Trehalose Monooleate: A Potential Antiaggregation Agent for

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Trehalose monooleate: a potential antiaggregation agent for stabilization of proteins Smita S. Kale, and Krishnacharya G. Akamanchi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00686 • Publication Date (Web): 11 Oct 2016 Downloaded from http://pubs.acs.org on October 12, 2016

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Molecular Pharmaceutics

Trehalose monooleate: a potential anti- aggregation agent for stabilization of proteins Smita S. Kale and Krishnacharya G. Akamanchi* Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Mumbai, India 400019

Corresponding Author * -Prof. K. G. Akamanchi, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Mumbai, India-400019 .Tel: +91-022-33612214, Fax: +91-22-33611020 E. Mail: [email protected], [email protected]

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GRAPHICAL ABSTRACT

AGGREGATION

TMO

TABLE OF CONTENTS 1. Introduction 2. Experimental 2.1 Materials 2.2 Methods 2.2.1 Molecular Docking 2.2.2 Synthesis of TMO 2.2.3 Surface tension measurements 2.2.4 Hemolytic activity 2.2.5 Cytotoxicity study 2.2.6 Sample preparation 2.2.7 Analytical techniques for evaluation 2.2.7.1 CD spectroscopy 2.2.7.2 Fluorescence spectroscopy 2.2.7.3 SEC-HPLC 2.2.7.4 Native PAGE 2.2.8 Molecular dynamics 3. Results and Discussion 4. Conclusions Acknowledgement References

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ABSTRACT Protein aggregation is a major problem of therapeutic proteins because aggregation decreases their therapeutic activity, shelf life and induces immunogenicity. Stabilization against aggregation is commonly attained by addition of different excipients like sugars, surfactants, buffers, salts, amino acids, polymers etc. Generally these excipients are required in combination for stabilization. Sugars are required at a higher concentration and commonly used surfactants like polysorbates have shortcomings due to oxidative degradation. With a view to have a multipurpose excipient to be effective at a lower concentration, we designed anti-aggregation agents (AAAs) which would encompass the functionalities of two or more conventional excipients and would curtail the number of excipients to be added for stabilization. Our first designed AAA, trehalose monooleate (TMO) is a sugar-fatty acid derivative. It has been evaluated in-silico by docking on aggregation prone regions of model protein Bovine Serum Albumin (BSA) and experimentally its effectiveness has been validated as stabilizer against agitation and thermal stress. TMO has a lower CMC of 6mg/L, is non-hemolytic and was found to be non-toxic by sulforhodamineB (SRB) colorimetric assay in Human Hepatoma Cell Line (Hep-G2) using adriamycin as positive contol. Various spectroscopic and separation analytical techniques were employed to monitor the aggregation profile of BSA in presence and absence of TMO. CD spectroscopy showed complete retention of helical structure at concentration as low as 0.05% of TMO, while fluorescence spectroscopy provided vital insights into conformational stability rendered by TMO. Native-PAGE and SEC-HPLC studies demonstrated absence of aggregates. Molecular dynamics study on BSA-TMO docked complex further substantiated the stabilization effect. Overall, it can be said that TMO has good anti-aggregation property. The present work is a preliminary attempt towards understanding protein excipient interactions and chemistry to provide rational basis for designing a single excipient for stabilization of protein formulations.

KEYWORDS Bovine serum albumin, Trehalose monooleate, Aggregation, Stabilization

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1. INTRODUCTION

Aggregation is characterized by formation of dimeric or polymeric aggregates with loss of native protein structure. It is the most common cause of protein instability. Protein aggregation usually occurs during fermentation, refolding, purification, formulation, and storage.1,2 Proteins are sensitive to various external factors like temperature, pH, cosolutes, shaking, shearing, metal ions, pressure etc. 3 Although protein aggregation can be inhibited by structural modifications however more convenient and common practice is addition of protein stabilizing excipients like sugars, polyols, surfactants, salts, PEGs, polymers, metal ions, and amino acids.

4,5

These

excipients stabilize by different mechanism at a specific concentration.6 Therefore, a combination of different classes of excipients are included at various concentrations in therapeutic protein formulations to ensure stability. Instead of using multiple excipients, we hypothesized that an excipient which can encompass the functionalities of two or more such stabilizers would better serve the purpose as anti-aggregation agent (AAA). It is reported that crowding reduces the conformational entropy of proteins and can speed up the conformational search for the folded state. AAAs would also provide a crowded environment by binding at various aggregation prone regions and aid in stabilization.7 Aggregation is considered to be mediated by short ‘aggregation prone’ peptide segments. Numerous software tools like AGGRESCAN, PAGE, TANGO, PASTA, SALSA, Zyggregator, AMYLPRED are available for prediction of aggregation prone regions (APRs) of proteins.8, 9 Topp et.al have described a strategy wherein APRs are predicted and excipients are selected based on their interactions with these regions. An ideal excipient binds with aggregation prone regions on the protein to limit interaction of that region with another protein molecule.10,

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Proteins are stabilized by excipients by different non-covalent interactions and therefore, we considered molecular docking studies to understand these non covalent interactions as well as binding affinity of various excipients and designed AAAs at APRs. To protect proteins from shaking/shearing-induced aggregation, surfactants are most commonly used. They inhibit protein aggregation by competing with proteins at hydrophobic surfaces.12 However, polysorbate 20 and polysorbate 80 that are commonly employed surfactants undergo auto-oxidation yielding reactive peroxides which cause degradation.13-16 Sugars in general 4


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protect proteins against dehydration by forming hydrogen bonds with the protein by serving as water substitute, increasing the thermal unfolding temperature and inhibiting irreversible aggregation of protein molecules.17 Sugars are added at high concentrations to prevent aggregation. Therefore, we hypothesized that a combination of sugar and surfactant in one and the same molecule as sugar- fatty acid derivative would be able to play a dual role and act as an AAA. As a hind sight to understand interactions of various classes of excipients and thereof design AAAs docking analysis was carried out with model protein Bovine Serum Albumin (BSA). Among designed and docked AAAs, Trehalose monooleate (TMO) which is a sugarsurfactant derivative has been synthesized and evaluated for its anti-aggregation potential using different analytical techniques. Further, Molecular dynamics study has been carried out to comprehend and validate the experimental results.

