High Resolution Capillary Isoelectric Focusing Mass Spectrometry

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins, And Monoclonal Antibodies with a Flow-through Microvi...
0 downloads 0 Views 1MB Size
Subscriber access provided by University of Sussex Library

Article

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides, Proteins and Monoclonal Antibodies with a Flow-Through Microvial Interface Lingyu Wang, Tao Bo, Zheng-xiang Zhang, Guanbo Wang, Wenjun Tong, and David D. Y. Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02175 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24 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

Analytical Chemistry

1

High Resolution Capillary Isoelectric Focusing Mass Spectrometry Analysis of Peptides,

2

Proteins and Monoclonal Antibodies with a Flow-Through Microvial Interface

3 4

Lingyu Wang1,2, Tao Bo3, Zhengxiang Zhang3, Guanbo Wang1, Wenjun Tong1*, and David Da

5

Yong Chen1,2*

6 7

1. College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, China 210023

8

2. Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1

9

3. Thermo Fisher Scientific, 7th Floor, Building F, Tower West, Yonghe Plaza, No. 28 Andingmen

10

Street East, Beijing, China 100007

11 12 13 14 15 16 17 18 19 20 21 22

* To whom correspondence should be addressed:

23

Wenjun Tong, Tel.: +8625 8589 1319, Email: [email protected]

24

David Da Yong Chen, Tel: +1 604 822 0878, Email: [email protected]

1

ACS Paragon Plus Environment

Analytical Chemistry 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 26

Abstract

27

Capillary isoelectric focusing directly coupled to high resolution mass spectrometry (cIEF−MS)

28

provides information on amphoteric molecules, including isoelectric point and accurate mass, which

29

enables structural interrogation of biopolymer pI variants. The coupling of cIEF with MS was

30

facilitated by a flow-through microvial interface, made by stainless steel with high chemical

31

resistance and mechanical robustness. Two on-column electrolyte configurations of cIEF−MS were

32

demonstrated using peptide and protein pI markers. The pI resolution was 0.02 pH unit in the pH

33

range of 5.5 to 7.0, with no anti-convective reagent (glycerol) added. High resolution Orbitrap

34

detector provides mass spectra for mid-sized proteins (< 30 kDa), enabling deconvolution with high

35

accuracy for IEF-focused low abundance species. Charge heterogeneity of therapeutic monoclonal

36

antibodies (mAb) is one of the most important attributes in the biopharmaceutical industry, and it is

37

routinely monitored by IEF and fractionation based methods. As a proof of concept, the commercial

38

formulation of infliximab was directly analyzed using cIEF−MS for separation and online

39

identification of mAb charge variants. The main intact antibody species along with two basic and

40

one acidic variants were observed, and their accurate molecular weights (Mw) recorded by MS

41

detector readily revealed the structural differences of these variants. Variants with 0.1 unit in pI

42

difference and 1 Dalton difference in molecular weight were readily resolved. The deconvoluted

43

intact Mw values showed ppm level accuracy compared to theoretical predictions.

44

2

ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24 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

45

Analytical Chemistry

1. Introduction

46 47

Capillary electrophoresis (CE) utilizes electric field to drive the separation of analytes based on their

48

electrophoretic mobilities, which are proportional to their charge-to-size ratio.1 With the same

49

instrument, capillary isoelectric focusing (cIEF) can be performed to separate amphoteric analytes

50

(e.g., peptides and proteins) based on their different isoelectric points (pI),2 and it is currently the

51

second most frequently performed working mode to capillary zone electrophoresis (CZE).3 Capillary

52

electrophoresis mass spectrometry (CE–MS) is a complementary technology to the widely used

53

liquid chromatography mass spectrometry (LC–MS) because of its low sample consumption, high

54

resolving power, and distinct separation mechanism which provides more information on more

55

hydrophilic molecules.4-7 In addition, high resolution MS provides additional structural interrogation

56

capability using accurate mass and tandem MS functionality.8 As the prime example of high

57

resolution full mass scanner, Orbitrap mass analyzers offer adjustable superb resolution with high

58

stability in mass accuracy.9

59 60

Because of the requirement for stable operations is different for liquid phase CE and gas phase

61

electrospray ionization (ESI), the online coupling of CE–MS is not as straightforward as that of LC–

62

MS. Since the first demonstration of CE-MS in 1987, which employed a coaxial configuration with a

63

sheath liquid, there has been 3-decades of effort to improve the robustness and sensitivity of the

64

hyphenated instrument.4,5,7 Some recent work has shown that the limit of detection (LOD) of

65

approximately 600 peptide molecules, which is close to the LOD of laser-induced fluorescence, can

66

be achieved.10,11 A sheathless design with an etched porous tip directly served as the ESI sprayers

67

has also shown remarkable sensitivity and have found many applications in biopharmaceutical

68

research.12,13 Microfluidic CE–MS has also been demonstrated, and has been used for intact antibody

69

analysis.14 We developed a beveled-tip non-symmetrical emitter with a flow-through microvial

3

ACS Paragon Plus Environment

Analytical Chemistry 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

70

interface, which utilized a stainless steel needle as the ESI sprayer.15 The hydrodynamic flow pattern

71

inside the microvial retained peak shape characteristics of eluted analytes while the beveled emitter

72

geometry maintains stable electrospray over a wide flowrate range.16,17 Several CE–MS systems to

73

online couple cIEF–MS have been demonstrated since 1995.18,19 The flow-through microvial

74

interface has been used for cIEF–MS analysis of proteins, and the buffer composition including the

75

amount of glycerol and ampholyte were optimized.20 Similar conditions were used by another group

76

recently to demonstrate that monoclonal antibodies can be characterized by cIEF-MS using a pulled

77

tip glass sprayer,21 which was also used for cIEF tandem MS analysis of digested protein lysates and

78

intact proteins has also been reported.22-24

79 80

The combination of cIEF separation and ESI-MS detection is analogous to 2-dimensional gel

