Effects of arginine in therapeutic protein formulations: a decade review and perspectives

Abstract

Arginine (Arg) is a natural amino acid with an acceptable safety profile and a unique chemical structure. Arg and its salts are highly effective in enhancing protein refolding and solubilization, suppressing protein–protein interaction and aggregation and reducing viscosity of high concentration protein formulations. Arg and its salts have been used in research and 20 approved protein injectables. This review summarizes the effects of Arg as an excipient in therapeutic protein formulations with the focus on its physicochemical properties, safety, applications in approved protein products, beneficial and detrimental effects in liquid and lyophilized protein formulations when combined with different counterions and mechanism on protein stabilization and destabilization. The decade literature review indicates that the benefits of Arg overweigh its risks when it is used appropriately. It is recommended to add Arg along with glutamate as a counterion to high concentration protein formulations on top of sugars or polyols to counterbalance the negative effects of Arg hydrochloride. The use of Arg as a viscosity reducer and protein stabilizer in high concentration formulations will be the inevitable future trend of the biopharmaceutical industry for subcutaneous administration.

Statement of Significance: Beneficial and detrimental effects of arginine in therapeutic protein formulations are reviewed and perspectives are provided.

INTRODUCTION

Protein therapeutics, also known as biologics, are a rapidly growing class of therapeutics for many serious diseases, such as cancer, metabolic disorders, cardiovascular conditions, genetic disorders and immunological diseases. Historically, most protein therapeutics have been administered to patients via the intravenous (IV) route. However, subcutaneous (SC) delivery, as an alternative to IV infusion, is of increasing interest as it provides expanded opportunity for self- or caregiver-assisted administration at home or in an office setting and it reduces the frequency of or eliminates hospital visits. For these reasons, SC delivery aids in improving patient experience and patient compliance, reducing patient and caregiver’s treatment burden and lowering treatment costs and healthcare resource utilization.

SC administration of protein therapeutics has traditionally been limited to injection volumes of 1–2 mL, with recent increases to volumes of about 3 mL. This injection volume limitation poses challenges for high-dose protein therapeutics. Overcoming injection volume limitations can be achieved either by enabling proteins to be formulated as high concentration injectables (≥100 mg/mL) or through facilitating the delivery of larger volumes of fluid (>3 mL) using innovative devices such as on-body delivery system or by locally and transiently modifying the SC space with hyaluronidase [1]. The high protein concentration approach may result in high propensity to aggregate and high viscosity, which can affect the stability, manufacturability, device functionality and administration of therapeutic proteins. As a result, the industry has been searching for viscosity reducers and protein stabilizers to achieve high concentration formulations for SC administration. Arginine (Arg) is one of the winners of the search and has become the most front-and-center excipient in high concentration protein formulations.

In this article, all amino acids are L-amino acids and are shortened with the standard three-letter abbreviations where possible. Arg is used as a general term for Arg and its salts, whereas the use of a specific Arg salt, primarily arginine hydrochloride (Arg.HCl) and arginine glutamate (Arg.Glu), is specified wherever applicable. Arg was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins [2]. This effect was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, Arg finds much wider applications than previously anticipated in the research and development of proteins, specifically in the biopharmaceutical industry [3, 4]. For example, Arg is used to suppress protein–protein and protein–surface interactions in solution, reduce viscosity and opalescence of high concentration protein formulations and prevent nonspecific adsorption to gel permeation chromatography columns. Arg facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns [3, 4]. However, there is a concern that Arg may be a protein denaturant, which may limit the expansion of its applications. Such concern arises from the facts that Arg decreases conformational and colloidal stability of certain proteins and contains a guanidinium group, which is a critical chemical structure for denaturing property of guanidine hydrochloride (Gdn.HCl) [5]. This review focuses on the various effects of Arg in liquid and lyophilized protein formulations and discusses the mechanism, controversy and concern of Arg as an excipient.

PHYSICOCHEMICAL PROPERTIES, SAFETY AND CLINICAL EXPERIENCE OF ARG

Arg is a naturally occurring α-amino acid that is used in the biosynthesis of proteins. It contains an α-carboxyl group, an α-amino group and a side chain consisting of a three-carbon aliphatic straight chain ending in a guanidino group (Figure 1) with pKa values of 2.18, 9.09 and 13.2 [6], and a water solubility of 182 mg/mL at 25 °C [7]. Arg and Arg.HCl, high purity, low endotoxin, low metal, USP/EP/JP/ChP grade are available for parenteral use from Pfanstiehl and Millipore [8, 9]. Arg is a Generally Recognized as Safe substance by the US Food and Drug Administration [10].

Figure 1.

Chemical structure of Arg in comparison with guanidine and glycine.

R-Gene 10®, Arg.HCl Injection, USP for IV use marketed by Pfizer, is indicated as an IV stimulant to the pituitary for the release of human growth hormone in patients where the measurement of pituitary reserve for human growth hormone can be of diagnostic usefulness [11]. The recommended adult dose is 30 g Arg.HCl (300 mL of R-Gene 10®) administered by IV infusion over 30 min. Adverse reactions associated with 1670 infusions in premarketing studies were as follows: nonspecific side effects consisting of nausea, vomiting, headache, flushing, numbness and local venous irritation were reported in ~3% of the patients. One patient had an allergic reaction that was manifested as a confluent macular rash with reddening and swelling of the hands and face. The rash subsided rapidly after the infusion was terminated and 50 mg of diphenhydramine was administered. One patient had an apparent decrease in platelet count from 150 000 to 60 000. One patient with a history of acrocyanosis had an exacerbation of this condition following infusion of R-Gene 10®.

Arg has been used in 20 approved protein injectable products, of which 17 are monoclonal antibody (mAb) formulations and three are non-antibody protein formulations as summarized in Table 1, based on the prescribing information available at Drugs@FDA [11] and the review articles [12, 13]. Arg concentrations in the 20 approved protein formulations range from 25 to 300 mM. The highest amount of Arg in a single IV dose is 3500 mg in Activase. The highest amount of Arg in a single SC dose is 78.3 mg if a dose of 6 mg/kg is chosen for a patient with a 75 kg body weight using the 150 mg/mL Hemlibra. Twelve (60%) out of the 20 Arg-containing protein products are approved in the last decade (2014–23), four in 2004–13 and four in 1987–2003. The approval uptrend is driven by booming biopharmaceutical product development and demand for high concentration formulations for SC administration.

