Discovery of unknown modifications in recombinant ...2015/04/10 · Discovery of Unknown...

Discovery of Unknown Modifications in Recombinant Monoclonal Antibodies

By Chris Chumsae

B.A. Chemistry, Assumption College

M.S. Chemistry, Northeastern University

A dissertation submitted to

The Faculty of

The College of Science of

Northeastern University

In partial fulfillment of the requirements

For the degree of Doctor of Philosophy

April 10, 2015

Dissertation directed by

Zhaohui Sunny Zhou

Associate Professor of Chemistry and Chemical Biology

ii

Abstract of Dissertation

Recombinant monoclonal antibodies have become one of the most important classes of

biotherapeutics today. Due to their complexity, they must be produced by cellular expression.

They are subject to various posttranslational modifications during manufacture, formulation and

storage. Occasionally, the antibody may encounter a reactive molecule which in turn, results in a

new variant. The application of modern analytical strategies including charge-based approaches

and mass spectrometry in a defined workflow can effectively elucidate the presence of new

species and help to understand the chemistry and underlying root cause. This dissertation will

discuss the discovery of three novel chemical modifications in a recombinant monoclonal

antibody therapeutic. First, methylglyoxal from cellular metabolism was found to react and

modify the side chains of susceptible arginine residues. The adduction increased the mass by 54

or 72 daltons and caused the antibody to elute earlier in weak cation exchange chromatography

(WCX). Second, the N-terminus of the antibody was determined to be modified by citric acid

used as an excipient in the formulation buffer during storage. The modification increased the

mass by 156 or 174 daltons and also resulted in an earlier elution of the modified species by

WCX. Lastly, the antibody was modified by the addition of vitamin C (ascorbic acid) added to

the cell culture as a supplement. It was determined that ascorbic acid degraded to xylosone

which in turn reacted with primary amines in the antibody. The modification increased the

molecular weight by 130 or 148 daltons and once again caused the antibody to elute earlier.

These results were confirmed using stable isotope labeled ascorbic acid. All of these variants

were initially observed as minor peaks in the chromatogram but the application of mass

spectrometry strategies and spiking studies proved that such unassuming changes are worth

further investigation.

iii

ACKNOWLEDGEMENTS

I thank my advisor, Professor Zhaohui Sunny Zhou, for his commitment to helping me

grow as a scientist and for his advice. I am indebted to him for introducing me to the Industrial

Ph.D. program at Northeastern University and encouraging me to pursue a doctoral degree at

Northeastern University. Sunny has helped me to stand tall as a scientist. . In addition, Sunny

has shown me by example what it means to be successful in science and in life.

I thank my co-advisor, Dr. Czeslaw Radziejewski, for his mentorship and support at

AbbVie. If not for his trust, I would not have been afforded the opportunities to contribute to

many projects which have given me the chance to flourish as a scientist. In addition, I want to

thank Czes for his friendship and guidance over the past eight years.

I thank Dr. Hongcheng Liu for his encouragement and belief in me to be successful in

pursuit of a Ph.D. degree. I am also appreciative to Dr. David Lee for encouraging words and

stimulating scientific discussions. I thank Gary Welch and Dr. Li Malmberg for supporting me

in my pursuit of a doctoral degree. I thank Drs. Peter Moesta, George Avgerinos and Ralph

Lambalot for believing in me and seeing the value in this endeavor.

I thank my committee members, Professors Barry Karger, Jeff Agar and Joe Zaia for their

helpful suggestions regarding my work.

I thank my colleagues, Dr. Anton Manuilov, Liqiang Lisa Zhou, Dr. Randall Burton, Dr.

Ivan Coreia, Dr. David Lee, Dr. Nathan Brown, David Ouellette, Leslie Alessandri, Dr.

Dongdong Wang, Yang Shen, Yun Zhang, Reema Raghevendra, Kathy Gifford, Georgeen Gaza-

iv

Bulseco, Karen Hurkmans, Michelle Deloreto and Aima Acquah. I want to give a special thanks

to Dr. Yu Zhou and Haly Raharimampionona for their hard work, dedication and their

friendship.

I thank my collaborators at AbbVie, Dr. Patrick Hossler, Sean McDermott, Chris Racicot,

Dr. Kartik Subramanian, Dr. Wei Lian, Dr. Chen Wang, Dr. Mia Wang, Dr. Mike Naill and

Lihua Yang.

I thank the members of SUNNYLAND, Dr. Tianzhu Indi Zang, Dr. Min Liu, Wanlu Qu,

Shanshan Liu, Kalli Catcott, Kevin Moulton and Mike Pablo.

I thank my parents for their support and belief in me. Their encouragement and pride

helped me to persevere. I only wish my dad was still alive to see me get here.

To my son Kyle, I hope my efforts will encourage him to pursue his own dreams, persist

when times get tough and show him that you are never too old to take on new challenges.

Lastly, I thank a very special person. Qing, you have encouraged me in so many ways in

life and have always believed that I would achieve this goal. I want to thank you for being by

my side on this journey. I look forward to many more journeys together.

v

Table of Contents

Abstract ii

Acknowledgements iii

Table of Contents v

List of Schemes x

List of Tables xi

List of Figures xii

List of Abbreviations and Symbols xv

Chapter 1 Introduction and Background 1

1.1 Introduction 1

1.2 Reactive Molecules 5

1.2.1 Free Thiols 5

1.2.2 Homocysteine and N-Homocysteinylation 7

1.2.3 Glycation 9

1.2.4 Advanced Glycation Endproducts 11

1.2.5 1,3-Bisphosphoglycerate 13

1.2.6 Peroxides and Lipid Peroxidation 15

1.2.7 Peroxide-mediated Sulfenylation 17

1.2.8 The complexity of a biological system 17

1.2.9 Intracellular influences and Reactive Oxygen Species 20

1.2.10 Detecting the Unexpected 21

1.3 Analytical Strategies to Discover the Unexpected 23

1.3.1 Analytical Detective Work 23

vi

1.3.2 Weak Cation Exchange Chromatography 23

1.3.3 Reduced Liquid Chromatography/Mass Spectrometry 25

1.3.4 Spiking Studies with Likely Molecules 26

1.3.5 Peptide Mapping with Detection by Mass Spectrometry 27

1.3.6 Agreement between the spiked agent and the initial findings 30

1.3.7 Stable Isotope Labels 31

1.3.8 Interpretation of all of the Findings 32

1.3.9 Summary 32

1.4 References 34

Chapter 2 Arginine Modifications by Methylglyoxal: Discovery in a Recombinant

Monoclonal Antibody and Contribution to Acidic Species 45

2.1 Abstract 45

2.2 Introduction 46

2.3 Materials and Methods 49

2.3.1 Materials 49

2.3.2 Weak cation exchange (WCX) chromatography 49

2.3.3 LC-MS analysis of light and heavy chains of antibody 49

2.3.4 Tryptic and Lys-C digestion 50

2.3.5 LC-MS analysis of peptides 50

2.3.6 Methylglyoxal reaction with monoclonal antibody 51

2.3.7 Global Analysis of MGO Modification 52

2.3.8 Quantification of Modified Peptides 52

2.4 Results and Discussion 53

vii

2.4.1 New acidic species induced by changes in cell culture 53

2.4.2 Localization of the modification sites 58

2.4.3 Deduction of the Structure of the Modification 61

2.4.4 Incubation with Authentic MGO to generate reference 61

2.4.5 Comparison of the in vitro references and the cell culture samples 64

2.4.6 Global Analysis of Modifications by MGO 64

2.4.7 Effects of MGO Modification on Charge 67

2.4.8 Relevance to other Contributing Factors to Acidic Species 70

2.4.9 Formation of MGO as a New Critical Attribute for Cell Culture 70

2.5 Conclusions 72

2.6 References 73

2.7 Supporting Information 78

Chapter 3 Discovery of a Chemical Modification by Citric Acid in a Recombinant

Monoclonal Antibody 87

3.1 Abstract 87

3.2 Introduction 88


3.3.1 Materials 90


3.3.3 LC-MS analysis of reduced antibody 91

3.3.4 Tryptic digestion 91


3.3.6 Reaction of citric acid buffer with the monoclonal antibodies 92

3.3.7 N-Terminal Variants and Additional IgG’s 93

viii


3.4.1 Unexpected covalent modifications by citric acid 96

3.4.2 Reduced LC/MS Analysis 98

3.4.3 Peptide Mapping and Determination of Sites of Modifications in antibody A 101

3.4.4 Elucidation of the chemical nature of the modifications 105

3.4.5 Reactions in citrate buffers (as compared to formulation) 105

3.4.6 Prevalence of the citrate modification 109

3.4.7 Influence of pH 111

3.4.8 Selectivity of amines 111

3.5 Conclusions 112

3.6 References 113

Chapter 4 When Good Intentions Go Awry: Modification of a Recombinant Monoclonal

Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbate

119

4.1 Abstract 119

4.2 Introduction 120


4.3.1 Materials 123


4.3.3 LC-MS analysis of reduced antibody 123

4.3.4 Fractionation of Acidic Species 124

4.3.5 Tryptic digestion 124


4.3.7 in vitro incubation of Monoclonal antibody with ascorbic acid 125

ix

4.3.8 Regio-labeled ascorbate to elucidate the mechanism of the modification 126


4.4.1 Ascorbate supplement in cell culture induced new acidic variants 127

4.4.2 Reduced LC/MS analysis of antibody stored in ascorbate buffer 129

4.4.3 Tryptic mapping and LC/MS/MS detection 134

4.4.4 Elucidation of the modification agent as xylosone 148

4.4.5 Incubation of antibody with 13

C Regio-labeled ascorbate 150

4.4.6 Chemical Nature of the Modifications 154

4.4.7 Cell Culture Media Additives 158

4.4.8 Acidic Species 161

4.5 Conclusions 161

References 163

Chapter 5 Perspectives and Future Directions 168

5.1 Perspectives and Future Directions 168

5.2 References 173

x

List of Schemes

Scheme 2-1: Chemical modification of arginine by methylglyoxal (MGO). 48

Scheme 3-1: (I) Formation of a citric acid anhydride intermediate from citric acid and the

subsequent reaction of the N-terminal amine with the anhydride. 95

Scheme 4-1: The formation of xylosone from ascorbic acid and subsequent reaction with

susceptible primary amines. 149

Scheme 4-2: Reaction scheme of cyclic xylosone with a protein primary amine. 156

Scheme 4-3: Reaction scheme of acyclic xylosone with a protein primary amine. 157

xi

List of Tables

Table S2-1: Results of manual search of methylglyoxal modified peptides found in the

recombinant antibody. 84

Table 3-1: The percentage of citric acid modification found in the N-terminus in different

antibodies. 110

Table 4-1: Peptides identified with modifications by xylosone. 135

Table 4-2: List of relevant cell culture media compounds. 159

xii

List of Figures

Figure 1-1: Representation of some common post-translational modifications which may

occur in recombinant monoclonal antibodies. 2

Figure 1-2: Structures of free thiols found in biological systems including recombinant cell

culture. 4

Figure 1-3: Formation of homocysteine thiolactone and the subsequent product with

susceptible lysine residues. 6

Figure 1-4: Mechanism of glycation. 10

Figure 1-5: Common advanced glycation end products derived from glucose. 12

Figure 1-6: The product of a reaction between lysine and 1,3-bisphosphoglycerate. 14

Figure 1-7: The structures of reactive electrophiles following lipid peroxidation. 16

Figure 1-8: The structure of cysteine, cys-sulfenic acid amd cys-sulfinic acid. 19

Figure 2-1: WCX chromatogram of the recombinant monoclonal antibody after protein A

purification (top and bottom traces were from cell culture M (modified) and N (normal),

respectively, at day 9). 55

Figure 2-2: Deconvoluted mass spectra of the light chain and heavy chains in fraction 1 and

fraction 2. 56

Figure 2-3: Representative MS/MS mass spectra of peptides modified by MGO. 59

Figure 2-4: WCX chromatogram of a purified Lys-0 antibody incubated with and without

authentic MGO. 63

Figure 2-5: Peak intensity of all methylglyoxal-modified peptides generated by Lys-C

digestion of fraction 1 and fraction 2. 66

Figure 2-6: Calculated pKa of the core group of arginine and the two products of arginine

modification by methylglyoxal. 69

Figure 3-1: The weak cation exchange chromatogram of the recombinant monoclonal

antibody formulated with and without citrate. 97

Figure 3-2: Mass spectra of the light chain from reduced LC/MS analysis. 99

Figure 3-3: HPLC-MS analysis of a tryptic digest of the recombinant monoclonal antibody A following storage at 40 ºC for 6 months in citrate buffer pH 5.2. 102

xiii

Figure 3-4: De novo sequencing of doubly charged peptides corresponding to unique m/z’s of

2052.91 (Middle pane) and 2034.90. 103

Figure 3-5: MS/MS spectra of Antibody A light chain N-terminal peptide for the native, +174

Da, +156A Da, and +156B Da citrate modifications. 104

Figure 3-6: The weak cation exchange chromatogram of Antibody A incubated in with citric

acid (formulation and buffer alone). 107

Figure 4-1: WCX-10 chromatograms of the recombinant monoclonal antibody control (top,

no ascorbic acid) and supplemented with 0.1, 1 and 3 mg/mL of ascorbate in cell culture,

respectively. 128

Figure 4-2: Light Chain spectra from reduced LC/MS analysis of unfractionated Antibody A.

131

Figure 4-3: LC/MS analysis of the recombinant monoclonal antibody light chain after

reduction of antibody. 132

Figure 4-4: Deglycosylated heavy chain spectra from reduced LC/MS analysis of fractionated

Peak A. 133

Figure 4-5: Extracted ion chromatograms (XIC) from the recombinant monoclonal antibody

tryptic map corresponding to the doubly charged light chain N-terminal peptide and N-terminal

peptides with +148 Da and +130 Da adducts. 136

Figure 4-6: The MS/MS spectra of the light chain N-terminal tryptic peptides from Peak A

fractionated from the recombinant monoclonal antibody supplemeted with 3 mg/mL ascorbate.

137

Figure 4-7: Extracted ion chromatograms from the heavy chain N-terminal peptide. 139

Figure 4-8: MS/MS spectra from the heavy chain N-terminal peptide. 140

Figure 4-9: Extracted ion chromatograms of a lysine containing peptide. 141

Figure 4-10: MS/MS spectra of a lysine containing peptide. 142

Figure 4-11: Relative susceptabilities of representative peptides modified by xylosone. 143

Figure 4-12: Comparison of XIC from xylosone modified N-terminal peptides from light chain

formed during cell culture and during in vitro incubation with ascorbate. 145

Figure 4-13: Comparison of XIC from xylosone modified N-terminal peptides from heavy

chain formed during cell culture and during in vitro incubation with ascorbate. 146

xiv

Figure 4-14: Reduced LC/MS of light chain incubated with xylosone over time. 147

Figure 4-15: A: Structures of the four different isotopic isoforms of ascorbate used to probe the

structure of the +130 Da and +148 Da adducts. 151

Figure 4-16: MS/MS spectra of light chain modified by xylosone derived from ascorbic acid or

ascorbic acid with 13C at C1, C2 or C3, respectively. 152


ascorbic acid with 13

C at C1, C2 or C3, respectively. 152

xv

List of Abbreviations and Symbols

13C

13C labeled carbon

1.3-BPG 3-bisphosphoglycerate

4-HNE 4-hydroxynonenal

AGE advanced glycation end products

Arg arginine

Asn asparagine

Asp aspartic acid

C18 octadecylsilane

CDR1 complementary determining region 1



CH1 Constant heavy domain 1



CHO Chinese hamster ovary

CID Collision induced dissociation

Cys cysteine

Da Dalton

DDA data dependent acquisition

DHAP dihydroxy acetone phosphate

DTT Dithiothreitol

EIC extracted ion chromatogram

FA formic acid

G-3-P glyceraldehyde-3-phosphate

Gln glutamine

Glu glutamic acid

GRAS generally regarded as safe

G0F core fucosylated biantennary glycan with 0 terminal galactose

HC heavy chain

HCl hydroch

HCD hard collision dissociation

IgG1 immunoglobulin G

isoD isoaspartic acid

isoAsp isoaspartic acid

kDa kilodalton

LC light chain

LC/MS liquid chromatography/mass spectrometry

Lys-0 antibody with 0 C-terminal lysine



Lys-C endoproteinase Lys-C

mAbs monoclonal antibodies

mg milligram

m/z mass to charge ratio

mL milliliter

xvi

MaxEnt Maximum Entropy

MGO methylglyoxal

MOLD methylglyoxal lysine dimer

MS/MS tandem mass spectrometry

MWCO molecular weight cut off

nm nanometer

nM nanoMoles

pgk 3-phosphglycerol lysine

PNGaseF protein N-glycanase

PTM posttranslational modification

Pyro-Glu pyroglutamate

R arginine

Q1 quadrupole 1

Q2 quadrupole 2 (collision cell)

Q-Tof quadrupole-time of flight mass spectrometer

ROS reactive oxygen species

TFA trifluoroacetic acid

TIC total ion current

tRNA transfer ribonucleic acid

UPLC ultra high pressure liquid chromatography

UV ultraviolet

VH variable heavy domain

WCX weak cation exchange chromatography

XIC extracted ion chromatogram

1

Chapter 1: Introduction and Background

1.1 Introduction

Recombinant monoclonal antibodies have become one of the most important classes of

biotherapeutics due to their target specificity and the ease of manufacture using standard

protocols1-2

. Thus, they have been widely studied due to their tremendous growth and

applicability across broad therapeutic areas3. As a consequence, the field of bioanalytical

chemistry as it applies to the analysis and characterization of these proteins has evolved

significantly over the last twenty years. In particular, techniques which delve into the primary,

secondary, tertiary and quaternary structure have uncovered many chemical changes which occur

due to multiple factors.

There are four sub-classes of immunoglobulin G, however, the most common sub-class

used in the generation of antibody drugs is IgG1. Recombinant monoclonal antibodies are

comprised of two heavy chains and two light chains which are assembled together by four

interchain disulfide bonds. The heavy chain is comprised of four immunoglobulin domains

stabilized by intrachain disulfide bonds. Three of these domains (CH1, CH2 and CH3) are

conserved and the binding domain is variable (VH) with the greatest variability occurring in the

binding regions known as the complementary determining regions or CDR1, CDR2 and CDR3.

In addition, the heavy chain has a glycosylation site in the CH2 domain. The presence of this

glycosylation serves to provide stability to the domain and may influence the domain

conformation4. The light chain consists of a variable and constant domain, the VL and CL,

respectively. The heavy chain is approximately 450 amino acids in length and the light chain is

2

approximately 215 amino acids in length. The structure of a recombinant monoclonal antibody

is depicted in Figure 1-1.