2. EXPERIMENTAL

2.1 Materials Oleic acid was procured from Sigma, USA. TBTU was obtained from Spectrochem, India. Trehalose was purchased from Sigma Aldrich (Sigma Aldrich Chemicals Pvt. Ltd., Mumbai, India). BSA with a purity of >96% was obtained from Hi Media, India. Dry pyridine and all other chemicals used in the study were of analytical reagent grade and were purchased from s d fine Chemicals, India. High-purity Milli-Q water (Millipore, Billerica, USA); with a resistance value of 18.2 MV/cm was used for all solutions. BSA solutions were freshly prepared just before the experiments by dissolving the protein in 0.1M phosphate buffer of pH 7.4. TLC was performed on precoated aluminum plates of silica gel 60 F254 (0.25 mm, E. Merck).

2.2 Methods 2.2.1 Molecular Docking BSA (pdb-3v03) was selected as model protein. AGGRESCAN (http://bioinf.uab.es/aggrescan/) was used to predict aggregation prone regions (APRs) on BSA. Molecules selected for docking consisted of sugars, surfactants, amino acids, denaturants and designed AAAs. Docking studies were performed using GLIDE, version 5.6, Schrodinger suite, LLC, New York, NY, 2010.18, 19 The structures of the molecules were drawn using LigPrep (2.3) module (Ligprep, Version 2.3, 2009), and geometry was optimized by means of Optimized Potentials for Liquid Simulations5



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2005 (OPLS-2005) force field. Finally, the ligand conformations with minimum energy were chosen. Protein preparation wizard of Schrodinger suite has been used to prepare protein. Docking studies were carried out on BSA at the APRs. The prepared protein was employed to build energy grids using the default value of protein atom scaling (1.0 Å) within a cubic box, centered around the centroid of the selected APRs. After Grid generation, the ligands were docked with the protein by using GLIDE 5.6 module in extra precision mode (XP).

2.2.2 Synthesis of TMO (6-o-oleoyl-α, α-trehalose) Synthesis of TMO was carried out as described by Grindley et al.20 (Scheme 1). Reaction was conducted under a dry nitrogen atmosphere. To a premixed solution of oleic acid (0.739 g, 2.6 mmol) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (0.834g, 2.6 mmol) in anhydrous pyridine (15 mL) stirred for 30 min, was added a solution of trehalose (0.900g, 2.4 mmol) in anhydrous pyridine (10 mL). The reaction mixture was stirred for 60 h at room temperature. Pyridine was removed under vacuum and the crude product was purified by silica gel (60-120 mesh size) column chromatography using a solvent gradient of 5−25% methanol in EtOAc−DCM (1:1). Pure TMO (650 mg, 41%) was obtained as sticky white mass. IR (KBr): ν (cm-1) 3379, 2926, 1735, 1651, 1593, 1400, 1109, 991, 941, 918, 806. 1H NMR (CD3OD): δ 0.80 (t, 3H), 1.19-1.3 (m, 20H), 1.49-1.51(m, 2H), 1.89-1.94 (m, 4H), 2.27(t, 2H), 3.18-3.29 (m, 2H), 3.36, 3.37 (2dd, 2H), 3.56 (dd, 1H), 3.6-3.89 (m, 4H), 3.94 (ddd, 1H), 4.12 (dd, 1H), 4.24-4.28 (dd, 1H), 4.97 (d, 1H), 4.99 (d, 1H), 5.29 (t, 2H).

Scheme 1. Synthesis of TMO

2.2.3 Surface tension measurements Surface tensions were measured using K100 MK2 tensiometer (Krüss, Hamburg, Germany) equipped with a platinum plate. The system was validated by measurement of surface tension of water. TMO (1g/L) was placed in a glass vessel and surface tension was measured over an interval of 1 h. CMC was determined from sharp breaks in surface tension versus logarithm of 6


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TMO concentration plots. Binding ratio was calculated by measuring surface tensions of binary mixtures of BSA-TMO. 0.001 M solution of BSA and 0.01M solution of TMO were mixed to obtain various binary solutions of BSA-TMO with constant BSA concentration.

2.2.4 Hemolytic activity Hemolysis studies were carried out for TMO by a reported in-vitro method.21 Freshly collected human blood was washed three times with an isotonic 0.1 M phosphate buffer saline (PBS) solution (pH 7.4) by centrifugation at 2800 rpm for 5 min. Surfactant solution was diluted with 0.1 M PBS up to a concentration of 0.05 mg/mL and 0.1 mg/mL for each sample. The RBC suspension (0.2 mL) was added to 1.8 mL of each sample. After incubation at 37 °C for 30 min; the samples were centrifuged at 3000 rpm for 10 min, the supernatant was collected and analyzed for hemoglobin release by spectrophotometric determinations at λmax 416 nm. To obtain 0 % and 100 % hemolysis, the same procedure was followed by adding 0.2 mL of the RBC suspension to 1.8 mL of PBS and distilled water, respectively. The degree of hemolysis was calculated using following equation:

where, A100 and A0 are the absorbances of the solution at 100 % and 0 % hemolysis, respectively.

2.2.5 Cytotoxicity study Cytotoxicity of TMO was evaluated by sulforhodamineB (SRB) colorimetric assay at concentrations 10, 20, 40 and 80 µg/ml in Human Hepatoma Cell Line (Hep-G2) using adriamycin as positive control. 2.2.6 Sample Preparation BSA solutions of 1mg/mL with and without 0.01%, 0.05% and 0.1% (w/v) of TMO were prepared in 0.1M phosphate buffer with pH 7.4. BSA solutions of 1mg/mL with 0.1% (w/v) of trehalose and oleic acid were also prepared in 0.1M phosphate buffer with pH 7.4. Absorption at 280 nm was measured to determine the protein concentration. BSA solutions were incubated at 75 °C for 4 h and shaken at 200 rpm for 24 h to induce thermal and agitation stress, respectively. A set of BSA solutions kept at 37 °C were used as control. All the solutions were filtered through 7



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0.2µm filters. For all the spectroscopic and chromatographic analysis, aliquots of the same freshly prepared sample were used. For CD and fluorescence measurements, samples were appropriately diluted with 0.1M phosphate buffer with pH 7.4.

