Micellar Morphology of Polysorbate 20 and 80 and Their Ester Fractions in Solution via Small-Angle Neutron Scattering

Journal Pre-proof Micellar morphology of Polysorbate 20 and 80 and their ester fractions in solution via Small Angle Neutron Scattering Jannatun Nayem, Zhenhuan Zhang, Anthony Tomlinson, Isidro E. Zarraga, Norman J. Wagner, Yun Liu PII:

S0022-3549(19)30824-X

DOI:

https://doi.org/10.1016/j.xphs.2019.12.016

Reference:

XPHS 1833

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 6 July 2019 Revised Date:

5 December 2019

Accepted Date: 18 December 2019

Please cite this article as: Nayem J, Zhang Z, Tomlinson A, Zarraga IE, Wagner NJ, Liu Y, Micellar morphology of Polysorbate 20 and 80 and their ester fractions in solution via Small Angle Neutron Scattering, Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.xphs.2019.12.016. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

1

Micellar morphology of Polysorbate 20 and 80 and their ester fractions in solution via Small Angle Neutron

2

Scattering

3

Jannatun Nayem , Zhenhuan Zhang , Anthony Tomlinson , Isidro E. Zarraga , Norman J. Wagner

4 5 6 7 8

1

9 10 11 12 13 14 15 16 17 18 19

1,2

1,2

3

3†

1,4*

, Yun Liu

1,2,4*

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, 19716, U.S.A., Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland, 20899, U.S.A., 3 Genentech Inc., South San Francisco, CA, 94080, U.S.A. 4 Department of Physics and Astronomy, University of Delaware, Newark, Delaware, 19716, U.S.A. † Current address: Sanofi Genzyme, 5 Mountain Road, Framingham MA 01701 USA 2

* Corresponds to: Yun Liu (Telephone: +1 301 975 6235; Fax: +1 301 921 9847) E-mail address: [email protected], [email protected] Norman J. Wagner (Telephone: +1 302-831-8079) E-mail address: [email protected]

ABSTRACT:

20

Surfactants are commonly used in therapeutic protein formulations in biopharmaceuticals to impart

21

protein stability; however, their solution morphology and the role of the individual components in these

22

structurally heterogeneous commercial grade surfactants at physiologically and pharmaceutically relevant

23

temperatures have not been investigated systematically. The micellar morphologies of Polysorbate 20 and

24

Polysorbate 80 and their primary components monoester fractions as well as the diester fractions, are evaluated at

25

4, 22 °C, 40 °C, and 50 °C using small angle neutron scattering to determine the aggregation number, radius of

26

gyration, core radius, critical micelle concentration, shell thickness, and shell hydration. The sizes and aggregation

27

numbers of the diester fractions of PS20 above 80 °C and PS80 above 50 °C exhibit significant changes in shape.

28

The analysis of the SANS data of PS20 confirms that the critical micellar concentration of the monoester fraction is

29

significantly higher at 4 °C compared with the diester fraction and their original material, all laurate PS20. Overall,

30

these experiments identify the dominant components responsible for the temperature dependent behavior of

31

these surfactants in pharmaceutical protein formulations.

32

Keywords: Protein formulation, polysorbate 20, polysorbate 80, fractions, polysorbate degradation,

33

hydrophobicity, CMC (critical micellar concentration), Guinier, aggregation number, thermal stability.

34

Abbreviation (goes in footnote after their first use): PS20, polysorbate 20; PS80, polysorbate 80; CMC, critical

35

micelle concentration; HLB, hydrophilic-lipophilic balance; POE, polyoxyethylene; FFA, free fatty acid; SANS, small

36

angle neutron scattering; SLD, scattering length density.

37

Introduction:

38

Polysorbate 20 (PS20) and polysorbate 80 (PS80), also known as Tween 20 and Tween 80, are two of the

39

most widely used surfactants to stabilize therapeutic proteins against surface induced denaturation, adsorption,

40

and aggregation observed in manufacturing processes such as storage, agitation, filtration, lyophilization, freeze-

41

thawing, spray drying, as well as clinical use of drugs . Although the exact molecular mechanism of the

42

stabilization process is not fully understood yet, polysorbate surfactants are known to protect proteins in

43

therapeutic formulations via two major mechanisms: (1) preferential localization at the air-water and liquid-solid

44

interfaces due to amphiphilic properties of surfactants and (2) modulation of protein’s interfacial behavior by

45

monomer-protein and micelle-protein complexes that minimize undesirable interactions that lead to irreversible

46

protein aggregation via association

47

concentration (CMC), and high hydrophilic-lipophilic balance (HLB) enable surfactants to protect the surface active

48

ingredients in pharmaceutical formulations against interfacial stresses

49

amphiphilic compounds composed of a hydrophilic polyoxyethylene (POE) sorbitan headgroup and hydrophobic

50

fatty acid ester tails with varying tail lengths according to their trade names (Figure 1).

1–3

1,2,4,5

. Their biocompatibility, inert nature, low toxicity, low critical micelle

3,6,7

. These surfactants are nonionic and

2

51

The compendial grade polysorbate surfactants used in protein formulations are inherently heterogeneous

52

and contain complex molecular entities which are formed due to their complex synthesis processes and the

53

structural variants of the chemical species (e.g. fatty acids) used in the synthesis process

54

of the head groups originates from dehydration of the sorbitol group, differences of the tail groups are due to the

55

distribution of fatty acids used during esterification, which can vary in terms of chain length and the number of

56

ester bonds such as mono-, di-, tri-, and tetra esters . Additional heterogeneity comes from the byproducts

57

generated during production of the polysorbate surfactants which include sorbitol, non-esterified

58

sorbitan/isosorbide POEs, POE chains, etc . Table 1 shows the fatty acid ester contents of the compendial grade

59

PS20 and PS80 molecules listed in the US and European Pharmacopoeia . Though there are many advantages that

60

lead to the widespread use of polysorbate surfactants in biopharmaceuticals, their structural heterogeneity,

3,8,9

. While heterogeneity

3,9

3

a,b

1,3,8–10

61

synthesis byproducts, and impurities make them susceptible to chemical and physical degradation

62

Polysorbate surfactants can degrade via two major pathways: hydrolysis (chemical or enzymatic) and auto-