Table 1.

Arg or Arg.HCl used in the approved protein formulations

Approved product	Route of administration	Arg amount per dose	Arg Conc.	Protein Conc. (mg/mL)	Other formulation composition	Year approved in USA
Leqembi (lecanemab) 500 mg/5 mL or 200 mg/2 mL vial	IV	210.7 mg (5 mL diluted into 250 mL 0.9% NaCl injection) or 84.3 mg (2 mL diluted into 250 mL 0.9% NaCl injection)	200 mM (42.1 mg/mL Arg.HCl)	100	25 mM histidine/histidine hydrochloride monohydrate (0.18 mg/mL histidine, 4.99 mg/mL histidine hydrochloride monohydrate), 0.05% (0.50 mg/mL) PS80, pH 5.0	2023
Spevigo (spesolimab) 450 mg/7.5 mL vial	IV	79.0 mg (15 mL diluted into 0.9% NaCl Injection)	25 mM (5.3 mg/mL Arg.HCl)	60	45.1 mM acetate (0.3 mg/mL glacial acetic acid, 3.3 mg/mL sodium acetate), 5.1% (51 mg/mL) sucrose, 0.04% (0.4 mg/mL) PS20, pH 5.0–6.0	2022
Aduhelm (aducanumab) 170 mg/1.7 mL or 300 mg/3 mL vial	IV	53.7 mg (1.7 mL diluted into 0.9% NaCl Injection) or 94.8 mg (3 mL diluted into 0.9% NaCl injection)	150 mM (31.6 mg/mL Arg.HCl)	100	20 mM histidine/histidine hydrochloride monohydrate (0.60 mg/mL histidine, 3.39 mg/mL histidine hydrochloride monohydrate), 10 mM (1.49 mg/mL) methionine, 0.05% (0.50 mg/mL) PS80, pH 5.5	2021
Evkeeza (evinacumab) 345 mg/2.3 mL or 1200 mg/8 mL vial	IV	111.0 mg (7.5 mL diluted into 0.9% NaCl injection or 5% dextrose injection)	70 mM (14.8 mg/mL Arg.HCl)	150	10 mM histidine/histidine hydrochloride (0.74 mg/mL histidine, 1.1 mg/mL histidine hydrochloride), 216 mM (30 mg/mL) proline, 0.1% (1 mg/mL) PS80, pH 6.0	2021
Jemperli (dostarlimab) 500 mg/10 mL vial	IV	42.2 mg (20 mL diluted into 0.9% NaCl injection or 5% dextrose injection)	100 mM (21.1 mg/mL Arg.HCl)	50	25 mM citrate (0.48 mg/mL citric acid monohydrate, 6.68 mg/mL trisodium citrate dihydrate), 31 mM (1.81 mg/mL) NaCl, 0.02% (0.2 mg/mL) PS80, no pH available in the prescribing information	2021
Margenza (margetuximab) 250 mg/10 mL vial	IV	495 mg (45 mL diluted into 0.9% NaCl injection)	52.2 mM (11 mg/mL Arg.HCl)	25	10 mM phosphate (0.58 mg/mL sodium phosphate dibasic heptahydrate), 1.1 mg/mL sodium phosphate monobasic monohydrate, 50 mM (2.9 mg/mL) NaCl, 0.01% (0.1 mg/mL) PS80, pH 6.1	2020
Enspryng (satralizumab) 120 mg/mL prefilled syringe	SC	26.1 mg (1.0 mL injection volume)	150 mM (26.1 mg/mL Arg)	120	20 mM (3.1 mg/mL) histidine with aspartic acid for pH adjustment, 0.05% (0.5 mg/mL) P188, pH 6.0	2020
Dupixent (dupilumab) 300 mg/2 mL prefilled syringe or prefilled pen	SC	10.5 mg (2 mL injection volume)	25 mM (5.3 mg/mL Arg.HCl)	150	20 mM (3.1 mg/mL) histidine, 12 mM (1 mg/mL) sodium acetate, 5% (50 mg/mL) sucrose, 0.2% (2 mg/mL) PS80, pH 5.9	2017
Dupixent (dupilumab) 200 mg/1.14 mL prefilled syringe or prefilled pen	SC	12 mg (1.14 mL injection volume)	50 mM (10.5 mg/mL Arg.HCl)	175	20 mM (3.1 mg/mL) histidine, 12 mM (1 mg/mL) sodium acetate 5% (50 mg/mL) sucrose, 0.2% (2 mg/mL) PS80, pH 5.9	2017
Kevzara (sarilumab) 150 mg/1.14 mL or 200 mg/1.14 mL prefilled syringe or prefilled pen	SC	8.9 mg (1.14 mL injection volume)	45 mM (7.8 mg/mL Arg)	132 or 175	21 mM (3.3 mg/mL) histidine, 5% (50 mg/mL) sucrose, 0.2% (2 mg/mL) PS20, pH 6.0	2017
Hemlibra (emicizumab) 30 mg/mL, 60 mg/0.4 mL, 105 mg/0.7 mL, or 150 mg/mL vials	SC	78.3 mg (3.0 mL injection volume for high dose)	150 mM (26.1 mg/mL Arg)	30 or 150	20 mM (3.1 mg/mL) histidine with aspartic acid for pH adjustment, 0.05% (0.5 mg/mL) P188, pH 6.0	2017
Entyvio (vedolizumab) 300 mg vial	IV	131.7 mg (5 mL diluted into 0.9% NaCl injection or lactated Ringer’s injection)	125 mM (26.3 mg/mL Arg.HCl)	60a	50 mM histidine/histidine monohydrochloride, 10% (100 mg/mL) sucrose, 0.06% (0.6 mg/mL) PS80, pH 6.3	2014
Benlysta (belimumab) 200 mg/mL prefilled autoinjector	SC	5.3 mg (1.0 mL injection volume)	25 mM (5.3 mg/mL Arg.HCl)	200	10 mM histidine/histidine monohydrochloride (0.65 mg/mL histidine, 1.2 mg/mL histidine monohydrochloride), 115 mM (6.7 mg/mL) NaCl, 0.01% (0.1 mg/mL) PS80, pH 6.0	2011
Actemra (tocilizumab) 162 mg/0.9 mL prefilled syringe or autoinjector	SC	19 mg (0.9 mL injection volume)	100 mM (21.1 mg/mL Arg.HCl)	180	20 mM histidine/histidine hydrochloride monohydrate, 30 mM (4.5 mg/mL) methionine, 0.02% (0.2 mg/mL) PS80, pH 6.0	2010
Arzerra (ofatumumab) 100 mg/5 mL or 1000 mg/50 mL vials	IV	1000 mg (100 mL diluted into 0.9% NaCl injection)	57 mM (10 mg/mL Arg)	20	50 mM (6.8 mg/mL) sodium acetate, 51 mM (2.98 mg/mL) NaCl, 0.07 mM (0.019 mg/mL) EDTA, 0.02% (0.2 mg/mL) PS80, pH 5.5	2009
Kesimpta (ofatumumab) 20 mg/0.4 mL prefilled pen or prefilled syringe	SC	4 mg (0.4 mL injection volume)	57 mM (10 mg/mL Arg)	50	50 mM (6.8 mg/mL) sodium acetate trihydrate, 51 mM (2.98 mg/mL) NaCl, 0.06 mM (0.018 mg/mL) EDTA, 0.02% (0.2 mg/mL) PS80, pH 5.5	2009
Xolair (omalizumab) 75 mg/0.5 mL prefilled syringe or 150 mg/mL prefilled syringe	SC	21.1 mg (0.5 mL injection volume) or 42.1 mg (1.0 mL injection volume)	200 mM (42.1 mg/mL Arg.HCl)	150	20 mM histidine/histidine hydrochloride monohydrate, 0.04% (0.4 mg/mL) PS20, no pH available in the prescribing information	2003
TNKase (Tenecteplase) 50 mg vial	IV	522 mg (50 mg dose)	300 mM (52.2 mg/mL Arg)	5a	163 mM (16 mg/mL) phosphoric acid, 0.04% (0.4 mg/mL) PS20, pH 7.3	2000
Enbrel (etanercept) 50 mg/mL prefilled syringe and autoinjector, 25 mg/0.5 mL prefilled syringe, 25 mg/0.5 mL vial, 50 mg/mL prefilled cartridge for reusable autoinjector	SC	5.3 mg (50 mg dose)	25 mM (5.3 mg/mL Arg.HCl)	50	120 mM NaCl, 1% sucrose, pH 6.3	1998
Activase (alteplase) 50 or 100 mg vial	IV	1700 or 3500 mg (50 or 100 mg dose, respectively)	201 mM (35 mg/mL Arg)	1a	102 mM (10 mg/mL) phosphoric acid, 0.01% (0.1 mg/mL) PS80, pH 7.3	1987