81

electrophoresis (2D GE).18 Meanwhile, it eliminates the needs for hands-on operations, and has

82

accurate mass measurement stemming from state-of-the-art mass spectrometers.19 Earlier works

83

showed that the catholyte can be manually changed to acidic CE–MS sheath liquid, and volatile

84

acids and bases (e.g., acetic acid, formic acid, ammonium hydroxide, etc.) can be used instead of

85

inorganic ones for MS compatibility.18,25-27 The online coupling of cIEF to Fourier transform ion

86

cyclotron resonance (FT-ICR) MS demonstrated for the first time the benefit of the highly accurate

87

isotopic resolving power in protein analysis.25 Later, a sandwich configuration with the analyte

88

segment sandwiched by anolyte and catholyte was developed for full automation.20,28,29 Conventional

89

IEF additives (e.g., methyl cellulose) minimize the convection/diffusion-related band broadening to

90

maintain IEF resolution during mobilization of focused bands to the MS detector, but common anti-

91

convective reagents inevitably diminished ionization efficiency, and introduced unacceptable MS

92

contamination.19 Glycerol was found to be suitable as it can maintain the IEF resolution and is

93

largely MS compatible.28

94

4

ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24 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

Analytical Chemistry

95

Therapeutic monoclonal antibodies (mAb) that are proven effective in the treatment of malignant

96

cancers and chronic autoimmune diseases significantly benefit patients.30,31 Unlike synthesis-based

97

production of small molecule drugs, recombinant mAbs are expressed by biologically engineered

98

hybridoma cell lines in vitro.32 Stemming from the complex nature of biologics production and

99

purification, various in vivo and in vitro modifications result in the existence of micro-heterogeneity,

100

including N-terminal cyclization, C-terminal lysing clipping, N-glycosylation, glycation, oxidation

101

of methionine/tryptophan, asparagine degradation and cysteine-related post translational

102

modifications (PTM).33 It gives rise to concerns about diminished potency of antigen binding and

103

increased risk of immunogenicity.33,34 Even though the modifications seem very small compared to

104

the tremendous size of intact mAbs, most of them cause observable shifts on isoelectric points (pI)

105

of typically 0.1 to 0.2 pH unit.35 To guarantee the safety and reliability of therapeutic biologics,

106

analysts in industry often use cIEF with point or whole column optical detection for charge

107

heterogeneity characterization.36 cIEF is the current gold standard for mAb characterization, but the

108

absorbance traces provide no structural information, requiring laborious fractionation for further

109

structural interrogation of separated charge variants. cIEF–MS analysis can readily fulfill the needs

110

of online micro variant identification.

111 112

In this report, the latest progress of cIEF−MS analysis facilitated by the flow-through microvial

113

interface is described. Peptide pI markers were used to demonstrate that resolution of 0.02 pH unit

114

with chemical mobilization can be achieved without the use of glycerol, and 0.08 pH unit with

115

glycerol added using combined hydrodynamic and chemical mobilization. With spectrum

116

deconvolution, the isotopically resolved charge envelopes of protein pI markers can be obtained.

117

Infliximab, a therapeutic mAb, was directly subjected to the cIEF–MS analysis without desalting.

118

With the Orbitrap MS, four mAb charge variants were successfully profiled on the total ion

5

ACS Paragon Plus Environment

Analytical Chemistry 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

119

chromatograph (TIC) and infliximab variant structures of 13 unique Mw values were identified with

120

ppm-level mass accuracy.

121 122

2. Experimental

123 124

2.1. Chemical and materials

125 126

Deionized water (18.0 MΩ/cm) was collected from a Milli-Q ultrapure water purification system

127

(Millipore, Bedford, MA, USA) and utilized for the preparation of all aqueous solution. Methanol

128

(LC-MS grade), formic acid (FA, 99%, ACS grade), acetic acid (AcOH, 99%, ACS grade),

129

ammonium hydroxide (NH3, ACS grade, 25%, w/w), sodium hydroxide (NaOH, ACS grade),

130

concentrated hydrogen chloride solution (HCl, ACS grade) arginine (Arg, >99% ACS grade),

131

iminodiacetic acid (IDA, >99%), hydroxyl propyl cellulose (HPC, averaged Mw 80,000), and

132

glycerol (ACS grade) were all purchased from Sigma Aldrich (Nepean, ON, Canada). Two brands of

133

carrier ampholyte (CA) were used in this work: Fluka (pH 3~10, 40% w/w in water, purchased from

134

Sigma Aldrich) and ampholyte (pH 3~10, 0.36 meq/mL·pH, GE Healthcare, Chicago, IL, USA).

135

Bare silica capillaries (50 µm i.d.) were purchased from Polymicro Technologies (Molex, IL, USA).

136

Agilent Technologies (Beijing, P. R. China) kindly provided the polyvinyl alcohol (PVA) coated

137

capillaries (50 µm i.d.) in this work. The IEF capillaries were neutrally coated with hydrophilic

138

polymers (PVA or HPC) to suppress electro-osmotic flow (EOF). The HPC coating protocol was

139

slightly modified from what was reported by Shen.37

140 141

Peptide pI marker kit was purchased from SCIEX Separations (Brea, CA, USA) containing five

142

synthetic peptide markers (5.0 mg/mL in water) with pI values of 10.0, 9.5, 7.0, 5.5, and 4.1. The

143

protein pI markers purchased from Sigma Aldrich (Shanghai, China) were ribonuclease A from

6

ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24 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

Analytical Chemistry

144

bovine pancreas (RNase-A, pI 9.6), myoglobin from equine heart (Myo I pI 6.8, Myo II pI 7.0),

145

carbonic anhydrase I human (HCA I, pI 6.5), bovine carbonic anhydrase (BCA, pI 6.1) and β-

146

lactoglobulin (Beta-lac, pI 5.1). Another protein mixture commercialized as a slab gel IEF reference

147

was purchased from Bio-Rad Laboratories (Hercules, CA, USA), consisting of cytochrome C (Cyt,

148

pI 9.6), human hemoglobin (HH, type C pI 7.5, type A pI 7.1), equine myoglobin (Myo I pI 7.0, Myo

149

II pI 6.8), human carbonic anhydrase (HCA, pI 6.5), Bovine carbonic anhydrase (BCA, pI 6.0) and

150

β-lactoglobulin (Beta-lac, pI 5.1). The prescription drug infliximab (Remicade, 100 mg/vial

151

formulation) was kindly provided by Agilent Technologies (Beijing).