2.2.7 Analytical techniques for aggregation evaluation 2.2.7.1 Circular Dichroism spectroscopy CD measurements were performed on Jasco spectropolarimeter, J 815 (Jasco Co. Tokyo) with a temperature controller system. All CD spectra were collected with a scan speed of 100 nm/min, resolution of 0.5nm, response time of 1s and a bandwidth of 1nm at 25 °C. Spectra were averaged with three scans and corrected with buffer and buffer plus TMO blanks. BSA solutions of 0.1 mg/mL with 0.1 cm path length cuvette were scanned in Far UV-CD region (190- 260 nm). MRE (Mean Residue Ellipticity) in deg cm2 .d mol-1 was calculated using following equation:

where θ is the CD in millidegree at 222 nm, MRW is mean residue weight (molecular weight of protein, 66,400 divided by total number of amino acids, 583), l is the path length of the cell and C is the protein concentration in mg/mL.22,23 Percent helical content was calculated using the following equation.

2.2.7.2 Fluorescence spectroscopy Intrinsic fluorescence measurements of BSA solutions were performed on a Jasco F-250 fluorescence spectrophotometer. The excitation wavelength was set at 280 nm and emission spectra were recorded in the range of 300-400 nm. A 1.00 cm quartz cell was used for these studies. The measurements were made using a resolution of 1nm at a scanning speed of 100 nm/min. Background correction was done with buffer and buffer plus TMO.

2.2.7.3 Size exclusion chromatography (SEC-HPLC) Formation of aggregates was investigated via SEC. Agilent1200 series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a UV detector and a TSK 3000 SWXL gel 8


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filtration column (Tosoh Bioscience, Tokyo, Japan) along with TSK SWXL guard column was used for this experiment. Fifty microliters of protein sample was injected into the HPLC. The mobile phase was pre-filtered phosphate buffered saline solution (PBS) flowing at a rate of 0.5 mL/min. Detection was carried out at 280 nm.

2.2.7.4 Native PAGE BSA samples (1mg/mL) were separated on a 7.5% PolyAcrylamide Gel Electrophoresis (PAGE) leaving out the Sodium Dodecyl Sulfate (SDS) and reducing agent, Dithiothreitol (DTT) from the standard SDS-PAGE protocol. Samples were electrophoresed towards the anode at a constant voltage of 100 V on a vertical electrophoresis unit purchased from BioRad, Hercules, CA, USA. The gels were stained with silver nitrate.

2.2.8 Molecular Dynamics study The MD simulation was carried out using Desmond Molecular Dynamics system, version 2.4, D. E. Shaw Research, New York, N. Y. 2010.24 The docked complex of protein and ligand were employed after Impact minimization. The complex was neutralized with 17Na+ ions and immersed in TIP3P water system in an orthorhombic box. The default relaxation protocol was used to slowly relax the system before the MD simulations. NPT ensemble was used at 300K and simulation was run for 10ns. The trajectory was stored and analyzed by calculating the root mean square deviations (RMSD), root mean square fluctuations (RMSF) and radius of gyration.

3. Results and Discussion Proteins in aqueous solution are immediately denatured at high temperature due to disruption of weak non covalent interactions including, ionic interactions, hydrogen bonds and hydrophobic interactions. APRs are the sites for initiation of unfolding, disruption of non-covalent interactions and further denaturation. Therefore, reinstating the non-covalent interactions at APRs would improve thermodynamic stability. In case of protein stabilization, it is hypothesized that an excipient which binds favorably with APRs will prevent unfolding of these APRs and subsequently prevents aggregation of the protein. Molecular docking is widely used in drug discovery to find binding orientation of small molecule drug candidates to target proteins, understand the interactions and predict activity of small molecules.25 Hence, we employed

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docking analysis to understand the interactions and binding of excipients at various APRs and the information was used for AAAs design. AGGRESCAN revealed total 17 APRs in BSA (Figure 1). Excipients like sugars (trehalose, mannitol, sucrose, maltodextrin), surfactants (PS 80, choline, carnitine), amino acids (arginine, histidine) and denaturants (urea, guanidinium chloride) were docked at all 17 APRs. Amongst the sugars, trehalose showed good docking score at multiple APRs. Trehalose is well accepted kosmotrope with a high glass transition temperature and is widely used as bioprotectant.26-30 Hence, AAAs were designed with trehalose as the head group and different functionalities as tail group.

Figure 1. Structure of BSA with all 17 APRs depicted as ribbons in different colours; APR’s residue numbers are 19-32, 44-51, 70-75, 134-139, 152-160, 225-237, 325-331, 340-351, 370376, 402-413, 421-428, 449-461, 500-505, 508-513, 526-533, 546-556, 576-583.

Stabilizers were observed to establish hydrogen bonding, salt bridges and electrostatic interactions with the APRs and possess good docking scores with high binding energy. In contrast, the denaturants like urea and guanidinium hydrochloride did not have good docking score and had very low binding energy. Sugars showed hydrogen bonding interactions mostly with acidic amino acids; surfactants demonstrate mostly hydrophobic interactions as well as hydrogen bonding with basic amino acids. Therefore, an excipient comprising of a sugar moiety as a head group and a fatty acid as tail would have favorable interactions at APRs and can play a dual role of a sugar and surfactant and act as AAA to stabilize a protein. As seen in Figure 2, sugar moiety forms hydrogen bonds, carbonyl functionality interacts with basic amino acids through hydrogen bonding and the fatty acid tail establishes hydrophobic interactions with the 10


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amino acids. Among the designed AAAs, TMO docked well with favorable interactions at most of the APRs; hence, it was selected for synthesis and evaluation (Table 1).

a)

b) 11



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Figure 2. a) Interaction diagram of TMO at APR-2 of BSA (hydrogen bonding interactions with GLU 45, LYS 64, ASP 72, GLU 73, LYS 76); b) Interaction diagram of TMO at APR-10 of BSA (hydrogen bonding interactions with GLN 389, GLN 393, ARG 409, SER 488, LEU 490

Table 1 Docking score, Glide energy (Evdw +Ecoulomb) and Glide emodel (electrostatic and Van der Waals energies) of TMO at all APRs of BSA. APR No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