63

oxidation

64

monoesters, oxidation preferentially cleaves the poly-ester (di-, tri-, and tetra esters) head groups, partially or

65

completely, at the unsaturated alkyl sites

66

depending on the enzyme

67

(FFA) esters, short chain organic acids, POE esters, aldehydes, and ketones3,9. The possible consequences of

68

degradation of polysorbate surfactants on formulation quality are twofold: (1) changes in the both the unimer and

69

micelle polysorbate structures

70

phenomena lead to the loss of surfactant functionalities and the formation of sub-visible and visible particles in

71

therapeutic formulations, which can adversely affect product quality

72

pharmacopeia established specific limits to the level of impurities for polysorbate surfactants used in parenteral

73

formulations . Characterization of the degraded PS systems in pharmaceuticals using compendial analytical

74

methods is extremely difficult as these systems produce inconsistencies in both degradation and stability

75

profiles

76

produce different signals in different characterization techniques; moreover, certain degradants from hydrolysis

77

and oxidation show significantly different behavior . This becomes even more challenging in drug formulations

78

due to matrix effects encountered by proteins and excipients . Recent studies by McShan et al. and Labrenz et al.

79

confirm that the primary components of the commercial grade PS20 have different degradation propensities due

80

to differences in their chemical compositions and, accordingly, promote different solution physicochemical

81

behaviors9,12. Given the relationship between heterogeneity of polysorbate surfactants and their related

82

degradation profiles, it is crucial to understand the degradation and stability behaviors of their primary

83

components. As the structural diversity of the compendial grade PS20 and PS80 used in biopharmaceuticals adds

84

an additional degree of complexity, we used all laurate (C12) (~99%) PS20 and all oleate (C18:1) (~98%) PS80 in our

85

studies for a more accurate analysis. It is becoming a more common practice in experimental studies to only use all

86

laurate PS20 and all oleate PS80 since these are the major constituents of the heterogeneous PS mixtures and can

87

predict their solution behavior well without additional degrees of complexity and biases in experimental studies .

1,3,9,11

.

. While chemical hydrolysis preferentially cleaves the POE sorbitan head groups off of the

3,8,9,11

. Enzymatic hydrolysis on the other hand can cleave either

3,12

. Both degradation pathways produce insoluble degradants such as free fatty acid

3,4,11

3,11,13,14

, and (2) accumulation of insoluble degradants in solution

3,10,13–16

. These

. As a result, the US and European

3

9,10

. A recent study by Lippold et al. confirms that the poly-ester subspecies of PS20 and PS80 in placebos

17

3

3,9

88

In recent years, most studies on polysorbate surfactants in pharmaceuticals focused on the relationship 3,8,10,11,13,14

89

between surfactant degradation and formation of particles during the storage of protein formulations

90

Previously, the CMC and aggregation behaviors of commercial grade PS20 and PS80 in water have been

91

investigated at different temperatures via static and dynamic light scattering, surface tensiometry, and

92

densitometry, but such measurements lack molecular level structural understanding

93

dynamic simulation study by Lapelosa et al. determined molecular information such as aggregation number and

94

radius of gyration of micelles formed using the monoester and diester components of PS2020. Mahajan et al.21,

95

Wadsater et al.

96

PS20 and PS80 micelles in different types of aqueous solvent using SANS and small angle X-ray scattering (SAXS).

97

However, no experimental studies on the actual nanoscale morphology and solution physicochemical properties of

98

the either all laurate PS20, all oleate PS80, and their individual primary components have been reported till date.

99

Therefore, the main objectives of this work are: (1) to characterize micellar morphologies of the individual

100

monoester and diester fractions, along with their original components, all laurate PS20 and all oleate PS80 to

101

understand their solution properties, and (2) to investigate the structural properties of micelles formed by each

102

fraction and their mixtures at a wide range of temperatures from the storage temperature (4 °C) to the

103

temperature above the body temperature. SANS is particularly suitable for this study because it can provide a

104

detailed quantitative description of the molecular structure of nonionic micelles in solution

105

demonstrates that simple structural analysis of the SANS data provides a clear understanding of the solution

106

physicochemical behavior of monoester and diester fractions of PS20 and PS80 and their mixtures. This work

107

investigates differences in micellar aggregates of the monoester and diester fractions of PS20 and PS80 at

108

pharmaceutically relevant temperatures (4-40 °C) and at higher temperatures (50-80 °C) to understand the trend

109

of the structure change when increasing the temperature.

110

pharmaceutical temperatures can potentially act as a useful predictor of micelle morphologies and solution

111

physicochemical behavior of PS20 and PS80 in therapeutic formulations. The higher temperatures are studied to

112

explore the stability limit of the morphologies found at the pharmaceutical temperatures.

113

Materials & Methods

114

Polysorbate Fractionated Samples

18,19

22

23

and Hideki Aizawa

.

. A recent molecular

have investigated the morphologies of commercial grade heterogeneous

21,22

. Our work here

Understanding the morphological behavior at

115

The two components of all laurate PS20 and all oleate PS80 used in this study consist of a sorbitan head

116

group and fatty acid chains (e.g. laurate and oleate esters) containing mono- and diesters. Figure 1 shows the

117

chemical structures of the monoester and diester fractions of all laurate PS20 and all oleate PS80 respectively.

118

These fractionated samples were provided by Genentech, Inc. (South San Francisco, CA, USA). Detailed information

119

about their preparation, purification, and characterization will be given in an upcoming paper by Tomlinson et al.

120

at Genentech Inc. Custom grade polysorbate raw materials were fractionated to obtain the major individual

121

component of PS20 and PS80. PS20 containing ~99% laurate esters (C12) was obtained from BASF (Ludwigshafen,

122

Germany) and PS80 containing 98% oleate esters (C18:1) was obtained from NOF (Irvine, CA). Note that the all

123

laurate PS20 and all oleate PS80 contain surfactant subspecies, such as POE sorbitan and isosorbide headgroups

124

with mono-, di-, tri- and tetra-ester as well as free hydrophilic head groups and impurities, derived during the

125

synthesis and manufacturing process. All the PS samples were stored at 4°C protected from light. Deuterium oxide

126

(D2O) was purchased from Cambridge Isotope.