^aProtein concentration is post reconstitution of the lyophilized powder for injection. All other products are solutions for injection. Arg: L-arginine, Arg.HCl: L-arginine hydrochloride, EDTA: Ethylenediaminetetraacetic acid, NaCl: sodium chloride, PS20: Polysorbate 20, PS80: Polysorbate 80, P188: Poloxamer 188.

Among the 17 approved antibody injectables, nine are solutions for SC injection, seven solutions for IV infusion and one lyophilized powder for IV administration with an Arg or Arg.HCl concentration ranging from 25 to 200 mM [11, 12]. There are 11 (65%) high concentration (≥100 mg/mL) antibody formulations for SC or IV administration containing Arg or Arg.HCl. Four antibody formulations (Enspryng, Hemlibra, Actemra and Xolair) for SC administration contain Arg or Arg.HCl, but do not contain NaCl nor a sugar or polyol, thus, in those solutions, Arg or Arg.HCl may also function as a tonicity adjuster (see Table 1).

Glutamate alone has been used in Hulio, Siliq, VariVax and Streptase at 11, 30, 5.9 and 29.6 mM buffer concentration, respectively [4, 11, 12]. But the combination of Arg and glutamate has not been used in approved protein therapeutics as of today. The effects of an equimolar mixture of Arg and glutamate on cell viability and cellular stress using in vitro cell culture systems were examined by Kheddo et al. with reference to sodium chloride (NaCl), in the context of mAb formulation [14]. Cells relevant to SC administration, the human monocyte cell line THP-1 and adherent human primary fibroblasts, were selected. The study results demonstrated that Arg.Glu had no further detrimental effects on THP-1 viability in comparison to NaCl at equivalent osmolalities, and that both salts at higher concentrations caused cell death by apoptosis. For adherent fibroblasts, both salts caused significant toxicity at ~ 400 mOsm/kg, although Arg.Glu caused a more precipitous subsequent decline in viability than did NaCl. These data indicate that Arg.Glu is of equivalent toxicity to NaCl and that the mechanism of toxicity is such that cell death is unlikely to trigger inflammation upon SC injection in vivo.

EFFECTS OF ARG IN THERAPEUTIC PROTEIN FORMULATIONS

Arg in liquid protein formulations

Arg has been widely used in liquid protein formulations as an excipient or additive in multiple publications. The effects of Arg in liquid protein formulations can be categorized into (i) enhancing protein refolding and solubility, (ii) stabilizing protein and suppressing aggregation, (iii) reducing viscosity of high concentration protein formulations and (iv) reducing nonspecific surface adsorption.

Enhancing protein refolding and solubility

The effect of Arg in protein refolding, solubilization and purification was reviewed by Tsumoto et al. [15]. In vivo and in vitro refolding of recombinant proteins, solubilization of insoluble bodies obtained after lysing Escherichia coli, elution of antibodies from Protein-A affinity chromatography and suppression of protein aggregation during storage were summarized and examples were provided in the publication. The review article by Lange and Rudolph [3], the pioneers in the Arg field, provided an extensive overview of the discovery, applications and mechanisms of Arg as an aggregation suppressor using the literature up to 2008. This article mainly reviews the effects of Arg that were studied over the past decade.