APR residues 19-32 44-51 70-75 134-139 152-160 225-237 325-331 340-351 370-376 402-413 421-428 449-461 500-505 508-513 526-533 546-556 576-583

Glide score Glide energy Glide emodel 0 -5.084 -6.061 -7.183 -4.846 -8.724 -10 -11.132 -5.701 -9.726 -6.205 -5.87 -5.606 -7.114 -7.059 -7.942 -7.266

0 -44.888 -45.240 -48.582 -52.627 -49.198 -54.338 -57.677 -39.482 -61.924 -52.618 -42.983 -49.790 -49.604 -51.116 -57.364 -42.053

0 -72.176 -61.839 -68.956 -72.416 -63.982 -69.282 -71.323 -51.812 -86.660 -73.288 -56.652 -64.126 -59.967 -64.511 -62.098 -43.295

The CMC value indicates the amount of surfactant required to reach maximum surface tension reduction. Lower the CMC, lesser amount of surfactant is required to effectively emulsify, solubilize and disperse.31 Trehalose monoesters of capric acid and lauric acid are reported to have CMC of 1.92 mg/mL and 0.33 mg/mL, respectively.32 CMC of TMO was found to be 0.0061mg/mL with surface tension value of 31.07mN, obeying the general rule that a two carbon atoms longer fatty acid results in an approximately 10-fold lower CMC.33 Surfactants are known to have concentration dependent effects on proteins i.e, at lower concentration they act as stabilizers and at higher concentration can destabilize the proteins.34 Therefore, TMO with a very low CMC with reference to CMC of polysorbate 20 and polysorabate 80 i.e. 0.15 mg/mL and 0.014mg/mL can be used at lower concentrations as stabilizer. Binding ratio of BSA-TMO system was measured at CMC of binary mixture at pH 4.0 and 7.4. In presence of BSA, CMC of TMO increased 5 and 25 times at pH 4.0 and 7.4, respectively.35 In 12


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Figure 3, the first inflection is called as cac (critical aggregation concentration), corresponding to beginning of BSA-TMO complex. The point at which surface tension becomes constant is the cmc of the complex. Binding ratio at cmc was determined for BSA-TMO complex at pH 4.0 and pH 7.4 using the following equation.36

where, CBSA is the concentration of BSA. Binding ratio of BSA-TMO complex at pH 4.0 and 7.4 was found to be 7.9 and 40.8, respectively.

Figure. 3 Surface tension isotherms of BSA-TMO binary mixtures containing 6 ×10–6 M BSA

Charge on BSA seems to modulate the BSA-TMO binding behavior at different pH values. At pH 4.0, which is slightly below the isoelectric point (pI) of BSA (pI of BSA is 4.8), TMO shows low binding ratio and it could be due to slight net positive charge on BSA. At pH 7.4, i.e. above the isoelectric point, BSA would have sufficiently high net negative charge, where TMO shows good binding ratio i.e. TMO interacts well. Also at pH 7.4, due to high net negative charge electrostatic repulsion may come into play between BSA molecules37. These two combined effects viz better interaction with TMO and electrostatic repulsion between BSA molecules might have resulted in stabilization. 13



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Substances are categorized as hemolytic or non-hemolytic based on the percent hemolysis caused. A test substance with a value of 10% or lower hemolysis is generally considered as safe and hemocompatible.38 Surfactants are known to rupture cellular membrane hence evaluation of hemolytic activity becomes necessary. As indicated in Table 2, with hemolysis of 0.22% and 0.054% at 0.1 mg/mL and 0.05mg/mL, respectively, TMO is proved to be non- hemolytic and safe. Also, TMO is biodegradable being made of natural components linked through bio-labile ester linkage.39

Table 2 Surfactant properties of TMO Property tested

Result

CMC ( mg/mL)

0.006

HLB

10.96a

Hemolysis (%)

0.22 and 0.054 at 0.1 and 0.05 mg/mL, respectively

a

Calculated using Griffins method

TMO showed a high cell survival rate of more than 100 % and did not exhibit any cytotoxicity at concentration of even 80µg/mL. The relative cell viability for Hep-G2 cell line in the presence of TMO at different concentrations is presented in Figure 4.

Figure 4. Growth curve: Human Hepatoma cell line Hep-G2

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In order to investigate changes in secondary and tertiary structures of BSA, in presence and absence of TMO, in stressed and control samples, far UV-CD spectra were analyzed. The far-UV CD spectrum of native BSA was characterized by the presence of two minima at 208 nm and 222 nm, characteristics of α-helical structures in proteins.40 Although, both control and stressed solutions of BSA retained characteristic helical features in CD spectrum, a significant sigmoidal decrease in ellipticity was observed in stressed BSA solutions. Upon heating the helical content in BSA solution without TMO was only 44.2%, whereas in solutions containing TMO it was 56%. After agitation, BSA samples in absence of TMO and with TMO had 49.9% and ≈56% helical content, respectively. The values for helical content of all solutions are presented in Table 3. Preservation of structural integrity and stabilizing effect of TMO in stressed BSA solutions is clearly evident from the overlapped CD spectra presented in Figure 5. One important indication of helical structures is the maximum ellipticity at 190-192 nm. This feature was particularly observed in case of native BSA and BSA solutions containing TMO. As seen in Figure 5 and Table 3, the stabilizing effect of TMO in both thermally stressed and agitated solutions seemed to increase with the increase in concentration of TMO, with 0.1% w/v being the most effective concentration.

a

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b

Figure 5. a) Overlay of Far UV-CD spectra of BSA samples with or without TMO upon subjected to thermal stress; b) Overlay of Far UV-CD spectra of BSA samples with or without TMO upon subjected to agitation stress Table 3 Spectral Characteristics of BSA under various conditions Heat Stressed BSA

Spectral Characteristics

MRE at 222 nm (deg 2

BSA

control

Agitation stressed

BSA+

BSA+

BSA+

0.01%

0.05%

TMO

BSA

BSA+

BSA+

BSA+

0.1%

0.01%

0.05%

0.1%

TMO

TMO

TMO

TMO

TMO

-22437.6

-17692.3

-22151.6

-22558.2

-22425.1

-19984.4

-22192.3

-22367.1

-22450.1

56.09

44.23

55.38

56.39

56.06

49.96

55.48

55.92

56.13

334

333

332

337

334

334

335

329

336

21.34

80.42

66.31

28.26

26.08

65.27

42.28

38.27

32.01

−1

cm dmol ) α-Helical content (%) Emission maximum wavelength Fluorescence intensity at 334nm