127

The naming conventions for monoester and diester of PS20 and PS80 are kept the same as in the

128

simulation study by Lapelosa et al.: (a) PS20M– POE sorbitan monolaurate ester, (b) PS20D– POE sorbitan dilaurate

129

ester (c) PS80M– POE sorbitan monooleate ester, and (d) PS80D – POE sorbitan dioleate ester. These fractions are

130

particularly chosen for this study because they are considered as the major subspecies

2,3

in PS20 and PS80.

131 132

Sample Preparation

133 134

Stock solutions of 10 mg/mL or 1% (w/v) were prepared in 99.8% D2O (Cambridge Isotope, MA) for the

135

components of the neat all laurate PS20 and all oleate PS80. These stock solutions were diluted to 2 mg/mL or

136

0.2% (w/v). For the fractions, a 0.2% (w/v) sample was prepared directly. D2O was used to reduce the incoherent

137

background of solvent molecules in neutron scattering experiments and to achieve good contrast between the

138

solvent and PS20 and PS80 molecules. For the mono- and diester mixtures, a 1:1 mass ratio was used, and the

139

concentration is fixed to 2 mg/mL. Subsequently, all the samples were filtered using PTFE syringe filters with 0.2

140

μm pore size (MicroSolv, NJ) to remove any particulates and impurities prior to the SANS experiments. For the

141

temperature studies, each sample was measured at 4, 22, 40, and 50 °C. The CMC of PS20M is significantly higher

142

than that of the other fractions. Thus, to do an accurate size and aggregation number analysis of the SANS data of

143

the PS20M micelles, the scattering contribution from monomers just below the CMCs were measured at the

144

relevant temperatures of PS20M which was later subtracted from each scattering data set.

145 146

Small Angle Neutron Scattering (SANS)

147 21

148

SANS can measure microstructures from 10Å to ~1000Å . When the incident neutron beam interacts

149

with the sample, it leads to a change in momentum, which is measured as a function of scattering angle, θ.

150

Subsequently, the scattered neutrons at a certain momentum transfer vector, Q, is collected at the detector.

151

Therefore, the structures observed in SANS are inversely related to the real space features. The scattering vector Q

152

is defined as:

153 154

Q=

sin ;

(1)

155 156

here λ is the wavelength of the neutrons.

157 158

For a simple monodispersed colloidal system, the scattering intensity, I(Q), as a function of Q is given by:

159 160

I Q = nV ∆ρ P Q S Q + B

(2)

161 162

n = number density of colloidal particles in solution

163

V = skeletal volume of one individual particle

164

∆ρ = contrast factor

165

P Q = normalized single particle form factor

166

S Q = effective inter-particle structure factor

167

B = background

168 169

The form factor represents the shape and size of a particle and the structure factor contains information

170

about the interaction between particles in solution. The contrast factor arises from the difference of the scattering

171

length density (SLD), ρ, of particles in solution and solvent. The background, B, has the contribution from the

172

incoherent scattering of hydrogen in the bulk. If the CMC is too high, B also contains the non-negligible

173

contribution from free monomers in solution. At low micelle concentration, S(Q) →1 and coherent scattering

174

intensity can be expressed as:

175 176

I Q = I Q − B = nV ∆ρ P Q ;

(3)

177 178

SANS measurements are performed on the 10m SANS instrument operated by nSoft Consortium at the

179

Center for Neutron Research (NCNR) in National Institute of Standards and Technology (NIST), Gaithersburg, MD.

180

The experiments are performed at two detector settings, (1) high Q: 1.2 m sample to detector distance with 5 Å

181

neutrons with 1200 s count time and (2) low Q: 5.2 m sample to detector distance with 10 Å neutrons with 1800 s

182

count time. This results in a Q range from 0.009 Å to 0.53 Å and a wavelength spread of ∆ / = 0.12. The

183

samples are contained in quartz cells with 1 mm path length. The scattered neutrons are collected at a 2D

184

detector. The 2D raw data are reduced using the standard IGOR NCNR data reduction program . First, the

185

scattering intensity is corrected for detector background scattering, transmission, empty cell scattering, and

186

detector efficiency. Subsequently, the corrected 2D data are azimuthally averaged to produce 1D scattering

187

intensity. The SANS experiment is performed at 4°C, 22°C, 40°C, 50°C, 70°C, and 80°C using a thermal bath to

188

control the temperature. About 40 mins is allotted for thermal and kinetic equilibration. The background scattering

189

is removed from all SANS signals by simply subtracting scattering of the buffer sample which is 99.8% D2O. The

190

instrument resolution is considered when analyzing data and the fits are assessed by the quality of the fitting

191

parameter chi-squared, χ , values.

-1

23

192 193

-1

Modeling of SANS Data

Guinier analysis is used to obtain the radius of gyration and the forward intensity, I

194 195

,!"#$%&"' ,

at Q → 0.

The Guinier law used for our system is given by:

196 197

,

I!"#$%&"' Q = I

,!"#$%&"'

exp − Q R/

where I

= nV

ρ01 2 − ρ34 .

(4)

-

198 199

,!"#$%&"'

200 201

R / is the radius of gyration. The Guinier law is independent of the particle shape. For spherical particles with a

202

uniform density distribution, R/ = 5 R, where R is the particle radius. The micelle number density, n!78"99" , is

203

related to the total monomer mole concentration in solution:

204

concentration of PS monomers. >?1 @ is the SLD of D2O solvent and >AB is the average SLD of PSs, which can be

205

calculated using the known molecular composition and mass density of these molecules (see Table 2). The SLD

206

values are calculated using NIST NCNR SLD calculator.

6

207 208

n!78"99" = C34

;<

;<==

The aggregation number of the micelles, NAgg, can be calculated once I

; here, C34 is the molar

,!"#$%&"'

is known. In our

209

experimental system, both micelles and monomers are present. In general, the solution scattering is the

210

summation of the scattering intensity of the micelles and the monomers defined as I!78"99" Q → 0 and

211

I!

C !"&

Q → 0 respectively:

212 213

I

,!"#$%&"'

Q → 0 = I!78"99" Q → 0 + I!

C !"&

Q→0

(5)

214 215

where I!78"99" Q → 0 = n!78"99" V!78"99" ∆ρ!78"99" , and I!