152 153 154

2.2. Instrumentation

155

Two CE−MS platforms were used for cIEF−MS experiments. The first set used an Agilent 7100 CE

156

system and an Agilent 6530 QTOF mass spectrometers, which was a Quadrupole Time of Flight MS

157

(Q-TOF MS). The chemical modifier solution was delivered by an additional Agilent 1260 NanoLC

158

pump. The flow-through microvial interface was installed on a home-made 3D-adjustable stage. The

159

second platform consists of a Sciex PA800+ CE system and a Thermo Scientific Orbitrap Fusion

160

Lumos mass spectrometer which is a tribrid platform of quadrupole, Orbitrap and linear ion trap. The

161

modifier was delivered by a syringe pump. The flow-through microvial interface was directly fixed

162

on the Thermo Scientific Nanospray Flex Ion Source. All systems realized fully automation, with

163

exception of cIEF-MS using the second configuration (vide infra). The protein concentration was

164

measured by a Thermo NanoDrop 2000 spectrophotometer at 280 nm or was based on analytical

165

balance. The charge envelope deconvolution relied on the algorithm of Thermo Scientific Protein

166

Deconvolution 4.0.

167 168

2.3. cIEF−MS configurations

169

7

ACS Paragon Plus Environment

Analytical Chemistry 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

170 171

Figure 1. (A) The first cIEF−MS configuration for protein and mAb analyses. The separation

172

capillary was sequentially filled with anolyte, sample and catholyte segments, forming a sandwich

173

structure. The flow-through microvial inside the ESI emitter was linked to a nanoLC or a syringe

174

pump for the chemical modifier delivery. (B) The focusing stage for the second configuration. The

175

capillary was filled with the ampholytes–analyte mixture. The CE inlet vial contained anolyte while

176

the flow-through microvial was filled by the basic catholyte. (C) The mobilization stage for the

177

second configuration. The pressure on the CE inlet and the migration of formate ions towards the

178

positive electrode initialized mobilization.

179 180

Two cIEF−MS configuration were used, as illustrated in Figure 1. The first configuration is fully

181

automated by employing a sandwich structure to eliminate the catholyte–modifier exchange, as

182

depicted in Figure 1(A).28 Three segments including the catholyte, sample and anolyte were

183

sequentially injected into the capillary and the segment lengths were controlled empirically by 8

ACS Paragon Plus Environment

Page 8 of 24

Page 9 of 24 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

Analytical Chemistry

184

injection time. Because of the shorter sample length (e.g., 37 to 40 cm), the focusing can be

185

completed in 30 min. Upon completion of focusing, mobilization was commenced by starting the

186

chemical modifier infusion and slightly pressurizing the CE inlet reservoir. The second configuration

187

is described in Figure 1(B and C). In this configuration, the whole capillary was filled with the IEF

188

sample while the sprayer flow-through microvial was filled with the basic catholyte by the syringe

189

pump. During the focusing step as illustrated by Figure 1(B), the catholyte was slowly pumped to

190

refresh the microvial which aims at washing away the electrolysis by-products. After the focusing

191

current was stabilized at a low value, the analyte mobilization was initialized by replacing catholyte

192

with the acidic chemical modifier composed of formic acid, methanol and water.

193 194

Capillary clean-up between the runs was crucial to the reproducibility and it was conducted prior to

195

and right after the cIEF–MS analysis. The rinsing procedure includes a mild water rinse and a 3.0 M

196

urea rinse. Acetic acid and ammonium hydroxide were utilized for MS compatibility. Glycerol (20%

197

v/v) used in place of the traditional anti-convention reagents, is mixed with anolyte, sample and

198

catholyte solutions.20,28,29 In the analysis of peptides which were less prone to surface adsorption,

199

glycerol was eliminated to maximize ESI efficiency.

200 201

2.4. cIEF−MS of peptide pI markers, protein pI markers and infliximab samples

202 203

Both methods were used for the analysis of peptide markers with the CE–Orbitrap MS platform with

204

HPC coated capillaries (1st configuration 80 cm and 2nd configuration 70 cm). The anolyte solution

205

was 1.0% (w/v) ammonia (0.59 M) in water, and the catholyte solution was 1.0% (v/v) acetic acid

206

(0.17 M) in water. The sample consisted of 2.0 mM IDA as the anolyte stabilizer, 2.5 mM arginine

207

as the catholyte stabilizer, as well as the Fluka ampholyte (2.5% v/v, stock solution) and five

208

synthetic peptide pI markers (pI 10.0, 9.5, 7.0, 5.5, 4.1) with the concentration of 50 µg/mL each. All

9

ACS Paragon Plus Environment

Analytical Chemistry 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

209

freshly prepared samples were vortexed and centrifuged by 12,000 rpm for 10 min at 10 ℃ prior to

210

analysis. The focusing stage utilized 30 kV (400 or 429 V/cm) for 45 min. During focusing, 4.5 kV

211

ESI voltage was turned off and 0.20 µL/min catholyte solution was pumped to refresh the flow-

212

through microvial. The chemical modifier solution was 0.50% (v/v) formic acid (0.13 M) in 80%

213

methanol (v/v) and its infusion (0.60 µL/min) was started right after the completion of focusing. The

214

anolyte-sample-catholyte length ratio for the sandwich configuration was 4:10:6, and its mobilization

215

utilized 25.5 kV plus 0.5 psi. There was 20% (v/v) glycerol added in the anolyte, sample and

216

catholyte segments using the sandwich configuration, but there was no glycerol for the other one.