The fluorescence property of BSA is mainly due to the tryptophan residues, Trp 134 and Trp 212 present on the surface in the Ist domain and inside the IInd domain, respectively.41 As protein goes 16


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from a native (folded) to a denatured (unfolded) state, the local environment surrounding the Trp residue changes, which affect the fluorescence properties. The maximum emission wavelength is very useful in estimating the hydrophobicity around the Trp residues. The shift in the position of fluorescence emission maximum corresponds to changes of the polarity around the chromophore molecule. A blue shift of λmax means that the amino acid residues are located in a more hydrophobic environment, and are less exposed to the solvent, while a red shift of λmax implies that the amino acid residues are in a polar environment and are more exposed to the solvent 42-46. The intrinsic fluorescence emission maximum (λmax) of BSA was found to be 334 nm. After heat treatment and agitation, the intrinsic fluorescence of BSA increased drastically indicating the exposure of Trp residues to more hydrophobic environment. BSA samples containing TMO did not show an extreme rise in fluorescence as depicted in the emission spectra in Figure 6 a. After heating at 75°C for 4h, the emission wavelength decreased to 333nm, due to less polar environment. BSA solution containing 0.01% TMO had high fluorescence intensity; however, those containing 0.05% TMO and 0.1% TMO did not show much rise in fluorescence (Figure 6 b). The emission wavelength of agitated BSA solution remained same as control, but fluorescence intensity showed extreme elevation. BSA solution with 0.05% and 0.1% TMO did not undergo an extreme increase in fluorescence intensity but the emission wavelength did show slight blue shift and red shift, respectively. Although a change in emission maxima in stressed BSA solutions did not occur, but a drastic increase in fluorescence intensity did take place. In presence of TMO, the significant change in fluorescence intensity was not observed. It reveals that under the stress conditions, TMO was able to interact with BSA without modifying its hydrophobic environment.

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a

b

Figure 6. a) Overlay of fluorescence spectra of BSA Samples after heating; b) Overlay of fluorescence spectra of BSA Samples after agitation. SEC-HPLC was carried out to detect aggregates and estimate percent monomer in the BSA samples after heat treatment and agitation. As shown in Figure 7a, BSA control solution displayed three peaks, major peak corresponding to monomer at 18.21 min (78.5%), small peaks due to aggregates at 16.08 min. and 14.82 min. After heating at 75 °C for 4h, it was observed that the amount of monomer (20%) decreased greatly and large aggregates were formed in higher 18


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amount. The larger aggregates eluted at 12.15 min. This observable fact is in accordance with results of SEC-MALLS analysis of BSA aggregation as a function of heating temperature.47 BSA samples with 0.01% and 0.05 % TMO also displayed larger aggregate peaks than the monomer after heat treatment. However, BSA solution with 0.1% TMO was seen to retain monomer (66%) in high quantity with comparatively less amount of aggregates. Unlike TMO, BSA solution with 0.1% of trehalose underwent massive aggregation and monomer (5%) was almost lost. BSA solution with 0.1 % oleic acid also showed lot of aggregation and was only able to retain very small amount of monomer (30%). Hence, TMO at 0.1% concentration was seen to prevent BSA from thermal aggregation. This phenomenon was in agreement with the spectral studies performed by us. Like thermally stressed solutions, agitation of BSA solution did result in formation of large aggregates as seen from Figure 7 b. BSA solution after agitation underwent a decrease in monomer content (76 %) with a small elevation in the dimer amount and small aggregates. BSA solution with 0.01% did show higher amount of aggregates, but 0.05% and 0.1% TMO containing BSA solutions had a higher monomer recovery (≈78 %) and with fairly less aggregates. In BSA solutions with 0.1% trehalose and 0.1% oleic acid aggregation after agitation was evident with formation of larger aggregates and decrease in monomer (74% and 73%, respectively) content. Calculated percent monomer content of BSA solutions is graphically represented in Figure 7 c. Trehalose and oleic acid individually failed to prevent aggregation in BSA stressed solutions in comparison to TMO.

a

BSA BSA after heating BSA + 0.01 % TMO BSA + 0.05 % TMO BSA + 0.1 % TMO BSA + 0.1 % Trehalose BSA + 0.1 % Oleic acid

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b

BSA BSA after heating BSA + 0.01 % TMO BSA + 0.05 % TMO BSA + 0.1 % TMO BSA + 0.1 % Trehalose BSA + 0.1 % Oleic acid

c

Figure 7. a) Overlay of SE-HPLC chromatograms of thermally stressed BSA solutions; b) Overlay of SE-HPLC chromatograms of agitation stressed BSA solutions; c) Graph showing monomer content in BSA solutions calculated from SEC-HPLC analysis; BSA control (blue), BSA after heat treatment (red), BSA after agitation (green) BSA is known to form covalent dimers and oligomers.48, 49 As shown in Figure 8, Native BSA loaded in lane 2, shows prominent monomer band at 66KDa similar to the molecular weight ladder band, as well as bands due to dimer and aggregates. After heat treatment at 75 °C for 4 h, BSA solution shows prominent bands for large aggregates as also seen in SEC-HPLC chromatogram. Similarly, lower band intensity of monomer band and trimer and large aggregate bands were observed in BSA solutions with 0.01% and 0.05% TMO. However, 0.1% TMO 20


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containing BSA solution did not show large aggregate band and has an intense monomer band. BSA solution containing 0.1 % trehalose had very small amount of monomer and large amounts of larger aggregates; in presence of 0.1 % of oleic acid also aggregation was clearly visible with decrease in monomer band. Agitation caused comparatively less aggregation compared to heat treatment. BSA solution in presence of 0.01%, 0.05% and 0.1% TMO showed an intense monomer band with regular dimer and oligomer bands even after agitation. However, individually 0.1 % trehalose and 0.1 % of oleic acid failed to prevent aggregation during agitation. Overall, the gel image clearly demonstrates the anti-aggregation prospective of TMO as corroborated by the previous analytical techniques.