C !"&

Q → 0 = n!

C !"& V! C !"&

216 217

The aggregation number, NF// =V!78"99" /V! NF// =

C !"& ,

n#99 − n!

I

which can be estimated by: ,!"#$%&"'

C !"&

− I

V !

,! C !"&

C !"&

ρ01 2 − ρ34

∆ρ!

C !"&

.

218 219

For the samples with mixtures of two different monomers, NAgg can be written as:

220 221

NF// =

GH,IJKLMNJO P GH,IHQHIJN

1 CKRR PCIHQHIJN S∑VWX,1 %! 9"IHQ,V Y IHQHIJN,V Z [\1 ] P[^_ 1 X

(6)

222 223

where n#99 = n"` = NF// n!78"99"$ + n!

224

micellar and monomeric forms and %mole!

225

assumed the mass density of different types of monomer to be the same.

C !"& is C,`

the number density that counts for all the PS molecules in both

is the molar composition of each type of monomer. Here, we have

226

When the CMC of PS sample is much smaller than the concentration of the sample, one can neglect the

227

scattering intensity from the small fraction of the PS monomers in solution. Even though both micelles and

228

monomers are present in solution together, the measured scattering intensities are dominated by the larger

229

micellar aggregates in this case. Therefore, equation 6 can be simplified as:

230 231

NF// =

GH,IdeJRRJ

1 CKRR S∑VWX,1 %! 9"IHQ,V Y IHQHIJN,V Z [\1 ] P[^_ 1 X

(7)

232 233

To study the morphology of micelles in solution, the SANS data of micelles are modelled with different

234

shape models. Note that different types of models are used to study micelles. The limitations of modeling are

235

discussed in details in the supporting information. Here, the oblate ellipsoidal core-shell model is used. This model

236

describes a core-shell particle with ellipsoidal shape where the main structural parameters are equatorial radius of

237

the core (Rc,eq), thickness of the equatorial shell (Ts,eq), polar radius of the core (Rc,po), thickness of polar shell (Ts,po),

238

axial ratio of the core, axial ratio of the shell, SLD of the core (ρ8

239

(ρ01 2 ). The general equation for the ellipsoidal core-shell form factor is represented by:

240 241 242

P Q =

$8#9" , h iF YLgJRR o

Q, r!7Cd , r!#ld , α i dα (8)

&" ),

SLD of the shell (ρ$f"99 ), and SLD of the solvent

244

intra-particle density correlation function, u8

245

r!#lx 1 − α w

,⁄

246

polar

radius,

247

radius. V8

248

the ellipsoidal particle respectively, j, u = − sinx − xcosx /x is the first order spherical Bessel function

249

Scale is the particle volume fraction.

inner &"

&"

&" j,

− ρ$f"99 V$f"99 j, u$f"99 ⁄u$f"99 is the

Where FSQ, r!7Cd , r!#ld , αZ = 3 ρ8

&"

− ρ$f"99 V8

⁄u 8

243

u8

&"

&"

+ 3 ρ8

&"

= Qtr!7Cu α + r!#lu 1 − α w

,⁄

and u$f"99 = Qtr!7Cx α +

. For an oblate core-shell ellipsoidal particle, r!#lu is the equatorial inner radius, r!7Cu is the r!#lx

is

the

equatorial

outer

radius,

and

r!7Cx

is

the

polar

outer

= 4⁄3 πSr!7Ce r!#le Z and V$f"99 = 4⁄3 πSr!7CL r!#lL Z are the volume of the core and shell of 23,27,28

.

250

The ellipsoidal core-shell analysis of the reduced SANS data was performed by the SASVIEW 3.1.2

251

software (www.sasview.org). The SLD was determined via NIST SLD calculator by accounting for scattering length

252

of each component and the volume it occupies (https://www.ncnr.nist.gov/resources/activation/). The SLD values

253

of the solvents and the micellar core were fixed during the data analysis with the assumption that the core was

254

only comprised of surfactant tails and was considered to be solvent free. The SLD of the head group layer, >~ , was

255

chosen as a free parameter which was fitted to account for solvent penetration in the shell. Therefore, the shell

256

SLD is an average of the head group and the solvent in the micellar corona which results in the decrease of

257

contrast. The average SLD of the shell, >~ , is determined from the core-shell model. The deuterium exchange of the

258

hydroxyl (-OH) groups were taken into consideration during the calculation. From a simple material balance of the

259

micellar shell, the hydration level in terms of the volume fraction of solvent (D2O) in the micelle shell, χ~•€•• , is

260

determined by the following equation:

261

χ~•€•• = 1 −

262

The polar core size was initially held at the approximate length of the hydrocarbon chains: 16.7 Å for laurate ester

263

and 24 Å for oleate ester, which were predicted from the bond lengths. Then, they were varied until no change in

264

the quality of the fitting parameter was observed. No polydispersity contribution was considered for out model

265

fits. Therefore, the fitted results are average values of our system.

‚ƒ1 „…‚†

‚ƒ1 „…‚‡†

(9)

266 267

Results and Discussion:

268

Critical micelle concentrations in aqueous solutions:

269

CMC values of surfactants can be determined using techniques such as surface tension, conductivity, 29–32

270

fluorescence measurements, and scattering techniques

. Among the CMCs of all samples, only the CMC value

271

of PS20M is considerably high. And the CMC values of all other fractions determined by fluorescence intensity at a

272

fixed excitation and emission are found to be less than 0.02 mg/ml, whose details will be reported in a different

273

report in future. Thus, the SANS intensity of monomer contributions of most samples can be ignored except the

274

PS20M sample.

275

Because D2O is used in our samples, the CMCs of PS20M in D2O are further evaluated using SANS at

276

different temperatures. A detailed description of how the CMCs were determined is provided in the supporting

277

info (S.I Figure S1-S3). The results are reported in Table 3. The CMC (S.I. Figure S3) at different temperatures for

278

PS20M decreases as the temperature increases over the range of temperatures studied here. The CMC values of

279

PS20M determined via SANS (Table 3) are 2.05 mg/ml at 4 °C, 1.50 mg/ml at 22 °C, 1.28 mg/ml at 40 °C, and 1.22

280

mg/ml at 50 °C. This is surprising because the sample concentration, 2 mg/mL, is higher than the typical CMC (0.06

281

mg/mL) for compendial grade heterogeneous PS20 .