Stabilizing protein and suppressing aggregation

Effects of Arg on photostability and thermal stability of four human immunoglobulin G1 (IgG1) mAbs were studied by Maity et al. [16]. The study demonstrated that Arg was a highly effective excipient that significantly reduced the light induced aggregation of the four IgG1 mAbs under the International Conference on Harmonization (ICH) light condition as measured by size exclusion chromatography (SEC), although thermal unfolding transition temperature (T_m) of one mAb (IMC-1A) was decreased by ~3.3 °C in the presence of Arg.HCl as compared with in NaCl. It is very interesting to note that Arg.HCl decreases the thermal stability (lower T_m) of IMC-1A considerably but increases the photostability of this protein significantly.

Stability of fibroblast growth factor 20 (FGF-20) was examined by Maity et al. [17] in the absence and presence of Arg. Thermal stability of FGF-20 measured by DSC was increased with an increase in Arg sulfate concentration up to 500 mM. Yoshizawa et al. [18] investigated the processes of the thermal aggregation of IgG in the presence of additives including Arg.HCl, NaCl, sodium thiocyanate, sodium sulfate, Gdn.HCl, urea, glucose, trehalose dihydrate, xylitol, ethanol, glycerol and ethylene glycol. Arg was the most effective additive to suppress the formation of insoluble aggregates during the heat treatment at 75 °C. Protein stabilizing sugars were able to suppress the formation of soluble aggregates and thereby increased the percent monomers after the heat treatment. These results indicate that the thermal aggregation of IgG occurs in a two-step process. The first step is the formation of soluble oligomers, which is triggered by the unfolding process that can be stabilized by typical sugars, and the second step is the formation of insoluble aggregates through weak cluster–cluster interactions, which can be suppressed by Arg. Therefore, the rational approach to design an antibody formulation is to combine a sugar or polyol and Arg on top of other excipients, such as buffer species and surfactant. Fan et al. [19] studied the effects of Arg on protein stability of SaeR, a transcription factor in Staphylococcus aureus. Arg enhanced the stability of SaeR. The thermal denaturation temperature of SaeR in 10 mM Arg buffer was significantly increased compared with the buffer without Arg.

Opalescence can pose challenges to high concentration formulation of proteins and is indicative of aggregation of a formulation. The effect of additives, including Arg.HCl, Gdn.HCl, NaCl and other amino acids, on opalescence of IgG solutions was studied by Oki et al. [20]. Arg was most effective in reducing opalescence of high concentration IgG solutions among the additives tested. Comparative analysis of the chemical structures of the additives showed that the effects of Arg.HCl on opalescence of IgG solutions resulted from its unique structure, which comprises an amino acid main chain, a guanidinium group and a counter ion. A few years later, the same group [21] investigated the mechanism of various types of small molecular additives including Arg.HCl, in suppressing opalescence. The opalescence was resulted from the change in the polarity of the vicinity of aromatic amino acid residues without unfolding of proteins. Once again, Arg demonstrated the effectiveness in reducing opalescence of high concentration IgG solutions among the additives tested at a pharmaceutically relevant concentration of 200 mM. These results indicated that Arg.HCl was an effective excipient in antibody formulations for the suppression of protein aggregation and opalescence by inhibiting the attractive electrostatic interaction between antibody molecules.

Reducing viscosity of high concentration formulations

Arg and Arg.HCl appear to be very effective in viscosity reduction, and it is a predominant viscosity reducer in the approved protein formulations [13, 22].

Viscosity reducing effect of Arg.HCl was examined by Inoue et al. [23] with six proteins comparing with Gly, lysine hydrochloride (Lys.HCl), Gdn.HCl and NaCl. The viscosities of bovine gamma globulin solution at 250 mg/mL at the physiological pH and 25 °C were reduced from 59.6 to 40.0 cP, 35.5 cP and 42.8 cP with 200, 500 and 1000 mM of Arg.HCl, respectively. Arg.HCl also decreased the viscosity of bovine gamma globulin solution at acidic and alkaline pHs. Arg.HCl was the most effective viscosity reducer among the five substances examined. Interestingly, Arg.HCl decreased the viscosity of antibody solutions (bovine gamma globulin, human gamma globulin and human immunoglobulin G) but not globular protein solutions (α-amylase and α-chymotrypsin). These results provided evidence that specific interactions between Arg.HCl and antibodies were the molecular origins of its viscosity reducing effect. Viscosity reduction of concentrated mAbs with Arg.HCl and Arg.Glu was also investigated by Borwankar et al. [24]. The addition of high concentrations of Arg.Glu (225–860 mM Arg + 245–620 mM Glu) or Arg.HCl (825 mM) reduced the viscosity of an ~250 mg/mL mAb solution up to 6-fold. With Arg.Glu, the viscosity of the mAb solution was reduced to 30 cP and for a polyclonal sheep IgG solution to 17 cP both at an ~250 mg/mL protein concentration. In contrast, the viscosity was only weakly affected by NaCl or trehalose. The large viscosity reduction from Arg may be attributed to direct binding to the mAb, resulting in suppression of both hydrophobic and local anisotropic electrostatic attraction. Aggregate formation was negligible for the mAb solutions with high Arg content as measured by SEC even after 8 weeks of 25 °C storage. This study demonstrated that Arg.Glu and Arg.HCl were very effective in reducing viscosity of high concentration antibody formulations. However, Arg concentrations examined are higher than a typical concentration used in an injectable product (25–200 mM as presented in Table 1) and can make the osmolality out of the acceptable range for SC administration [25]. It is recommended that osmolality be taken into consideration when Arg is added to a protein formulation. Ideally, injectable products intended for SC administration should be formulated as isotonic solutions (osmolality of about 300 mOsm/kg) with the lower and upper osmolality limits generally controlled between 240 and 600 mOsm/kg in order to prevent injection pain [25–27].

Reducing protein adsorption

Shikiya et al. [28] examined additives including Arg.HCl, Lys.HCl, Gdn.HCl, NaCl, Gly and glucose, for the prevention of protein adsorption on polystyrene particles as a commonly used material for vessels such as disposable test tubes and microtubes. A protein solution at a low concentration of 0.067–0.25 mg/mL was mixed with polystyrene particles, and then the adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to polystyrene particles through electrostatic, hydrophobic and aromatic interactions, resulting in a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives tested, Arg.HCl was most effective in reducing the binding of proteins to polystyrene particles as well as activity loss. The effect of Arg.HCl in reducing protein adsorption to polystyrene particles was more pronounced at 500 mM than at 100 mM. Moreover, even after the mixing of protein and polystyrene particles, the addition of Arg.HCl at 500 mM caused desorption of the bound protein from polystyrene particles. This study demonstrated a new function of Arg.HCl as an adsorption reducer.