Figure 1-1: The structure of a recombinant monoclonal antibody

CH1

VH

CL

VL

CH2

CH3

CDRs

Heavy Chain

LightChain

N-linked glycosylation

Inter-chain disulfide bonds

Fc Region

Fab Region

3

Recombinant monoclonal antibodies are far more complicated than small molecule drugs

due to the sheer size plus additional posttranslational modifications. Such modifications may be

driven enzymatically or chemically and increase the molecular heterogeneity of recombinant

mAbs. These are discussed in detail in several recent reviews5-8

and include but are not limited

to deamidation9-14

, glycation15-20

, incomplete C-terminal lysine processing21-25

, N-linked

glycosylation26-34

and O-linked glycosylation35-40

, C-terminal amidation41

, oxidation42-45

and N-

terminal pyroglutamate formation5-8, 13, 46-51

. Representative posttranslational modifications are

depicted in Figure 1-2.

Recent reports have elucidated a new classification of posttranslational modification by

endogenous reactive molecules. In general, these are far less studied with respect to protein

analysis of recombinant biotherapeutics. Such species may be reactive metabolites, unreactive

species capable of forming a reactive intermediate or molecules which may degrade into reactive

species. In addition, such posttranslational modifications may be the result of the intracellular

conditions such as those which lead to reactive oxygen species (ROS).

4

Figure 1-2: Representation of some common post-translational modifications which may occur

in recombinant monoclonal antibodies.

5

1.2 Reactive Species

1.2.1 Free Thiols

One of the most familiar classes of reactive molecules is the free thiols52-53

. Reduced

thiols are very potent nucleophiles which are capable of forming disulfide bonds with other free

thiols in proteins or with intact disulfide bonds thereby disrupting the intended cysteine pairing

and inducing a scrambling event52-54

. Examples of products of free thiols include

cysteinylation55-57

and glutathionylation58-59

. Common free thiols are shown in Figure 1-3. The

reaction of one of these species with a catalytic cysteine resulting in a disulfide bond will ablate

protein function56, 60-61

. In addition, free thiols may promote disulfide bond scrambling which

has the potential of destabilizing the protein or altering its structure62-63

. In all of these cases, an

increase in molecular weight will be observed imparted by the addition of these reactive species.

6

Figure 1-3: Structures of free thiols found in biological systems including recombinant cell

culture.

7

1.2.2 Homocysteine thiolactone and N-Homocysteinylation

A precursor to cysteine biosynthesis is homocysteine which may either form methionine

following methylation or cystathionine, the precursor of cysteine, following a vitamin B6

mediated complex with serine64

. In addition to its potential to react with free cysteines within

the primary structure of a protein65

, it may also be transformed into homocysteine thiolactone

through the action of methyonyl tRNA synthetase66-67

(see Figure 1-4). Homocysteine

thiolactone may then react with primary amines in a protein, specifically lysine, resulting in N-

homocysteinylation which modifies the side chains of lysine and increases the mass by 117

daltons66-67

.

8

Figure 1-4: Formation of homocysteine thiolactone and the subsequent product with susceptible

lysine residues.

9

1.2.3 Glycation

Carbohydrates may react with proteins when their reducing end (electrophic aldehyde)

encounters a reactive primary amine to form a Schiff base (aldimine) as shown in Figure 1-515-16

.

An Amidori rearrangement will form a stable ketoamine exhibiting a mass increase of 162

daltons16, 68

. Although some level of glycation is unavoidable because glucose is added to the

cell culture as a primary energy source, the tertiary structure of the protein may facilitate the

Amidori rearrangement and lead to significant levels of glycation15

. To this point, reports of

histidine residues16

and acidic residues15

facilitating glycation suggest their side chains may

promote the abstraction of a proton from the glucose C2 atom which initiates a rearrangement of

the aldimine to a more stable ketoamine.

Glycation has also been reported when glucose was used in the formulation of a

biotherapeutic molecule69

. More surprisingly, glycation was associated with a protein drug

which was formulated in sucrose70-71

. In these reports, the glycosydic bond between the fructose

and glucose sub-units was hydrolyzed during storage thus freeing the reducing end of glucose

and enabling glycation to occur. Thus, glycation resulted from an unexpected source.

10

Figure 1-5: Mechanism of glycation. The epsilon primary amine of lysine attacks the reducing

end of glucose forming a Schiff base following dehydration. An Amidori rearrangement

produces the more stable ketoamine.

11

1.2.4 Advanced Glycation Endproducts

Advanced glycation end products (AGE) are another set of reactive molecules derived

from glucose which may contribute to molecular heterogeneity72

. Advanced glycation end-

products have their analytical roots based in the analysis of blood samples from patients with

diabetes73

. High glucose levels facilitate their formation74

. An increase in the oxidative stress

exerted on the cell may promote the formation of AGE’s as this may retard the glyoxylase I/II

pathway due to reduced glutathione75

. Common AGEs are shown in Figure 1-5 (I).

Specifically, methylglyoxal and glyoxal have been associated with chemical

modifications of proteins74, 76-77

. Methylglyoxal is a by-product of glycolysis75

. The two

glycolytic intermediates of the catalysis of fructose bisphosphate aldolase are glyceraldehyde-3-

phosphate (G-3-P) and dihydroxy acetone phosphate (DHAP)78

. If DHAP loses its phosphate

group before the catalysis of triose phosphate isomerase can occur then methylglyoxal will form.

In addition to arginine residues, MGO has been shown to have reactivity with lysine and cysteine

residues albeit to a lesser extent79

. Advanced glycation end products have been implicated in

protein cross-linking between arginine and lysine residues resulting in a pentosidine or between

two lysine residues forming a methylglyoxal lysine dimer (MOLD)80

. Representative products

of AGEs and protein are shown in Figure 1-6 (II).

12

Figure 1-6: I. Common advanced glycation end products derived from glucose. II.

Representative products of AGE’s with nucleophilic amino acid side chains.

13

1.2.5 1,3-Bisphosphoglycerate

The possibility that other reactive intermediates or metabolites prevalent in biological

systems may contribute to new posttranslational modifications is an intriguing one.

Interestingly, 1,3-bisphosphoglycerate (1.3-BPG), the glycolytic intermediate generated from the

catalysis of glyceraldehyde-3-phosphate dehydrogenase has been reported as another example of

a reactive metabolite81

. 1,3-BPG is a strong electrophile due to its acylphosphate moiety81-82

.

Specifically, 1,3-BPG has been shown to react with the lysine primary amine forming 3-

phosphglycerol lysine (pgk) as shown in Figure 1-7.

14

Figure 1-7: The product of a reaction with 1,3-bisphosphoglycerate and the epsilon primary

amine of lysine.

15

1.2.6 Peroxides and Lipid Peroxidation

Other examples of reactive electrophiles in biological systems are the products of lipid

peroxidation83

. Oxidative stress may lead to an increase in reactive oxygen species.

Consequently, peroxides have been shown to react with unsaturated fats forming a variety of

reactive electrophiles84-85

. The nucleophilic side chains of cysteine, histidine and lysine have

been reported to react with these species through Michael addition resulting in carbonylation86-87

.

Specifically, protein modifications by reactions with 4-hydroxynonenal (4-HNE),

malondialdehyde and oxononenal (see Figure 1-8) have been reported88-92

. Such modifications

may be far more ubiquitous following oxidative stress than previously thought. Recombinant

monoclonal antibodies which recognize protein adducts formed by these reactive electrophiles

have proven to be a valuable tool93-94

.

16

Figure 1-8: I. The structures of reactive electrophiles following lipid peroxidation. These

species may react with the side chains of lysine, cysteine and histidine. II. The product of

cysteine side chain attack on the electrophilic center of 4-HNE.

17

1.2.7 Peroxide-mediated Sulfenylation

Related to oxidative stress and the production of reactive oxygen species is peroxide-

mediated sulfenylation of free cysteine residues95

. Hydrogen peroxide may react with free

sulfhydryls to form Cys-sulfenic and Cys-sulfinic acid96-97

(see Figure 1-9). The formation of

these posttranslational modifications has emerged as another signaling mechanism available to

biological systems analogous to phosphorylation95

. These species have proven to be labile and

thus difficult to measure using today’s analytical approaches98

, thereby requiring the

implementation of selective trapping reactions for their detection. Specifically, trapping of

sulfenylated products may be achieved by stabilization with dimedone or iododimedone99

. The

stabilized products have subsequently been analyzed by proteomics workflows. These labile

PTM’s remind us that

1.2.8 The complexity of a biological system

Biological systems are extremely complicated with countless chemical processes in

constant flux. In addition, changes in the external environment can induce major shifts in the

cellular metabolic flow. It would be a Herculean feat to catalogue all of these processes and in

turn identify a comprehensive list of all reactive molecules and species. What is possible,

however, is to gain an understanding of general chemical reactivity that may occur between

proteins and chemical functionalities. By taking a diligent approach and open mind towards

seemingly insignificant changes in standard analytical assessments, analytical protein scientists

can continue to expand the list of these variants and their underlying chemistry. The discovery

of chemical modification which may impact the function of the therapeutic or increase the risk of

18

immunogenicity can mitigate potential risks to patients and ensure that biopharmaceutical

companies release drugs which meet the highest product quality.

19

Figure 1-9: The structure of cysteine, cys-sulfenic acid, cys-sulfinic acid and cys-sulfonic acid.

20

1.2.9 Intracellular influences and Reactive Oxygen Species

The intracellular environment of CHO cells has been implicated in the formation of

reactive oxygen species (ROS). Reactive oxygen species are normal by-products of cellular

metabolism100

. Specifically, the formation of superoxide in the mitochondria has been linked to

the presence of enzymes that can donate electrons to O2 thus forming O2·- (superoxide)

101. For

such transformations, the ratio of NADH/NAD+ is elevated facilitating an increase in the

abundance of enzymes reacting with O2. During periods of cellular stress, the levels of ROS

may increase dramatically102

. Reactive oxygen species have been associated with damage to

DNA, RNA, and proteins103

. In addition, reactive oxygen species have been linked to lipid

peroxidation and advanced glycation end products104-105

.

At high ROS levels, cells produce more glutathione and thioredoxin that can act to

scavenge reactive oxygen species106

. These mechanisms are regulated by the intracellular redox.

Specifically, glutathione disulfide and thioredoxin cannot control ROS accumulation, thereby

further raising ROS levels that lead to cellular damage. Upon oxidative stress, the cell will

attempt to convert GS-SG to GSH to increase antioxidant reserves. Enzymes such as glutathione

reductase facilitate this transformation107

.

Cysteine starvation has been linked to glutathione deficiency108

. The cysteine moiety is

the functional residue within glutathione making it a critical component. A lack of intracellular

cysteine due to cellular starvation thus leads to a depletion in glutathione. As a result, the levels

of intracellular ROS may increase.

Both glutathione depletion induced by high levels of reactive oxygen species and cysteine

starvation resulted in a similar cell culture phenotype. Both have a clear role with respect to the

accumulation of reactive oxygen species. In addition, both of these events may be directly

21

related where lower cysteine levels but not necessarily achieving complete starvation coupled to

increased demand of glutathione due to ROS can lead to a depletion of cysteine, glutathione or

both. As a result of these factors, the glyoxylase I/II pathway will be down regulated thereby

increasing cellular levels of methylglyoxyl109

.

The implementation of a chemically defined media, which is discussed in Chapter 2,

could play a role in either of these scenarios. Cellular starvation and cysteine depletion induced

by insufficient media components in a chemically defined media may explain depletion of

glutathione and thus the retardation in the glyoxylase I/II pathway. In addition, chemically

defined media has been shown to induce higher cell densities thus increasing the overall culture

metabolic demands. In these cases, the culture exhibited signs of oxidative stress and increased

reactive oxygen species. The formation of ROS may have been a result of cellular starvation or

may have directly induced through a futile attempt to arrest the oxidative stress leading to

depletion of reduced glutathione and cysteine. More studies would be necessary to better

understand how either of these scenarios contributes to the formation of methylglyoxal.

1.2.10 Detecting the Unexpected

All of these examples remind us that reactive molecules may arise from an unexpected

source and facilitate an increase in unwanted protein modifications.. Accordingly, the chemical

modification of a recombinant monoclonal antibody presents new challenges to the

biotechnology industry. These therapeutic ‘magic bullets’ are destined for administration to

patients therefore their subsequent product quality is held to a high bar. Even low levels of

heterogeneity may be problematic to the patient due to loss of potency, increased risk of

immunogenicity, or both. Chemical modifications to the complementary determining regions

22

can ablate antibody function. In addition, these chemical modifications are not limited to the

expressed biotherapeutic, albeit, they will exist as the most abundant protein in the cell culture.

Key host cell proteins may also be affected by modification of these reactive molecules. Such

events can affect the overall health and expression levels of the cell culture. It is therefore

important to understand the underlying product quality and to extrapolate root cause from the

analytical observations.

23

1.3 Analytical Strategies to Discover the Unexpected

1.3.1 Analytical Detective Work

Todays’ analytical strategies are well suited to perform deep characterization of proteins

and protein drugs. Technologies such as mass spectrometry, liquid chromatography and

capillary electrophoresis are capable of measuring very subtle differences in the primary

structure of a recombinant monoclonal antibody. Applied work flows consisting of

complimentary methodologies can be used to uncover molecular heterogeneity and root cause

through a stepwise approach. Specifically, chromatographic approaches can separate a

recombinant monoclonal antibody with a chemical modification from the native antibody.

Subsequently, analytical tools may be applied to understand the nature of the variant. Of all

these tools, mass spectrometry has emerged as the most powerful analytical tool enabling the

assessment of the entire primary structure of a recombinant monoclonal antibody. Discrete

changes can be determined at the residue level facilitating the identification of new discoveries in

this important class of biotherapeutics.

1.3.2 Weak Cation Exchange Chromatography

Proteins have a global charge which is due to the cumulative effects of its protonated

basic residues and its deprotonated acidic residues in the primary structure and is represented as

the isoelectric point. The pH and local environment can influence these charges. Additionally,

one must distinguish between those charged residues which are located on the surface of the

protein and thus exposed. In addition to local environment, chemical modifications can

influence the surface charge of the protein6, 8, 20

. For instance, if a basic amino acid is covalently

modified, the side chain pKa may become depressed potentially leading to a deprotonation at

24

that site16, 66

. A protein in this scenario would exhibit a reduced number of basic charges on its

surface.

A very powerful technique in discerning changes in a recombinant monoclonal antibody

primary structure is weak cation exchange chromatography21, 25

. Weak cation exchange columns

contain polymeric beads coated with carboxylate groups110

. It is the electrostatic interaction

between protonated basic residues on the protein surface (lysine, arginine, histidine) and these

carboxylates which cause the protein to bind to the column. At first glance, it appears to be a

low resolution technique as compared to today’s modern mass spectrometry workflows.

However, it is remarkably sensitive to any site specific changes in the number of surface charges

on the antibody. For instance, it has been shown that a single site of glycation on a hyper-

susceptible lysine in a recombinant monoclonal antibody resulted in a decrease in pI presumably

due to the loss of this protonation site15

.

The interpretation of low levels of a chemical modification is hampered by the presence

of the acidic species which comprise a highly heterogeneous population5-6, 8, 19, 21

. Contributors

include terminal sialylation39, 111-113

, asparagine deamidation13-14

, and glycation15-16

just to name a

few. The appearance of a new species may exist as a slight increase in the UV profile which

may be difficult to discern from the already complex unresolved chromatographic region. In

addition, it may be challenging to differentiate an unresolved low abundance chromatographic

peak from differences due to fluctuations in the chromatographic performance.

Often, a variant may only exist in trace amount making the discovery and analysis of the

variant difficult. Isolating minor peaks or regions of subtle changes in the chromatographic

separation has proven to be a valuable strategy.43, 114

. A specific region which may be changing

can be isolated for subsequent analysis by mass spectrometry. In this way, the overall

25

complexity will be reduced and the variant which maybe present can be enriched. It is important

to note that other endogenous contributors within this region may also be present therefore it is

prudent to compare LC/MS spectra against a control. Additionally, the shift in the retention time

may provide clues about the distribution of a modification on a recombinant monoclonal

antibody114

. For example , if more than one peak is isolated, the relative retention time shifts and

corresponding levels of a variant may begin to establish the effects that multiple modifications

may have on the surface charge.

1.3.2 Reduced Liquid Chromatography/Mass Spectrometry

Reduced LC/MS analysis is a key step in determining root cause of differences observed

in the weak cation exchange chromatogram. The antibody sample is treated with a reducing

agent such as 10 mM DTT which breaks the interchain disulfide bridges between the heavy and

light chains. The sample is introduced into the mass spectrometer using reversed phase

chromatography which will resolve the light chain from the heavy chain. Within the

electrospray source, the sample will become desolvated and stripped of negative counter ions to

enhance ionization. A quadrupole-time of flight mass spectrometer (Q-Tof) used in MS mode

will measure a distribution of multiply charged species corresponding to the eluting light chain

or heavy chain. The spectral pattern is deconvoluted using the Maximum Entropy (MaxEnt)

algorithm which reconstructs the single charged spectrum.

The technique provides the molecular weight of the recombinant monoclonal antibody

light chain and heavy chain as well as any other molecular weights due to the modifications

associated with the primary structure. For example, mass shifts of +16 daltons or +162 daltons

can be attributed to the formation of methionine sulfoxide43

or glycation16

, respectively. In

26

addition, there are databases which exist and have extensive lists of changes in mass and a

reported root cause. Two such databases are Deltamass

(http://www.abrf.org/index.cfm/dm.home) and Unimod (http://www.unimod.org). It is possible

to make an initial assignment to an observed variant based on mass shift especially if the analysis

was performed on a high mass accuracy instrument.

1.3.4 Spiking Studies with Putative Reactive Species

From a quote; “analytical scientist are good at finding what they know”. This statement

is quite true. In other words, it is easy to find something if you know what to look for.

However, should the observed mass shift in the recombinant protein drug be unfamiliar and not

easily recognized, then identifying the underlying change to the primary structure can be quite

challenging. It is now up to the analytical scientist to perform some detective work and try to

pinpoint specific differences which may have occurred in the recombinant monoclonal antibody

during cell culture or long term storage.

One must keep an open mind when it comes to such challenges. If a potential candidate,

i.e., reactive species, can be identified from what is known about the changes in molecular

weight and the conditions from the cell culture, storage, etc, then a spiking study of the candidate

or panel of candidates may be performed. Specifically, if the reactive molecule or its stable

precursor is available, it can be spiked into a pure sample of the recombinant monoclonal

antibody. In practice, the ideal species of this “pure sample” will be the major peak which

typically consists of the recombinant monoclonal antibody without C-terminal lysine on both of

its heavy chains and devoid of the acidic species. Following an appropriate incubation with the

http://www.unimod.org/

27

spiked agent, the sample may be analyzed by weak cation exchange chromatography and by

reduced LC/MS. The results can be compared to the initial observations. In addition, any shifts

in the chromatographic profile may be directly attributed to changes in the primary structure

induced by the presence of the spiked agent. If the retention times between the initial sample

which showed subtle changes and the stressed sample are in good agreement, the possibility

increases that this reactive molecule is the root cause.

1.3.5 Peptide Mapping with Mass Spectrometry

A peptide map is often referred to as the protein fingerprint. The digestion of a reduced

and alkylated recombinant monoclonal antibody with a highly specific protease will generate a

predicatble and reproducible peptide map. Trypsin is commonly used to digest the reduced and

alkylated protein at the C-terminus of lysine and arginine residues generating peptides which can

be separated on a reversed phase C18 (octadecylsilane) column. High efficiency separations

may be achieved by using small particle size (1.7 µ) and the application of ultra-high pressure

liquid chromatography (UPLC). The peptides will be separated based on their relative

hydrophobicity as the percent of organic mobile phase increases. Eluting peptides may be

detected by UV absorbance at 214 nm before being introduced into the electrospray source.