32

282 283

Microstructures of PS20 in aqueous solutions:

284

Three types of PS20 samples were chosen for study: PS20M (sorbitan POE monolaurate), PS20D (sorbitan

285

POE dilaurate), and all laurate PS20. Even though the exact compositions of PS20M and PS20D in all laurate PS20

286

can vary, the HPLC compositional analysis confirms that the fractions make up about ~40% of the all laurate PS20

287

samples. Shapes and sizes of the self-assembled micelles in solution are determined via SANS experiments. The

288

chemical formulas of the PS20M and PS20D are shown in Figure 1(a) and Figure 1(b) respectively. The schematics

289

drawn in Figure 1 are guided by the results from SANS.

290

Figure 2 shows the measured SANS scattering intensity patterns, I(Q) VS. Q, of PS20M (Figure 2(a)), PS20D

291

(Figure 2(b)), all laurate PS20 (Figure 2(c)), PS80M (Figure 2(d)), PS80D (Figure 2(e)), and all oleate PS80 (Figure

292

2(f)) at 4, 22, 40, and 50 °C. Note that, only the PS20 results will be discussed in this section and PS80 results will be

293

discussed in the subsequent section. The concentrations of all PS20 samples are fixed at 2 mg/ml. The minimum

294

concentration of surfactant required to protect proteins is typically determined by testing formulations during

295

accelerated degradation studies and thus, can vary profoundly . In this study, surfactants were studied at

3

3

296

concentrations that are above the typical concentrations (0.1-1 mg/mL) used in pharmaceutical formulations in

297

order to have enough scattering intensity for the SANS measurements. PS20 samples used here have only lauric

298

acid chains. Note that the commonly used commercial grade PS20 in protein formulations is composed of mostly

299

laurate esters (~ 40-60%) . Therefore, this study can provide insights into the differences in the micellar self-

300

assembly

301

of compendial grade heterogeneous PS20 as a function of temperature.

3

302

The SANS patterns of the PS20 samples exhibit an overall increase in scattering intensity with

303

temperature rise which identifies the temperature dependence of the micellar structure of different fractions. The

304

low-Q scattering intensity of PS20M increases 49% between 4 °C and 22 °C and 60% between 22 °C and 40 °C.

305

However, between 40 °C and 50 °C, the change is very small. Note that, the micellar transition is rather gradual for

306

PS20M between 4 °C to 22 °C. The flat shape and slightly upward turn of the scattering curve at 4 °C indicate that

307

the PS20M remains a little below CMC while slowly transitioning into a micellar aggregate. As the temperature

308

increases to 22 °C, changes in the scattering intensity as well as the shape of the curve confirm the transition from

309

monomers to micelles. The large increase in the low-Q scattering intensity from 22 °C to 40 °C indicates that

310

micelles at 40 °C has a larger aggregation number. The micellar structure between 40 °C and 50 °C does not change

311

much. A consistent increase in scattering intensity is observed for PS20D fraction as it was heated from 4 °C to

312

50 °C. This suggests that the PS20D fraction forms micelles with larger aggregation number as the temperature

313

rises. In addition, we explored PS20D micellar structure at higher temperatures (70 °C and 80 °C) and the results

314

will be discussed with PS80D results later for relevancy. For the all laurate PS20 samples, an increase in the low-Q

315

scattering intensity is observed between 4 °C to 22 °C. A steady increase in the scattering intensities is observed

316

from 22 °C to 50 °C.

317

The NAgg and Rg values at different temperatures for PS20M, PS20D, and all laurate PS20 are determined

318

by applying Guinier law to the SANS data (see Figure 3(a) and 3(b). The incoherent background at high Q was

319

subtracted prior to analyzing the SANS data. Note that, the sample concentration of PS20M is a little below the

320

CMC at 4 °C where micelles start to form gradually. Therefore, the aggregation number for PS20M is not reported

321

at 4 °C in Fig. 3(b). For the Guinier analysis of PS20M data at temperatures larger than 4 °C, the contribution of the

322

monomers to the scattering pattern is subtracted first.

323

As stated earlier, the weak scattering intensity of the PS20M fraction at 4 °C is due to monomers in

324

solution. Our analysis shows that the PS20M monomer has an Rg of 16.7 ± 2 Å at 4 °C. Based on Tanford’s

325

formula

326

the contribution from both the head and tail which implies that the monomers are not in their extended form in

327

solution. At 22 °C, Rg of the PS20M fraction increases to 27.2 ± 2 Å suggesting formation of micelles in solution.

328

With temperatures increasing to 50 °C, Rg increases to 30.2 ± 1.5 Å. The NAgg of PS20M micelle at 22 °C is

329

determined to be 22.0 ± 0.6. According to the MD simulation study performed by Lapelosa et al., PS20M forms a

330

micelle with 18 monomers and a Rg of 21 Å at 300 K (~27 °C) , which is in agreement with our experimental

331

results. In accordance with the Rg values, the NAgg values also increase as temperature increases. For instance, the

332

increase of NAgg from 22.0 ± 0.6 at 22 °C to 28.6 ± 0.5 at 50 °C is about 27% which is in line with the increase of Rg

333

by ~11% leading to approximately 30% change of the volume, which is in very good agreement with the change in

334

NAgg. Compared with PS20M, the Rg values of PS20D micelles at different temperatures are slightly smaller but the

335

NAgg values are significantly larger. In addition, PS20D exhibits an unusually large increase of NAgg from 36.0 ± 0.3 at

336

4 °C to 50.2 ± 0.7 at 50 °C. At the same time, Rg increases from 24.9 ± 0.4 Å at 4 °C to about 27.7 ± 0.9 Å at 50 °C.

337

Particularly, micellar aggregation of PS20D becomes more sensitive to temperatures above 40 °C. The two laurate

338

tails of the PS20D micelles potentially affect the structure and physicochemical properties of the micelles in two

339

ways: (1) their packing geometry decreases the curvature of the micelles and (2) two hydrophobic tails cause an

340

increase in hydrophobicity. According to the simulation study by Lapelosa et al. on PS20M and PS20D, micellar

341

clustering is predominantly governed by the overall free energy which is directly proportional to the exposed

342

surface area .