Figure 8. Gel image of BSA samples; a) Thermally stressed BSA solutions; Molecular weight ladder: lane 1; BSA control :lane 2; BSA (stressed) : lane 3; BSA + 0.01%TMO :lane 4; BSA + 0.05%TMO :lane 5; BSA + 0.1%TMO: lane 6; BSA + 0.1% Trehalose: lane 7; BSA + 0.1% Oleic acid: lane 8; b) Agitation stressed BSA solutions; Molecular weight ladder: lane 1; BSA control: lane 2; BSA (stressed):lane 3; BSA + 0.01%TMO: lane 4; BSA + 0.05%TMO: lane 5; BSA + 0.1%TMO: lane 6; BSA + 0.1% Trehalose: lane 7; BSA + 0.1% Oleic acid: lane 8.

After the wet lab studies, it was important for us to get more insights into conformational changes and stability of BSA in presence of TMO, for which Molecular dynamics study was taken up. The simulation trajectories of BSA and BSA-TMO complex were analyzed based on Root Mean Square Deviation (RMSD), Radius of Gyration (ROG), Root Mean Square Fluctuations (RMSF). Figure 9(a), shows the RMSD evolution of BSA and BSA-TMO complex. Monitoring the RMSD of the protein can give insights into its structural conformation throughout the simulation.50 RMSD value for BSA was around 7Å. Initially the value increases 21



and attains equilibrium after 5ns. BSA-TMO complex posses lower RMSD value of 3Å and the stage of equilibration was reached early at around 4.5ns. Therefore, lower RMSD values evidently indicate that BSA-TMO complex has undergone least structural changes during the simulation and hence is stable. The radius of gyration (Rg) is a measure of overall compactness and size of protein.51 Average Rg values for BSA and BSA-TMO were 3.71nm and 3.73nm respectively. From the Figure 9(b), it is seen that Rg values for BSA initially was 3.64 nm and then increased upto 3.77 nm in 2 ns. In case of BSA-TMO complex, Rg value increased from 2ns to 5ns and became steady after 6 ns. At the end of 10 ns, Rg values were 3.71 and 3.69 for BSA and B SA-TMO complex, respectively. Insignificant deviations observed in Rg values of BSATMO compared to BSA prove that TMO does not cause considerable changes in the secondary structure of BSA.

BSA BSA-TMO

a

38.0

BSA BSA-TMO

b

37.8 8

37.6 37.4

6

Rg

37.2 4

37.0 36.8

2

36.6 36.4 0

36.2 0

2000

4000

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0

10000

2000

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2

1

0 0

200

400

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22


8000

10000

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Figure 9. a) The root-mean-square deviation (RMSD in angstrom) of BSA backbone vs simulation time for BSA free and BSA with TMO; b) The radius of gyration (Rg in angstrom) of BSA vs simulation time for BSA free and BSA with TMO; c) Plot of Root mean Square Fluctuation (RMSF) of C-alpha value as a function of residue no. for BSA free and BSA with TMO Root Mean Square Fluctuation (RMSF) is useful for characterizing local changes along the protein chain. On this plot, peaks indicate areas of the protein that fluctuate the most during the simulation. Surfactants mostly bind with subdomain IIIA. RMSF values around subdomain IIIA are 2.2Å for BSA and 1.82Å for BSA-TMO system [Figure 9(c)]. Subdomain IIIA of BSA is the region where seven APRs with high aggregation propensity are present. Docking studies demonstrate that TMO has good docking scores in subdomain IIIA. The flexibility of this region and almost all the residues is lowest in BSA-TMO proving its stabilizing potential.

Favorable interactions and binding of TMO at various APRs of BSA result in prevention of aggregation of BSA. TMO helps in prevention of adsorption of BSA and restricting molecular motion due to crowding. Hence, both conformational and colloidal stability of BSA has been conserved. In comparison to polysorbate which is usually added in concentration between 0.0017 – 0.16 % w/v

52–55

, TMO offers stabilization at concentration of 0.1% w/v. In spite of being the

most successful excipient PS 80 still suffers from dilemma of auto oxidation and subsequent degradation of product. In comparison a polyoxyethylene free AAA like TMO is definitely a beneficial alternative, as demonstrated by the present study.

4. Conclusion Despite numerous excipients and techniques available to tackle protein aggregation, choice of excipient, concentration and evaluation techniques is still a complex and challenging task. Number of approved excipients is limited and some are having degradation issues. Most of the research in protein stabilization is focused on studying the effect of these already existing excipients on different proteins at various conditions. Taking this scenario into consideration, we have introduced anti-aggregation agent, which is chemically fortified with functionalities making it a multipurpose excipient. Employing a multipurpose excipient would help to reduce the number of excipients required for stabilization in a formulation.

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As a preliminary attempt, TMO (a sugar-fatty acid derivative) as AAA has been designed, synthesized and evaluated by in-silico and wet lab studies. Unlike polysorbates, TMO is a single molecular entity and molecular structure is less prone to oxidation. Aggregation potential of TMO has been evaluated at very high temperature i.e. 75 °C and after 24h agitation at 200rpm and it successfully prevented aggregation of BSA at 1mg/mL concentration. Both the computational and experimental results are in synergy and prove that TMO has a good stabilizing potential and confirms to be a promising AAA. Present study also demonstrates that in-silico studies can be very useful guide for design and selection of AAAs. Synthesis and evaluation of other AAAs is in progress.

ACKNOWLEDGEMENTS Authors are thankful to the University Grants Commission, Government of India for financial support under UGC-SAP. Authors are thankful to National Institute for Research in Reproductive Health (NIRRH), and DBT-CEB Centre for Energy Biosciences at ICT, Mumbai for availing the facility of Circular dichroism spectrometer and SEC-HPLC, respectively.

SUPPORTING INFORMATION IR and 1H NMR spectra of TMO are provided.

REFERENCES 1.

Wang, W. Protein Aggregation and Its Inhibition in Biopharmaceutics. Int J Pharm. 2005, 289 (1-2), 1–30.

2.

Wang, W.; Nema, S.; Teagarden, D. Protein aggregation—Pathways and Influencing Factors. Int. J. Pharm. 2010, 390 (2), 89–99.

3.