Q-TOF and LTQ-Orbitrap mass spectrometers may be used to perform peptide mapping.

Both of these instruments are capable of performing MS and MS/MS. In MS full scan mode, Q-

TOF type instruments direct the ion flow through the first quadrupole and collision cell to a

pusher where the ions are cooled. The pusher then repells the ion packet into the time of flight

tube where following a refocusing in the reflectrons, they are directed to the detector. The

28

software measures the time the ions were in flight to determine the mass using the formula:

KE=1/2mv2. In MS/MS mode, typically data dependent acquisition (DDA) is used. A defined

number of the most abundant ions (e.g. 5) are each isolated by the quadrupole or Q1 region. The

isolated ion passes to the collision cell or Q2 where it encounters inert argon. Collisions with the

inert argon cause the peptides to lose residues from the C-terminus or N-terminus resulting in b

ions or y ions, respectively. Energy may be applied to accelerate the ions through this region to

induce greater fragmentation. The fragmented peptide is once again detected in the TOF. The

fragmentation pattern may be compared to a predicted pattern either manually or with processing

software, which can determine the amino acid sequence of the peptide. The same process will be

applied to the second most intense ion and so forth until all five have been measured. These ions

may be excluded from the determination of the next 5 most abundant ions in order to evaluate

less abundant peptides.

Using an LTQ-Orbitrap platform in a high resolution mode, the ions are passed through

the linear ion trap and accumulate in the C-trap. The ions are then directed into the orbitrap

where the ions orbit and oscillate around a central electrode. Once the ions have achieved the

specific orbit based on their mass to charge ratio, the ions are directed to the detector. A fourier

transformation is applied to the data which is used to construct the peptide mass spectra. For

MS/MS, data dependent acquisition may also be applied. One at a time, the most abundant

peptides are isolated sequentially using the linear ion trap. Each ion is then fragmented in the ion

trap by increasing the applied energy resulting in collisionally induced dissociation. The

fragments are then directed into the C-trap for subsequent entry into the orbitrap. The fragments

are separated and detected. This mode of MS/MS sometimes provides better b ion coverage that

with the use of a collision cell but suffers from the one third rule. To this point, the LTQ-

29

Orbitrap Velos has an additional collision cell analogous to that discussed with the Q-TOF and

fragments parent ions by collision with inert gas molecules (denoted as hard collisionally

induced dissociation by the manufacturer). Furthermore both of the activation modes may be

used in a single analysis. In this configuration, one could perform alternating CID and HCD

fragmentation so as to have complimentary fragmentation patterns significantly improving the

depth of the data. Of course, the drawback of this approach is that it reduces the number of

MS/MS scans which may be acquired over a given time frame.

The peptide mapping data may be examined chromatographically by evaluating the UV

and TIC traces for obvious differences. Of course, evaluating the mass data provides the most

pertinent information with regards to changes in the primary structure of the recombinant

monoclonal antibody, however, often necessitates a detailed search of the suspected mass shift.

The data may be submitted to a search algorithm which seeks to measure and assign amino acid

sequences to the fragmentation pattern. In addition, if part of the primary structure is evident in

a pattern but there is a mass shift, the software will attempt to assign a defined variant to a

specific residue in the peptide. Carboxymethylation of cysteine residues is a typical example of

this. The one limitation to using this strategy to assign variants is that you must A priori assign

what potential variants you may expect to see. Assigning oxidation to methionine, deamidation

to asparagine, or glycation to lysine are some common applications of this approach. The big

challenge is that if you are working with an unknown variant, you do not know its mass and you

do not know the susceptible residues in the primary structure.

To the previous point, localizing the target residue of the modification may be the most

critical step in determining the source of the observed heterogeneity. Many of the computational

search engines require that you set a target residue and the expected mass shift. If the analytical

30

scientist has a good idea of what the adduction may be then speculating the target residue or

residues based on nucleophilicity and other favorable chemical properties may be possible.

Performing a non-specific search will be quite computationally intensive and often provides so

many false positives that the data loses value.

Sometimes it is prudent to perform a manual search for peptides which may exhibit the

observed mass shift from previous analytics. This can take time, but if identification can be

made, it results in significant progress to solving the source of the modification. Once the site of

modification is determined, the chemistry of the candidate reactive molecule and residue can be

hypothesized. The chemical modification will likely increase the overall size of the target

residue. If lysine or arginine are modified the reactive molecule, the steric effects of the

molecule will likely occlude the residue from the trypsin active site. Therefore, if lysine or

arginine residues are suspected as the site of modification, miscleaved tryptic peptides must also

be considered. In addition, alternate proteases may be employed with different specificity than

trypsin resulting in fully cleaved peptides. Being able to make a direct comparison between the

native and modified peptide is vital to gaining any quantitative information. This is not possible

when trying to compare a modified miscleaved peptide to fully cleaved unmodified peptides.

1.3.6 Agreement between the spiked agent and the initial findings

Once the site of the unknown modification has been determined and a likely candidate

has been identified, it is important to apply the analytical strategies to determine if all of the

generated data is in good agreement. Of course, all of these techniques should be applied to both

the spiking studies. The generation of a more pronounced peak in the weak cation exchange

31

chromatogram which has high similarity to the initial observations provides evidence the spiked

agent has the same influences on charge as the unknown species.

The mass spectral data will be of particular value in assessing the similarities between the

unknown and the forced product. Specifically, the MS and tandem MS data should be compared

between the two with an emphasis on mass accuracy and spectral profile. For instance, highly

similar fragmentation profiles between the same peptide from both conditions suggest that not

only do two samples have the same primary structure but that both have the same site of

modification. In addition, the modification may influence the fragmentation and ionization of

specific ion series. Specifically, modification of arginine residues by malondialdehyde resulted

in a fragmentation pattern that was rich in B ions due to the influence the adduction had on the

peptide backbone115

. The generation of equivalent MS and MS/MS spectra provides solid

evidence that the spiked material is the reactive molecule which resulted in the variant.

1.3.7 Stable Isotope Labels

The use of a stable isotope labeled analog of the spiked agent can help to identify

conclusively the source of a chemical modification in the recombinant monoclonal antibody.

The incorporation of the heavy label serves to unambiguously assign a specific molecule as the

source of the adduction. This technique can be particularly powerful because the labeled

molecule may co-elute with the reactive species due to their equivalent chemical properties but

can easily be discerned by a mass spectrometer. Stable isotope labeling has proven valuable in

the identification of a glycation site using heavy labeled glucose116

. In addition, cross-linked

peptides were identified apriori by using the incorporation of 18

O during the digestion process117

.

32

1.3.8 Interpretation of all of the Findings

The final things to consider pertain to the origination of the reactive species and the

influence the cell culture conditions, storage conditions, etc. may have had on its appearance,

reactivity or both. Understanding the chemistry of the reactive molecule is a must. The

influence of redox and pKa on the reactive molecule and the recombinant monoclonal antibody

can not only help to elucidate the mechanism but may also provide clues about conditions which

can prevent the modification.

Understanding the global prevalence of the modification will not only add to the overall

knowledge of the reactive molecule but will also provide a better understanding to which

residues may be the most susceptible, ie reactive in the recombinant monoclonal antibody on a

broad basis. Such information can be applied to future studies as potential hotspots. Of

particular importance is the consideration of the Structure-Function relationship as it pertains to

the chemical modification. Variants which exist in the complimentary determining regions

(CDR’s) may ablate recognition of the epitope creating the most significant functional liabilities

therefore particular attention should be taken discerning the fidelity of these regions118-119

.

1.3.9 Summary

Altogether, a comprehensive analytical approach which utilizes techniques capable of

discerning minor changes in the primary structure along with state of the art mass spectrometry

techniques can provide an effective strategy in determining whether and unexpected change to

33

the primary structure has occurred. Enhanced understanding of the biology of the expression

system can provide for better guided hypotheses to what may be the underlying reasons for the

observed changes. The implementation of high resolution mass detectors, peptide mapping,

spiking studies, sample enrichment and stable isotope labeling can provide conclusive support

for the nature of an unknown variant. Lastly, communication of this information to the scientific

community will build the overall knowledge within the field and facilitate the identification of

these unwanted variants.

This dissertation presents the discovery of three unique chemical modifications which

occurred in a recombinant monoclonal antibody. These variants are all reported for the first time

with respect to recombinant protein drugs. In all three cases, the observed mass change was not

a familiar one, at least in the literature compiled for recombinant protein drugs. In addition, the

previously described analytical workflows were necessary to determine the nature and

prevalence of each of the specific variants. This work serves to expand the knowledge base of

protein modifications which can occur in protein drugs. In addition, it validates these analytical

strategies as viable approaches for assigning identities to ambiguous molecular weight variances.

Lastly, it underscores the utility of weak cation exchange chromatography. Although it is not a

high resolution method, its ability to discern minor changes in the protein drug is quite valuable

and an important first step in determining the true nature of changes to the primary structure.

34

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45

Chapter 2 Arginine Modifications by Methylglyoxal: Discovery in a Recombinant

Monoclonal Antibody and Contribution to Acidic Species

This chapter is based on a published paper with the same title

Analytical Chemistry 2013, 85(23):11401-9

2.1 Abstract:

Heterogeneity is common among protein therapeutics. For example, the so-called acidic

species (charge variants) are typically observed when recombinant monoclonal antibodies

(mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein

posttranslational modifications have been established as contributors, but still cannot completely

account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal

(MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in

Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to

two adducts (dihydroxyimidazolidine and hydroimidazolone) with increase of molecular weights

of 72 and 54 Daltons, respectively. In addition, the modification by methylglyoxal causes the

antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to

which an antibody was modified at multiple sites corresponds to the degree of shift in elution

time. Furthermore, cell culture parameters also affected the extent of modifications by

methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other

metabolic pathways. Our findings again highlight the impact that cell culture conditions can

have on the product quality of recombinant protein pharmaceuticals.

46

2.2 Introduction

Recombinant biotherapeutics are associated with an inherently increased level of

structural complexity as compared to traditional small molecule drugs. Various protein post-

translational modifications (PTM’s) have been well documented as major contributors to

heterogeneity in recombinant monoclonal antibodies 1-5

. Some of these processes occur during

fermentation, such as glycosylation and sialic acid incorporation; 6-8

while others can occur

through purification, storage and even sample preparation, such as oxidation and disulfide bond

scrambling9-10

. Yet the known modifications still cannot explain all the variants.

A group of extensively studied charge variants include the so-called acidic species that

are observed when recombinant monoclonal antibodies are analyzed by weak-cation exchange

(WCX) chromatography, see Figure 1. One major contributing factor is the removal of the C-

terminal lysine of the heavy chain by cell-produced carboxypeptidease, reducing the overall

positive charge 11

; these variants are commonly referred to as Lys-0, Lys-1 and Lys-2 species.

C-Terminal amidation 12

is another enzymatic process during fermentation. Spontaneous non-

enzymatic transformations include the formation of pyroglutamate (Pyro-Glu) from an N-

terminal glutamine (Gln) that removes the positive charge of the free N-terminus 13

, and the

deamidation of asparagine (Asn) to aspartic (Asp) or isoaspartic acid (isoAsp or isoD) that

introduces negatively charged carboxylic acids 14-19

. Other modifications without altering the

formal charges can shift the retention time of an antibody on weak cation exchange

chromatography, likely due to perturbation of local charge and conformation, such as incomplete

glycosylation 20

and the presence of free cysteinyl thiols instead of disulfide10, 21

. It is worth

noting that some modifications are imparted by metabolites, such as glycation by glucose2, 22-23

,

methionine oxidation by reactive oxygen species (ROS)24

, cysteinylation by cysteine 25

, and N-

47

homocysteinylation by homocysteine thiolactone26-27

. Again, it is interesting to note that

although many modifications have been reported; the observed heterogeneity of recombinant

monoclonal antibodies on weak cation exchange chromatography still cannot be explained

completely, suggesting more modifications are yet to be identified.

As report herein, we observed two well-defined acidic species under certain cell culture

conditions. Detailed analyses have revealed that several arginine (Arg) residues were modified

by methylglyoxal (MGO), further confirmed by comparing native antibody treated with authentic

MGO. As illustrated in Scheme 1, the resulting dihydroxyimidazolidine and hydroimidazolone

adducts increase molecular weights by 54 and 72 Daltons, respectively; these modifications

cause the antibody to elute earlier in the weak cation exchange chromatogram. Consequently,

the extent to which an antibody was modified at multiple sites corresponds to the degree of shift

elution time. While protein modification by MGO is known in biology, our discovery is the first

for a recombinant protein product. Furthermore, cell culture parameters also affect the extent of

modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose,

lipids or other metabolic pathways.

48

Scheme 2-1. Chemical modification of arginine by methylglyoxal (MGO). The guanidine of the

arginine side chain reacts with the dicarbonyls to form a dihydroxyimidazolidine with a mass

increase of 72 Da, and the subsequent loss of water leads to a hydroimidazolone with a mass

increase of 54 Da.

49

2.3 Materials and Methods

2.3.1 Materials

The recombinant monoclonal antibody was produced by stably transfected Chinese

hamster ovary (CHO) cells cultured in a bioreactor and purified at AbbVie Bioresearch Center

(Worcester, MA). Dithiothreitol (DTT) was from Sigma (St. Louis, MO). Acetonitrile and

trifluoroacetic acid (TFA) were from J.T.Baker (Phillipsburg, NJ). Formic acid (FA) was from

EMD (Gibbstown, NJ). Trypsin was from Worthington (Lakewood, NJ). Endoproteinase Lys-C

was from Roche (Indianapolis, IN). Methylglyoxal was from MP Biomedicals (Solon, OH).

Guanidine-HCl was from Thermo Scientific (Rockford, IL).

2.3.2 Weak cation exchange (WCX) chromatography

The antibody in low salt buffer was loaded onto a ProPac 4 x 250 mm WCX-10 column

(Dionex, Sunnyvale, CA) at 94% mobile phase A (10 mM sodium phosphate, pH 7.5) and 6%

mobile phase B (10 mM sodium phosphate and 500 mM sodium chloride, pH 5.5) at a flow-rate

of 1 mL/min. The percentage of mobile phase B was increased from 6% to 16% over 20 min to

elute the antibody monitored by UV absorbance at 280 nm. The column was then washed using

100% mobile phase B and then equilibrated using 6% mobile phase B for 9 minutes between

injections. Fractionation was performed across the WCX-10 chromatogram. Distinct peaks

were collected as individual fractions. The collected fractions were concentrated using Amicon

Ultra-15 centrifugal filter with a molecular weight cut-off of 10 kDa (Millipore, Billerica, MA)

and a table-top centrifuge operated at 4 ºC.

2.3.3 LC-MS analysis of light and heavy chains of antibody

50

Light chain and heavy chain of the antibody from different fractions were analyzed using

an HPLC (Agilent 1260, Santa Clara, CA) with a reversed phase column (Vydac, C4, 1 x 150

mm i.d., 5µ particle size) coupled to a Q-TOF mass spectrometer (Agilent, 6510). Antibody was

reduced using DTT (10 mM final concentration). Ten microliters of each sample was loaded at

95% mobile phase A (0.08% formic acid and 0.02% TFA in Milli-Q water) and 5% mobile phase

B (0.08% formic acid and 0.02% TFA in acetonitrile) and then eluted using a gradient from 5%

mobile phase B to 35% mobile phase B in 20 min. The column was washed using 90% mobile

phase B and equilibrated using 5% mobile phase B for 10 min. The flow rate was 50 µL/min

and column oven temperature was 60 ºC. The mass spectrometer was operated in positive ion

mode with a scan range from m/z 600 to 3200. Ion spray voltage was 4500 volts and the source

temperature was 350 ºC.

2.3.4 Tryptic and Lys-C digestion

Protein factions from WCX were denatured using 6 M guanidine hydrochloride in 100

mM Tris, pH 8.0 and reduced using 10 mM DTT at 37 ºC for 30 min. Alkylation was performed

using 25 mM iodoacetic acid at 37 ºC for 30 min. The samples were buffer exchanged to 10 mM

Tris pH 8.0 using NAP-5 columns (GE Healthcare, Piscataway, NJ). The samples were digested

either with trypsin or Lys-C at 1:20 (w:w, enzyme:antibody) and incubated at 37 ºC for 4 hours.

2.3.5 LC-MS analysis of peptides

A UPLC (Acquity, Waters, Milford, MA) equipped with a UPLC C18 reversed phase

column (Waters, 1 x 150 mm i.d., 1.7µ particle size) and a Thermo Scientific LTQ-Orbitrap

Velos mass spectrometer (Thermo Fisher, West Palm Beach, FL) were used to analyze peptide

51

samples. Forty µL of each sample was loaded at 98% mobile phase A (0.08% formic acid and

0.02% TFA in Milli-Q water) and 2% mobile phase B (0.08% formic acid and 0.02% TFA in

acetonitrile) and then eluted using a gradient from 2% mobile phase B to 35% mobile phase B in

80 min. The column was washed using 90% mobile phase B and equilibrated using 2% mobile

phase B for 10 min. The flow rate was 50 µL/min and column oven temperature was 60 ºC. The

mass spectrometer was operated in positive ion mode with a scan range from m/z 400 to 2000

with alternating collision induced dissociation (CID) and hard collision dissociation (HCD) of

the three most intense parent masses. Ion spray voltage was set at 4500 volts and the source

temperature was set at 350 ºC. The data was were analyzed by searching extracted mass traces

of cleaved and subsequently miscleaved tryptic peptides containing peptides with mass increases

of 54 and 72 Daltons. The data were also searched against the theoretical primary structure

using the Sequest algorithm (Thermo Scientific, West Palm Beach, FL).

2.3.6 Methylglyoxal reaction with monoclonal antibody

Authentic methylglyoxal (MP Biomedical, Solon, OH) was incubated with the isoform of

the recombinant monoclonal antibody without C-terminal Lys (denoted as Lys-0), collected from

the WCX-10 chromatography. Lys-0 antibody at 1 mg/mL in 10 mM sodium phosphate at pH

7.0 was incubated with 2.8 mM methylglyoxal at 35 ºC for 0, 1, 2, 3, 4 and 5 hours. Lys-0

antibody incubated under the same conditions without methylgloxal was used as a control. The

samples were analyzed by WCX as described in the previous section. Peaks that appeared in the

acidic region of the chromatogram in the sample treated with methylglyoxal for 2 hours were

fractionated for further analysis as described for the samples from cell culture, and the data from

the MGO spiking fractions and the cell culture fractions were compared.