Controversial effects of Arg

Destabilizing effects of Arg in protein formulations have been reported. Effect of Arg on conformational stability was studied by Thakkar et al. [29] using DSC and intrinsic Trp fluorescence spectroscopy. Arg (up to 300 mM) was found to decrease T_m of both model mAbs (mAb-A and mAb-B) in an Arg concentration-dependent manner, the higher the Arg concentration, the lower the T_m. Arg destabilized mAb-B by influencing its tertiary structure stability, potentially because of the chaotropic nature of guanidinium group in Arg, resulting in a lower hydration contribution to the apparent compressibility of mAb-B. In contrast, sucrose (up to ∼500 mM) showed a concentration-dependent stabilization effect on mAb-A and mAb-B as measured by increased T_m. Similarly, the effect of charged amino acids Arg, Asp, Glu and Lys on the stability of ribonuclease A and alpha-lactalbumin was examined by Anumalla and Prabhu [30, 31]. The thermal stabilities of the proteins in the presence of the amino acids were monitored by T_m, and the structural changes were analyzed using fluorescence quenching and near-UV circular dichroism (CD). Arg destabilized both the proteins whereas Asp, Glu and Lys stabilized the proteins. Furthermore, the quenching and CD results suggested that the addition of amino acids did not alter the structure of the proteins significantly. The effects of Arg reducing conformational stability by decreasing T_m and perturbing the local structure of proteins had raised concerns to limit the broad application of Arg as an excipient. Ishibashi et al. [5] provided rationales and concluded that Arg differed from Gdn.HCl in the mode of interactions with proteins, and Arg was not a protein denaturant. Arg lowered the T_m of certain proteins, but the extent was insufficient to cause denaturation of proteins at or below room temperature.

Alternative forms of Arg salts

An alternative salt form of Arg in combination with Glu (Arg.Glu) was suggested in 2004 [32] as a way to increase the solubility limit and long-term stability of several diverse proteins prone to aggregation. Since then, Arg.Glu has been widely adopted in protein structural and functional studies. In fact, several studies established that on a per-mole basis, Arg.Glu was much more effective at reducing protein–protein association and aggregation than Arg.HCl [33]. Recently, the stabilizing effect of high concentrations (up to 500 mM) of Arg.Glu versus Arg.HCl on a selected IgG1 was explored, which suggested that having Glu (or Asp) as counterions counteracted the potentially negative destabilizing effects of Arg.HCl [34]. The stabilization effect of Arg.Glu was also compared with Arg.HCl by Kheddo et al. [33] with four model mAbs at commonly used pH values for mAbs formulations. Arg.Glu at a concentration of 50–200 mM had the potential to increase the physical stability of mAbs formulated at pHs of 5–7 as measured by aggregation temperature (T_agg). Under accelerated stability conditions at a protein concentration of 30–50 mg/mL, Arg.Glu at 200 mM reduced monomer loss for the least stable mAb to a greater extent than observed for Arg.HCl, and suppressed aggregation at both pH 5.5 and pH 7.0. The different effects of Arg salts formed by HCl, Glu and Asp on stability of an IgG1 were also observed by Fukuda et al. [34] in a mechanistic study, further confirming that the destabilization effect of Arg.HCl can be counteracted by combination of Arg with equimolar Glu or Asp.

The influence of Arg salts with acetate, glutamate, chloride and sulfate as the counterions, in comparison to NaCl and sodium sulfate, was investigated by Zhang et al. [35]. Thermal stability of the CH₂ domain for an IgG1 mAb at 1 mg/mL and its aggregation kinetics were systematically studied at pH 4.8 in individual solutions of Arg salts. Melting temperatures were measured by both DSC and differential scanning fluorimetry. The aggregation kinetics was determined by assessing reversibility for the CH₂ domain in the DSC repetitive scans and then cross-examined by the isothermal aggregation study measured by SEC. The effect of Arg on the thermal stability and aggregation kinetics of the antibody was shown to be strongly anion-dependent: both Arg acetate and Arg.Glu improved the antibody stability, whereas both Arg sulfate and Arg.HCl decreased it. This finding indicates that Arg prefers the protein surface, rather than the exposed backbone upon unfolding and that other mechanisms are also involved in the observed impacts of different counterions to the thermal stability and aggregation kinetics of the antibody. The counteracting abilities of Asp, Glu and Lys against Arg’s destabilizing effect on the stability of ribonuclease A and alpha-lactalbumin were found to be different [30, 31]. Glu could counteract Arg at the lowest fraction in the mixture. Lys required nearly equimolar concentration, whereas Asp needed almost double the concentration to counteract Arg-induced destabilization of the proteins. The results further suggest that the addition of Glu could be an effective strategy to increase the protein stability.

Another potential option to counterbalance the detrimental impact of Arg.HCl was revealed by Svilenov et al. [36] in a study comparing the effects of different additives on therapeutic protein stability. The physical stability of a model mAb IgGκ was investigated at pH 5.0 and 6.5 in the presence of 280 mM sucrose, 140 mM Arg.HCl, 70 mM Arg.Glu, 140 mM Gdn.HCl and 140 mM NaCl. In low ionic strength of 10 mM histidine buffer, the addition of salts reduced the antibody colloidal and thermal stability. The presence of glutamate ion in the Arg salt partially reduced the damaging effect of increased ionic strength. The addition of 280 mM sucrose shifted the thermal protein unfolding to a higher temperature. The Arg salts can have a detrimental effect on the protein colloidal stability in protein formulations where addition of sucrose may stabilize the protein and counterbalance the detrimental effect of Arg salts. Arg.Glu may therefore be a potential alternative to Arg.HCl when it is coupled with sucrose in mAb formulation development [33].

Arg in lyophilized protein formulations

Arg exhibits cryo- and lyoprotective effects similar to those of sugars. There are three lyophilized protein products: Entyvio, TNKase and Activase, containing Arg.HCl or Arg at a concentration of 125, 300 and 201 mM, respectively, as presented in Table 1. Applications of Arg in lyophilized (freeze drying) protein formulations were reviewed by Startzel in 2018 [37]. Since then, several research works exploring the effects of Arg on lyophilized protein formulations have been published. This review is to provide a summary of the publications not covered by Startzel.