Krishnan, S.; Randolph, T. W.; Carpenter, J. F. Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces in Nonnative Protein Aggregation. Pharm. Res. 2003, 20 (9), 1325-1336.

4.

Manning, M. C.; Chou, D. K.; Murphy, B. M.; Payne, R. W.; Katayama, D. S. Stability of Protein Pharmaceuticals: An Update. Pharm. Res. 2010, 27 (4), 544–575.

24


Page 25 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5.

Kamerzell, T. J.; Esfandiary, R.; Joshi, S. B.; Middaugh, C.; Volkin, D. B. Proteinexcipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev. 2011, 63, 1118–1159.

6.

Challener, C.; Excipient Selection for Protein Stabilization. Pharm Tech APIs, Excipients, and Manufacturing Supplement. 2015, 39 (18), s35–s39.

7.

Senske, M.; Törk, L.; Born, B.; Havenith, M.; Herrmann, C.; Ebbinghaus S. Protein Stabilization by Macromolecular Crowding through Enthalpy Rather Than Entropy. J. Am. Chem. Soc. 2014, 136, 9036−9041.

8.

Tsolis, A. C.; Papandreou, N. C.; Iconomidou, V. A.; Hamodrakas, S. J. A Consensus Method for the Prediction of “Aggregation-Prone” Peptides in Globular Proteins. PLoS One 2013, 8 (1), e54175.

9.

Agarwal, N. J.; Kumar, S.; Wang, X.; Helk, B.; Singh, S. K.; Trout, B. L. Aggregation in Protein-Based

Biotherapeutics:

Computational

Studies

and

Tools

to

Identify

Aggregation-Prone Regions. J. Pharm. Sci. 2011, 100, 5081–5095. 10. Shulda, S.; Ashley, J.; Li, Y.; Topp, E. M.; Camarda, K. V. Excipient Design for Protein Drug Formulation via Molecular Simulation and Computational Molecular Design. In 18th European Symposium on Computer Aided Process Engineering – ESCAPE 18, ed. B. Braunschweig and X. Joulia, Elsevier, 2008; pp. 1-6. 11. Roughton, B. C.; Pokphanh, A. I.; Topp, E. M.; Camarda, K. V. Optimizing ProteinExcipient Interactions for the Development of Aggregation-Reducing Lyophilized Formulations. In 11th International Symposium on Process Systems Engineering; Elsevier B.V., 2012; Vol. 31, pp 1351–1355. 12. Balcão, V. M.; Vila, M. M. D. C. Structural and Functional Stabilization of Protein Entities: State-of-the-Art. Adv. Drug Deliv. Rev. 2014. 13. Maggio, E. T. Polysorbates, peroxides, protein aggregation, and immunogenicity – a growing concern. J. Excipients and Food Chem. 2012, 3, 45–53. 14. Kerwin, B. A. Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways, J. Pharm. Sci. 2008, 97(8), 29242935. 15. Tomlinson, A.; Demeule, B.; Lin, B.; Yadav, S. Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

Particulates, and Insights into the Degradation Mechanism. Mol. Pharm. 2015, 12, 3805−3815. 16. Doshi, N.; Demeule, B.; Yadav, S.; Understanding Particle Formation: Solubility of Free Fatty Acids asPolysorbate 20 Degradation Byproducts in Therapeutic Monoclonal Antibody Formulations. Mol. Pharm. 2015, 12, 3792−3804. 17. Ohtake, S.; Kita, Y.; Arakawa, T. Interactions of Formulation Excipients with Proteins in Solution and in the Dried State. Adv. Drug Deliv. Rev. 2011, 63 (13), 1053–1073. 18. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicicm J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem. 2004, 47, 1739-1749. 19. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening J. Med. Chem. 2004, 47, 1750-1759. 20. Paul, N. K.; Twibanire, J. A. K.; Grindley, T. B. Direct Synthesis of Maradolipids and Other Trehalose 6 - Monoesters and 6,6 ′ -Diesters. J. Org. Chem.2013, 78, 363−369. 21. Kalhapure, R. S.; Akamanchi, K. G. Oleic Acid Based Heterolipid Synthesis, Characterization and Application in Self-Microemulsifying Drug Delivery System. Int. J. Pharm. 2012, 425 (1-2), 9–18. 22. Tan, C. Y.; Rahman, R. N. Z.; Kadir, H. A.; Tayyab, S. Calcium-induced stabilization of α-amylase against guanidine hydrochloride denaturation. Afr J Biotechnol. 2010, 9, 7934–41. 23. Kondo, A.; Murakami, F.; Higashitani, K. Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles. Biotechnol. Bioeng. 1992, 40, 889–894. 24. Bowers, K. J.; Chow, E.; Xu, H.; Dror, R. O.; Eastwood M. P.; Gregersen B. A.; Klepeis, J. L.; Kolossvary I.; Moraes, M. A.; Sacerdoti, F. D.; Salmon, J. K.; Shan, Y.; Shaw, D. E. Scalable Algorithms for Molecular Dynamics Simulations on Commodity Clusters, In Proceedings of the ACM/IEEE Conference on Supercomputing (SC06), Tampa, Florida, 2006; November 11-17.