217

The mobilization was based on 15 kV voltages only for the whole-column filling configuration. The

218

Orbitrap resolution was set at 60,000 and the ion transfer tube temperature was set as 300 ℃. In the

219

analyses using GE Healthcare ampholyte, the sample solution contained 1.0 mM IDA, 5.0 mM

220

arginine, and 2.5% (v/v) ampholyte stock solution. The focusing step was 30 kV for 35 min. All

221

other experimental details were the same as those when the Fluka ampholyte was used.

222 223

For the protein pI markers, the sandwich configuration using a 75.0 cm PVA coated capillary was

224

used on the CE–Q-TOF MS platform. Glycerol (20%, v/v) was added into the anolyte, sample and

225

catholyte. The anolyte and catholyte were 1.0% (v/v) acetic acid and 1.0% (w/v) ammonium

226

hydroxide. The sample contained 5.0 mM IDA, 5.0 mM arginine and 2.5% (v/v) Fluka ampholyte.

227

The protein markers were RNase-A (0.40 mg/mL), Myo (0.40 mg/mL), HCA I (0.40 mg/mL), BCA

228

(0.40 mg/mL) and Beta-lac (0.37 mg/mL). The length ratio of anolyte, sample and catholyte was

229

4:10:6. The focusing step utilized 30 kV (400 V/cm) for 25 min. The chemical modifier was 1.0 %

230

(v/v) formic acid in 80% methanol with a flowrate of 1.0 µL/min and it started 25 min after the

231

focusing was started. After another 5 min, a 50 mbar (0.73 psi) pressure was applied to accelerate the

232

mobilization. The ESI voltage, Fragmentor voltage and Skimmer voltage were 5.0 kV, 300 V and 65

233

V, respectively. The 300 ℃ drying gas flowrate was 4 L/min.

10

ACS Paragon Plus Environment

Page 10 of 24

Page 11 of 24 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

Analytical Chemistry

234 235

For protein analysis using the Orbitrap MS, 70.0 cm HPC coated capillaries were used for the

236

sandwich configuration and 55.0 cm HPC coated one for the other configuration. For the second

237

configuration, the catholyte solution was 1.0% (w/v) ammonia in 39% water and 60% methanol, and

238

the added methanol was to facilitate electrospray at the beginning of MS detection. The Bio-Rad

239

protein mixture concentration was about 0.10 mg/mL for each of the 6 proteins. The focusing step

240

was 30 kV for 30 to 45 min, depending on the sample segment length. The modifier was 2.0% (v/v)

241

formic acid (0.52 M) in 80% methanol with a flowrate of 0.60 µL/min. The chemical mobilization

242

was achieved by 25.5 kV. The ESI voltage, ion transfer tube temperature and Orbitrap resolution

243

were 4.5 kV, 350 ℃ and 300,000. Unless noted, all were same to the peptide analysis.

244 245

In the analyses of infliximab, the sandwich configuration and an 80 cm PVA coated capillaries were

246

utilized with the CE–Orbitrap MS system. In the 40-cm sample segment, the concentration of the

247

solid formulation was 0.50 mg/mL, and mAb concentration was measured to be 0.042 mg/mL. The

248

preservative salt and medicinal additive occupied 91.6% (w/w) of the infliximab formulation. Five

249

peptide pI markers of 10 µg/mL were then added to the solution. The 0.60 µL/min modifier pumping

250

was initialized right after the 30 min, 30 kV focusing step. After another 5 min, 0.5 psi (34 mbar)

251

was applied for combined hydrodynamic/chemical mobilization. The Orbitrap MS continuously

252

switched the m/z ranges between 100 to 1,500 for pI marker detection (Resolution 120,000) and

253

1,500 to 5,000 (Resolution 30,000) for mAb detection. The ESI voltage, ion transfer tube

254

temperature and in-source fragmentation were 4.5 kV, 350 ℃ and 100 V. All other parameters were

255

from the same as the cIEF–MS analysis of proteins.

256 257

3. Results and discussion

258

11

ACS Paragon Plus Environment

Analytical Chemistry 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

259

3.1. cIEF−MS analysis of peptide pI markers

260 261

To demonstrate the feasibility of our online CE–MS coupling strategies and to determine the pI

262

resolution of the proposed configurations, five synthetic peptide pI markers were subjected to cIEF–

263

MS analysis. It is a good practice to use these peptide for a test run to ensure the separation and

264

detection system is functional properly, especially because adding these markers in the real sample

265

has no adverse effect for the analysis. The second configuration involved the change from the

266

catholyte to the modifier in the flow-through microvial, because it replaced the distal end reservoir of

267

catholyte with microvial chamber inside the ESI emitter. The volume needed for the complete

268

solution exchange was measured to be 2.90 ± 0.15 µL (n = 6) and it brought a circa 3-min delay in

269

mobilization. Upon the completion of solution exchange by the acidic modifier, chemical

270

mobilization and electrospray ionization were initialized because the formate ions begun to replace

271

the hydroxide ions and titrate the pH gradient, and the methanol in the modifier facilitate the

272

desolvation of IEF eluents. The second coupling strategy demonstrated highest pI separation power

273

cIEF-MS can provide under optimal conditions. Because it used the whole separation capillary for

274

the sample segment, the volume injected was approximately three times higher than that of the

275

routine cIEF−UV protocol with sample segment of only 30 cm. Summarized in Table 1, pI

276

resolution as low as 0.021 pH unit can be achievable. Longer focusing and mobilization time is

277

needed, which were typically 45 min and 80 min using a 70 cm HPC coated capillary.