30,31

, the maximum extended chain length of lauric acid chain is 16.7 Å. The estimated Rg, however, includes

20

20

343

The temperature dependence of the micellar aggregation, shapes, and sizes of all laurate PS20 are also

344

investigated and compared with the temperature dependence of the individual fractions (see Figure 2(c), 3(a), and

345

3(b) and Table S1(c)). Both the Rg and NAgg of all laurate PS20 have an abrupt increase from that at 4 °C to that at

346

22 °C. Rg increases from 25.1± 0.9 Å to 29.4± 0.7 Å and NAgg increases from 22.1± 0.2 to 27.5 ± 0.2. PS20M also

347

exhibits notable changes in size and aggregation between 4 °C and 22 °C which potentially contribute to the

348

observed structural changes of all laurate PS20. Once the temperature is over 22 °C, Rg stabilizes to 29.4± 0.7 Å at

349

22 °C to about 30.2 ± 0.7 Å Å at 50 °C and NAgg increases from 27.5± 0.2 to 31.1 ± 0.3. Since the Rg and NAgg of

350

PS20M and PS20D are similar at temperatures between 22 °C and 50 °C, it is difficult to determine the dominant

351

component responsible for the changes in Rg and NAgg between these temperatures. Note that NAgg of all laurate

352

PS20 can only be estimated approximately as only the major components of all laurate PS20 are considered.

353

The oblate ellipsoidal core-shell model is used to fit our SANS data. Here, we have particularly explored

354

the effects of temperature on the micellar morphologies of PS20M, PS20D, and all laurate PS20. The compositions

355

of their core and shell are listed in the supporting information (Table S1(a-c)). Although the absolute scattering

356

intensities increase for all PS20 samples with increasing temperatures, the shape of the curves do not change

357

significantly which indicates that the shape of the micelles remain similar. The aspect ratios (equatorial to polar

358

lengths) of these oblate shaped ellipsoidal micelles range from 1.4 to 1.9. A prior literature study on similar PS20

359

systems also indicated that they tend to form oblate ellipsoids . This model analysis reports the hydration of the

360

micellar shell which decreases slightly with increasing temperature. The micellar coronas for all PS20 samples are

361

always found to be highly hydrated.

32

362 363

Microstructures of PS80 in aqueous solutions:

364

The SANS patterns for PS80M, PS80D, and all oleate PS80 at the four temperatures of 4, 22, 40 and 50 °C

365

are shown in Figure 2(d-f). The shape of the scattering patterns of PS80 are similar to that of PS20. Similar to the

366

PS20 samples, the concentration is fixed at 2 mg/ml for all PS80 samples. The absolute scattering intensity of

367

PS80M at low-Q in Figure 2(d) is ~6 times higher than that of PS20M fraction, which is reasonable given that the

368

PS80M fraction has a higher molar mass than PS20M due to the longer hydrophobic tail group. The PS80M fraction

369

only exhibits a weak increase with increasing temperatures. In contrast, the scattering data for PS80D in Figure

370

2(e) shows a large increase in the low-Q scattering intensity at temperatures between 40 °C to 50 °C. The shape of

371

the scattering curve also changes indicating that PS80D undergoes a significant microstructural transition.

372

Similarly, the scattering intensity of the all oleate PS80 sample in Figure 2(f) increases noticeably with increasing

373

temperature. In particular, the intensity increases by 44% between 40 °C to 50 °C. This suggests that the PS80D

374

fraction may contribute to the overall physicochemical properties of all oleate PS80 that lead to the observed

375

aggregation property at higher temperatures.

376

Similar to the all laurate PS20 SANS data analysis, we have obtained the Rg and NAgg using the Guinier

377

analysis for all oleate PS80 and its fractions (Figure 3(c-d)). Note that, the contribution of the PS80 monomers to

378

the scattering intensity can be safely ignored because the CMCs are much smaller compared to the sample

379

concentration. The Rg values of the PS80 micelles are generally larger than those of the PS20 micelles due to the

380

difference in their hydrophobic tail since the oleate tail group has a longer hydrocarbon chain with a kink. For an

381

instance, Rg of PS80M is 28.8 ± 0.6 Å at 4 °C, 30.0 ± 0.6 Å at 22 °C, 32.8 ± 1 Å at 40 °C, and 33.3 ± 2 Å at 50 °C.

382

Compared to PS80M, the Rg values of PS80D increase slightly between at 4 °C and 40 °C but increases from 34.2 ±

383

0.4 Å at 40 °C to a significantly larger value of 101 ± 3 Å at 50 °C which is in accordance with the change of their

384

scattering pattern. The Rg values of all oleate PS80 are 31.1 ± 0.5 Å at 4 °C, 32.0 ± 0.4 Å at 22 °C and increases

385

from 38.0 ± 0.6 Å at 40 °C to 52.8 ± 1 Å at 50 °C. At 4 °C, NAgg for PS80M, PS80D, and all oleate PS80 samples are

386

39.4 ± 0.3, 59.8 ± 0.3, and 65 ± 0.4, respectively. NAgg for PS80M, PS80D, and all oleate PS80 increase to 48.9 ± 1,

387

69.9 ± 3, and 93.9 ± 0.6 at 40 °C, and 50.6 ± 1, and 238 ± 3, and 141 ± 1 at 50 °C. Similarly, the fitted NAgg values

388

also indicate that PS80M has relatively weak temperature dependence while PS80D shows strong temperature

389

dependence above 40 °C. The hydrophobic portions of PS80D surfactant monomers have two oleic acid chains that

390

have double bonds with a kink. This increased hydrophobicity may play an important role in the strong

391

temperature dependence observed for this sample above 40 °C.

392

The NAgg values of PS80M are lower than those of PS80D due to the steric effect of the bulky sorbitan

393

head group. The significant change in micellar aggregation for PS80D at 50 °C (Rg of 101 ± 3 Å and NAgg of 240 ± 3)

394

corresponds to an ~225% increase of Rg and ~300% increase of NAgg compared with the micelles formed at 22 °C.

