Controlling protein stability: Mechanisms revealed using formulations of arginine, glycine and guanidinium HCl with three globular proteins

Platts, Lauren; Falconer, Robert J.

doi:10.1016/j.ijpharm.2015.03.051

Cited by 56 publications

(39 citation statements)

References 33 publications

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“…There are various theories that have been postulated to explain alteration of a protein's structural stability and solubility by excipients in terms of the modification of the hydration layer around the protein. ‘Preferential interaction’ explains alteration of the hydration layer in terms of waters displacement by the excipient; ‘preferential hydration’ suggests additional water is introduced into the hydration layer because of the presence of the excipient in the bulk phase; and ‘competition for water’ suggests the excipient's own hydration layer competes for water with the protein's hydration layer . What has previously hampered research into the ‘hydration layer’ surrounding a protein and verification of the preferential interaction, preferential hydration and competition for water theories was the paucity of suitable analytical techniques.…”

Section: Introductionmentioning

confidence: 99%

Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy

Wallace

Férachou

Ke³

et al. 2015

Journal of Pharmaceutical Sciences

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Terahertz time-domain spectroscopy (THz-TDS) has been shown to detect overlapping extended hydration layers around proteins. Here, we used THz-TDS to detect modulation of the extended hydration layer around monoclonal antibodies (mAbs) by the introduction of commonly used excipients. Proline and sucrose altered the hydration layer around a mAb (mAb1), which was observed as a negative shift in the plateau in absorbance above ~100 mg/mL mAb1 (~70,000 water molecules per mAb); arginine had no effect. At lower concentrations of ~10 mg/mL mAb1 (~700,000 water molecules per mAb) proline and arginine modulated the hydration layer, which was observed as a negative shift in the relative absorbance, whereas sucrose had no effect. The changes in the extended hydration layer were not translated to shifts in the thermal stability or protein:protein interaction parameter. The hydration layer of a second mAb (mAb2) was further shown to be modulated by more complex formulations composed of two or more excipients; although the differences in terahertz absorbance were not predictive of viscosity or long-term stability. THz-TDS promises to be a useful tool for understanding a protein's interaction with excipients in solution and the challenge will be to determine how to apply this knowledge to protein formulation.

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Section: Introductionmentioning

confidence: 99%

Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy

Wallace

Férachou

Ke³

et al. 2015

Journal of Pharmaceutical Sciences

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“…which modify the ionic strength of the solution, can also influence the direction and the magnitude in which arginine salts impact protein physical stability. 5,[8][9][10][11] Especially arginine can have complex effects on the protein unfolding, aggregate formation, and aggregate growth, depending on the conditions and the nature of its counterion. 7,12 The concentration of the additive is also essential but limited by the target osmolality of the formulation that typically should be close to physiological.…”

mentioning

confidence: 99%

“…13 Many of the studies with sucrose and arginine salts observe effects on protein stability that depend on the additive concentration. 5,8,9,11 Often, 0.5-1 M of sucrose or arginine hydrochloride (ArgHCl) has a beneficial impact on protein stability, 8,[14][15][16][17] whereas for arginine glutamate (ArgGlu), the optimal working concentration is typically much lower, 50-200 mM. 5,6,18 High concentrations of additives make the solutions hypertonic and thus unsuitable for therapeutic protein formulations that will be injected undiluted in patients.…”

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confidence: 99%

Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine Salts on Monoclonal Antibody Physical Stability and Aggregation During Long-Term Storage

Svilenov

Kulakova

Zalar

et al. 2020

Journal of Pharmaceutical Sciences

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Keywords:monoclonal antibody(s) pH sucrose arginine physical stability protein aggregation protein formulation fluorescence spectroscopy light scattering (dynamic) nuclear magnetic resonance (NMR) spectroscopy a b s t r a c tUnderstanding the effects of additives on therapeutic protein stability is of paramount importance for obtaining stable formulations. In this work, we apply several high-and medium-throughput methods to study the physical stability of a model monoclonal antibody at pH 5.0 and 6.5 in the presence of sucrose, arginine hydrochloride, and arginine glutamate. In low ionic strength buffer, the addition of salts reduces the antibody colloidal and thermal stability, attributed to screening of electrostatic interactions. The presence of glutamate ion in the arginine salt partially reduces the damaging effect of ionic strength increase. The addition of 280 mM sucrose shifts the thermal protein unfolding to a higher temperature. Arginine salts in the used concentration reduce the relative monomer yield after refolding from urea, whereas sucrose has a favorable effect on antibody refolding. In addition, we show 12-month long-term stability data and observe correlations between thermal protein stability, relative monomer yield after refolding, and monomer loss during storage. The monomer loss during storage is related to protein aggregation and formation of subvisible particles in some of the formulations. This study shows that the effect of commonly used additives on the long-term antibody physical stability can be predicted using orthogonal biophysical measurements. IntroductionOne fundamental aim during the development of therapeutic proteins is finding formulations that provide sufficient protein stability during long-term storage. Some critical variables of these formulations are solution pH, ionic strength, and the presence of additives. The additives usually belong to the group of sugars, polyols, amino acids, or surfactants. 1,2 Among these, sucrose is the most frequently used in marketed therapeutic protein formulations. 1 From the amino acids, arginine is of considerable interest, as in some cases, it can suppress protein aggregation or reduce the viscosity of highly concentrated protein solutions. 3,4 In addition, the use of different arginine salts is a topic of intense research because the arginine counterion can determine the effect on protein stability. [4][5][6][7] Sucrose and arginine salts can affect the thermal protein unfolding and aggregation differently depending on the protein molecule. The presence of other buffer components, such as NaCl, Abbreviations used: A 2 , second virial coefficient; ACF, autocorrelation function; AF STD , protein-additive saturation transfer difference amplification factors from NMR; D, mutual diffusion coefficient from DLS; DLS, dynamic light scattering; D max , maximum dimension from SAXS; FI350/FI330, intrinsic protein fluorescence intensity ratio 350nm/330nm; IP1, inflection point of the first thermal unfolding (at a lower temperature); IP2, inflection...

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“…Pro has pyrrolidine ring which allows it to have least conformational states and low conformational entropy restricting the configuration of preceding amino acids thus it is more common on rigid and turn conformations and hence reported to be higher in thermophilic protein 116 . Pro has been used to increase protein thermo-stability and can be considered, here, a potential hotspot to enhance thermostability of BGLs 124 . Similarly, Met, Asn, and Ser are thermo-labile that undergo either oxidation or deamination (Asn) at elevated temperature and are therefore less common in the thermostable protein 125,126 .…”

Section: Comparative Analysis Of Amino Acids Compositionmentioning

confidence: 99%

In silico Approach to Elucidate Factors Associated with GH1 β-Glucosidase Thermostability

Ahmed¹,

Sumreen²,

Bibi³

et al. 2019

J Pure Appl Microbiol

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Controlling protein stability: Mechanisms revealed using formulations of arginine, glycine and guanidinium HCl with three globular proteins

Cited by 56 publications

References 33 publications

Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy

Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy

Orthogonal Techniques to Study the Effect of pH, Sucrose, and Arginine Salts on Monoclonal Antibody Physical Stability and Aggregation During Long-Term Storage

In silico Approach to Elucidate Factors Associated with GH1 β-Glucosidase Thermostability

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