278

12

ACS Paragon Plus Environment

Page 12 of 24

Page 13 of 24 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

Analytical Chemistry

279 280

Figure 2. (A) cIEF−MS analysis of five peptide pI markers using the Fluka ampholyte. The three

281

insets were the zoomed in peaks for pI 5.5 marker in three consecutive experiments. (B) cIEF−MS

282

analysis of 5 peptide markers using the GE Healthcare ampholyte. The inset was the extracted ion

283

chromatogram (EIC) for ampholyte molecules with m/z 294.166. The pI values of ampholyte EIC

284

peaks were assigned according to the mobilization times of adjacent peptide markers.

285 286

Two different carrier ampholytes (pH 3 to 10) were utilized, and anti-convective reagent was not

287

needed for the cIEF–MS analysis of peptides. With Fluka ampholyte and chemical mobilization

288

voltage of 15 kV, very sharp EIC peaks of the pI 7.0, 5.5 and 4.1 markers were observed. As to the

289

sharpest pI 5.5 peptide marker, the smallest full width at half maximum (FWHM) was 2.7 seconds,

290

and 4.1 ± 0.8 seconds in average (n = 11 in 4 days). The pI 5.5 peak standard deviation was 0.21

291

seconds (0.0035 min), which is the highest reported cIEF-MS resolution to date. cIEF using

292

fluorescent detection has demonstrated sharper peaks with peak standard deviation of 0.13 s (0.0021

293

min) of the pI 6.5 marker.38 The peak symmetry in this work was 0.99 ± 0.35 (n = 11), demonstrating

294

that hydrodynamic flow patterns in the flow-through microvial contribute little to the peak distortion,

295

hence maintaining the IEF resolution with post-column detection. The pI resolution near the 13

ACS Paragon Plus Environment

Analytical Chemistry 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 14 of 24

296

migration time of pI 5.5 marker was as high as 0.021 pH, and in general 0.033 ± 0.007 pH (n = 11).39

297

To our best knowledge, this is the highest resolution achieved for online cIEF−MS analysis. As to

298

the pI 7.0 marker, the minimum FWHM was 4.1 seconds, and the average was 8.3 ± 3.1 seconds (n =

299

11). The smallest standard deviation was 0.24 seconds (0.0040 min). The corresponding pI resolution

300

was 0.024 pH, and on average 0.034 ± 0.009 pH. In the sandwich configuration with glycerol

301

addition, slightly lower pI resolution was observed using the combined hydrodynamic and chemical

302

mobilization. For pI 7.0 marker, even though the FWHMs were similar, the pI resolution of 0.08 pH

303

unit was 2.4 times lower. It’s been reported that discrete mAb variants were typically spaced out

304

with 0.2 pH intervals.35 With the system presented in this work, a 0.1 pH difference was sufficient to

305

be separated for the charge heterogeneity analysis of mAb therapeutics.

306 Table 1. Resolving power of the cIEF-MS method for peptides Ampholyte pI markers min. ∆pH ∆pH FWHM (s) 7.0 0.024 0.034 ± 0.009 8±3 Fluka1 5.5 0.021 0.033 ± 0.007 4.1 ± 0.8 9.5 0.055 0.07 ± 0.02 7.8 ± 0.3 2 GE 7.0 0.030 0.04 ± 0.01 3.7 ± 0.5 5.5 0.037 0.05 ± 0.02 3.7 ± 0.5 9.5 0.11 0.12 ± 0.03 13 ± 3 3 Fluka 7.0 0.06 0.08 ± 0.02 9±2 5.5 0.09 0.12 ± 0.03 9±1 1,2 3

n 11

3

3

70-cm sample plug, 2nd configuration, chemical mobilization

40-cm sample plug, 1st configuration, chemical mobilization and 0.5 psi pressure

307 308

As to the GE Healthcare ampholyte, it provided linear pH gradient with adequate pI resolution at

309

high pH regions. Sharp peaks of pI 10.0 and pI 9.5 markers were observed with slightly higher

310

amount of arginine spacer. The R2 between mobilization time and pI values was 0.99961 and 0.996 ±

311

0.004 in triplicate tests, respectively. By extracting ampholyte cations with m/z 294.166, nine peaks

312

appeared on the EIC profile as shown in the inset of Figure 2(B), which were structural isomers with

14

ACS Paragon Plus Environment

Page 15 of 24 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

Analytical Chemistry

313

pI differences. It is worth noting that the averaged m/z of GE ampholyte cations was approximately

314

400 while in the other types it was 500.

315 316

3.2. cIEF−MS analysis of protein pI markers

317 318

To minimize the convection-induced band broadening and prevent protein aggregation and

319

precipitation, solutions inside the separation capillary were mixed with 20% v/v glycerol for protein

320

analysis. The effect of amount of glycerol on protein separation has been studied previously.20,21

321

Because of the increased viscosity, a small pressure was applied to accelerate the analyte

322

mobilization.

323 324

With the CE–Q-TOF MS, one complete run of six protein markers can be finished within 90 min

325

with the sandwich configuration, including sample loading and IEF separation. The mobilization

326

stage needed an extra 50 mbar (0.73 psi) pressure to be completed within 30 min. As shown in

327

Figure 3(A), myoglobin I (pI 6.8) and myoglobin II (pI 7.0) were baseline separated, showing the

328

proper IEF resolution at the neutral region. Two minor myoglobin peaks were revealed, showing the

329

complexity of its charge variants. Two partially separated peaks for human carbonic anhydrase I

330

were detected, indicating two isoforms. The EICs were based on three to five most abundant signals

331

in the charge envelopes, and triplicate tests all have highly similar profiles. Compared with the

332

previous study of the identical marker cohort without the small pressure applied, the pI resolution

333

with pressure-assisted mobilization was well preserved at the basic and neutral regions, while the

334

peaks were somewhat broadened at the acidic region.20 The sandwich configuration is easy to

335

operate and completely automated. Therefore, it was used for the analysis of monoclonal antibodies

336

shown in the following section.

