Table 2.

Summary of the available information on the potential alternatives to PSs for the stabilization of protein biotherapeutic formulations.

Excipient		Protein Stabilization Efficiency	Physicochemical Stability	Regulatory Status and Safety	Comparative Studies to PS
	Subcategory
Surfactants	Sucrose fatty acid esters	remains to be investigated. As in the case of other surfactants, protection of the protein integrity through direct interaction as well as competitive interface adsorption is expected.	are susceptible to chemical and enzymatic cleavage of ester bond and enzymatic degradation of the sugar moiety	- GRAS - FDA-approved for food and cosmetic use - sucrose stearate and sucrose palmitate: FDA-approved for oral and topical formulations - sucrose monopalmitate (0.5 g/kg): resulted in hemolytic reactions [183,184]	Not available
	Sugar monoesters	remains to be investigated. As in the case of other surfactants, protection of the protein integrity through direct interaction, as well as competitive interface adsorption being expected.	are susceptible to chemical and enzymatic cleavage of ester bond and enzymatic degradation of the sugar moiety	nontoxic and biodegradable emulsifiers for use in the food industry [190]	Not available
	Polyethylene glycol (PEG) stearates and PEG fatty esters	polyoxyl 15 hydroxy stearate and polyoxyl 35 castor oil (Kolliphor® HS 15 and Kolliphor® EL): protect amylase and bovine serum albumin (BSA) from chemical and mechanical stresses [204]. As in the case of other surfactants, protection of the protein integrity through direct interaction therewith as well as competitive interface adsorption is expected.	are susceptible to chemical and enzymatic cleavage of ester bond and oxidation of POE [203]	- PEG 2 stearate: FDA-approved for topical application - PEG 8 and 100 stearates: FDA-approved for oral and topical application - PEG 40 stearate: FDA-approved for ophthalmic, oral, and topical applications - polyoxyl 15 hydroxy stearate and polyoxyl 35 castor oil: FDA approval for oral, parenteral, and ophthalmic application - potential PEG-mediated immunogenicity and hypersensitivity	- polyoxyl 15 hydroxy stearate: superior toxicity profile to PS80 [209] - polyoxyl 15 hydroxy stearate and polyoxyl 35 castor oil: comparable efficiency in protecting BSA against mechanical stress for 7 days [204] - polyoxyl 35 castor oil: superior efficiency to stabilize amylase in the presence of H2O2 over a period of two months [204]
	Block polyethylene-propylene glycols (Poloxamers®)	protect the protein through direct interaction as well as competitive interface adsorption	are susceptible to thermal oxidation of PPO block in solid and liquid states	- P188: FDA-approved for intravenous formulations, part of commercial biotherapeutic formulations such as Gazyva®, Orencia®, Norditropin®, and Hemlibra® - potential PEG-mediated immunogenicity and hypersensitivity	P188: Comparable efficiency with PSs in stabilizing mAb formulations, production of PDMS during long-term storage due to interactions with the silicone oil in the stopper in case of P188 [241], increase of glide force in prefilled syringes by PS, but not P188
	Polyoxyethylene fatty ethers (Brij®)	protect the protein through direct interaction as well as competitive interface adsorption [247,248]	might be susceptible to metal- and photo-induced oxidation [78,251]	- FDA-approved for topical administration - Brij® 58: no side effects when administrated in a concentration of 20 mg/mL in animal toxicological studies [249]	- Brij® 92: comparable efficiency to PSs in stabilizing rhGH [247] - Brij® 35: performed inferior to PSs in improving the mechanical and thermal stability of mAb, but superior in protecting the protein against photo-oxidation [250] - Brij® 58: superior inherent stability of the surfactant in protein formulations when compared to PS20 and PS80 [249]
	Non-ester sugar-based surfactants	n-dodecyl-β-D-maltoside: serves as cryoprotectant for D-alanine Peptide T-amide [253], reduces the aggregation and immunogenicity of human IFN- β [254], prevents insulin self-association under mechanical stress in liquid state [253]	susceptible to enzymatic degradation of the sugar moiety	- n-dodecyl-β-D-maltoside: classified by EPA as non-mutagenic, non-toxic, and non-irritating [252] - biodegradable and biocompatible	Not available
	N-alkyl amino acid polyether amides	N-myristoyl phenylalanine Jeffamine M1000 diamide (FM1000): protects proteins from thermal and mechanical stresses [255,256]	FM1000: potential susceptibility of the amide bond to hydrolysis, through to a significantly lower extent compared with ester bond in ester surfactants	Not available	FM1000: 3-fold more reduction of thermal-induced abatacept aggregation compared to PS20 and PS80 [255], inferior, and superior IgG protection from agitation-mediated stress compared to PS80 and PS20, respectively [256], protein stabilization from agitation-mediated stress in IV bags was superior to both PS20 and PS80 [256]
Carbohydrates	Disaccharides and sugar alcohols	- protect the proteins against thermal and to some extent mechanical stresses in solid and liquid state, act mostly by altering protein–solvent interactions, also serve as molecular crowders [259,272] - mostly improve the thermal stability of the protein - act as cryoprotectants - are associated with inadequate protein protection necessitating the co-application of surfactants - chemical modification, e.g., esterification with fatty acids, etc. improves the protein stabilization efficiency	are susceptible to enzymatic degradation	FDA-approved, GRAS, established excipients in the formulation of intravenously injected formulations	are often used in combination with PSs to confer proteins adequate stability