52

2.3.7 Global Analysis of MGO Modification

Methylglyoxal can modify arginine, lysine and cysteine residues28-29

. To this point,

pertinent structures for potential modifications were searched for both manually and with the

Sequest Algorithm (Thermo Scientific, West Palm Beach, FL) to understand the degree to which

the antibody has been modified. Both lysine and arginine must be considered to result in mis-

cleavages when trypsin is used as the digest agent. In contrast, modified cysteine residues will

lie within a tryptic peptide, but its reactivity is hampered by the fact that all cysteines in a

recombinant monoclonal antibody should be involved in disulfide bonds. Structures considered

in the analysis are adducts of arginine (dihydroxyimidazolidine, hydroimidazolone and

argpyrimidine), lysine (N-epsilon (1-carboxyethyl) lysine28

, and cysteine (carboxyethyl

cysteine)28-29

.

2.3.8 Quantification of Modified Peptides

Quantification of MGO modified peptides was performed on endoprotease Lys-C

generated peptides. Comparison of Lys-C peptides eliminates MGO-induced mis-cleavages

allowing for direct quantification between the matching modified and unmodified peptides.

These data were generated using the parameters outlined in the LC/MS analysis of peptides

section discussed previously. Extracted mass chromatograms corresponding to Lys-C peptides

encompassing identified MGO sites from the tryptic mapping experiments were compared for

the native peptide and +54 and +72 Dalton increases. The integrated areas of the confirmed

extracted ion chromatogram (EIC) peaks of the modified and native Lys-C peptides were used to

quantify the percent of methyglyoxal modification at each of the susceptible sites.

53

2.4 Results and Discussion

2.4.1 New acidic species induced by changes in cell culture

Under culture conditions M (modified), an increase in acidic species occurred during

protein expression. In Figure 2-1, the weak cation exchange chromatogram shows two well-

defined peaks with considerable shorter retention time than that of the main peak (Lys-0

isoform), which were absent under conditions N (normal). Although, it was determined later by

a spiking study that after reduction, light chain with 2% modification of the 54 Da species can be

detected and 5% modification can be unambiguously assigned, fractions were collected to enrich

materials with such modifications. Each fraction was reduced to generate heavy chain and light

chains, which were analyzed by LC/MS; the resulting mass spectra are shown in Figure 2-2. The

molecular weight of 23408.24 Da for the major peak was in agreement with the theoretical

molecular weight of 23408.13 Da for the light chain (LC) based on its amino acid sequence. In

addition, two distinct peaks with molecular weights of 23462.44 Da and 23480.26 Da were also

observed, corresponding to increases of approximately 54 Da and 72 Da over that for the native

light chain. Moreover, a ladder of additional peaks with mass increase of 54 or 72 Da

increments, albeit with lower intensity, was also observed, likely due to the same modifications

occurring at multiple sites. A similar pattern of modifications was also observed for the heavy

chain as shown in Figure 2-2. The major peak with a molecular weight of 50637.95 Da is in

agreement with the theoretical molecular weight (50636.78 Da) of the heavy chain without the

C-terminal Lys (Lys-0) and with a core-fucosylated biantenary complex oligosaccharide without

the terminal galactose (G0F). Similar to the light chain, two additional peaks with molecular

weights of 50692.06 Da and 50709.12 Da were also observed (Fraction 2), which are

54

approximately 54 Da and 72 Da greater than the theoretical molecular weight. Again, the

additional ladder of peaks with lower intensity are likely due to the same modification occurring

at more than one site.

It is worth noting that these mass changes are not among the reported protein

modifications in recombinant monoclonal antibodies. Additionally, searching ABRF Delta Mass

database (www.abrf.org/index.cfm/dm.home) of common protein modifications did not yield

plausible explanation for the observed mass increases of 54 Da or 72 Da either. Furthermore, it

was not initially clear whether these two modifications were related. To elucidate the chemical

nature and site of modifications, peptide mapping was carried out first to identify modified

peptides.

http://www.abrf.org/index.cfm/dm.home

55

Figure 2-1. The top panel is a typical WCX chromatogram of the recombinant monoclonal

antibody after protein A purification (top and bottom traces were from cell culture M (modified)

and N (normal), respectively, at day 9). The peaks labeled as Lys-0, Lys-1 and Lys-2 are

antibody isoforms without the C-terminal Lys, with one C-terminal Lys and with two C-terminal

Lys on the heavy chains, respectively. Peaks at 2.2 and 2.8 min were observed in antibody

expressed in cell culture M and are denoted by Fractions 1 and 2, respectively, which are the

focus of this study. The bottom panel shows the time dependence of the formation of these two

species.

56

Figure 2-2. Deconvoluted mass spectra of the light chain and heavy chains in fraction 1 (A and

B) and fraction 2 (C and D). The theoretical molecular weight of the light chain is 23408.13 Da

(observed 23408.24); 23462.44 and 23480.66 Da in Pane A represent increase of mass of 54 and

72 Da, respectively. The theoretical molecular weight of the heavy chain is 50636.78 Da

(observed 50637.95); 50691.04 and 50710 Da represent increase of mass of 54 and 72 Da,

respectively. In antibody treated by authentic MGO (2.7 mM, 5 h), a series of peaks with

A

G

E

C

B

H

F

D

LC

Fraction 1

LC

Fraction 2

LC

Authentic

MGO

LC

Control

HC

Control

HC

Authentic

MGO

HC

Fraction 1

HC

Fraction 2

A

G

E

C

B

H

F

D

LC

Fraction 1

LC

Fraction 2

LC

Authentic

MGO

LC

Control

HC

Control

HC

Authentic

MGO

HC

Fraction 1

HC

Fraction 2

57

incremental mass increase of 54 and/or 72 were observed (see E and F). These additional peaks

are absent from the control (G and H).

58

2.4.2 Localization of the modification sites

Peptide mapping was performed on fractions 1 and 2 in the weak cation exchange

chromatogram. Initially, all theoretical tryptic peptides with mass increase of either 54 Da or 72

Da were manually searched but none were found. We surmised that the modifications might

perturb proteolysis; hence, the manual search was expanded to tryptic peptides with one single

mis-cleavage at either lysine or arginine, and several such peptides were observed. Two

representative MS/MS spectra corresponding to the same peptide with +54 Da and +72 Da mass

increase are shown in Figure 2-3. The internal arginine residue should be cleaved by trypsin but

was not, suggesting it was likely modified. MS/MS was performed using an alternating

fragmentation activation of CID performed in the ion trap and HCD performed in the collision

cell to produce complimentary fragmentation spectra to provide better coverage. The major

fragment ions in the spectra are from the b-ion series. A mass increase of 54 Da associated with

b6 and b15-ions localized the modification to the first six amino acids that include the only

arginine residue in this peptide. In addition to b-ions, several y-ions were also observed. Most

important was that while the y12 ion showed the predicted mass of the unmodified peptide

fragment, the y13 ion (refer to Figure 2-3, Pane A) displayed a mass increase of 54 Da thus

confirming that the modification was on the arginine. Similarly, for light chain peptide T3 (a

doubly charged precursor ion of m/z of 1090.5), a mass increase of 72 Da was associated with

both b8 and b15 ions that localized the modification to the first eight amino acids. Again, the

observation of y12 ion with the predicted mass and y13 ion with a mass increase of 72 Da

confirmed that the modification was on the Arg residue (refer to Figure 2-3, Pane B).

59

A

0

20

40

60

80

100 1128.58 1314.66

1018.01

1861.921477.72

1605.78

1734.84848.46

944.46

1218.66781.39668.37429.28

B

0

20

40

60

80

100 1128.58 1314.66

1057.54

1862.931018.01

1477.72

1606.78

1733.83848.46

944.46

1286.67781.40668.37429.28

1971.52

b15+1

b14+1

b13+1

b12+1

b11+1b9

+1b10

+1

b7+1

b8+1

b6+1

y13+1

y10+1

y7+1

y4+1

y12+1

b15+1

b14+1

b13+1

b12+1

b11+1

b9+1 b10

+1

b7+1

b8+1

b6+1

y13+1

y10+1

y7+1

y4+1

y12+1

C

0

20

40

60

80

100 1073.05

1332.661018.011146.59

1495.73

1880.931624.80848.46

734.37606.26

1219.67

411.27

D

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

20

40

60

80

100 1073.05

1018.01

1332.661146.59

1496.74

1624.79 1880.94

848.46

989.50

1218.66

429.28 734.36

y7+1

b14+1

b10+1

b9+1

b11+1

b15+1b12

+1

b13+1

y12+1

y13+1

y4+1

y7+1

b14+1

b10+1

b9+1

b11+1

b15+1b12

+1

b13+1

y12+1

y13+1

y4+1

Cell Culture

Cell Culture

Authentic MGO

Authentic MGO

ASQGI R* N Y L A W Y Q Q K PGKy13 y12

b7 b8 b15b14b13b12b11b10b9b6

y10 y4y7


b7 b8 b15b14b13b12b11b10b9b6

y10 y4y7

ASQGI R** N Y L A W Y Q Q K PGKy13 y12

b15b14b13b12b11b10b9

y4y7


b15b14b13b12b11b10b9

y4y7

A

0

20

40

60

80

100 1128.58 1314.66

1018.01

1861.921477.72

1605.78

1734.84848.46

944.46

1218.66781.39668.37429.28

B

0

20

40

60

80

100 1128.58 1314.66

1057.54

1862.931018.01

1477.72

1606.78

1733.83848.46

944.46

1286.67781.40668.37429.28

1971.52

b15+1

b14+1

b13+1

b12+1

b11+1b9

+1b10

+1

b7+1

b8+1

b6+1

y13+1

y10+1

y7+1

y4+1

y12+1

b15+1

b14+1

b13+1

b12+1

b11+1

b9+1 b10

+1

b7+1

b8+1

b6+1

y13+1

y10+1

y7+1

y4+1

y12+1

A

0

20

40

60

80

100 1128.58 1314.66

1018.01

1861.921477.72

1605.78

1734.84848.46

944.46

1218.66781.39668.37429.28

A

0

20

40

60

80

100 1128.58 1314.66

1018.01

1861.921477.72

1605.78

1734.84848.46

944.46

1218.66781.39668.37429.28

B

0

20

40

60

80

100 1128.58 1314.66

1057.54

1862.931018.01

1477.72

1606.78

1733.83848.46

944.46

1286.67781.40668.37429.28

1971.52

B

0

20

40

60

80

100 1128.58 1314.66

1057.54

1862.931018.01

1477.72

1606.78

1733.83848.46

944.46

1286.67781.40668.37429.28

1971.52

b15+1

b14+1

b13+1

b12+1

b11+1b9

+1b10

+1

b7+1

b8+1

b6+1

y13+1

y10+1

y7+1

y4+1

y12+1

b15+1

b14+1

b13+1

b12+1

b11+1

b9+1 b10

+1

b7+1

b8+1

b6+1

y13+1

y10+1

y7+1

y4+1

y12+1

C

0

20

40

60

80

100 1073.05

1332.661018.011146.59

1495.73

1880.931624.80848.46

734.37606.26

1219.67

411.27

D

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

20

40

60

80

100 1073.05

1018.01

1332.661146.59

1496.74

1624.79 1880.94

848.46

989.50

1218.66

429.28 734.36

y7+1

b14+1

b10+1

b9+1

b11+1

b15+1b12

+1

b13+1

y12+1

y13+1

y4+1

y7+1

b14+1

b10+1

b9+1

b11+1

b15+1b12

+1

b13+1

y12+1

y13+1

y4+1

C

0

20

40

60

80

100 1073.05

1332.661018.011146.59

1495.73

1880.931624.80848.46

734.37606.26

1219.67

411.27

C

0

20

40

60

80

100 1073.05

1332.661018.011146.59

1495.73

1880.931624.80848.46

734.37606.26

1219.67

411.27

D

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

20

40

60

80

100 1073.05

1018.01

1332.661146.59

1496.74

1624.79 1880.94

848.46

989.50

1218.66

429.28 734.36

D

400 600 800 1000 1200 1400 1600 1800 2000

m/z

400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

20

40

60

80

100 1073.05

1018.01

1332.661146.59

1496.74

1624.79 1880.94

848.46

989.50

1218.66

429.28 734.36

y7+1

b14+1

b10+1

b9+1

b11+1

b15+1b12

+1

b13+1

y12+1

y13+1

y4+1

y7+1

b14+1

b10+1

b9+1

b11+1

b15+1b12

+1

b13+1

y12+1

y13+1

y4+1

Cell Culture

Cell Culture

Authentic MGO

Authentic MGO


b7 b8 b15b14b13b12b11b10b9b6

y10 y4y7


b7 b8 b15b14b13b12b11b10b9b6

y10 y4y7


b15b14b13b12b11b10b9

y4y7


b15b14b13b12b11b10b9

y4y7

60

Figure 2-3. Representative MS/MS mass spectra ([1081.5]2+

at 31 min and [1090.5]2+

at 32 min,

respectively) of peptides containing Arg residues modified by MGO forming a

hydroimidazolone in cell culture (A and C) or from incubation with authentic methylglyoxal

forming a dihydroxyimidazolidine (B and D). Modifications that resulted in the molecular

weight increases of both 54 Da and 72 Da were localized to Arg based on the MS/MS

fragmentation pattern. R* and R** denote the modified arginine by mass increases of 54 Da and

72 Da, respectively.

61

2.4.3 Deduction of the Structure of the Modification

Chemically reactive, arginine can be modified by a host of biological molecules and

chemical reagents, the most common being carbonyls (aldehydes and ketones). The carbonyl-

guanidyl adducts are formed via an initial addition mechanism (the mass of the adduct equals the

combined mass of the carbonyl and arginyl peptide) and possibly a subsequent condensation

(elimination of a water molecule of the initial addition adduct; thus, a further mass decrease of

18 Da). Based on these mechanisms, the molecules that modified arginine in our antibody

should have an intact molecular weight of either 54 or 72 (54 +18) Da (for the +54 Da adduct),

72 or 90 (72+18) Da (for the +72 Da adduct). Furthermore, since both adducts were induced by

changes in cell culture conditions, we surmised that one or more metabolites were the culprits.

The following carbonyl molecules match the mass values: for 54 Da, 2-propynal; for 72 Da,

methylglyoxal, malonaldehyde, glycidaldehyde, butanal and butanones; and for 90 Da,

glyceraldehyde and dihydroxyacetone (glycerone). Methylglyoxal was our top candidates for the

following considerations: (1) being a dicarbonyl, it forms fairly stable adducts with arginine; (2)

production of methylglyoxal is known to be dependent on cell culture conditions30-34

,

reminiscing autoinducer-2 (AI-2, another dicarbonyl metabolite)35-37

and (3) the two adducts

shown in Scheme 1 can account for both observed mass increase. A similar approach has been

used to identify unknown protein crosslinking in an antibody38

.

2.4.4 Incubation with Authentic MGO to generate reference.

In order to confirm that the modification of the monoclonal antibody in cell culture was

indeed by MGO, Lys-0 antibody shown in Figure 2-1 was isolated using weak cation exchange

fractionation and was subsequently incubated with authentic MGO at 35 ºC; then, the reaction

62

products were analyzed by weak cation exchange chromatography. As shown in Figure 2-4,

peaks corresponding to fractions 1 and 2 in the weak cation exchange chromatogram (labeled as

Peaks A and B, respectively) increased with the prolonged incubation. In comparison,

incubation of the Lys-0 fraction without MGO under the same conditions for the entire 5 hours

did not result in any observable changes in peak profile (data not shown). Peaks A and B (Figure

2-4) were collected from the sample incubated for 2 hours using weak cation exchange

chromatography and the samples were analyzed by LC-MS in the same manner as for the

samples from cell culture. As shown in Figure 2-2 (panes E and F), peaks with molecular

weights of 23462 Da and 23480 Da correspond to molecular weight increases of 54 Da and 72

Da, respectively, compared to theoretical molecular weight of 23408 Da for the light chain.

Similarly, peaks with molecular weights of 50691 Da and 50709 Da with molecular weight

increases of 54 Da and 72 Da, respectively, as compared to theoretical molecular weight of

50637 Da for the unmodified heavy chain were also observed. Additional ladders of peaks with

increments of 54 and/or 72 Da were also observed, as for the samples from cell culture. Similar

to other PTM’s, formation of the initial MGO-adducts may alter protein conformation and

dynamics, thus altering the kinetics of modifications at other sites. As a result, the formation of

different adducts are interdependent, as indicated by the adduct distribution.

63

Figure 2-4. WCX chromatogram of a purified Lys-0 antibody incubated with and without

authentic MGO (2.8 mM at 35 ºC) for various durations as labeled. The bottom trace shows the

chromatogram of the antibody generated from cell culture. Acidic peaks derived from

incubation with authentic MGO are labeled as Peak A and Peak B, respectively. The retention

times of these peaks are in good agreement with Fraction 1 and Fraction 2 displayed in the

bottom trace which are the subject of this study.

0-Lys

T = 0 Hrs

T = 5 Hrs

T = 4 Hrs

T = 3 Hrs

T = 2 Hrs

T = 1 Hrs

Cell Culture

Peak A

Peak B

Fraction 2Fraction 1

0-Lys

T = 0 Hrs

T = 5 Hrs

T = 4 Hrs

T = 3 Hrs

T = 2 Hrs

T = 1 Hrs

Cell Culture

Peak A

Peak B

Fraction 2Fraction 1

64

2.4.5 Comparison of the in vitro references and the cell culture samples.

The peptide maps of the fractionated acidic species from the MGO spike in phosphate

buffer and the corresponding cell culture acidic fractions were compared by LC/MS/MS.

Modified tryptic peptides from both samples exhibit comparable retention times within the

normal range of variation from run to run, e.g., 31.5 minutes and 32.3 minutes for 1081.5

[M+2H]2+

corresponding to the peptide with a mass increase of 54 Da and 1090.5 [M+2H]2+

corresponding to the peptide with a mass increase of 72 Da, respectively. In addition, MS/MS

analyses indicated that the modification for both samples can be localized to the same arginine

residues in both the cell culture derived antibody and the MGO stressed 0-Lys samples.

The MS/MS fragmentation patterns of the spectra from the antibody spiked with MGO in

aqueous buffer and antibody modified during cell culture are shown in Figure 2-3 and are nearly

identical; e.g., both spectra exhibit many b ions as well as some key y ions that allow conclusive

localization of the modification as MGO-modified arginine.

2.4.6 Global Analysis of Modifications by MGO

After MGO was confirmed to be responsible for the modifications at the site described

above, the entire primary structure of the antibody was assessed for both the

dihydroxyimidazolidine (+72 Da) and the hydroimidazolone (+54 Da) adducts. Several sites in

both variable and conserved domains were identified. All modified peptides exhibited the

modification at arginine residues and subsequently resulted in mis-cleavages. The distribution

was quantitated based on endoprotease Lys-C digest analyzed by LC/MS/MS peptide mapping.

65

Lys-C was chosen for it only cleaves at lysine but not at arginine or MGO-modified arginines, so

peptide counterparts with and without MGO-modifications can be directly compared. The

distribution and peak intensity of the modified arginine-containing peptides (both the +54 and

+72 Da adduct) are shown in Figure 2-5. The differences are evident and likely to affect the

elution behavior seen in the WCX chromatography.

66

Figure 2-5.: Peak intensity of all methylglyoxal-modified peptides generated by Lys-C

digestion of fraction 1 (black) and fraction 2 (red), which are normalized to the peak intensity of

the corresponding native (unmodified) peptides. The checkered bars represent the +54 Da

adduct and the solid bars represent the +72 Da adduct.