337

15

ACS Paragon Plus Environment

Analytical Chemistry 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

338 339

Figure 3. (A) The cIEF−MS analysis of a 6-protein mixture using the 1st configuration. The signal

340

intensity of RNase-A and Myo were multiplied by 10 and 0.5, respectively, for clarity of

341

presentation. (B) Analysis of a Bio-Rad protein marker mixture using the 2nd configuration on the

342

Orbitrap platform. The left inset demonstrated the isotopic pattern of Myo I + 13 H+, while the one

343

on the right was for Myo EIC. (C) Analysis of the Bio-Rad protein mixture with pure chemical

344

mobilization. The insets demonstrated the BCA EIC and the BCA + 25 H+ isotopic pattern. The

345

peaks with assigned numbers were listed in Table 2. Peaks of the same protein were labeled with the

346

same number across Panels A, B and C.

16

ACS Paragon Plus Environment

Page 16 of 24

Page 17 of 24 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

Analytical Chemistry

347 348

The Bio-Rad reference mixture contains six proteins and eight pI species, and it was slightly

349

different from the protein mixture analysis on the Q-TOF platform. The Bio-Rad sample was

350

subjected to IEF analysis using two configurations on the Orbitrap MS platform, as shown in Figure

351

3(B) and 3(C). The whole column filling configuration resulted in high pI resolution, while the

352

Orbitrap MS provided sensitive detection with up to 300,000 mass resolution. Synthetic peptide pI

353

markers were mixed with the proteins. By linear regression of the mobilization times to pI values of

354

peptide markers, the pI of other IEF peaks in Figure 3(B) was assigned and summarized in Table 2.

355

Two pI isoforms of β-lactoglobulin were observed, and the highly similar charge envelopes indicated

356

their charge variation with negligible mass shift, such as asparagine deamidation. In Figure 3, the

357

FWHM of BCA EICs were 22.0 seconds, 18.6 seconds and 7.0 seconds in Panel A, B and C,

358

respectively, and the sharpest BCA peak still had 15 points with the threshold set as 3% intensity of

359

peak maximum. The FWHM was larger in Figure 3(B) relative to Figure 3(C), suggesting that high

360

pI resolution required gentle chemical mobilization without the assisting pressure. With 300,000 MS

361

resolution, all proteins within 30,000 Da were isotopically resolved. Therefore, deconvoluted spectra

362

of all TIC peaks reliably revealed in-depth structural information by providing intact masses. Table 2

363

demonstrated the complexity of the Bio-Rad protein marker mixtures as revealed by the Orbitrap

364

detector, which is higher than that of the of 6-protein mixture analyzed on the Q-TOF platform. For

365

example, Peak 4 contained five intact masses with relative intensity > 5%. The right inset in Figure

366

3(B) was a summed EIC of myoglobin using the five highest isotopic peaks (∆ m/z < 0.5) of the

367

eight peaks of the charge envelope (+9 to +16), and it unambiguously showed a 3rd charge variant

368

with intensity of only 3.8% of the most abundant one. The Peak 2 and Peak 3 belonged to

369

hemoglobin A and C. Hemoglobin proteins dissociated into subunits (15,129 Da, 15,869 Da and

370

15,744 Da) during the ESI process in the acidic environment. We did not observe signals from intact

371

phycocyanin (Mw 232,000, pI 4.6) with the Orbitrap MS detector.

17

ACS Paragon Plus Environment

Analytical Chemistry 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 18 of 24

372

peak # 1 2

Table 2. Deconvolution results of cIEF-MS protein peaks name pI native mass (Da) and relative intensity Cyt 10.1 12361.08 90% 12347.04 10% HH-A/C

7.5

a a

3

Myo I

7.1

Myo II HCA

6.8 6.4

6

BCA

5.9

15128.72

b c

4 5

15128.73

44% 33%

15744.38

6%

16954.22 17569.88 28784.91

69% 9% 100%

d

15868.72 29% c

a

15744.38

16%

16954.24 29%

15869.66

20%

15128.73 12% 16977.23 5%

16995.28

10%

18301.7 18209.3

6% 6%

18279.69

57%

29028.43 43%

β-lac 5.8 18279.69 β-lac 4.7 18189.29 a, b, c, d The labels denote similar masses.

72% 47%

18604.05 18% 19237.65 40%

7 8

d

b

373 374

3.3. cIEF−MS analysis of infliximab, a monoclonal antibody pharmaceutical formulation

375 376

With the first configuration, we detected the charge envelopes of intact antibody in IEF eluents as

377

shown in Figure 4, demonstrating the feasibility of cIEF–MS analysis using the flow-through

378

microvial interface for analytes up to 150,000 Da. The infliximab sample with its formulation

379

containing salt and preservative additives was dissolved with deionized water and directly mixed

380

with carrier ampholyte and pI markers. The sample preparation step takes less than 20 min, including

381

the centrifugation step for particulate removal. The formulated sample contained 91.6% (w/w) salts

382

and additives, but their interference was alleviated by the desalting nature of IEF. Because half of the

383

separation capillary was assigned to the sample segment, 0.79 µL sample–ampholyte mixture (40 cm

384

× 50 µm) was injected, and the mAb consumption was only 33 ng for one complete analysis.

385 386

A combination of chemical and hydrodynamic mobilization (i.e., 25.5 kV and 0.50 psi) was applied

387

for analyte mobilization. The chemical modifier flowrate could be as low as 0.30 µL/min, but a 0.60

388

µL/min was routinely used to maintain the stable electrospray. During the focusing stage, the flow of

18

ACS Paragon Plus Environment

Page 19 of 24 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

Analytical Chemistry

389

chemical modifier brought no interference on the IEF separation. Many ionic species, such as Na+

390

from the preservative salts, could penetrate the catholyte segment and enter the interface microvial,

391

but they were washed out by the continuously flowing chemical modifier. Therefore, MS

392

contamination stemming from excipients was alleviated. By simultaneously monitoring the m/z

393

range between 100 to 1,200 and 1,500 to 5,000, both peptide markers and intact mAb was observed.