The effect of Arg.HCl on the aggregation of BSA in lyophilized formulations containing sugars and polyols, including mannitol, trehalose, lactose and sucrose, was assessed by Hackl et al. [38, 39]. The screening of the sugars/polyols was performed in order to select the best combination of excipients that can produce freeze-dried cakes with elegant appearance, adequate mechanical properties and reconstitution times, and minimize the moisture sorption as Arg.HCl has a tendency to absorb moisture. The best combinations of Arg.HCl, mannitol and trehalose were selected for a subsequent study as mannitol in a combination with Arg.HCl can reduce moisture sorption, whereas trehalose provides a degree of lyoprotection. A total of 10 formulations, which comprised BSA with five different ratios of trehalose-to-mannitol with and without Arg.HCl, were lyophilized and subsequently assessed for internal structures, moisture sorption at 75% relative humidity storage condition and protein aggregation. The study results demonstrated that Arg.HCl included in lyophilized formulations leaded to a significant decrease in BSA aggregation. High hygroscopicity of Arg.HCl could be significantly compensated by introducing mannitol into a protein formulation. The formulation consisting of Arg.HCl, mannitol and trehalose at the mannitol/trehalose ratio > 1 had acceptable physical appearance, moisture resistance and favorable stability.

A series of studies by Seifert et al. [40–42] explored the effects of counterions, amorphous stabilizer, crystalline bulking agents and moisture contents to a model lyophilized antibody formulation. Seifert and Friess [40] first explored Arg citrate-, Arg.HCl- and Arg lactobionate-based lyophilized formulations aiming for elegant cake appearance using 2 and 50 mg/mL of a model IgG1 mAb without additional excipients and in combination with sucrose as amorphous stabilizer as well as mannitol and phenylalanine as crystalline bulking agents. Poor cake appearance for the pure Arg.HCl-based formulation with 2 mg/ml mAb was observed. The combination with sucrose, mannitol and phenylalanine at selected ratios as well as a high mAb concentration of 50 mg/mL were able to improve cake appearance. Overall, the crystalline bulking agent phenylalanine in combination with Arg.HCl rendered elegant cakes with minimal protein aggregation. The alternative citrate and lactobionate salts yielded elegant cake quality but mAb stability was inferior to Arg.HCl. The combination with mannitol showed reduced stability as expected because of mannitol crystallization. Thus, the combination of Arg.HCl with phenylalanine was superior to any other formulations tested including the sucrose based and may allow for substantial process time reduction because of the crystalline scaffold delivering pharmaceutically elegant cakes.

The same group [41] investigated cake appearance and protein aggregation and fragmentation of the model IgG1 mAb lyophilized formulations. Arg.HCl formulations collapsed during lyophilization because of its low glass transition temperature (T_g) and partially crystallized during storage, but provided the best protein stability at low antibody concentration of 2 mg/mL, followed by Arg succinate. Arg citrate, Arg phosphate and Arg lactobionate formulations resulted in amorphous elegant cakes, but inferior protein stability. Addition of sucrose improved cake appearance and protein stability. Arg phosphate with sucrose resulted in similar protein stability as the sucrose reference. Mixtures of sucrose with Arg.HCl, Arg lactobionate or Arg succinate provided better stability than sucrose alone. While 50 mg/mL antibody improved the cake appearance, only Arg lactobionate provided sufficient protein stability next to sucrose. Arg.HCl partially crystallized after 3-month storage at 50 °C but crystallization was suppressed in the sucrose mixtures or at 50 mg/mL mAb. Overall, sugar-free Arg.HCl and Arg lactobionate lyophilized formulations stabilized the antibody comparably or better than sucrose alone depending on antibody concentration. The best protein stability was found for mixtures of Arg.HCl, Arg lactobionate or Arg succinate with sucrose.

Seifert and Friess [42] subsequently tested stability of the model mAb lyophilized with Arg salts at residual moisture levels of <0.5 and ~2.5% for up to 6 months at 40 °C. The mAb aggregation formulated with Arg.HCl and Arg succinate was similar or even less compared with a sucrose reference formulation. Arg citrate, Arg lactobionate and Arg phosphate resulted in lower protein stability. Overall, Arg formulations with high residual moisture levels resulted in better protein stabilization despite decreased T_g. Arg.HCl and Arg succinate, especially at high residual moisture levels, could be promising alternatives to sucrose for stabilization of mAb in lyophilized protein formulations. The low T_g of Arg can be increased by increasing protein concentration as the T_g increases linearly with increasing protein concentration [43]. It should be noted that there is no surfactant added to the formulations investigated in this study. Addition of one of the commonly used surfactants, such as polysorbates 20, polysorbate 80 or poloxamer 188, may further improve the antibody stability during lyophilization process, storage and reconstitution.

Mechanism of Arg effects

The mechanisms of Arg’s effects on proteins were reviewed by Arakawa et al. [44, 45]. Since then, the mechanisms of Arg interacting with proteins have further been investigated and additional mechanisms have been proposed based on experimental data and/or computational simulations. This article classifies the mechanisms into five recognized categories for easy understanding: (i) solubilization of aromatic amino acid side chains, (ii) neutral crowder, (iii) structural dual effect, (iv) cluster formation, and (v) preferential interactions as evidenced by the following studies.

Solubilization of aromatic amino acid side chains

Tsumoto et al. [15] analyzed the three relevant interactions of Arg with proteins, namely, effect on surface tension, preferential interaction and effect on aromatic amino acid solubility, proposing that only the interactions between the guanidinium group of Arg and the π electrons of aromatic amino acid side chains may be responsible for the observed effect of Arg in protein refolding, solubilization and suppression of protein aggregation. Arakawa et al. [44] experimentally determined the solubility of amino acids in aqueous Arg solution. From this study, it was evident that Arg bound more favorably to aromatic side chains, especially Trp and Tyr. The mechanisms governing the stabilization and destabilization of IgG1 of human IL-6 receptor by Arg was studied by Fukuda et al. [34] at a concentration of 100 mg/mL with different Arg concentrations ranging from 0 to 500 mM at three different pH values of 4.5, 5.2 and 6.0. Thermodynamic and fluorescence analyses revealed that the suppression of aggregation by Arg could be attributed to the shielding of hydrophobic regions on the IgG1 surface by the interaction between Arg and hydrophobic amino acid residues, resulting in decreased hydrophobicity of IgG1. On the other hand, the facilitation of aggregation and degradation by Arg was presumably because of the conformational destabilization of IgG1 by the interaction between the guanidinium group of Arg and some acidic residues.