0

0.1

0.2

0.3

0.4

0.5

LC30 LC93 LC108 HC16 HC259 HC359 HC420

Re

lati

ve In

ten

sity

Site of Arginine Modification

Fraction 1 (+54)

Fraction 2 (+54)

Fraction 1 (+72)

Fraction 2 (+72)

67

MGO may also react with lysine resulting in Nε-(carboxyethyl)lysine with a mass

increase of 72 Da39

, but the reaction is considerably slower and the product much less stable than

with arginine. A thorough examination for MGO modifications on lysine residues did not result

in any matches, indicating the MGO modification is selective toward arginine under our

conditions.

The majority of the modifications occur at the CDR region, which is more flexible than

the other parts of the antibody. Arg30 of the light chain is particularly solvent accessible. These

factors may contribute to its propensity for modification. Certainly, specific interactions with

other residues, local environment and other factors may be involved as well 40-41

.

2.4.7 Effects of MGO Modification on Charge

When an ionizable amino acid is chemically modified, its pKa value may be significantly

perturbed. Using the Advanced Chemistry Development pKa prediction program accessed

through SciFinder, the following values were obtained: guanidinium (12.55, experimental value),

dihydroxyimidazolidine (7.13 +/- 0.6, calculated) and hydroimidazolone (6.93 +/- 0.4,

calculated)42

as shown in Figure 2-6. The lower pKa values imparted by MGO modifications 43

translate to more acidic species, consistent with an earlier elution by weak cation exchange

chromatography. Additionally, the steric bulkiness conferred by MGO modifications may also

interfere with the stationary phase interaction and reduce the binding strength. Conformational

changes are another possible contributor to changes in chromatographic behavior. To this point,

we compared the charge envelopes of both modified and unmodified heavy and light chains

68

obtained by mass spectrometry with respect to charge distribution44-45

. No apparent differences

were seen (data not shown), suggesting no major alteration in high-order structures.

69

Figure 2-6.: Calculated pKa of the core group of arginine and the two products of arginine

modification by methylglyoxal. The pKa of the guanidinium core group is significantly

depressed to 7.13 and 6.93 for the dihydroxyimidazolidine and hydroimidazolone core groups,

respectively

70

2.4.8 Relevance to other Contributing Factors to Acidic Species

The observation of acidic species formation over time and at elevated pH and

temperature is fairly well understood. Several studies have shown that asparagine deamidation

to aspartate and isoaspartate introduces an additional negative charge on proteins 14-19, 46-47

. The

observation of an increase in acidic species occurring as the cell culture progresses appears to be

due to something other than an increase in deamidation as all the samples underwent equivalent

storage and the altered chromatograms differ from those generated in forced deamidation.

Previously, some studies have shown that the introduction of a glycated lysine residue in a

protein can result in an acidic shift seen in weak cation exchange chromatography by perturbing

the charge on the protonated primary amine23

. In many cases, however, these known

modifications cannot fully explain all the acidic species. Our findings suggest that arginine

modification by MGO should be considered.

2.4.9 Formation of MGO as a New Critical Attribute for Cell Culture

Complicated metabolic pathways for methylglyoxal exist in CHO cells. MGO can be

formed enzymatically or non-enzymatically from glucose, e.g., the ene-diol intermediate of

dihydroxyacetone phosphate (DHAP)30, 32, 48-49

. Not surprisingly, many reports of protein

modifications by methylglyoxal come from research related to diabetes in which blood glucose

levels are elevated; and MGO may be involved in the formation of advanced glycation end

products (AGEs). In addition, because of the high reactivity and toxicity of MGO, dedicated

catabolic mechanisms also exist 48-51

. Previous studies highlighted that cells have biochemical

pathways to effectively remove methylglyoxal32

. The glyoxalase pathway is a glutathione-

mediated process which converts methylglyoxal to D-lactate by the glyoxylase I and glyoxylase

II enzymes. In addition, MGO can be reduced to propanediol through the NADPH-dependant

71

aldose reductase pathway. Indeed, Chaplen and coworkers have observed that methylglyoxal

can accumulate in CHO cell cultures due to perturbed regulation in MGO metabolism30-34, 40

.

Under our N (normal) and M (modified) conditions, no detectable differences in glucose

levels were observed; but presumably, other difference did result in the accumulation of

methylglyoxal. Changes in the flux of the glyoxalase pathway may be the cause of this

unexpected appearance of methylglyoxal in the modified conditions. To the best of our

knowledge, this is the first report of such a modification taking place in a CHO cell expression of

a recombinant monoclonal antibody, affecting the quality of the protein products.

It is also important to consider that MGO not only affects the protein product being

expressed, but also broadly affects many proteins in the host cells and possibly their biological

functions. At the cellular level, vigor, viability, cell density and protein production yield may

also be negatively affected. Conversely, elevated MGO formation also reflects a change in

metabolic flex of competing pathways30, 32-33, 48-49, 51-53

. In summary, MGO can be considered a

biomarker of a less than optimal cell culture.

72

2.5 Conclusions

Triggered by changes in cell culture media, a new modification of a recombinant

monoclonal antibody by methylglyoxal (MGO) was identified by a combination of weak cation

exchange chromatography, peptide mapping and mass spectrometry. Modification by MGO,

e.g., the corresponding mass increase of 54 Da and 72 Da for the arginine derivatives, should be

considered when assessing heterogeneity in recombinant proteins. In addition, our finding serves

as a reminder that other unreported modifications are likely to exist in protein pharmaceuticals.

Furthermore, the results demonstrate the critical role of cell culture conditions and downstream

processes in controlling product quality.

73

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78

SUPPORTING INFORMATION

Table of Contents

Figure S2-1 Comparison of peptide MS data between acidic peaks from cell culture and acidic

fractions from methylglyoxal incubation 79

Figure S2-2 Comparison of peptide MS/MS data between acidic peaks from cell culture and

acidic peaks from methylglyoxal incubation 80

Figure S2-3 Reduced analysis of light chain from peaks 1 and 2 formed during cell culture

81

Figure S2-4 Reduced analysis of heavy chain from peaks 1 and 2 formed during cell culture

82

Figure S2-5 Formation of Peak 1 and Peak 2 during cell culture 83

Table S2-1 Results of manual search of methylglyoxal modified peptides found in the

recombinant antibody 84

Table S2-2 Raw data values of Lys-C peptide peak intensity from Cell Culture WCX

fractions 1 and 2 85

Table S2-3 Relative Intensity of Lys-C peptides from Cell Culture WCX fractions 1 and 2

85

Figure S2-6 Detection limit of reduced LC/MS to detect MGO (+54 Da) 86

79

Figure S-1.: Comparison of peptide MS data between acidic peaks from cell culture and acidic

fractions from methylglyoxal incubation. The data shows the corresponding mass spectra of a

3+ ion representing a mis-cleaved peptide with a mass increase of 54 daltons.

ASQGIR(MGO)NYLAQYQQKPGK

Cell Culture Fraction 1

MGO Spike Fraction 1

M+H=2162.1 da

M+H=2162.1 da

720.0 720.5 721.0 721.5 722.0 722.5 723.0 723.5 724.0 724.5

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100R

ela

tive A

bun

dance

721.71

721.38

722.05

722.38

722.71

723.05720.71 721.04720.37 723.39

721.24

721.85

722.18

721.58

722.52

723.72 724.06 724.40

721.71

721.38

722.05

722.38

722.71

723.05721.04 723.38

721.65721.32

722.11

721.78

720.82722.44

720.43 723.85720.14 724.40

M+3H

M+3H

ASQGIR(MGO)NYLAQYQQKPGK

Cell Culture Fraction 1

MGO Spike Fraction 1

M+H=2162.1 da

M+H=2162.1 da

720.0 720.5 721.0 721.5 722.0 722.5 723.0 723.5 724.0 724.5

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100R

ela

tive A

bun

dance

721.71

721.38

722.05

722.38

722.71

723.05720.71 721.04720.37 723.39

721.24

721.85

720.0 720.5 721.0 721.5 722.0 722.5 723.0 723.5 724.0 724.5

m/z

0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100R

ela

tive A

bun

dance

721.71

721.38

722.05

722.38

722.71

723.05720.71 721.04720.37 723.39

721.24

721.85

722.18

721.58

722.52

723.72 724.06 724.40

721.71

721.38

722.05

722.38

722.71

723.05721.04 723.38

721.65721.32

722.11

721.78

720.82722.44

720.43 723.85720.14 724.40

M+3H

M+3H

80

Figure S-2 : The figure shows the total ion current (top), the native peptide (second pane), the

peptide modified by one MGO (+54 da) and lastly, a trace showing no evidence of the second

arginine within this peptide being modified. The site was identified by previous tryptic digest

and the degree of modification was determined by the percent of the respective peaks in the

XIC’s of the native and MGO modified peptides

Da )

81

Figure S-3.: The figure displays a mass spectrum of a heavy chain Lys-C peptide showing the

isotopic distributions of the +5 charge state (Arg93 internalized). In this manner, the native and

the two MGO products of a single modified arginine can be seen within the same spectrum.

82

Figure S-4.: Mass spectra of the light chain for a pure 0 Lys are shown over the five hour period.

The additional peaks of +54da and +72da have formed and have greatly increased the observed

mass heterogeneity. The formation of the additional peaks is in agreement with the observed

peaks from the acidic fractions isolated from cell culture. The control was incubated for the

entire five hour period but without the addition of methylglyoxal and subsequently showed no

added complexity or formation of additional peaks.

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

Control

5 hours

4 hours

3 hours

2 hours

1 hour

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

Control

5 hours

4 hours

3 hours

2 hours

1 hour

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

Control

5 hours

4 hours

3 hours

2 hours

1 hour

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

Control

5 hours

4 hours

3 hours

2 hours

1 hour

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

6x10

0

523407.77

23388.39 23426.77

6x10

0

2

4 23407.75

23479.4323388.41 23426.96 23615.7423551.5323515.41 23581.90

6x10

0

2

23407.69

23461.44

23551.5423443.4623388.48 23515.35 23617.1923586.92 23641.47

6x10

0

1

223407.69

23461.67

23479.5223551.55

23515.3523443.19 23605.1223388.49 23641.85

6x10

0

1

2 23407.67 23461.69

23479.5323551.56

23515.44 23605.5923443.0923388.48 23569.87 23642.29

6x10

0

1

23461.6923407.67

23551.5423479.5423515.42 23605.7523442.95 23569.8023388.47 23642.04

23340 23360 23380 23400 23420 23440 23460 23480 23500 23520 23540 23560 23580 23600 23620 23640

Control

5 hours

4 hours

3 hours

2 hours

1 hour

83

Figure S-5.: Mass spectra of the heavy chain for a pure 0 Lys are shown over the five hour

period. The additional peaks of +54da and +72da have formed and have greatly increased the

observed mass heterogeneity.

6x10

0

2

50634.32

50796.3450730.59 50958.81

6x10

0

1

2 50635.37

50707.31 50797.5450869.05

6x10

0

1

50633.1150705.05

50795.21 50867.05 50914.46 51005.15

5x10

0

5

50635.53 50707.51

50779.1650848.22 50917.67 50988.53 51061.96

5x10

0

5

50707.9750636.05

50779.74

50849.6850919.01 50989.13 51061.18

5x10

0

550707.8050635.74

50779.47

50849.9450919.84

50989.58 51060.14

50500 50550 50600 50650 50700 50750 50800 50850 50900 50950 51000 51050 51100

Control

5 hours

4 hours

3 hours

2 hours

1 hour

6x10

0

2

50634.32

50796.3450730.59 50958.81

6x10

0

1

2 50635.37

50707.31 50797.5450869.05

6x10

0

1

50633.1150705.05

50795.21 50867.05 50914.46 51005.15

5x10

0

5

50635.53 50707.51

50779.1650848.22 50917.67 50988.53 51061.96

5x10

0

5

50707.9750636.05

50779.74

50849.6850919.01 50989.13 51061.18

5x10

0

550707.8050635.74

50779.47

50849.9450919.84

50989.58 51060.14

50500 50550 50600 50650 50700 50750 50800 50850 50900 50950 51000 51050 51100

6x10

0

2

50634.32

50796.3450730.59 50958.81

6x10

0

1

2 50635.37

50707.31 50797.5450869.05

6x10

0

1

50633.1150705.05

50795.21 50867.05 50914.46 51005.15

5x10

0

5

50635.53 50707.51

50779.1650848.22 50917.67 50988.53 51061.96

5x10

0

5

50707.9750636.05

50779.74

50849.6850919.01 50989.13 51061.18

5x10

0

550707.8050635.74

50779.47

50849.9450919.84

50989.58 51060.14

50500 50550 50600 50650 50700 50750 50800 50850 50900 50950 51000 51050 51100

Control

5 hours

4 hours

3 hours

2 hours

1 hour

84

Table S-1.: Results of manual search of methylglyoxal modified peptides found in the

recombinant antibody

MGO Modified Tryptic Peptides (mis-cleavages) ASQGIR*NYLAWYQQKPGK

YNR*APYTFGQGTK

R*TVAAPSVFIFPPSDEQLK

EVQLVESGGGLVQPGR*SLR

DTLMISR*TPEVTCVVVDVSHEDPEVK

EPQVYTLPPSR*DELTK

SR*WQQGNVFSCSVMHEALHNHYTQK

85

Table S-2: Raw data values of Lys-C peptide peak intensity from Cell Culture WCX fractions 1

and 2

Fraction 1 Peak Intensities Fraction 2 Peak Intensities

Site Unmod. MGO(+54) MGO(+72) Unmod. MGO(+54) MGO(+72)

LC30 273429695 138486086 27697217 398154884 86277387.5 19843799

LC93 637430527 54150819 10830164 517745203 52710066.9 12123315

LC108 567693294 2761976.3 552395.27 293643660 980063.272 225414.55

HC16 517056804 73797731 14759546 672482015 90574849.9 20832215

HC259 478982501 47025077 9405015.5 614438486 64274080 14783038

HC359 321068113 41433990 8286798.1 451460954 53981224.7 12415682

HC420 359124190 14302232 2860446.4 416919964 15076683 3467637.1

Table S-3: Relative Intensity of Lys-C peptides from Cell Culture WCX fractions 1 and 2

Fraction 1 Peak Intensities Fraction 2 Peak Intensities

Site Unmod. MGO (+54) MGO (+54) Unmod. MGO (+72) MGO

(+72)

LC30 1.00 0.506477855 0.216693028 1.00 0.101295571 0.0498394

LC93 1.00 0.08495172 0.101806963 1.00 0.016990344 0.0234156

LC108 1.00 0.004865262 0.003337594 1.00 0.000973052 0.00076765

HC16 1.00 0.142726546 0.134687394 1.00 0.028545309 0.0309781

HC259 1.00 0.098177026 0.104606208 1.00 0.019635405 0.02405943

HC359 1.00 0.129050469 0.119570085 1.00 0.025810094 0.02750112

HC420 1.00 0.039825309 0.036162056 1.00 0.007965062 0.00831727

86

A. Reduced Light Chain

B. Expanded view of the +54 Da modified light chain

Figure S-6: Detection limit of reduced LC/MS to detect MGO (+54 Da) . The deconvoluted

mass spectrum of reduced light chain spiked with MGO modified light chain (top pane is full

view and bottom pane focuses on the +54 Da species). Modified light chain was spiked into

native light chain to deduce the level at which the +54 Da peak could be detected without an

enrichment step. Tha data shows overlaid traces at 10%, 7.5%, 5%, 2.5%, 2%, 1%, 0.5%, 0.1%

and 0% modified antibody spikes. The +54 Da peak was detected at 2% modification and was

unambiguously identified at 5% modification on our instrument.

Light Chain

+54 Da (MGO)

10%

7.5%

5%

2.5%

2%

1%(0.5, 0.1, 0)

+54 Da (MGO)

87

Chapter 3 Discovery of a Chemical Modification by Citric Acid in a Recombinant

Monoclonal Antibody

This chapter is based on a published paper with the same title

Analytical Chemistry 2014, 86(18):8932-6

3.1 Abstract

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity

that can arise from various post-translational modifications. The formulation for a protein

product is to maintain a specific pH and to minimize further modifications. Generally

Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a

range between 3 and 6, and is generally considered chemically inert. However, as we reported

herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a

phosphate/citrate-buffered formulation at pH 5.2, and led to the formation of so-called “acidic

species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed

that the modification occurred at the N-terminus of the light chain. Three additional antibodies

also showed the same modification but displayed different susceptibilities of the N-termini of the

light chain, heavy chain or both. Thus, ostensibly unreactive excipients under certain conditions

may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies.

By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and

capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again

remind us that it is prudent to consider formulations as a potential source for chemical

modifications and product heterogeneity.

http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CB8QFjAA&url=http%3A%2F%2Fwww.fda.gov%2Ffood%2FIngredientspackagingLabeling%2FGRAS%2F&ei=yObkU8b1KJT-yQTElYKwCw&usg=AFQjCNFU0gixNsgl5DmqrLo0kiTKq1pd_w&bvm=bv.72676100,d.aWw


88

3.2 Introduction

As most protein pharmaceuticals, recombinant monoclonal antibodies have a higher

degree of inherent complexity as compared to traditional small molecule drugs. Various protein

post-translational modifications (PTM’s) have been well documented as major contributors to

heterogeneity observed in recombinant monoclonal antibodies1-6

. Some of these processes occur

during cell culture, such as modifications by reactive metabolites (e.g. methylglyoxal and

homocysteine thiolactone)7-8

, glycosylation and sialic acid incorporation9-17

; while others can

occur through production, purification and storage, such as oxidation18-21

, deamidation22-27

,

crosslinking28-29

, protein-protein interactions30

and fragmentation31-34

.

An important part of drug development is to optimize formulation for a given

biotherapeutic35-36

. The formulation should minimize unwanted modifications or degradation

during storage3, 37

. For example, polysorbate80 may be added to mitigate aggregation38-41

. Free

methionine may reduce the formation of methionine sulfoxide in proteins42-45

. A critical aspect

of formulation is the control of pH. One major reason is to minimize the deamidation of

asparagine, a spontaneous non-enzymatic process that occurs in all monoclonal antibodies and

the vast majority of protein pharmaceuticals16, 22, 24-26, 46-47

. Specifically, mildly acidic pH has

been shown to reduce deamidation of asparagine22-27

.

While almost all excipients added to the biotherapeutic formulation are Generally

Recognized as Safe (GRAS) and considered chemically inert (i.e., free from reactions with the

protein products), they may nonetheless display unexpected reactivities. For example,

autoxidation of polysorbate 80 generated radicals that in turn increased the oxidative liabilities of

the formulation, e.g., increases in methionine sulfoxide48

. Photo-oxidation also induces

cleavage, crosslinking and aggregation13, 29, 34, 49-50

. Glycation has been reported when glucose (a



89

reducing sugar with a hemiacetal or aldehyde group) was added to a lyophilized protein drug51

.

As a result of this finding, sucrose (devoid of hemiacetal or aldehyde group) was used instead to

reduce aggregation52

. Yet, in other studies, the glycosidic bond of non-reducing sucrose was

shown to hydrolyze into glucose and fructose, resulting in glycation during storage53-54

.