394

To maximize the sensitivity of mAb, the high mass range started at 1,500 was chosen to eliminate

395

the interference from carrier ampholyte with an averaged m/z of approximately 500, and one mAb

396

spectrum was composed of three microscans to enhance S/N.

397

398 399

Figure 4. (A) cIEF−MS TIC and five EICs of infliximab mixed with peptide pI markers on the CE–

400

Orbitrap MS platform. Five colored EICs belonged to the peptide pI markers. The markers traces

19

ACS Paragon Plus Environment

Analytical Chemistry 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 20 of 24

401

were magnified for clarity. (B) Raw EIC data of infliximab obtained with the m/z range of 3,000 to

402

5,000. The inset on the right was the averaged mass spectrum for the main IEF peak (#3). The

403

zoomed-in inset on the left demonstrated the details of the +39 peak cluster. (C) The deconvoluted

404

mass spectrum for the main peak.

405

Table 3. Deconvolution results and identified variant structures cIEF−MS peak

#1 K2

#2 K1

#3 K0 #4 Deamidation

glycoforms G0F/G0F-Man G0F/G0F G1F/G0F G1F/G1F G0F/G0F G1F/G0F G1F/G1F unknown G0F/G0F-Man G0F/G0F G1F/G0F G1F/G1F G2F/G1F unknown G1F/G0F

deconvoluted mass (Da) 148563.08 148768.77 148931.27 149094.39 148640.52 148800.44 148963.23 149103.47 148309.50 148512.86 148675.14 148837.39 148996.20 149059.89 148678.63

theoretical mass (Da) 148565.37 148768.56 148930.70 149092.84 148640.38 148802.52 148964.66 n. a. 148309.01 148512.20 148674.34 148836.48 148998.62 n. a. 148675.34

mass error (ppm) -15.4 1.4 3.8 10.4 0.9 -14.0 -9.6 n. a. 3.3 4.4 5.4 6.1 -16.2 n. a. 22.1

406 407

The maximum mAb signal (m/z 3376.35, 1.22E5) belonged to the +39 peak cluster, and it was only

408

0.014% of the highest co-eluted ampholyte signal (m/z 275.15, 8.72E8). It is the highly sensitive

409

Orbitrap MS that enabled the detection of intact mAb co-eluted with ESI-interfering carrier

410

ampholytes. In the m/z range of 3,000 to 5,000, no predominant carrier ampholyte signals were

411

observed and the mAb charge envelope was obtained with a clean baseline. Therefore, this wide EIC

412

can be considered the mAb TIC profile. At least four mAb charge variants were observed, as

413

demonstrated by Figure 4(B). The pI values of the resolved mAb peaks was assigned based on the

414

linear regression of peptide pI values to their mobilization times. In addition to the main peak, Peak 3

415

showing the intact mAb, and the two basic variants originating from C-terminal lysine clipping (∆m

20

ACS Paragon Plus Environment

Page 21 of 24 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

Analytical Chemistry

416

128 Da), an acidic variant peak (Peak 4) appeared with a small mass shift on its deconvoluted

417

spectrum relative to the main peak.40 Asparagine deamidation was responsible for this variation as it

418

caused a +1 Da mass shift together with an acidic pI shift.41 Highly sensitive detection provided

419

detailed mass spectra and the following deconvoluted intact masses were in accordance with the

420

theoretical predictions, as summarized in Table 3. In total, 15 intact molecular weight values

421

originating from glycosylation heterogeneity and charge variation have been observed following

422

strict deconvolution settings (noise rejection 99% confidence, abundance threshold 3% and mass

423

tolerance 20 ppm), and 13 of them have been unambiguously correlated to the mAb microstructures

424

by theoretical prediction of Mw values. The averaged absolute mass error was only 8.7 ppm.

425 426

4. Conclusion

427 428

In this work, capillary isoelectric focusing was directly coupled to high-resolution mass

429

spectrometers with a stainless-steel flow-through microvial ESI interface. Two cIEF–MS

430

configurations had been demonstrated. The first one sequentially introduced the anolyte, sample and

431

catholyte segments into the neutrally coated capillary in a fully automated process, and the second

432

one used the whole capillary for IEF separation. Two instrumental platforms were used for the

433

evaluation of the two cIEF−MS configurations, including a CE–Q-TOF MS system and a high-

434

resolution CE–Orbitrap MS system. With the second configuration, the pI resolution reached 0.021

435

pH unit for peptide pI markers. As to protein markers such as BSA, the FWHM was as low as 7.0

436

seconds with isotopic pattern observed, showing the high resolving power delivered by both cIEF

437

and Orbitrap MS. Based on the aforementioned cIEF−MS methodology, we characterized an intact

438

monoclonal antibody sample, infliximab with its formulation. With only 30 nano grams of mAb

439

sample consumed in a single injection, four clearly defined TIC peaks demonstrated the

440

effectiveness of the IEF separation, while thirteen unique intact mAb Mw values were identified by

441

charge envelope deconvolution and theoretical Mw prediction. The results showed that this

21

ACS Paragon Plus Environment

Analytical Chemistry 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

442

methodology offered a real alternative for the structural interrogation of mAb charge heterogeneity

443

from the currently used methods based on fractionation and following LC–MS analysis. In addition,

444

the stainless steel sprayer is robust and easy to install, potentially providing a worry free

445

environment for cIEF-MS operation.

446 447

5. Acknowledgment

448 449

The study was supported by Nanjing Normal University, National Natural Science Foundation of

450

China (Grant Number 21475061), and Natural Sciences and Engineering Research Council of

451

Canada (NSERC). LW acknowledges a Mitacs Accelerate Fellowship sponsored by PromoChrom

452

(Richmond, BC). The access to Agilent CE−MS instrumentation and the infliximab formulation

453

were kindly provided by Agilent Technologies, Beijing, China.

