingredients Database for injectable formulations, and also in the Canadian List of Acceptable Non-medicinal Ingredients [291]. The safety of these ingredients has been established based on the lack of evidence signifying their systemic toxicity, irritation, sensitization, and adverse clinical reports [291]. While mannitol is partially metabolized by the gut microbiota, it is not degraded following intravenous administration. Both mannitol and the products of its partial metabolism are cleared through renal excretion in a few hours [298,299]. Sorbitol, on the other hand, is rapidly metabolized through normal glycolytic pathways, ultimately creating carbon dioxide and water [299]. While both excipients have been shown to be safe, literature holds a few reports about the hypersensitivity towards intravenous administration of mannitol [300,301,302]. Even in cases where mannitol has been used as an excipient in lower concentrations, hypersensitivity reactions have been still reported [291].
While sugar alcohols offer the advantage of safety and appropriate regulatory status, they often cannot provide the protein with adequate protection from all sources of stress, in particular the interface-induced stresses. As a result, these often need to be used in combination with a second, more surface-active excipient to ensure the adequate stability of the protein during all stages of production and storage. They can also provide a backbone for further chemical modification to reconcile the advantages of these molecules with the surface-active properties that can be provided through conjugation with other more lipophilic moieties.
Cyclodextrins
According to the Inactive Ingredient Database of the FDA, four different types of cyclodextrins (CDs) have already been approved for use in pharmaceutical formulations. These include 2-Hydroxypropyl-γ-cyclodextrin (HP-γ-CD) for ophthalmic and topical formulations, α-cyclodextrin (α-CD) or Alfadex for liquid and solid intracavitary formulations, β-cyclodextrin (β-CD) or Betadex for oral, topical, intramuscular, and intravenous formulations, and sulfobutylether-β-cyclodextrin (SBE-β-CD) for intravenous and subcutaneous formulations [161]. CDs are cyclic oligosaccharides ( c) and the diversity in the ring size and in the sidechain of CDs confer them different properties that can be used for a variety of applications [303]. CDs are produced through the enzymatic degradation of starch and possess a hydrophilic outer surface and a hydrophobic cavity, rendering them water-soluble and biocompatible [304]. The most popular natural CDs are α, β, and γ, which contain 6, 7, and 8 glucopyranose units, respectively [304]. These are non-hygroscopic, crystalline, and homogeneous substances. Given their ideal cavity size for accommodating small molecules of the size of an amino acid, efficient drug complexation and loading, availability, and low cost, β-CDs provide an interesting candidate for the complexation with various drugs, mainly for the purpose of enhancement of the drug water solubility, improvement of the drug stability, increase of the drug dissolution rate and bioavailability, reduction of the side effects, and manipulation of the drug’s physicochemical characteristics [305]. To elaborate on the diverse role of CDs in drug delivery systems and the variety of CD-based systems for parenteral use is out of the scope of the current paper. For more information, the reader is referred to a well-structured review by Ferreira et al. (2022) for further information [306].
Interestingly, some CDs, e.g., HP-β-CDs (Hydroxypropyl-β-cyclodextrin) and Mβ-CDs (Methyl-β-cyclodextrins) have been shown to inhibit protein aggregation in a manner similar to non-ionic surfactants [307,308,309,310]. This can be either a by-product of the preferential adsorption of the CDs to the interface or else originate from the interaction of the CD molecules with the protein itself. It seems that both the nature and the concentration of the protein and the properties of the CDs determine which of the two aforementioned mechanisms will be dominant. Samra et al. (2010), who investigated the stabilizing effect of seven different CDs on three pharmaceutically relevant proteins, reported that almost all investigated CDs could successfully inhibit protein aggregation at a pH of 5.5. They argued that this potentially pertains to the characteristics (isoelectric point, molecular weight, and detailed tertiary structure) of the proteins, which in turn influences the nature of the interaction of the CDs therewith [311]. Concurringly, CDs have been shown to successfully stabilize rhGH through direct interaction therewith, for the unusually high proportion of solvent-accessible aromatic amino acids favors such interactions. It appears that stabilization due to the direct interaction between the CDs and the protein is often the dominant mechanism in case the protein has substantial solvent exposure to hydrophobic amino-acid residues [311]. Protein stabilization as a result of the preferential adsorption to the interface, however, is governed by the degree of the surface activity of the CD-derivative under investigation rather than the structure of the protein. For instance, HP-β-CD has been shown to reduce the interface-induced precipitation of porcine growth hormone [307]. HP-β-CD has also been shown to be a beneficial PS alternative for the protection of the IgG formulations from aggregation at the air–water interface [312,313].