	Cyclo- dextrins	protect protein either through direct interaction (in case of certain proteins with substantial solvent exposure to hydrophobic amino-acid residues [311]) or competitive interfacial adsorption (in case of CD with higher surface-active properties [307]), dominant mechanism also depends on protein concentration	are potentially susceptible to enzymatic degradation	- 2-hydroxypropyl-γ-cyclodextrin; FDA-approved for ophthalmic and topical administration - α-cyclodextrin: FDA-approved for intracavitary administration - β-cyclodextrin and sulfobutylether-β-cyclodextrin: FDA-approved for oral, topical, intramuscular, subcutaneous, and intravenous administration (GRAS)	hydroxypropyl-β-cyclodextrin: reduced the interface-induced precipitation of porcine growth hormone through a mechanism similar to PS20 [307], protects PS20 from enzymatic degradation [324] thereby combination of the two might be beneficial for overcoming the drawbacks associated with PS degradation
	Hydroxypropyl methyl cellulose (HPMC)	efficiently stabilizes mAb formulations such as cetuximab and abatacept [330]	is potentially susceptible to enzymatic degradation	FDA-approved, GRAS excipient for oral, buccal, vaginal, and nasal administration	stabilization of cetuximab comparable with PS80 [330]
	Dextran	stabilize the proteins through a crowding effect [332], reduce the surface adsorption of the proteins in the liquid state [53], maintain protein structural integrity during freezing, drying, and storage in the solid state [333]	potential susceptibility to enzymatic degradation	used in several injectable formulations	Not available
Amino acid-based stabilizers	Amino acids	- certain amino acids promote protein solubilization and refolding mostly by altering protein–solvent interactions, also serve as molecular crowders, stabilizing effect of amino acids is protein dependent - arginine protects protein in solution, and acts as a cryoprotectant [255,256]	are not prone to degradation under formulation and storage conditions	- GRAS - FDA-approved for injectable (intramuscular, intravenous), and oral formulations	Not available
	Polyamines	protect proteins against thermal aggregation and denaturation	Not available	- oral acute toxicity dose of 2000, 600, and 600 mg/kg body weight for putrescine, spermidine, and spermine, respectively [363] - no-observed adverse effect level (NOAEL) following oral administration was 2000 ppm (180 mg/kg body weight/day) for putrescine, 1000 ppm (83 mg/ kg body weight/day) for spermidine and 200 ppm (19 mg/kg body weight/day) for spermine [363] oral administration of spermidine in human and animal models has been well-tolerated [362]	Not available
	Albumin	protects protein through direct interaction as well as competitive interface adsorption [365,366,368]	is potentially prone to forming aggregates with itself or other proteins	- is marketed as an injectable product on its own, and is approved as an excipient for injectable formulations - hypersensitivity and immunological reactions to albumin-containing IFN formulations have been reported [23] - concerns regarding blood-transmitted diseases in case of human and bovine serum albumin	Not available
Synthetic amphiphilic polymers	Polyether polyols (PEG) and Polypropylene glycols (PPG)	- PEGs: increase protein stability through covalent binding, protection of the free protein from thermal and lyophilization stresses [373,374] - PPGs: protect cetuximab from agitation-induced stress, potentially through direct interaction with the protein [330]	are potentially susceptible to oxidation	- PEGs: FDA-approved for intravenous administration, immunogenicity, and hypersensitivity to formulations containing PEGs and their derivatives have been reported [226] - PPGs: FDA-approved for oral and topical formulations, cross-reactivity of anti-PEG antibodies with PPGs is possible [378]	PPGs: comparable efficiency with PS80 in protecting cetuximab from agitation-induced stress [378]
	Polyampholytes	protect proteins from thermal and agitation-induced stress [380]	Not available	Not available	Not available
Ionic liquids		- can protect the proteins against thermal and storage-mediated unfolding [387,390,391,392] - can induce refolding of denatured proteins [388,389] - stabilizing effects depends on the concentration, the length of the alkyl chain and the nature and size of the anion [385]	are generally recognized as highly stable [414]	Toxicity studies have been limited to bacteria, fungi, plants, small animals, and human cell lines [408,409]	Not available

BSA: bovine serum albumin, CD: cyclodextrin, EPA: environmental Protection Agency, FDA: Food and Drug Administration, FM100: N-myristoyl phenylala-nine Jeffamine M1000 diamide, GRAS: generally recognized as safe, HPMC: hydroxypropyl methyl cellulose, IFN-β: interferon beta, IgG: immunoglobulin G, IV: intravenous, mAb: monoclonal antibody, NOAEL: no-observed adverse effect level, P188: poloxamer 188, PEG: polyethyelene glycol, PDMS: protein–polydimethylsiloxane, ppm: parts per million, PSs: polysorbates, POE: polyethylene oxide, PPG: polypropylene glycol, PPO: polypropylene oxide, rhGH: recombinant human growth hormone.