454 455

6. References

456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476

(1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (2) Hjerten, S. J. Chromatogr. A 1985, 346, 265-270. (3) Rodriguez-Diaz, R.; Wehr, T.; Zhu, M. D. Electrophoresis 1997, 18, 2134-2144. (4) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230-1232. (5) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574A-584A. (6) Maxwell, E. J.; Chen, D. D. Y. Anal. Chim. Acta 2008, 627, 25-33. (7) Kleparnik, K. Electrophoresis 2015, 36, 159-178. (8) Yates, J. R. Nat. Methods 2011, 8, 633-637. (9) Zubarev, R. A.; Makarov, A. Anal. Chem. 2013, 85, 5288-5296. (10) Cheng, Y. F.; Dovichi, N. J. Science 1988, 242, 562-564. (11) Sun, L.; Zhu, G.; Zhao, Y.; Yan, X.; Mou, S.; Dovichi, N. J. Angew. Chem. Int. Ed. 2013, 52, 13661-13664. (12) Moini, M. Anal. Chem. 2007, 79, 4241-4246. (13) Faserl, K.; Sarg, B.; Kremser, L.; Lindner, H. Anal. Chem. 2011, 83, 7297-7305. (14) Redman, E. A.; Batz, N. G.; Mellors, J. S.; Ramsey, J. M. Anal. Chem. 2015, 87, 2264-2272. (15) Maxwell, E. J.; Zhong, X.; Zhang, H.; van Zeijl, N.; Chen, D. D. Electrophoresis 2010, 31, 1130-1137. (16) Maxwell, E. J.; Zhong, X.; Chen, D. D. Anal. Chem. 2010, 82, 8377-8381. (17) Zhong, X.; Maxwell, E. J.; Chen, D. D. Anal. Chem. 2011, 83, 4916-4923. (18) Tang, Q.; Harrata, A. K.; Lee, C. S. Anal. Chem. 1995, 67, 3515-3519.

22

ACS Paragon Plus Environment

Page 22 of 24

Page 23 of 24 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

477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510

Analytical Chemistry

(19) Huhner, J.; Lammerhofer, M.; Neususs, C. Electrophoresis 2015, 36, 2670-2686. (20) Zhong, X.; Maxwell, E. J.; Ratnayake, C.; Mack, S.; Chen, D. D. Anal. Chem. 2011, 83, 87488755. (21) Dai, J.; Lamp, J.; Xia, Q. W.; Zhang, Y. R. Anal. Chem. 2018, 90, 2246-2254. (22) Zhu, G.; Sun, L.; Keithley, R. B.; Dovichi, N. J. Anal. Chem. 2013, 85, 7221-7229. (23) Sun, L.; Zhu, G.; Dovichi, N. J. Anal. Chem. 2013, 85, 4187-4194. (24) Zhu, G.; Sun, L.; Dovichi, N. J. J. Sep. Sci. 2017, 40, 948-953. (25) Yang, L.; Lee, C. S.; Hofstadler, S. A.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 1998, 70, 3235-3241. (26) Jensen, P. K.; Pasa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D. Anal. Chem. 1999, 71, 2076-2084. (27) Kuroda, Y.; Yukinaga, H.; Kitano, M.; Noguchi, T.; Nemati, M.; Shibukawa, A.; Nakagawa, T.; Matsuzaki, K. J. Pharm. Biomed. Anal. 2005, 37, 423-428. (28) Mokaddem, M.; Gareil, P.; Varenne, A. Electrophoresis 2009, 30, 4040-4048. (29) Lecoeur, M.; Gareil, P.; Varenne, A. J. Chromatogr. A 2010, 1217, 7293-7301. (30) Chames, P.; Van Regenmortel, M.; Weiss, E.; Baty, D. Br. J. Pharmacol. 2009, 157, 220-233. (31) Weiner, L. M.; Surana, R.; Wang, S. Z. Nat. Rev. Immunol. 2010, 10, 317-327. (32) Gaughan, C. L. Mol. Divers. 2016, 20, 255-270. (33) Liu, H.; Ponniah, G.; Zhang, H. M.; Nowak, C.; Neill, A.; Gonzalez-Lopez, N.; Patel, R.; Cheng, G.; Kita, A. Z.; Andrien, B. mAbs 2014, 6, 1145-1154. (34) Liu, H.; Gaza-Bulseco, G.; Faldu, D.; Chumsae, C.; Sun, J. J. Pharm. Sci. 2008, 97, 2426-2447. (35) Hosken, B. D.; Li, C.; Mullappally, B.; Co, C.; Zhang, B. Anal. Chem. 2016, 88, 5662-5669. (36) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Van Dorsselaer, A.; Sanglier-Cianférani, S. Anal. Chem. 2013, 85, 715-736. (37) Shen, Y. F.; Smith, R. D. J. Microcolumn Sep. 2000, 12, 135-141. (38) Dada, O. O.; Ramsay, L. M.; Dickerson, J. A.; Cermak, N.; Jiang, R.; Zhu, C.; Dovichi, N. J. Anal. Bioanal. Chem. 2010, 397, 3305-3310. (39) Righetti, P. G. Isoelectric focusing: theory, methodology and application; Elsevier, 2000; Vol. 11. (40) Antes, B.; Amon, S.; Rizzi, A.; Wiederkum, S.; Kainer, M.; Szolar, O.; Fido, M.; Kircheis, R.; Nechansky, A. J. Chromatogr. B 2007, 852, 250-256. (41) Huang, L.; Lu, J.; Wroblewski, V. J.; Beals, J. M.; Riggin, R. M. Anal. Chem. 2005, 77, 14321439.

511

23

ACS Paragon Plus Environment

Analytical Chemistry 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

512

For TOC Only

513

24

ACS Paragon Plus Environment

Page 24 of 24