26


Page 27 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25. Meng, X.-Y.; Zhang, H.-X.; Mezei, M.; Cui, M. Molecular Docking: A Powerful Approach for Structure-Based Drug Discovery. Curr. Comput. Aided. Drug Des. 2011, 7 (2), 146–157. 26. Jain, N. K.; Roy, I.; Effect of trehalose on protein structure. Protein Science 2009, 18, 2436. 27. Elbein, A. D.; Pan, Y. T.; Pastuszak, I.; Carroll, D. New insights on trehalose: a multifunctional molecule. Glycobiology. 2003, 13, 17R–27R. 28. Garg, A. K.; Kim, J-K.; Owens, T. G.; Ranwala, A. P.; Choi, Y. D.; Kochian, L. V.; Wu, R. J. Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA. 2002, 99, 15898–15903. 29. Green, J. L.; Angell, C. A. Phase relations and vitrification in saccharide-water solutions and the trehalose anomaly. J Phys Chem. 1989, 93, 2880–2882. 30. Chen, T.; Fowler, A.; Toner, M. Literature review: supplemented phase diagram of the trehalose–water binary mixture. Cryobiology 2000, 40, 277–282. 31. http://www.jrhessco.com/critical-micelle-concentration-measuring-surfactant-efficiency (accessed May 2015) 32. Schiefelbein, L.; Keller, M.; Weissmann, F.; Luber, M.; Bracher, F.; Friess, W. Synthesis, Characterization and Assessment of Suitability of Trehalose Fatty Acid Esters as Alternatives for Polysorbates in Protein Formulation. Eur. J. Pharm. Biopharm. 2010, 76 (3), 342–350. 33. Aveyard, R.; Binks, B. P.; Chen, J.; Esquena, J.; Fletcher, P. D. I. Surface and Colloid Chemistry of Systems Containing Pure Sugar Surfactant. Langmuir, 1998, 14, 4699– 4709. 34. Otzen, D. Protein–surfactant Interactions: A Tale of Many States. Biochim. Biophys. Acta - Proteins Proteomics 2011, 1814 (5), 562–591. 35. Aihua, Z.; Jing, L.; Ying, J.; Fang, L. and Bozhong M. Interaction Between Surfactin and Bovine Serum Albumin. J Disper Sci Technol. 2014, 35:1, 48-55. 36. Jean, B.; Lee, L.T. and Cabane, B. Effects of Sodium Dodecyl Sulfate on the Adsorption of Poly (N-isopropylacrylamide) at the Air−Water Interface. 1999, Langmuir, 15: 7585– 7590.

27



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Page 28 of 29

37. Vetri, V.; D'Amico, M.; Foderà, V.; Leone, M.; Ponzoni, A.; Sberveglieri, G.; Militello V. Bovine Serum Albumin protofibril-like aggregates formation: solo but not simple mechanism. Arch Biochem Biophys. 2011, 508, 13-24. 38. Amin, K.; Dannenfelser, R. In Vitro Hemolysis: Guidance for the Pharmaceutical Scientist. J. Pharm. Sci., 2006, 95, 1173–1176. 39. Banno, T.; Toyota, T.; Matsumura, S. Creation of Novel Green Surfactants Containing Carbonate Linkages, Biodegradation - Life of Science, Dr. Rolando Chamy (Ed.), 2013, ISBN:

978-953-51-1154-2,

InTech,

DOI:

10.5772/56163.

Available

from:

http://www.intechopen.com/books/biodegradation-life-of-science/creation-of-novelgreen-surfactants-containing-carbonate-linkages 40. Kandagal, P. B.; Ashoka, S.; Seetharamappa, J.; Shaikh, S. M. T.; Jadegoud, Y.; Ijare, O. B. Study of the interaction of an anticancer drug with human and bovine serum albumin: spectroscopic approach. J. Pharm. Biomed. Anal. 2006, 41, 393–399. 41. Gelamo, E. L.; Silva, C. H. T. P.; Imasato, H.; Tabak, M. Interaction of Bovine (BSA) and Human (HSA) Serum Albumins with Ionic Surfactants: Spectroscopy and Modelling. Biochim. Biophys. Acta - Protein Struct. Mol. Enzymol. 2002, 1594 (1), 84–99. 42. Miller, J. N. Recent advances in molecular luminescence analysis. Proc. Anal. Div. Chem. Soc.1979, 16, 203–208. 43. Chen, G. Z.; Huang, X. Z.; Xu, J. G.; Zheng, Z. Z.; Wang, Z. B. Method of Fluorescence Analysis, 2nd ed.; Science Press: Beijing, China, 1990; pp. 112. 44. Hu, Y. J.; Liu, Y.; Wang, J. B.; Xiao, X. H.; Qu, S. S. Study of the interaction between monoammonium glycyrrhizinate and bovine serum albumin. J. Pharm. Biomed. Anal. 2004, 36, 915–919. 45. Zhang, G.; Wang, Y.; Zhang, H. ; Tang, S.; Tao, W. Human serum albumin interaction with paraquat studied using spectroscopic methods. Pestic. Biochem. Physiol. 2007, 87, 23–29. 46. Fan, J.-C.; Chen, X.; Wang, Y.; Fan, C.-P.; Shang, Z.-C.; Binding interactions of pefloxacin mesylate with bovine lactoferrin and human serum albumin. J. Zhejiang Univ. Sci. 2006, 7, 452–458. 47. Su, R.; Qi, W.; He, Z.; Zhang, Y.; Jin, F. Multilevel Structural Nature and Interactions of Bovine Serum Albumin during Heat-Induced Aggregation Process. Food Hydrocoll. 2008, 22 (6), 995–1005. 28


Page 29 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


48. Navarra, G.; Giacomazza, D.; Leone, M.; Librizzi, F.; Militello, V.; San Biagio, P. L. Thermal Aggregation and Ion-Induced Cold-Gelation of Bovine Serum Albumin. Eur. Biophys. J. 2009, 38 (4), 437–446. 49. Arakawa, T.; Kita, Y. Protection of Bovine Serum Albumin from Aggregation by Tween 80. J. Pharm. Sci., 2005, 89 (5), 646–651. 50. Sudhamalla, B.; Gokara, M.; Ahalawat, N.; Amooru, D. G.; Subramanyam, R. Molecular Dynamics Simulation and Binding Studies of β-Sitosterol with Human Serum Albumin and Its Biological Relevance. J. Phys. Chem. B 2010, 114, 9054–9062. 51. Lobanov, M. Yu.; Bogatyreva, N. S.; Galzitskaya, O. V. Radius of Gyration as an Indicator of Protein Structure Compactness. Mol. biol. 2008, 42 (4), 623-628. 52. Hawe, A.; Friess, W. Development of HSA-free formulations for a hydrophobic cytokine with improved stability. Eur. J. Pharm. Biopharm. 2008, 68 (2), 169–182. 53. Treuheit, M. J.; Kosky, A. A.; Brems, D. N. Inverse relationship of protein concentration and aggregation, Pharm. Res. 2002, 19 (4), 511–516. 54. Hawe, A.; Filipe, V.; Jiskoot W. Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations, Pharm. Res. 2010, 27 (2), 314–326. 55. Wang, W. Instability, stabilization, and formulation of liquid protein pharmaceuticals, Int. J. Pharm. 1999,185 (2), 129–188.

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Trehalose Monooleate: A Potential Antiaggregation Agent for

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