Neutral crowder

Baynes et al. [46] in the next year following the publication of Tsumoto et al. [15] presented the “neutral crowder” mechanism of stabilizing effect of Arg against aggregation and hypothesized that Arg was a neutral crowder. The term was proposed for an additive that exhibited properties of reducing the rate of aggregation without affecting the folding rate and equilibrium, and was preferentially excluded from the gap between the protein molecules in the association transition state resulting in an increase in the free energy of the protein–protein encounter complex. In other words, Arg selectively increased the energy barrier for protein–protein association.

Dual effect

The thesis of Shah [47] elucidated the molecular mechanism of Arg on protein stabilization against aggregation through the dual effect of the guanidinium group of Arg. The role of the guanidinium group of Arg was examined experimentally, using heat-induced aggregation of three model proteins, BSA, β-lactoglobulin and lysozyme, in the presence of Arg and guanidine. Arg could enhance heat-induced aggregation of concentrated protein solutions (0.3 mM) of BSA and β-lactoglobulin, but not for lysozyme. This finding indicated that Arg’s preferential interactions with certain residues over others could determine the effect of Arg on aggregation. The guanidinium group of Arg plays a dual role. It binds to the acidic residues to enhance protein aggregation, whereas it binds to the aromatic residues to suppress aggregation. The other segments of Arg, i.e. the aliphatic segment and the polar C and N terminals, protrude into the solution to aid in solvating the Arg-aromatic residue pair, thereby assisting suppression of aggregation of proteins.

Arg’s ability to modulate the thermal stability of proteins through three distinct interactions between Arg and a protein was elucidated by Platts and Robert using globular proteins, BSA, myoglobin and lysozyme [48]. The dual effect was presented as Arg’s effect on the protein unfolding behaves like the sum of its structural constituent parts, Gly and the guanidinium ion. Below 100 mM Arg acted like Gly, above 100 mM it showed destabilizing effects similar to Gdn.HCl. Gly could affect the thermal stability of a protein by direct interaction with the charged side chains and/or the peptide backbone of the protein observed at low Arg concentrations below 100 mM and by competing for water between the unfolding protein and the cosolute, thus increasing the energy required to hydrate the unfolding protein. On the other hand, the guanidinium ion of Arg acted by direct interaction with apolar regions exposed during unfolding reducing the energy required to hydrate the unfolding protein.

The influence of Arg.HCl on the solution structures of two model mAbs was analyzed by Lilyestrom et al. [49] using small angle X-ray scattering (SAXS). Results showed that Arg.HCl at 100–600 mM concentrations was able to change the overall shape toward the “open” conformation and increase the maximum size of both mAb1 and mAb2. The structural relaxation of mAbs induced by Arg.HCl seen in the SAXS experiments provided further evidence of the dual role of Arg.HCl, both ionic and hydrophobic, to affect protein conformational dynamics and stability in solution.

Cluster formation

The cluster formation mechanism reported by Vagenende et al. provided the molecular origins of Arg’s effects on protein association and folding [50]. Cluster formation was initiated by the association of Arg with specific protein surface loci through cooperative interactions with protein guanidinium and carboxyl groups. The clusters formed by protein-associated Arg ions significantly increased the apparent protein radius measured by SEC and were expected to affect the apparent charge properties and hydrophobicity of the protein surface. This resulted in the unique effects of Arg on many protein processes involving protein association and folding. Interestingly, this study also noted that the effects of Arg on protein conformational stability were concentration dependent: Arg destabilized proteins at low Arg concentrations (<0.5 M) but incrementally stabilized proteins at high Arg concentrations (>1 M) as demonstrated by the T_m changes measured by DSC. However, the Arg concentrations examined in this study are in a range of 0.2–2 M, and are too high to be applicable to a protein formulation for SC administration, except the low-end concentration of 0.2 M.

Preferential interaction

Preferential interaction is the manifestation of widely different modes of binding ranging from weak transient interactions to strong, stoichiometric interactions of solvent components with the protein surface in the native state and hence reflects the interactions of the additives with the amino acid side chains and peptide bonds as described by Arakawa et al. [44, 45]. Arg acted similarly to Gdn.HCl interacting favorably with most amino acid side chains but binding of Arg to the protein surface was different from the binding of Gdn.HCl. Such different binding of Arg made it suppress protein aggregation, but not destabilize proteins. The preferential binding behavior of Arg to protein, as well as the impact of Arg on the conformational and colloidal stability of protein solution, was investigated by Wen et al. [51] with the model proteins, BSA and ovalbumin, using fluorescence-based and dynamic light scattering techniques in the absence and presence of Arg. The result showed that Arg preferentially bound to the aromatic amino acids of proteins mainly through hydrogen bonds and Van der Waals’ forces. The fluorescence quenching, the decreased fluorescence lifetime and the red-shifted ANS (8-Anilino-1-naphthalenesulfonic acid) peak position revealed that Arg perturbed the local environment of Trp and Tyr residues. The electrostatic repulsion among BSA and ovalbumin molecules was attenuated after adding Arg as measured by reduced interaction parameter k_D values. These findings provided strong evidence that Arg possessed negative effects on tertiary conformational and colloidal stability of BSA and ovalbumin during the preferential binding process.