Pertinent to this work, photochemical degradation of citric acid led to acetonation of therapeutic

proteins55

. Therefore, it is important to thoroughly evaluate the protein drug integrity following

storage in the defined formulation and to screen for unexpected reactivity and modifications.

As reported herein, we observed an early eluting peak (i.e., acidic species) in the weak

cation exchange (WCX) chromatogram for an antibody in citric acid formulation. Peptide

mapping and mass spectrometric analysis revealed that covalent modifications by citric acid led

to the formation of amides (mass increase of 174 daltons) and/or imides (mass increase of 156

daltons) at the N-terminus of the light chain56

. Furthermore, three additional recombinant

monoclonal antibodies displayed a similar susceptibility of the N-termini of both the light and

heavy chains. To the best of our knowledge, this is the first report of a citric acid modification of

recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two

or more carboxylic acid groups and capable of forming an anhydride may exhibit similar

reactivities57-58

. Altogether, our findings again remind us that it is prudent to carefully consider

formulation excipients as a potential source for chemical modifications.

90

3.3 Materials and Methods:

3.3.1 Materials

The recombinant monoclonal IgG1 antibody was produced by stably transfected Chinese

hamster ovary (CHO) cells cultured in a bioreactor and purified at AbbVie Bioresearch Center

(Worcester, MA). Dithiothreitol (DTT) was from Sigma (St. Louis, MO). Acetonitrile and

trifluoroacetic acid (TFA) were from J.T. Baker (Phillipsburg, NJ). Formic acid (FA) was from

EMD (Gibbstown, NJ). Trypsin was from Worthington (Lakewood, NJ). Endoproteinase Lys-C

was from Roche (Indianapolis, IN). Guanidine-HCl was from Thermo Scientific (Rockford, IL).

Citric acid (monohydrate, cat.# 0110-01) and sodium citrate (dehydrate, cat. # 3647-05) were

from JT Baker (Phillipsburg, NJ). The citrate formulation consisted of a 1 mM sodium citrate,

6.5 mM citric acid combined with Na2HPO4, polysorbate 80 and mannitol with the pH adjusted

to 5.2 with sodium hydroxide. The 20X citrate buffer consisted of 20 mM sodium citrate, 130

mM citric acid combined with Na2HPO4 with the pH adjusted to 5.2 with sodium hydroxide.

Antibody A, Antibody A-S, Antibody B and Antibody C were all recombinant monoclonal

antibodies (IgG1’s) produced at AbbVie Bioresearch Center, Worcester, MA.



(ThermoFisher, Sunnyvale, CA) at 94% mobile phase A (10 mM sodium phosphate, pH 7.5) and

6% mobile phase B (10 mM sodium phosphate and 500 mM sodium chloride, pH 5.5) at a flow-

rate of 1 mL/min59

. The percentage of mobile phase B was increased from 6% to 16% over 20

min to elute the antibody monitored by UV absorbance at 280 nm. The column was then washed

91

using 100% mobile phase B and then equilibrated using 6% mobile phase B for 9 minutes

between injections.

3.3.3 LC-MS analysis of reduced antibody


an HPLC (Agilent 1260, Santa Clara, CA) with a reversed phase column (Vydac, C4, 1 x 150

mm, 5µ particle size) coupled to a Q-TOF mass spectrometer (Agilent, 6510). Antibody was

reduced using DTT (10 mM final concentration) at 37ºC for 30 minutes. Ten microliters of each

sample was loaded at 95% mobile phase A (0.08% FA and 0.02% TFA in Milli-Q water) and 5%

mobile phase B (0.08% FA and 0.02% TFA in acetonitrile) and then eluted using a gradient from

5% mobile phase B to 35% mobile phase B in 20 min. The column was washed using 90%

mobile phase B and equilibrated using 5% mobile phase B for 10 min. The flow rate was 50

µL/min and column oven temperature was set at 60 ºC. The mass spectrometer was operated in

positive ion mode with a scan range from m/z 600 to 3200. Ion spray voltage was 4500 volts and

the source temperature was 350 ºC.

3.3.4 Tryptic digestion

Proteins were denatured using 6 M guanidine hydrochloride in 100 mM Tris, pH 8.0 and

reduced using 10 mM DTT at 37 ºC for 30 min. Alkylation was performed using 25 mM

iodoacetic acid in 1M Tris pH 8.0 at 37 ºC for 30 min. The samples were buffer exchanged to 10

mM Tris pH 8.0 using NAP-5 columns (GE Healthcare, Piscataway, NJ). The samples were

digested with trypsin at 1:20 (w:w, enzyme:antibody) at 37 ºC for 4 hours.

92


An UPLC (Acquity, Waters, Milford, MA) equipped with a UPLC C18 column (Waters,

1 x 150 mm i.d., 1.7µ particle size) and a Thermo Scientific LTQ-Orbitrap Velos mass

spectrometer (Thermo Fisher, West Palm Beach, FL) were used to analyze peptide samples.

Forty µL of each sample was loaded at 98% mobile phase A (0.08% formic acid and 0.02% TFA

in Milli-Q water) and 2% mobile phase B (0.08% formic acid and 0.02% TFA in acetonitrile)

and then eluted using a gradient from 2% mobile phase B to 35% mobile phase B in 80 min. The

column was washed using 90% mobile phase B and equilibrated using 2% mobile phase B for 10

min. The flow rate was 50µL/min and column oven temperature was set at 60 ºC. The mass

spectrometer was operated in positive ion mode with a scan range from m/z 400 to 2000 with

alternating low energy collision induced dissociation (CID) and high energy collision induced

dissociation (HCD) of the three most abundant parent masses. Ion spray voltage was set at 4500

volts and the source temperature was set at 350 ºC. The data were analyzed by searching

extracted mass chromatograms (XIC) of tryptic peptides with mass increases of 156 and 174

daltons. The data were also searched against the theoretical primary structure using the Sequest

algorithm and selecting the observed variant increases (Thermo Scientific, West Palm Beach,

FL).

3.3.6 Reaction of citric acid buffer with the monoclonal antibodies

A 20X citric acid buffer (10 mM Na2HPO4, 20 mM Na citrate/130 mM citric Acid pH

5.2), in which the citric acid concentration was 20-fold of that of the formulation solution, was

prepared. Antibody A was diluted to 1 mg/mL in this 20X buffer and stored at 40 ºC for up to 30

days. Antibody A incubated under the same conditions in actual 1X formulation buffer was used

93

as a control. In addition, Antibody A was stored in the 20X citrate buffer with the pH adjusted

up to 7.0. Sample aliquots were removed at days 5, 10, 15, 20 and 30 and stored at -80 ºC until

analysis. The samples were analyzed by WCX-10 as described in the previous section.

3.3.7 N-Terminal Variants and Additional IgG’s

An analog of Antibody A was produced in which the N-terminal residues of the light

chain and heavy chain were swapped (see Table 1). The resulting antibody (Antibody A-S) had

a glutamate at the N-terminus of the light chain and an aspartate at the N-terminus of the heavy

chain. Two other recombinant IgG1’s, Antibody B and Antibody C were also used for the study.

The N-terminal sequences (first ten residues) of all the antibodies are shown in Table 1.

Antibody A-S and the recombinant IgG1’s were formulated into the 20X citrate buffer at pH 5.2

as described in the previous section. The samples were incubated at 40 ºC for 30 days.

Following the incubation, the samples (including Antibody A in 20X citrate) were analyzed by

reduced LC/MS and tryptic mapping with MS/MS detection for the presence and abundance of

the citric acid modifications.

94


As detailed below, we found that citric acid covalently modified the N-termini of either

or both the light and heavy chains in four different antibodies. Our analysis and results are

consistent with the mechanism depicted in Scheme 1 with the anhydrides of citric acid as key

intermediates.

95

Scheme 3-1: (I) Formation of a citric acid anhydride intermediate from citric acid and the

subsequent reaction of the N-terminal amine with the anhydride; (II) The four possible products

of the reaction; +174A and +174B represent adducts formed between the N-terminal amine and

the citric acid anhydride. The +156A and +156B represent the subsequent imide products (5 and

6-membered rings, respectively) resulting from the cyclization of the newly formed amide and

another carboxylic acid in citric acid. There are three carboxylic acids in citric acid: two are

equivalent as denoted by the red dots and the other by the blue dot.

I.

II.

96

3.4.1 Unexpected covalent modifications by citric acid

A recombinant monoclonal antibody (Antibody A) was stored at 40 ºC for 6 months in

two different formulations to determine if there were any major differences in the protein

stability. One formulation had 1 mM sodium citrate, 6.5 mM citric acid and the other

formulation was without sodium citrate/citric acid; both had mannitol and polysorbate 80 and

were at pH 5.2. Analysis of the samples by weak cation exchange (WCX) chromatography

revealed that significant degradation and accumulation of multiple acidic species in both samples

(see Figure 3-1). Most noticeably, the citrate formulation induced a very early eluting and well-

defined peak (Peak A in Figure 3-1) that was absent in the other sample. This finding prompted

us to perform subsequent analysis in order to determine the nature of these species.

97

Figure 3-1: The weak cation exchange chromatogram of the recombinant monoclonal antibody

formulated with and without citrate. The top trace shows an early eluting acidic peak (Peak A)

which is significantly smaller in the formulation without citrate. The control represents

Antibody A in the citrate formulation stored at 4 ºC.

98

3.4.2 Reduced LC/MS Analysis

Peak A fractions were examined by reduced LC/MS (see Figure 3-2). The major peak

(observed mass 23408 Da) corresponded to the native light chain (theoretical mass 23408 Da).

Two other masses of 23564 and 23582 were observed: increases of 156 and 174 Da,

respectively. Pertinent to the mechanism of formation discussed later, these two masses differ by

18 Da and are likely due to the loss of a water molecule. In addition, a mass of 23570 Da was

determined to be a glycation product (+162 Da).

99

Figure 3-2: Mass spectra of the light chain from reduced LC/MS analysis. Full scale spectrum is

shown at the left and expanded view is shown at the right. All samples were stored in citric acid

buffer at 40 ºC except the control which was stored at 4 ºC. (A) 1 mM sodium citrate, 6.5 mM

citric acid at pH 5.2 for 6 months; (B) 1 mM sodium citrate, 6.5 mM citric acid at pH 5.2 for 1

months; (C) 20 mM sodium citrate, 130 mM citric acid at pH 5.2 for 1 month (D) 20 mM sodium

100

citrate, 130 mM citric acid at pH 7.0 for 1 month (E) Light chain control (same as A except

stored at 4 ºC for 1 month).

101

3.4.3 Peptide Mapping and Determination of Sites of Modifications in antibody A

Peptide mapping with mass spectrometric detection revealed three tryptic

peptides present in the formulation with citrate but were absent in the formulation

without citrate. These peaks corresponded to doubly charged ions of peptides with masses of

2051.90 Da (Peptide B) and 2033.88 Da (Peptide C and D), respectively (Figure 3-3). Peptides

C and D were isobaric yet chromatographically resolved on the C18 RP-HPLC column and both

exhibited greater retention and thus greater hydrophobicity than Peptide B. The analysis of the

MS/MS spectra of Peptides B, C and D were in good agreement to each other with the exception

of the 18 Da mass shift between some of the b ions but clearly all three spectra were from the

same fragmentation series. Manual de novo sequencing (Figure 3-4) performed on these

peptides revealed high homology to the predicted y ion series of the N-terminal peptide of the

light chain (Peak A). A comparison of these MS/MS spectra against the experimental MS/MS

spectrum of the N-terminus of the light chain peptide showed high similarity between the

fragmentation patterns as shown in Figure 3-5. The y ion series between Peak A (Native), Peak

B (+174 Da), Peak C and Peak D (+156 Da) covered all residues in the peptide with the

exception of the N-terminal aspartate. The b ion series, although limited, showed strong signal

with coverage amongst the first three residues. Consequently, we were able to assign the

observed mass increases to the N-terminus of the light chain. Thus, the data confirmed that the

mass increases of 156 or 174 Da were from modifications on the light chain N-terminal amine

(i.e., Asp1). Subsequent analysis of the heavy chain N-terminal peptide of Antibody A did not

show any modification.

102

Figure 3-3: HPLC-MS analysis of a tryptic digest of the recombinant monoclonal

antibody A following storage at 40 ºC for 6 months in citrate buffer pH 5.2. The extracted ion

chromatograms corresponding the +2 charge ions for the native light chain peptide (A, m/z =

1877.89), the same peptide with +174A and +174B (B) and mass increases of +156A and +156B

(C), respectively.

103

Figure 3-4. De novo sequencing of doubly charged peptides corresponding to unique M/z’s of

2052.91 (Middle pane) and 2034.90 (Bottom pane). The data are compared to the light chain N-

terminal peptide of M/z 1878.89 (Top pane). Major fragmentation neutral losses corresponding

to a y ion series were in agreement and the deduced sequence is shown at the top of the figure.

104

Figure 3-5: MS/MS spectra of Antibody A light chain N-terminal peptide for the native, +174

Da, +156A Da, and +156B Da citrate modifications, respectively. Both y- and b-ion series

support the modification was at the N-termini. The top spectrum corresponds to the native

peptide while the second spectrum corresponds to the same peptide with the + 174 Da

modification on the light chain N-terminus. The bottom two spectra correspond to the two

products for the + 156 Da modification on the light chain N-terminus.

105

3.4.4 Elucidation of the chemical nature of the modifications

No protein modifications listed in either the ABRF Delta Mass database

(www.abrf.org/index.cfm/dm.home) or the Unimod database (www.unimod.org) could give rise

to the three observed species. As previously stated, citric acid was only present in the

formulation of the sample where these modifications were found. The molecular weight of citric

acid is 192 Da therefore the difference between the observed variants of +174 Da and +156 Da

suggests two successive losses of water from citric acid. As illustrated in Scheme 3-1, we

propose a mechanism that involves the initial formation of citric anhydride, the subsequent

formation of an amide with an amino group in the protein (e.g., the N-terminus) that results in a

molecular weight increase of 174 Da. Further condensation of the resulting amide and another

carboxylic group in covalently attached citric acid leads to the formation of imides (either five-

or six-membered), which confers a molecular weight increase of 156 Da. In addition, it is

reasonable to expect that these two products would form at different rates favoring the 5-

membered product and further supported by the two +156 isobaric peptides we observed (156A

and 156B shown in Figure 3-3). This mechanism is consistent with the results reported on citrate

modification of peptides and the propensity of citric acid to form an anhydride under acidic

conditions56-58, 60-61

.

3.4.5 Reactions in citrate buffers (as compared to formulation)

To isolate and narrow down the factors involved in the modification, antibody A was

incubated in citric acid buffer at the same pH (5.2) but without other formulation excipients (e.g.,


106

without mannitol and polysorbate 80) at 40 ºC for 1 month. Similar to the sample from citrate

formulation, the weak cation exchange chromatogram (Figure 3-6) shows a clear time-dependent

increase in the amount of acidic species. In addition, the formation of the distinct early eluting

peak has a comparable retention time to peak A from the sample formulated in citrate. Similarly,

the reduced LC/MS analysis of the light chain showed a major peak in good agreement with the

theoretical mass and also showed two higher molecular weight masses with increases of +156 Da

and +174 Da but at a higher abundance than the citrate formulation (Figure 3-2). And again,

tryptic mapping confirmed on the same adducts localized to the N-terminus of the light chain

(data not shown). Thus, these experiments supported our hypotheses that the citric acid was

indeed the modifying agent causing the heterogeneity on the N-terminus of the light chain. In

addition, we searched for the same modifications on the heavy chain N-terminal peptide and

found trace levels of the +174 Da adduct and no detection of the +156 Da adduct thus we saw

similar susceptibility as our stability sample (see Table 3-1).

107

Figure 3-6: The weak cation exchange chromatogram of Antibody A incubated in with citric

acid (formulation and buffer alone). A: Analysis of Antibody A stored in 20X citrate buffer for

0, 5, 10, 15, 20 and 30 days and Antibody A in the citrate formulation for 6 months, all at 40 ºC.

The data show the formation of a prominent early eluting acidic peak. In addition, the peak

108

aligns well with the Peak A from the sample stored in the citrate formulation for 6M/40C. B:

Time-dependent accumulation of peak A.

109

3.4.6 Prevalence of the citrate modification

To better understand the scope of this modification, several additional antibodies were

examined (see Table 3-1). One was a variant of Antibody A (Antibody A-S) in which the N-

terminus of the light chain had an aspartate substituted with a glutamate and the N-terminus of

the heavy chain had a glutamate substituted with an aspartate; in essence, the two termini were

swapped. LC/MS analysis of the light and heavy chains of Antibody A-S showed the same site

of modification and similar susceptibility as Antibody A (see Table 3-1), suggesting protein

structures (such as solvent accessibility) perhaps play a more dominant role than specific amino

acid residues62-64

. Additionally, as shown in Table 3-1, Antibody B and Antibody C were also

modified by citric acid at the N-termini of both the light chain and heavy chain. The

modification was also observed on a heavy chain N-terminal alanine residue (data not shown),

suggesting that this modification may occur on other residue at the N-terminus and the N-

terminal acidic side chains (Asp or Glu) are not obligatory. Thus, the modification of the N-

terminal primary amine by citrate appears to be common amongst recombinant IgG1 monoclonal

antibodies but may be influenced by other factors such as the antibody structure and

microenvironment7-8, 15, 62-63

. Furthermore, in all cases, the +174 Da species were more

prominent than the +156 Da species, indicating the former are likely the initial products as

proposed in Scheme 3-1.

110

Table 3-1: The percentage of citric acid modification found in the N-terminus of each chain in

different antibodies: Antibody A in citrate formulation for 6 months at 40 ºC; and Antibodies A,

A-S, B and C in 20X citrate buffer for 30 days at 40 ºC. The +156 Daa and +156 Da

b refer to the

two products formed after the second anhydride formation. The first ten residues of the N-

terminal framework of the heavy chains and light chains of antibodies are also listed (n.d.

denotes not detected).

Recombinant IgG1 LC N-terminus +174 Da +156 Daa +156 Dab HC N-terminus +174 Da +156 Daa +156 Dab

Antibody A (1X, 6M) DIQMTQSPSS 1.3 1.2 0.4 EVQLVESGGG n.d. n.d. n.d.

Antibody A (20X, 1M) DIQMTQSPSS 7.0 2.0 1.4 EVQLVESGGG 0.02 n.d. n.d.

Antibody A-S (20X, 1M) EIQMTQSPSS 8.7 1.3 0.6 DVQLVESGGG 0.8 n.d. n.d.

Antibody B (20X, 1M) EIVLTQSPDF 4.8 0.1 0.1 EVQLVQSGAE 6.4 0.4 0.6

Antibody C (20X, 1M) DVLVTQSPLS 1.8 0.2 0.2 EVKLVESGGG 3.2 0.2 0.3

111

3.4.7 Influence of pH

As shown in Scheme 1, a key intermediate for the modification is citric acid anhydride57-

58, 60-61. Citric acid has three pKa’s of 3.14, 4.75 and 6.39, so at pH 5.2, one of the carboxylic

acids will be predominantly protonated, a first step for anhydride formation. As reported,

formation of citric acid anhydride occurs between pH 3.0 to 6.0, with the maximum at pH 4.0 to

4.556

. At pH 5.2 for our formulation, citric acid anhydride can still accumulate to a significant

degree and modify the antibodies. Increasing the pH to neutral conditions, however, would

markedly diminish the formation of the anhydride, thus little modification of the antibodies (see

Figure 3-2, pH 7 data).