395

This is a signature of structural evolution which is believed to be induced by proximity to the stability limit of the

396

ellipsoidal PS80D micelles. In addition, we explored this stability limit for PS20D fraction which is observed at

397

temperatures above 70 °C (S.I. Figure S8). As is evident in the SANS plot in Figure 2(e), the micellar size and

398

aggregation of all oleate PS80 increase significantly with increasing temperature. We believe that PS80D fraction

399

partially contribute to such behavior. Note that in order to reach the Guinier region for PS80 samples at higher

400

temperatures, the samples are measured at a smaller Q range with one additional instrument configuration. The

401

Guinier plots for these samples are also included in the supporting information.

402

The aforementioned ellipsoidal core-shell model is also employed to model the micelles in the PS80

403

samples. The progressive dehydration of the POE groups in the micellar corona as a function of increasing

404

temperatures for each PS80 sample is reported in the supporting information (Table S2(a-c)). PS80M head groups

405

are highly hydrated ranging from 89-97% which is analogous to the PS20M results. Evidently, the overall

406

hydrocarbon makeup of the PS80D influences its aggregation propensity and plays a key role in instability behavior

407

at higher temperature. The micelles formed in this case is found to be significantly elongated. Furthermore, the

408

level of hydration of all oleate PS80 head groups are also comparable to those of the PS80M and PS80D fractions

409

which is anticipated because all oleate PS80 sample is a heterogeneous mixture that includes both PS20M and

410

PS20D fractions.

411 412

Microstructures of mixtures of PS20M and PS20D:

413 414

In an effort to understand the role of individual component in a mixture, the morphologies and

415

temperature dependence of mixtures of PS20M and PS20D fractions are investigated. The mixture sample

416

concentration and mixing ratios are fixed at 2 mg/mL and 1:1 mass ratio of PS20M:PS20D, respectively. SANS

417

measurements of the mixed components of PS20 at 4, 22, 40 and 50 °C are shown in Figure 4a. The scattering

418

results are analogous to those of the all laurate PS20 sample (Figure 2(c)) and exhibit similar temperature

419

dependence. The compositional information from the Guinier analysis of the mixed components of PS20 samples

420

are shown in Figure 4(b) and Table S4. Note that, the CMCs of these fractions are estimated based on the ideal

421

solution theory that predicts the CMC of a mixture of nonionic surfactants from the CMC of each component in

422

the mixture; the CMC of the mixture will just be the sum of the molar fraction of each component multiplied by

423

their individual CMC

424

the individual component PS20M and thereby, the anomalously high CMC of PS20M at 4 °C do not play a role in

425

the micelle formation. In addition, NAgg of micelles of each component can also be estimated based on the

426

aforementioned ideal solution assumption. The estimated NAgg values of PS20M + PS20D at 4, 22, 40, and 50 °C are

427

33, 34, 37, and 41 respectively which is in good agreement with the experimentally determined NAgg values of 31.8

428

± 0.2, 35.1 ± 0.2, 37.0 ± 0.2, and 38.2 ± 0.2 at the same respective temperatures. No significant temperature

33

34,35

. In our case, the calculated CMC of the mixed components is 42% lower than the CMC of

429

dependence on micellar sizes for PS20M + PS20D mixture is observed. Following the previous methodology, the

430

oblate ellipsoidal core shell model are used to fit these SANS patterns (Table S4), the average SLD values of the

431

micellar shell are comparable to all laurate PS80 and its individual components. The hydration values confirm

432

decrease of shell hydration with increasing temperature.

433 434

Microstructures of samples by mixing different components of PS80:

435 436

Figure 4b shows the scattering patterns for the mixture of different components of PS80 at 4, 22, 40 and

437

50 °C. The concentration for all samples is 2 mg/mL with 1:1 mass ratio between PS80M and PS80D. The overall

438

scattering intensities increase for these samples compared to the PS20M + PS20D fractions mixture since PS80

439

forms larger micelles due to the increased length of the hydrophobic oleate tail. Similar to the PS20M + PS20D

440

sample, this sample does not exhibit any substantial change in their scattering profiles with increasing

441

temperature. No low-Q upturn at 50 °C was observed in contrast to the all oleate PS80 at 50 °C. According to the

442

ideal solution mixing theory, the estimated NAgg values for PS80M + PS80D at 4, 22, 40, and 50 °C are 49, 56, 59,

443

and 136 respectively which is in good agreement with the experimentally determined NAgg values up to 40 °C (see

444

Figure 4d). Since a notable increase in aggregation is observed for all oleate PS80 at 50°C, the ideal solution theory

445

could not be applied at 50 °C. The fitted structural parameters of these mixture samples from Guinier analysis are

446

listed in Table S5. The micellar Rg and NAgg values do not exhibit any temperature dependence which is in line with

447

their scattering profiles.

448

Similar to PS20M + PS20D, the SANS results of the PS80M + PS80D mixture are also fitted using an

449

ellipsoidal core-shell model. The results are listed in Table S5. The shell hydration values of all the two-component

450

mixtures are comparable to the individual fractions and decrease slightly with increasing temperatures. Note that

451

we have not discussed the core and shell thickness parameters obtained from the oblate core shell models for any

452

of the polysorbate samples in this paper, as the error bars are significantly larger from the fit. However, an overall

453

qualitative morphological picture can be obtained from these fitted parameters.

454 455

456

Conclusions

457

The microstructure of micelles in aqueous solution of all laurate PS20 and all oleate PS80, as well as the

458

mono- and diester fractions and their mixtures, are measured and reported at temperatures pertinent to

459

biopharmaceutical formulation stability studies. The micelle size and aggregation number increase with increasing

460

temperature. For all laurate PS20, the monoester component, PS20M, shows large temperature dependence at

461

the temperature below 22 °C while the diester component, PS20D, has a slow but gradual increase in both Rg and

462

Nagg. We also observed an anomalously high CMC of the monoester fraction, PS20M compared to the CMC of

463

compendial grade PS20. For all oleate PS80, the diester component with two fatty acid chains exhibits a stronger

464

temperature dependence of the micelle size above 40 °C as compared with the monoester component, PS80M. As

465

the all oleate PS80 is the major component of compendial grade PS80 surfactants widely used in the

466

pharmaceutical industry, the diester fraction is expected to play an important role for the temperature

467

dependence of the PS80 samples, and in particular, the formation of undesired aggregates in commercial

468

formulations.