As a result of the duality of the mechanisms involved in the CD-mediated protein stabilization, i.e., reduction of the protein surface adsorption and protein stabilization through direct interaction, it seems that a CD derivative beneficial for one therapeutic protein may have little effect on, or even jeopardize, the stability of another protein [314]. CD-derivatives that inhibit protein particle formation caused by one stress condition may not be able to do so in case of a different stress condition. Moreover, the required CD to protein concentration or molar ratio that would yield adequate stabilization differs greatly depending on the nature of both components. Thus, for the selection of CD-derivatives for the stabilization of biotherapeutic protein formulations, several factors should be heeded. The first is the stabilizing role that CDs are supposed to play in the formulation, i.e., “direct” interaction with the protein versus prevention of “surface/interface-induced” aggregation. Stabilization through the former may only be possible in certain cases, for which the presence of the highly solvent accessible exposed hydrophobic residues on the proteins might be an important but not compelling prerequisite [314]. The use of CDs to prevent surface-induced aggregation, on the other hand, may be possible in many cases, for which the surface activity of the CD-derivative must be taken into account [303]. The concentration of the protein might also play an important part. In solutions with lower protein concentrations, CDs might be able to better interact with the interface, leading to more efficient protection of the protein against interface-induced denaturation and particle formation. In case the protein concentration in the solution is high, however, the interaction of the CDs with the protein molecules might gain more relevance, which, as previously elaborated, varies significantly depending on the type of the CD and the presence of hydrophobic residues in the protein structure [311].
While CDs offer the benefit of chemical stability in biotherapeutic formulations, the concern of their enzymatic degradation remains to be investigated. A recent study by Zhang et al. (2022) investigated the chemical stability of HP-β-CD in comparison to PS20 and PS80. When subjected to heat stress, autoclave, light, and oxidative stresses, HP-β-CD remains almost stable in contrast to PS20 and PS80 [315]. However, the degradation of CDs can be triggered by various enzymes mainly from the glycoside hydrolase family 13, including cyclodextrinase, α-amylase, glycoamylase, and amylase belonging to the bacteria and archaea domain involved in the metabolism of carbohydrates [316]. Some of these are currently in use in CDs, and their derivates have shown low toxicity and resistance to the enzymatic degradation [317]. The saccharide nature of CDs endows them with physicochemical properties similar to dextrins. An outstanding property of CDs is their resistance against β-amylase due to the lack of susceptible non-reducing end groups. Conversely, α-amylases are capable of hydrolyzing the CDs from the carbohydrate chain as in starch, but to a lower extent [318]. The degree of hydrolysis is directly proportional to the size of the free CD moiety. By burying oxygen bridges inside the CD cavity, the resistance to hydrolysis increases [319]. Accordingly, α- and β-CDs are resistant to salivary α-amylase, while γ-CDs are sensitive to pancreatic and salivary α-amylase [320,321]. Furthermore, Aspergillus oryzae α-amylase, a fungal amylase mainly used for saccharide fermentation, has shown enzymatic activity towards γ-CDs, leading to the production of maltooligomers [322]. Additionally, two more α-amylase enzymes with cyclo-malto-dextrinase specificity have been identified in thermophilic bacterial strains [323]. It should be, however, noted that the majority of the identified CD-degrading enzymes are of bacterial origin, and hence will not create any issues in case the protein is purified from a eucaryotic host. In case of bacterial hosts, quantification of the CD degrading enzyme traces in HCPs and the determination of the enzymes’ affinity for the molecule is necessary to present a fair comparison between CDs and PSs in terms of their potential sensitivity to HCP-mediated degradation.
CDs can also be used in combination with PSs to help mitigate their limitations. In an interesting patented work, Connolly et al. (2017) demonstrated that the use of HP-β-CD, together with PS20, improves the stability of the latter towards enzymatic degradation. This was confirmed based on the decrease of the sub-visible particle formation in PS-containing solutions in the presence of different HCPs. HP-β-CD has also been shown to protect PS20 in the presence of several enzymes (phosphotriesterase-like-lactonase, Candida antarctica lipase B, renin-like enzyme, and lipoprotein lipase) in a concentration-dependent manner [324]. It has been hypothesized that such protective effects might originate from the “direct” interaction of the CD with PS20, leading to the formation of an inclusion complex. This would in turn decrease the interaction between the host cell enzyme traces and the PS20, thereby protecting the latter against degradation. Furthermore, it is described that CDs can help solubilize the sub-visible particles originating from the enzymatic degradation of the PS20 [324]. Accordingly, using a combination of CDs and PSs for the stabilization of protein biopharmaceutics can be beneficial. An improved stability of the PS surfactants will help overcome some of the problems originating from the PS degradation, including the potential damage to the protein structure as well as the sedimentation of visible or sub-visible FFA particles. It should be, however, noted that combined use of CDs and PSs would increase the complexity of the formulation. Moreover, CDs might disturb established analytical methods to detect surfactants like PSs.