The preferential interactions of Arg.HCl with three therapeutically relevant, IgG1 mAbs were characterized by Sudrik et al. [52] using vapor pressure osmometry in comparison with trehalose and NaCl. The effect of Arg on the reversible self-association, aggregation and viscosity behavior of these mAb molecules was also characterized. Arg.HCl was excluded from the surface of all three IgG1 mAbs at all concentrations in the range of 0–0.4 molal (mole/kg). Arg.HCl decreased the colloidal stability of mAb-A and mAb-B. On the contrary, it enhanced the colloidal stability of mAb-C as measured by the interaction parameter k_D. Its effect of reducing colloidal stability at the low concentration of 100 mM was not through the preferential exclusion mechanism. The preferential interactions of Pro, Arg.HCl and NaCl with three therapeutically relevant IgG1 antibodies were probed by Cloutier et al. [53] through examining how excipients interacted with different types of surface patches in the variable region (Fv). The effects of Arg.HCl on aggregation and viscosity were highly dependent on the surface charge distribution and the extent of exclusion from highly hydrophobic patches. At pH 5.5, Arg.HCl tended to increase the aggregation of an antibody with a strongly positive charge on the Fv, whereas it reduced the viscosities of antibodies with either a hydrophobicity-driven mechanism or a charge-driven mechanism.

DISCUSSION AND PERSPECTIVES

Arg is a natural amino acid with an acceptable safety profile as demonstrated by the clinical experience of the 20 approved protein products, in which the highest amounts of Arg dosed to patients are 78.3 mg for a single SC administration and 3500 mg for a single IV infusion. Arg has a unique structure of guanidino group, which is a critical chemical structure of denaturing property of Gdn.HCl, and a carboxyl group, which is a structure of stabilizing property of glycine. In between is a three-carbon aliphatic chain that acts like a bridge (Figure 1). Arg is highly effective in enhancing protein refolding and solubilization, suppressing protein–protein interaction and aggregation and reducing viscosity of high concentration protein formulations. The destabilization effect of Arg has been reported in several studies based on the observation that Arg reduces melting temperature and perturbs the local structure of proteins. However, the extent of the destabilization effect is insufficient to cause denaturation of proteins at or below room temperature. From the references cited in this review, there is no correlation established between the reduced conformational stability by Arg and the long-term stability at 2–8 °C, which is the typical recommended storage condition for protein formulations, with the exception of the study by Svilenov et al. [36], in which some conformational stability parameters were correlated with the 12-month stability data at 25 °C. There are some studies carried out at high temperatures, such as 75 °C [18], or high Arg concentrations, such as 1–2 M [50]. But translating the findings at such high temperatures and/or high Arg concentrations to a much lower storage temperature and pharmaceutically relevant concentration in line with acceptable osmolality (240–600 mOsm/kg) for SC administration warrants further investigation. As for the 20 marketed Arg-containing protein products, it is evident that their stabilities have been thoroughly studied in supporting the biologics license applications and commercial shelf-life setting although the detailed stability data may not be publicly available for review. The favorable effect of Arg in therapeutic protein formulations can also be proved by product shelf-lives. For instance, Evkeeza, Enspryng, Dupixent and Kevzara all using Arg or Arg.HCl as a viscosity reducer are robustly stable and have a shelf-life of 36, 24, 36 and 36 months, respectively, when stored at 2–8 °C and protect from light [54–57].

In addition to Arg and Arg.HCl, glycine, proline, Lys.HCl and sodium chloride were also used as viscosity reducers in the approved high concentration antibody therapeutics [13, 22]. Numerous novel excipients, such as caffeine, nicotinic acid, ascorbic acid, hydroxyproline, tryptophan, L-ornithine monohydrochloride, phenylalanine, thiamine phosphoric acid ester chloride dihydrate, benzenesulfonic acid, pyridoxine hydrochloride, etc., and their combinations were evaluated as viscosity reducers in high concentration protein formulations and several patents were filed [54, 58–60].

Although Arg is currently a prevalent viscosity reducer, it is not universal. In fact, Zeng et al. [61] demonstrated that caffeine effectively lowered the viscosity of the two model mAb formulations (150 mg/mL infliximab and 200 mg/mL ipilimumab), whereas Arg.HCl and sodium chloride had either a negative effect (increase viscosity) or no impact on the viscosity. Likewise, Xu et al. [62] published comprehensive data proving the inability of Arg to decrease the viscosity of the NISTmAb model antibody.

The typical SC solution formulation proposed by Strickley and Lambert [12] consisting of 100 mg/mL antibody, 50 mg/mL sucrose, 0.01 M histidine, 0.01 mg/mL polysorbate 80 and 10 mg/mL Arg at pH 6 could serve as a good starting point in formulation development of a new antibody, but may not fit all antibodies. Among the marketed 46 high concentration antibody products, there were a total of 23 instances (50%) wherein viscosity reducers were used [22]. The remaining 23 instances not containing viscosity reducers have a protein concentration in the range of 100–150 mg/mL (inclusive) with a mean value of 117 mg/mL, implying that viscosity reducer(s) may not be necessary if protein concentration of a formulation falls within the range (unpublished data). If addition of viscosity reducer(s) is required, it should be selected on a case-by-case basis using design of experiment, one factor at a time and/or rational excipient selection approaches [63] in order to develop a stable and manufacturable high concentration product, not only considering the type of viscosity reducer, but also its concentration. Selection of excipients has been relying on prior knowledge and trial and error. Recently a computational approach based on the site identification by ligand competitive saturation (SILCS) technology, termed SILCS-Biologics was applied to rational excipient selection using two model proteins NISTmAb and CNTO607. The strong correlations observed between viscosity and the various SILCS metrics revealed the potential application of SILCS-Biologics in predicting viscosity of high concentration formulations.

Overall, it can be concluded that the benefits of Arg overweigh its risks when it is used appropriately. However, the application of Arg in protein formulations should be determined on a case-by-case basis. With that in mind and based on the various studies reviewed, it is recommended to add Arg along with glutamate as a counterion to high concentration protein formulations on top of sugars or polyols to counterbalance the negative effects of Arg.HCl. Without doubt, more and more high concentration protein formulations will be formulated with Arg as a viscosity reducer and protein stabilizer in the future.

AUTHOR CONTRIBUTIONS

Steven Ren (Conceptualization [equal], Data curation [equal], Formal analysis [equal], Writing—original draft [equal], Writing—review & editing [equal]).

FUNDING

None from WuXi Biologics or other organizations.

CONFLICT OF INTEREST STATEMENT

Steven Ren is an employee and shareholder of WuXi Biologics. The author reports no other potential conflicts of interest for this work.

DATA AVAILABILITY

The data that supports the review article is openly available.

ETHICS AND CONSENT STATEMENT

Consent is not required.

ANIMAL RESEARCH

This is not applicable.