469

Our SANS results show that PS20 and PS80 micelles are ellipsoidal shape with a core-shell structure, which

470

is predominantly governed by the packing geometry of large hydrophilic head groups and small hydrophobic tails

471

of the polysorbate species. The shells for all polysorbate samples are found to be highly hydrated. The analysis of

472

aggregation number and micellar size indicates that the diester fractions for both PS20 and PS80 have higher

473

aspect ratio with more micellar aggregation compared to the monoester fraction micelles. For the mixed

474

component samples, the CMCs are different from those of the individual components and no significant

475

temperature dependence is observed.

476 477

Acknowledgements: The authors are thankful for useful discussions with Dr. Paul Butler and Dr. Stijn Koshari.

478

Certain commercial equipment, instruments, or materials (or suppliers, or software, …) are identified in this paper

479

to foster understanding. Such identification does not imply recommendation or endorsement by the National

480

Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily

481

the best available for the purpose. This manuscript was prepared under the partial support of the cooperative

482

agreements 70NANB15H260 and 70NANB10H256 from NIST, U.S. Department of Commerce and NSF Graduate

483

Research Fellowship Program funding. Y. L. acknowledges the support by the Center for High Resolution Neutron

484

Scattering (CHRNS), a partnership between the National Institute of Standards and Technology and National

485

Science Foundation under Agreement No. DMR-1508249. Experimental data shown in the figures can be obtained

486

upon request.

487

References:

488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529

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Table 1: PS20 and PS80 Fatty Acid Contents by European Pharmacopoeia Fatty acid ester

% PS20

Caproic (C6)

≤1

CH3(CH2)4COOH

Caprylic (C8)

≤10

CH3(CH2)6COOH

Capric (C10)

≤10

CH3(CH2)8COOH

Lauric (C12)

40-60

Myristic (C14)

14-25

≤5

CH3(CH2)12COOH

Palmitic (C16)

7-15

≤16

CH3(CH2)14COOH

Palmitoleic (C16)

%PS80

a,b

Chemical Formula

CH3(CH2)10COOH

≤8

CH3(CH2)5CH=CH(CH2)7COOH

Stearic (C18)

≤7

≤6

CH3(CH2)16COOH

Oleic (C18:1)

≤11

≥58

CH3(CH2)7CH=CH(CH2)7COOH

Linoleic ( C18:2)

≤3

≤18

CH3(CH2)4CH=CH(CH2)CH=CH(CH2)7COOH

≤4

CH3(CH2)CH=CH(CH2)CH=CH(CH2)CH=CH(CH2)7COOH

Linolenic (C18:3) b

−65. Council of Europe. European pharmacopoeia (Ph. Eur.) European Medicines Agency, 5th ed., 2005; 2267−71.

a Con venti on USP. The Unite d State s Phar maco peia. USP 36 NF. 2013; 31: 2160

Table 2: Solution physicochemical parameters of PS20 and PS80 fractions Parameters

PS20M

PS20D

PS80M

PS80D

CPS (mg/mL)

2.00

2.00

2.00

2.00

CPS (mM)

1.63

1.42

1.53

1.26

1228

1410

1310

1574

MW (g/mol) ρPS (10 Å )

0.591

0.512

0.549

0.464

ρCore (10 Å )

-0.013

-0.013

0.041

0.041

ρShell(10 Å )

0.704

0.704

0.704

0.704

6

-2

6

6

-2

-2

Table 3: CMCs of PS20M in D2O measured via SANS. And % decrease refers to the decrease of CMC with increasing temperature.

Temperature (°C)

PS20M CMC

% Decrease

4

2.05 ± 0.10

-

22

1.50 ± 0.08

27

40

1.28 ± 0.05

38

50

1.22 ± 0.04

40

Figure 1: Chemical formulas (Hewitt et al., 2011;) and schematics of solution morphology of the mono- and diester components of all laurate (C12:1) PS20 and all oleate (C18:1) PS80. The fractions in this study are: (a) PS20M: POE sorbitan monolaurate and (b) PS20D: POE sorbitan dilaurate (c) PS80M: POE sorbitan monooleate and (d) PS80D: POE sorbitan dioleate. The monoester fraction contains one fatty acid tail and the diester fraction contains two fatty acid tails, (e) PS20M and PS80M form approximately ellipsoidal shaped micelles with lower aggregation numbers, (f) due to the two-tail geometry and higher hydrophobicity, PS20D and PS80D fractions form micelles with higher aggregation number compared to the monoester fractions and have a flatter geometry compared to the other fractions.

Figure 2: SANS patterns of (a) PS20M, (b) PS20D, (c) all laurate PS20, (d) PS80M, (e) PS80D, and (f) all oleate PS80 at pharmaceutically relevant temperatures: 4, 22, 40 and 50 °C indicated by different symbols: diamonds, triangle, square, and circle respectively. The black lines are the ellipsoidal core-shell model fits to the data. PS20M at 4 °C shows flatter curve. With increasing temperatures, the shape and the scattering intensity at low-Q change. PS20D demonstrates low Q upturn which indicates higher propensity of micellar aggregation. All laurate PS20 data show an overall increase in scattering intensity. PS80M fraction shows a slight change with increasing temperatures. PS80D shows a significant low-Q upturn at 50 °C which indicates micellar morphology change with higher aggregation number. All oleate PS80 sample also shows increase in absolute scattering intensities with increasing temperatures.

Figure 3: (a) Radius of gyration, Rg and (b) aggregation number, NAgg, of PS20M, PS20D, and all laurate PS20 at 4, 22, 40 and 50 °C are plotted on the left and (c) Rg and (d) NAgg of PS80M, PS80D, and all oleate PS80 at the aforementioned temperatures are plotted on the right. (Note: error bars are included in the tables)

Figure 4: SANS patterns of the mixed components of (a) PS20M + PS20D and (b) PS80M + PS80D at 4, 22, 40 and 50 °C. Different sample mixtures and temperatures are indicated by color and symbols. (c) Radius of gyration (Rg) and (d) aggregation number (NAgg) determined via Guinier law for PS20M + PS20D, and PS80M + PS80D.