3.4.8 Selectivity of amines

We investigated whether there were any citrate modifications to the primary amines of

lysine residues following the accelerated storage conditions. We searched the peptide mapping

data using the Sequest algorithm (ThermoFisher Scientific) and did not find any modification to

lysine residues. In general, N-terminal amines have a lower pKa (around 8) than those on the

side chains of lysine (around 10). Under mildly acidic conditions (e.g., pH 5.2), though the vast

majority of the N-terminal and lysyl amines are protonated, significant higher percentage of the

N-terminal amines are deprotonated, thereby nucleophilic and can react with anhydride.

Therefore, the observed selectivity of amines are consistent with the generally observed

reactivities of N-terminal amines65-66

.

112

3.5 Conclusions

Our results suggest the general susceptibility of the N-terminal amines to modifications

by citric acid. The reactivity is likely influenced by multiple factors, including pH, pKa at the N-

terminal amines and structural features, therefore the sites and extent of modification cannot be

precisely predicted and thus should be investigated experimentally. In addition, formulations

with elevated concentrations of citric acid would likely cause a greater extent of the modification

therefore it would be prudent to consider other excipients which may be better suited for the

desired pH range. In particular, other molecules containing two or more juxtaposed carboxylic

acid groups may exhibit analogous reactivities (via the formation of anhydrides). Examples from

the Generally Recognized as Safe (GRAS) list include adipic acid, malic acid, succinic acid and

tartaric acid. Altogether, our findings are yet a reminder that the unexpected reactivity of

excipients and formulation, though generally considered chemically inert and safe, should be

carefully scrutinized.


113

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Chapter 4 When Good Intentions Go Awry: Modification of a Recombinant Monoclonal

Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbate

This chapter is based on a submitted paper with the same title

4.1 Abstract

With the advent of new initiatives to develop chemically defined media, cell culture

scientists screen many additives to improve cell growth and productivity. However, the

introduction or increase of supplements—typically considered beneficial or protective on their

own—to the basal media or feed stream may cause unexpected detrimental consequences to

product quality. For instance, because cultured cells are constantly under oxidative stress,

ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture

media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell

culture was covalently modified by xylosone (molecular weight 148), an oxidative product of

ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-

termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148

Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that

ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as

acidic species. Our work serves as a reminder that additives to cell culture and their metabolites

may become reactive and negatively impact the overall product quality, and should be carefully

monitored with any changes in cell culture conditions.

120

4.2 Introduction

Until recently, cell culture scientists mostly focused on cell growth and protein

expression level; but as demonstrated herein, the quality of the final products has recently

emerged as another critical attribute to be considered. To this point, variations in product

quality, or microheterogeniety, are mostly attributed to a myriad of post-translational

modifications (PTMs)1-6

. In order to reduce variability in product quality, cell growth and

expression levels, the current trend in recombinant monoclonal antibody production has been to

move from complex undefined hydrolysate media to the utilization of chemically defined media7-

8. Furthermore, it has been shown that modulation of the supplemental feed can impact the

product quality of the protein drug9-14

. For example, the addition of manganese and galactose to

the medium can increase the amount of terminal galactosylation on the biantennary

oligosaccharide in the CH2 domain of a recombinant monoclonal antibody10

.

A common additive is the much-storied vitamin C (ascorbic acid). In addition to its

function as a cofactor for collagen synthesis15

, it has been implicated as an antioxidant

(biological reductant) and potent scavenger of reactive oxygen species (ROS)16-19

. Large scale

production of recombinant monoclonal antibodies require higher levels of oxygen to maintain

higher cell densities20

. Such conditions may generate reactive oxygen species21-22

. Therefore,

including antioxidants such as ascorbic acid is generally considered protective and desirable.

Such a view is also held for human nutrition and health, perhaps epitomized by the mega-dose

championed by Linus Pauling23

.

Pertinent to this study, media components or additives have been shown to affect product

quality and protein modifications24-25

. The best known example is arguably glycation of proteins

by glucose26-28

, an essential nutrient as the main energy source of cultured cells. While

121

unavoidable, glycation nevertheless is predictable. On the other hand, it is challenging to predict

and detect modifcations by secondary metabolites or by-products. For example, we previously

reported that a recombinant monoclonal antibody was unexpectedly modified by an

accumulation of methylglyoxal (MGO) during cell culture due to a change in media that was

perceived as beneficial29

. Methylglyoxal is a dicarbonyl compound that is generated as a by-

product of glycolysis29-30

. As a reactive metabolite, it modifies the side chains of susceptible

arginine residues forming adducts with mass increases of 72 Da and 54 Da, respectively. Under

optimal conditions, methylglyoxal is effectively removed by the glutathione dependent

glyoxylase I/II pathway30

. However, changes in the cell culture conditions, specifically the

redox state, can affect this important balance, ultimately leading to increased amounts of this

modification31

. Modifications from other reactive species include cysteinylation32

,

glutathionylation33-34

, and N-homocysteintylation from homocysteine thiolactone35

. Therefore,

changes to the cell culture medium may result in unexpected changes in product quality36-37

.

As reported herein, we observed an increase in acidic species in a recombinant

monoclonal antibody supplemented with ascorbic acid during cell culture. Additionally, we

observed two unidentified masses in the reduced LC/MS analysis of acidic fractions which

exhibited molecular weight increases of 130 and 148, respectively. Detailed analyses revealed

that these modifications occurred on the primary amines of the N-termini of heavy and light

chains and also susceptible lysine residues. This was confirmed by in vitro incubation of native

antibody with increasing concentrations of ascorbic acid. Given that the molecular weight of

ascorbate is 176, it was hypothesized that metabolites or degration produts of this nutrient were

the culprits. This was confirmed in more detailed mechanistic investigations using 13

C labeled

ascorbate. As illustrated in Scheme 1, ascorbate is first oxidized to dehydroascorbic acid.

122

Subsequent decarboxylation generates xylosone38-39

. Xylosone is highly reactive and is capable

of modifying susceptible primary amines, resulting in mass increases of 148 Da and 130 Da for

the hemiaminal and Schiff base, respectively (Scheme 1). To the best of our knowledge, this is

the first report of an ascorbate-originated xylosone modification of a recombinant monoclonal

antibody in vitro and in cell culture.

123

4.3 Materials and Methods

4.3.1 Materials

The recombinant monoclonal antibody (adalimumab) was produced by stably transfected

Chinese hamster ovary (CHO) cells cultured in a bioreactor and purified at AbbVie Bioresearch

Center (Worcester, MA). Dithiothreitol (DTT) was from Sigma (St. Louis, MO). Acetonitrile

and trifluoroacetic acid (TFA) were from J.T.Baker (Phillipsburg, NJ). Formic acid (FA) was

from EMD (Gibbstown, NJ). Trypsin was from Worthington (Lakewood, NJ). Ascorbic acid

was from Sigma (St. Louis, MO). 13

C labeled ascorbate reagents labeled at either the carbon 1,

carbon 2 or carbon 3 position were from Sigma (St. Louis, MO). Guanidine-HCl was from

Thermo Scientific (Rockford, IL).



(Thermo Scientific, Rockford, IL) at 94% mobile phase A (10 mM sodium phosphate, pH 7.5)

and 6% mobile phase B (10 mM sodium phosphate and 500 mM sodium chloride, pH 5.5) at a

flow-rate of 1 mL/min. The percentage of mobile phase B was increased from 6% to 16% over

20 min to elute the antibody monitored by UV absorbance at 280 nm. The column was then

washed using 100% mobile phase B and then equilibrated using 6% mobile phase B for 9

minutes between injections.

4.3.3 LC-MS analysis of reduced antibody

124


an HPLC (Agilent 1260, Santa Clara, CA) with a reversed phase column (WR Grace, C4, 1 x

150 mm i.d., 5µ particle size) coupled to a Q-TOF mass spectrometer (Agilent, 6510). Antibody

was reduced using DTT (10 mM final concentration). Two microliters of each sample were

loaded at 95% mobile phase A (0.08% FA and 0.02% TFA in Milli-Q water) and 5% mobile

phase B (0.08% FA and 0.02% TFA in acetonitrile) and then eluted using a gradient from 5%

mobile phase B to 35% mobile phase B in 20 min. The column was washed using 90% mobile

phase B and equilibrated using 5% mobile phase B for 10 min. The flow rate was 50 µL/min

and column oven temperature was 60 ºC. The mass spectrometer was operated in electrospray

positive ion mode with a scan range from m/z 600 to 3200. Ion spray voltage was 4500 volts and

the source temperature was 350 ºC.

4.3.4 Fractionation of Acidic Species

In order to understand the chemical nature of the new acidic peaks, ascorbate

supplemented samples were analyzed using weak cation exchange (WCX) chromatography, and

the distinct acidic peaks were fractionated. The pooled fractions were concentrated using an

Amicon Ultra 10 KDa MWCO (Millipore) and subjected to analysis by reduced LC/MS analysis

as described previously.

4.3.5 Tryptic digestion

Protein fractions from WCX chromatography were denatured using 6 M guanidine

hydrochloride in 100 mM Tris, pH 8.0 and reduced using 10 mM DTT at 37 ºC for 30 min.

Alkylation was performed using 25 mM iodoacetic acid at 37 ºC for 30 min. The samples were

125

buffer exchanged to 10 mM Tris pH 8.0 using NAP-5 columns (GE Healthcare, Piscataway, NJ).

The samples were digested with trypsin at 1:10 (w:w, enzyme:antibody) and incubated at 37 ºC

for 4 hours.


An UPLC (Acquity, Waters, Milford, MA) equipped with a UPLC C18 reversed phase

column (Waters, 1 x 150 mm i.d., 1.7µ particle size) and a Thermo Scientific LTQ-Orbitrap

Velos mass spectrometer (Thermo Fisher, Waltham, MA) were used to analyze peptide samples.

Forty µL of each sample was loaded at 98% mobile phase A (0.08% formic acid and 0.02% TFA

in Milli-Q water) and 2% mobile phase B (0.08% formic acid and 0.02% TFA in acetonitrile)

and then eluted using a gradient from 2% mobile phase B to 35% mobile phase B in 80 min. The

column was washed using 98% mobile phase B and equilibrated using 2% mobile phase B for 10

min. The flow rate was 50µL/min and column oven temperature was 60 ºC. The mass

spectrometer was operated in positive ion mode with a scan range from m/z 300 to 2000 with

alternating CID and HCD of the three most intense parent masses. Ion spray voltage was set at

4500 volts and the source temperature was set at 350 ºC. The data were analyzed by searching

extracted mass traces of tryptic peptides with mass increases of 130 and 148 daltons. The data

were also searched against the theoretical primary structure using the Sequest algorithm and

selecting the observed variant increases (Thermo Scientific, West Palm Beach, FL).

4.3.7 in vitro incubation of Monoclonal antibody with ascorbic acid

Ascorbic acid (Sigma, St Louis, MO) was dissolved in phosphate–buffered saline (20

mM Na2HPO4, 150 mM NaCl, pH 7.2) at a concentration of 3 mg/mL (17 mM) in order to

simulate the high ascorbate conditions from the cell culture experiments. The isoform of the

126

recombinant monoclonal antibody without any C-terminal Lys (denoted as Lys-0) was diluted

into the ascorbate solution and incubated in at 37 ºC for 14 days, the typical duration of the cell

culture experiments. The antibody was buffer exchanged to 10 mM Tris pH 7.0 following the

incubation. Lys-0 antibody incubated under the same conditions in PBS alone was used as a

control. The samples were analyzed by weak cation exchange chromatography and reduced

LC/MS as described in the previous sections. Specific peaks that appeared in the acidic region

of the chromatogram in the sample incubated with ascorbate were fractionated for further

analysis.

4.3.8 Regio-labeled ascorbate to elucidate the mechanism of the modification

Ascorbic acidthat was specifically regio-labelled with 13

C (at C1, C2 or C3 carbon,

respectively) was obtained from Sigma-Aldrich (see Figure 4-14). These ascorbic acid isomers

were used to elucidate and confirm the mechanism for the formation of the reactive degradant

(xylosone) and the final modifications to the antibody. The antibody was incubated with each of

the labeled ascorbate reagents for 14 days at 37 ºC. The samples were analyzed by reduced

LC/MS and by tryptic mapping with LC/MS/MS for analysis of the resulting adducts.

127


As detailed below, we have discovered that a recombinant monoclonal antibody

(adalimumab) was modified by ascorbic acid both in vitro and in cell culture. Furthemore, mas

spectrometric and mechanistic investigation has attributed the modifications to xylosone, a

degradation product of ascorbate. The modifications occurred on primary amines, including the

N-termini of both light and heavy chains and also susceptable lysine residues.

4.4.1 Ascorbate supplement in cell culture induced new acidic variants

A recombinant monoclonal antibody was expressed in shake flasks using various

supplementation and additives. Of specific interest were cell culture conditions under which the

concentration of ascorbate was increased (0, 0.1, 1 and 3 mg/mL, respectively). When

antibodies were analyzed by weak cation exchange chromatography, the formation of new acidic

species directly correlated with ascorbate concentration (Figure 4-1). This observation led us to

initiate a detailed structural analysis of the recombinant monoclonal antibody in order to

determine the chemical nature and cause of the increase in charge heterogeneity.

128

Figure 4-1: WCX-10 chromatograms of the recombinant monoclonal antibody control (top, no

ascorbic acid) and supplemented with 0.1, 1 and 3 mg/mL of ascorbate in cell culture,

respectively. The increase in ascorbate concentration corresponds to an increase in the early

eluting species and a well defined peak (Peak A).

129

4.4.2 Reduced LC/MS analysis of antibody stored in ascorbate buffer

Without separating antibody variants, reduced LC/MS analysis of the recombinant

monoclonal antibody heavy chain and light chain from the ascorbate supplemented cultures did

not show obvious differences in the mass spectra (Figure 4-2). Therefore, fractionation of the

earlier eluting peaks and the Lys-0 peak from weak cation exchange (Figure 4-1) was employed.

The analysis of the Lys-0 fraction by reduced LC/MS revealed a deconvoluted light chain mass

spectra which was in good agreement with the expected light chain mass (Figure 4-3, A).

Analysis of the mass spectra resulting from Peak A, however, exhibited discernible differences

as shown in Figure 4-3 (B). The deconvoluted light chain mass spectra showed the expected

mass as well as several lower abundance peaks with higher molecular-weight. Two of the

masses corresponded to mass increases of 148 Da and 130 Da.

In order to elucidate the nature of the additional species, a pure fraction of the Lys-0

species was treated with ascorbic acid in vitro. The deconvoluted spectrum (Figure 4-3, C) of

the in vitro sample was highly similar to that from cell culture (Figure 4-3, B). First, the

observed molecular weight of 23408.5 Da represented the unmodified light chain from Lys-0

incubated with ascorbate and was in good agreement with the theoretical value of 23408.1.

Secondly, two other lower intensity peaks were also observed with masses of 23538.3 Da and

23556.6 Da which corresponded to mass increases of 130 Da and 148 Da, respectively (Figure 4-

3, C). Furthermore, these two masses are in good agreement with the two masses representing

+130 Da and +148 Da from cell culture suggesting that they were generated due to the ascorbic

acid supplementation. Analysis of the heavy chain from the ascorbate supplementation

experiments was complicated by the presence of inherent glycosylation. Treatment with

130

PNGaseF and carboxypeptidase B simplified the heavy chain mass spectra (Figure 3-4), which

suggests the same ascorbate-related modifications are present although not as conclusive as

shown in the light chain data due to other low intensity peaks in the spectra.

131

Figure 4-2: Light Chain spectra from reduced LC/MS analysis of unfractionated Antibody A.

Two masses of +130 and +148 Da increase within the mass spectra as more ascorbate is added to

the cell culture.

132

Figure 4-3: LC/MS analysis of the recombinant monoclonal antibody light chain after reduction

of antibody. A: Deconvoluted mass spectrum of of the 0 Lys peak (theoritical MW = 23408.13

Da; Observed MW = 23408.34 Da). B: Deconvoluted spectrum of Peak A from cell culture.

The main peak (23408.44) agrees with the theoretical molecular weight of the light chain

(23408.13). Several lower intensity peaks are also observed including those with mass increases

of 130 Da and 148 Da, respectively. C: Deconvoluted mass spectrum of light chain from

Antibody A incubated in buffer containing 3 mg/mL ascorbate for 14 days at 35 ºC, showing the

same peaks as from cell cutlure.

133

Figure 4-4: Deglycosylated heavy chain spectra from reduced LC/MS analysis of fractionated

Peak A. Although two masses of +130 and +148 Da are observed, the heavy chain mass spectra

was less conclusive than the light chain mass spectra.

134

4.4.3 Tryptic mapping and LC/MS/MS detection

Tryptic mapping was performed on the recombinant monoclonal antibody produced in

cell culture supplemented with up to 3 mg/mL of ascorbate and on the 0 Lys isoform incubated

with ascorbate in vitro. It is important to note that the mass difference between these two species

is 18 daltons therefore they may be related and involve the loss of a water molecule, reminiscing

modifications of lysine or arginine residues by methylglyoxal (MGO) or other carbonyl-

containing molecules. Therefore, we searched for mis-cleaved trypic peptides and found none

with an internal arginine. Then, we examined potential modifications of primary amines,

specifically the N-termini and mis-cleaved lysine containing peptides, which did indeed produce

several possible sites of modification as shown in Table 4-1.

Specifically, the light chain N-terminal peptide had two chromatographically resolved

+148 Da peaks with close elution to the native peptide and a +130 peak that eluted later as

shown in Figure 4-5. The MS/MS spectra were analyzed for the native and modified +148 Da

and +130 Da peptides that all had y ion series which were in good agreement with the predicted

amino acid sequence and covered the entire peptide except the first two N-terminal residues as

shown in Figure 4-6. The b ion series was limited but had strong signal for the first three

residues at the N-termini. In addition, a strong signal for the native and modified a1 and a2 ions

was also present. All together, our data conclusively localized both modifications (+148 Da and

+130 Da) to the N-terminal amine of the light chain (Asp1).

135

Table 4-1. Peptides identified with modifications by xylosone (sites are denoted by asterick *

and NH2- denotes N-terminal amine).