In addition to the enzymatic degradation, the oxidative cleavage of CDs is another under-investigated potential instability mechanism for these excipients [325]. Within this context, the presence of OH-radicals generated by auto-oxidation processes can lead to the cleavage of glucosidic bonds of β-CD, producing thereby different oligosaccharides like D-xylose, D-threose, D-arabinose, and D-erythrose [325].
Overall, there seems to be a need for more in-depth research to provide further information regarding the ability of the CDs to stabilize protein formulations. Furthermore, the degradation profile of the excipient, along with its consequences for the quality of the final product, particularly upon long-term storage, should be examined. The available information at this point depicts a promising picture, where CDs, alone or in combination with PSs, can help overcome the drawbacks of the latter in the formulation of injectable protein biotherapeutics.
Hydroxypropyl methylcellulose
Hydroxypropyl methylcellulose (HPMC) ( d) is a semi-synthetic cellulose ether, well-known for its use as a pharmaceutical excipient in oral dosage forms [326]. HPMC is a unique excipient in the sense of the malleability of its physicochemical characteristics, which supports the molecule diverse applications. Within this context, HPMC has been used for the formulation of mucoadhesive systems for the delivery of small molecules, as an excipient in amorphous solid dispersions to enhance the bioavailability of poorly water-soluble drugs, or as a component of various nanocarrier formulations [327,328]. HPMC and other cellulose derivatives have been shown to be non-toxic following pulmonary, oral, intraperitoneal, subcutaneous, or dermal application [329]. These have been shown to be non-irritating to mildly irritating, non-sensitizing, and non-mutagenic in clinical studies [329].
Interestingly, HPMC has been tested as a stabilizer for mAb formulations [330]. In one study, different cellulose polymers, including methylcellulose, HPMC, hydroxypropyl cellulose, and hydroxyethyl cellulose, were used to stabilize 1 mg/mL cetuximab. Among these, low molecular weight HPMC could efficiently stabilize the Ab, with an efficiency comparable with PS80 [330]. Similarly, the ability of low molecular weight HPMC to protect abatacept has been demonstrated [330].
HPMC is classified as GRAS, included in the FDA Inactive Ingredients Database for use in ophthalmic and nasal formulations, oral capsules, suspensions, syrups, and tablets, and listed in the Canadian list of acceptable non-medicinal ingredients [161,291]. A high oral dosage of HPMC has been evaluated for the treatment of postprandial insulinemia [329] and metabolic syndromes such as hypertension, proinflammatory or inflammatory state, prothrombotic state, atherogenic dyslipidemia, and insulin resistance [331].
Based on the above-debated information, more profound research is worth being dedicated to the investigation of the ability of HPMC to stabilize protein formulations, degradation of the molecule, and the safety of the excipient following intravenous administration.
Dextrans
Dextrans ( e), complex branched glucans, are another group of carbohydrate-based polymers capable of protein stabilization through molecular crowding effect [332]. Dextrans can reduce the adsorption of the proteins to the liquid/air interface [53], thus preventing the aggregation and denaturation thereof in liquid formulations. They can also help maintain protein structural integrity during freezing, drying, and storage in the solid state [333]. Within the context of the last point, the molecular weight of dextran is a determining factor for the extent of protein stabilization. In a study comparing the efficiency of dextrans with different molecular weights ranging from 12 kD to 2000 kD on the structural stability of freeze-dried protein, the medium molecular weight of 512 kD was shown to be most effective [334]. The reasons behind the differences in protein stability among various molecular weight dextrans remain unknown [334].
The drawback of dextrans for protein stabilization lays in their tendency to form polymerprotein aggregates. This occurs through a Maillard reaction between the protein’s primary amines and dextran’s reducing ends. Typically, these reactions are observed following storage at temperatures over 50 °C within the course of days. For some proteins, such as those of blood origin, the reaction can occur even following around 6 months of storage at room temperature or a month at 37 °C. Dextranol, a form of reduced dextran, has been shown to solve this problem, thereby serving a more efficient lyoprotectant [335].
Dextrans are FDA-approved and already part of several FDA-approved injectable formulations. However, based on our literature search, there have been a few reports of side effects in patients. For instance, infusion of dextran 40 (average molecular weight: 40,000 D) has been reported to induce renal diseases in patients with reduced urine flow. Infusion of dextran 70 (average molecular weight: 70,000 D) might induce hypersensitivity reactions, such as urticaria, hypotension, and bronchospasm, and milder reactions like fever and nasal congestion. Despite not being so common, anaphylactic reactions are still possible when dextran is administered [294]. In fact, a case of anaphylactic reaction to dextrans as a component of the BCG (Bacillus Calmette–Guérin) vaccine and remimazolam formulation has been reported [336,337].
Despite the potential benefits, it is unclear whether the use of dextrans alone can confer adequate stability to the protein biopharmaceuticals. Here again, the polymers can be used, potentially in combination with a more surface-active excipient, as potential PS alternatives, or else serve as a backbone for further chemical modifications to enhance their surface-active properties.
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