Chain Peptide Sequence Increase in Mass (Da) Location

_______________________________________________________________________

LC *NH2-DIQMTQSPSSLSASVGDR 148, 130 N-terminus

LC NYLAWYQQK*PGK 148 Variable Region

LC APK*LLIYAASTLQSGVPSR 148 Variable Region

LC APYTFGQGTK*VEIK 148 Variable Region

LC VEIK*R 148 Constant Region

LC EAK*VQWK 148 Constant Region

LC VQWK*VDNALQSGNSQESVTEQDSK 148 Constant Region

LC DSTYSLSSTLTLSK*ADYEK 148 Constant Region

LC HK*VYACEVTHQGLSSPVTK 148 Constant Region

LC VYACEVTHQGLSSPVTK*SFNR 148 Constant Region

HC *NH2-EVQLVESGGGLVQPGR 148, 130 N-terminus

HC DNAK*NSLYLQMNSLR 148 Variable Region

HC GPSVFPLAPSSK*SGSGGTAALGCLVK 148 Variable Region

HC DYFPEPVTVSWNSGALTSGVHTFPAV

LQSSGLYSLSSVVTVPSSSLGTQTYIC

NVNHK*PSNTK 148 Constant Region

HC VSNK*ALPAPIEK 148 Constant Region

HC ALPAPIEK*TISK 148 Constant Region

HC DELTK*NQVSLTCLVK 148 Constant Region

HC TTPPVLDSDGSFFLYSK*LTVDK 148 Constant Region

HC LTVDK*SR 148 Constant Region

136

Figure 4-5: Extracted ion chromatograms (XIC) from the recombinant monoclonal antibody

tryptic map corresponding to the doubly charged light chain N-terminal peptide and N-terminal

peptides with +148 Da and +130 Da adducts. The +148 Da adduction resulted in two

chromatographically resolved species in the XIC.

137

Figure 4-6: The MS/MS spectra of the light chain N-terminal tryptic peptides from Peak A

fractionated from the recombinant monoclonal antibody supplemeted with 3 mg/mL ascorbate.

The top spectra is for the unmodified peptide; the two middle spectra show the two +148 Da

peptides, respectively; and the bottom spectra is for the +130 Da peptide. In all cases, the a1 ions

definitively localize the modifications to the N-termini of the peptides.

138

The N-terminal peptides of the heavy chain also exhibited a parent peak in the native

extracted mass chromatogram, two isobaric peaks in the extracted mass chromatogram

corresponding to a mass increase of +148 Da and two isobaric peaks in the extracted mass

chromatograms corresponding to a mass increases of +130 Da (Figure 4-7). Similar to those of

the light chain N-termini, the MS/MS spectra for all five of the peaks again showed a very strong

y ion series with good agreement with the expected amino acid sequence as shown in Figure 4-8.

Once again, the b ion series was used to definitively assign the +148 Da and +130 Da

modification to the N-termini of the two sets of resolved modified peptides.

The analysis of other peptides from the recombinant monoclonal antibody showed the

modification of mis-cleaved lysine residues (see Figures 4-9 and 4-10). However, lysines

residues exhibited lower susceptibility to modification as compared to the N-terminal primary

amines as shown in the analysis of the in vitro ascorbate incubation over time and at increasing

concentrations of ascorbate (Figure 4-11). The observation is in good agreement with the lower

pKa of the N-terminal amines (~ 8) as compared to the pKa of the lysyl amine on the side chain

(~ 10)40-41

; and of course , these modifications are also likely to be influenced by other factors

such as the antibody structure and microenvironment24, 29, 35, 42-44

. In addition, it is important to

note that only a +148 Da species was seen for all modified lysine residues in the antibody, again

suggesting local environment is likely to affect the nature and distribution of various chemical

forms, as discussed in greater details later.

139

Figure 4-7: Extracted ion chromatograms from the heavy chain N-terminal peptide. The XIC

for the +148 mass produced two peptides which elutes slightly later than the unmodified XIC.

The ratio is approximately 2:1 suggesting one of the isomers is favored over the other. The XIC

for the +130 mass also produced two peaks which also had a 2:1 ratio which suggests both

species resulted from a Schiff base formation as shown in Scheme 1.

140

Figure 4-8: MS/MS spectra from the heavy chain N-terminal peptide. The data conclusively

localizes the +148 Da and +130 Da modifications from the peaks shown in S-2 to the N-terminus

of the heavy chain.

141

Figure 4-9: Extracted ion chromatograms of an unmodified (top) peptide with an internal lysine

and the same peptide modified by a xylosone adduction. Only the +148 Da modification was

observed for all modified lysine containing peptides.

142

Figure 4-10: MS/MS spectra of the unmodified (top) and modified (bottom) lysine containing

peptides shown in S-5. The data localizes the +148 xylosone adduction to the internal lysine

residue.

143

Figure 4-11. Relative susceptabilities of representative peptides modified by xylosone

144

The same exercise was performed for the tryptic maps of the pure 0 Lys isoform treated

with ascorbate in vitro (0.1, 1.0 and 3.0 mg/mL) over time (3, 5, 11 and 14 days). The same

modifications of the heavy chain and light chain N-terminal peptides were observed by the

presence of mass increases of +130 Da and +148 Da on these peptides, respectively (Figure 4-12

and Figure 4-13). Additionally, the MS/MS data once again localized the increase to the

respective N-terminal residues (data not shown). Furthermore, miscleaved lysine containing

peptides were also observed to a similarly lesser degree. The relative susceptibilities and rate of

the modification to these representative peptides are shown in Figure 4-11. Reduced LC/MS

analysis of the light chain also showed a correlation between the duration of the in vitro

incubation and the extent of these modifications (Figure 4-14). Thus, the modifications observed

in the cell culture samples supplemented with ascorbate were in good agreement with the

modifications seen from in vitro treatment, narrowing down the modification species to ascorbic

acid or its derivatives.

145

Figure 4-12: Comparison of XIC’s of modified light chain N-terminal peptides generated in cell

culture (Peak A) or during in vitro incubation with ascorbate. The overall chromatographic

profiles between the two samples are in good agreement suggesting the same chemical

modifications are generated in both conditions.

146

Figure 4-13: Comparison of XIC’s of modified heavy chain N-terminal peptides generated in

cell culture (Peak A) or during in vitro incubation with ascorbate. Once again, the overall

chromatographic profiles between the two samples are in good agreement suggesting the same

chemical modifications are generated in both conditions.

147

Figure 4-14: Reduced LC/MS of light chain from recombinant mAb incubated with xylosone

over time. The data show that the levels of the +130 and +148 Da modifications increase over

time.

148

4.4.4 Elucidation of the modification agent as xylosone

The mass of ascorbate is 176 Da which is 28 Da greater than the +148 modification

observed, so it was unlikely that ascorbate itself modified the antibody, but rather its degradation

product(s) was the likely culprit(s). Another hint is the 18 Da difference (130 vs 148 Da)

between the two observed modifications, suggesting a dehydration (elimination of a water

molecule) following the initial reaction; this is reminiscent of modifications of lysine or arginine

residues by methylglyoxal (MGO) or other carbonyl-containing molecules. All together, we

postulated that the bona fide reactive species should be degraded from ascorbate and also contain

reactive carbonyl group(s). Xylosone thus emerged as a likely candidate as it has been reported

as a degradation product of ascorbate (see Scheme 4-1 for its formation pathway) 38

.

Furthermore, xylosone has a mass of 148 Da and two carbonyl groups. The 130 Da adduct may

be due to the loss of a water molecule following the initial addition reaction between the

carbonyl group of xylosone and the amines in the protein (hemiaminal to Schiff base as shown in

Scheme 4-1).

149

Scheme 4-1:

150

4.4.5 Incubation of antibody with 13

C Regio-labeled ascorbate

Isotopic labeling and tracing often provides detailed mechanistic insights45-47

. In order to

confirm that ascorbate was degraded to xylosone (losing the carbon atom at 1 position), which in

turn modified the antibody, we used ascorbate regio-specifically labeled with 13

C (at C1, C2 or

C3 carbon, respectively, as shown in Figure 4-15, A). Following incubation with unlabeled

ascorbate or one of the three regio-labeled ascorbate molecules, the antibody was analyzed by

tryptic peptide mapping with mass spectrometry detection. The results (Figure 4-15, B) lended

conclusive evidence supporting the mechanism in Scheme 4-1: labeling at the C1 position (13

C

or 12

C) produced the same mass spectra; in contrast, ascorbate with 13

C labeling at C2 or C3

shifted the peaks to 1 m/z higher compared to 12

C ascorbate (+149 vs +148 and 131 vs 130,

respectively). In addition, MS/MS localized the modification to the light chain N-terminal

residue or heavy chain N-terminal residue including the heavy label for the C2 and C3 regio-

labeled ascorbates as shown in Figure 4-16 and Figure 4-17, respectively. Thus, the data have

verified that the C1 carbon in ascorbate is lost but neither C2 nor C3. Therefore, our data

confirmed that ascorbate was converted to xylosone with the concomitant loss of C1 carbon atom

as illustrated in Scheme 4-1.

151

Figure 4-15: A: Structures of the four different isotopic isoforms of ascorbate used to probe the

structure of the +130 Da and +148 Da adducts. The red dot denotes carbon-13 labeling at a

given position. B: The mass spectra of the doubly charged modified light chain N-terminal

trypic peptide of Antibody A incubated with 3 mg/mL of ascorbate. The pattern of mass shift,

indicates that the carbon atom at 1 position in ascorbate is cleaved off in the final adducts,

consistent with the proposed mechanism of xylosone being the reactive intermediate (see

Scheme 4-1).

152


ascorbic acid with 13C at C1, C2 or C3, respectively. The heavy label is not retained in the

ascorbate labeling at C1 but is retained when labeled at C2 and C3 thus conforming that

ascorbate is degrading to xylosone which subsequently modifies susceptible primary amines.

153

Figure 4-17: MS/MS spectra of heavy chain modified by xylosone derived from ascorbic acid or

ascorbic acid with 13C at C1, C2 or C3, respectively. The data supports the conversion of

ascorbate to xylosone and subsequent modification to the heavy chain N-terminal primary amine.

154

4.4.6 Chemical Nature of the Modfications

When a carbonyl reacts with an amine, two products may form: the initial addition

reaction leads to a hemiaminal (see Scheme 4-1) with a mass equals to the total of the masses of

the two reactants (amine and carbonyl; for xylosone, 148 Da); a subsequent elimination of a

water molecule (18 Da) leads to a Schiff base (imine, see Scheme 4-1) with a mass that is 18 Da

less than the hemiaminal (148-18=130 Da). Hence the masses of the two adducts match with the

observed masses.

The underlying chemistry, including stereo-, regio- and positional isomers of the adducts,

also explains the multiple isobaric peaks observed (see Figures 4-5 and 4-7). For instance, the

formation of the hemiaminal can result in two stereoisomers (the chiral center is denoted with an

* in Scheme 4-1). Two other factors further complicate the situation: first, two carbonyls exist in

xylosone; and second, xylosone may exist in both cyclic and acyclic forms (see Schemes 4-2 and

4-3), and each may lead to the hemiaminal and Schiff base forms described above. Furthermore,

the Schiff bases can undergo further cyclization as well (Scheme 4-3).

For this antibody, no modification of arginine was observed; at first, it was some what

surprising to us as both xylosone and methylglyoxal contain two adjacent carbonyl groups.

However, upon closer inspection, as shown in Scheme 4-2, several hydroxyl groups in xylosone

can readily form stable cyclic hemiacetal or hemiketal with one of the carbonyl group, thereby

leaving only one reactive carbonyl group for further reactions with amines that is similar to that

of glycation of amines. Of course, protein structures and local environments can certainly affect

the stability of the products described above24

. For example, for some other proteins,

modification of arginine by xylosone was reported48

.

155

Ascorbate oxidative degradants have been reported as a source of chemical modifications

in human eye lens and were shown to modify lysine and arginine residues in model systems38-39

.

However, neither a +148 Da or +130 Da mass deviation nor are listed as a xylosone modification

in the ABRF Delta Mass database (www.abrf.org/index.cfm/dm.home) or the Unimod database

(www.unimod.org). To the best of our knowledge, this is the first report of a xylosone

modification of a recombinant monoclonal antibody.


http://www.unimod.org/

156

Scheme 4-2. Reaction scheme of cyclic xylosone with a protein primary amine.

157

Scheme 4-3: Reaction scheme of acyclic xylosone with a protein primary amine.

158

4.4.7 Cell Culture Media Additives

Cell culture scientists are under constant pressure to increase titers, enhance cell culture

performance and improve product quality. The practice of modifying the cell culture medium is

the predominant tool used to address these goals10, 49-54

. However, it is difficult to predict

whether a specific additive will address a specific biochemical need of the host cell with the

desired outcome or whether these good intentions will go awry (see Table 4-2). Occasionally, a

cell culture additive may have negative implications on product quality55-58

. In our study, a

recombinant monoclonal antibody (adalimumab) exhibited a difference in product quality

following a change to the cell culture conditions. Our initial observations were confounded by

the fact that ascorbate unexpectedly degraded quite rapidly to xylosone that in turn exhibited

reactivity with susceptible primary amines. It has been well established that increasing glucose

levels in a cell culture will increase the potential for glycation to occur through well understood

chemistry26-27

. However, the discovery that ascorbate supplementation to the cell culture of a

recombinant monoclonal antibody induced a novel glycation-like modification by xylosone was

quite surprising and is a reminder that product quality is another parameter that must be

considered when making changes to the cell culture feeding strategies. Futhermore, it is worth

noting that xylosone almost certainly modifies a myriad of proteins of the host cells, thereby

directly affecting a broad range of biological activities ultimately impacting the culture viability.

159

Table 4-2: List of cell culture additives shown to affect the product quality

1. Wong, D.C.F., et al., Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation

quality in CHO cell cultures. Biotechnology and Bioengineering, 2005. 89(2): p. 164-177.

2. Jing, Y., et al., Identification of cell culture conditions to control protein aggregation of IgG fusion proteins expressed

in Chinese hamster ovary cells Process Biochemistry 2012. 47(1): p. 69-75.

3. Crowell, C.K., et al., Amino acid and manganese supplementation modulates the glycosylation state of erythropoietin

in a CHO culture system. Biotechnol Bioeng, 2007. 96(3): p. 538-49.

4. Banks, D.D., et al., The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated

formulation studies. J Pharm Sci, 2009. 98(12): p. 4501-10.

5. Fischer, S., J. Hoernschemeyer, and H.C. Mahler, Glycation during storage and administration of monoclonal antibody

formulations. Eur J Pharm Biopharm, 2008. 70(1): p. 42-50.

6. Hossler, P., et al., Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the

targeted shifting of protein glycosylation profiles of recombinant protein therapeutics. Biotechnol Prog, 2014.

7. Gramer, M.J., et al., Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and

galactose. Biotechnol Bioeng, 2011. 108(7): p. 1591-602.

8. Hayter, P.M., et al., Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human

interferon-gamma. Biotechnol Bioeng, 1992. 39(3): p. 327-35.

9. Yuk, I.H., et al., Controlling glycation of recombinant antibody in fed-batch cell cultures. Biotechnol Bioeng, 2011.

108(11): p. 2600-10.

10. Tachibana, H., et al., Changes of monosaccharide availability of human hybridoma lead to alteration of biological

properties of human monoclonal antibody. Cytotechnology, 1994. 16(3): p. 151-7.

11. Baker, K.N., et al., Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol

Bioeng, 2001. 73(3): p. 188-202.

12. Gu, X. and D.I. Wang, Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding

of N-acetylmannosamine. Biotechnol Bioeng, 1998. 58(6): p. 642-8.

13. Kaufman, R.J., M. Swaroop, and P. Murtha-Riel, Depletion of manganese within the secretory pathway inhibits O-

linked glycosylation in mammalian cells. Biochemistry, 1994. 33(33): p. 9813-9.

14. Chaderjian, W.B., et al., Effect of copper sulfate on performance of a serum-free CHO cell culture process and the

level of free thiol in the recombinant antibody expressed. Biotechnol Prog, 2005. 21(2): p. 550-3.

Compound Type Product Quality Impact Reference

Gln Amino Acid Low glutamine decreased sialylation and increased

hybrid and high mannose N-glycans [1]

Cystine Amino Acid Reduced protein aggregation [2]

Cys, Ile, Leu, Trp,

Val, Asn, Asp, Glu Amino Acid Increased sialylation [3]

Sucrose Sugar Increased glycation [4, 5]

Increased high mannose N-glycans [6]

Galactose Sugar Increased G1/G2 N-glycans [7]

Glucose Sugar

Low glucose reduced full glycosylation [8]

Low glucose reduced glycation [9]

Changes in antigen binding due to changes in

glycosylation [10]

ManNAc Sugar Increased sialylation [11, 12]

GlcNAc, uridine,

ManNAc Sugar Increased glycan antennarity [11]

Mn Trace Metal Increased G1/G2 N-glycans [7]

Low Mn inhibited O-glycosylation [13]

Cu Trace Metal Promoted disulfide bond reformation [14]

Fe Trace Metal Increased protein oxidation [15]

Co Trace Metal Increased G1/G2 N-glycans [16]

Ascorbate Vitamin Increased peptide bond breakage and deamination [17]

DMSO Non-nutrient Reduced sialylation [18]

Glycerol Non-nutrient Increased sialylation, decreased aggregation [18]

Dexamethasone Non-nutrient Decreased aggregation [19]

160

15. Kim, K., S.G. Rhee, and E.R. Stadtman, Nonenzymatic cleavage of proteins by reactive oxygen species generated by

dithiothreitol and iron. J Biol Chem, 1985. 260(29): p. 15394-7.

16. Hossler, P., C. Racicot, and S. McDermott Targeted shifting of protein glycosylation profiles in mammalian cell culture

through media supplementation of cobalt. Journal of Glycobiology, 2014. 3.

17. Richheimer, A. and A.B. Robinson, Degradation of transferrin in the presence of ascorbic acid and oxygen.

Orthomolecular Psychiatry, 1977. 6(4): p. 290-299.

18. Rodriguez, J., et al., Enhanced production of monomeric interferon-beta by CHO cells through the control of culture

conditions. Biotechnol Prog, 2005. 21(1): p. 22-30.

19. Qian, Y., Y. Jing, and Z.J. Li, Glucocorticoid receptor-mediated reduction of IgG-fusion protein aggregation in

Chinese hamster ovary cells. Biotechnol Prog, 2010. 26(5): p. 1417-23.

161

4.4.8 Acidic Species

The modification of a recombinant monoclonal antibody by xylosone led to an increase

in acidic species. Acidic species has been widely studied with regards to recombinant

biotherapeutics. Common contributors include asparagine deamidation59-62

, glycation of primary

amines26-27, 45

, incorporation of sialic acid63

, etc. and our recent studies showing methylglyoxal

(MGO) modification of arginine residues29

and covalent adduction of the N-terminal primary

amines by citrate64

. In all these cases, there is a change in formal charge either due to the

introduction of a carboxylate into the molecule or the perturbation of a basic residue protonation

due to a depression in its ionizable group’s pKa. The adduction of the N-terminus of heavy and

light chains and lysine primary amines of the recombinant monoclonal antibody by xylosone

appears to follow this second case. Thus, in depth studies of the underlying cause of acidic

species helps analytical scientists establish general chemical susceptibilities that provide insight

and may ultimately allow the assignment of all molecular variants which exist in this highly

heterogeneous region65

.

4.5 Conclusions

Cell culture supplementation with ascorbic acid caused an unexpected change to the

product quality of a recombinant monoclonal antibody, therefore the use of ascorbic acid as a

supplement should be taken with caution. The recombinant monoclonal antibody was modified

by xylosone, a highly reactive species generated as an oxidative degradation product of

ascorbate, but not directly introduced into the culture. In addition, xylosone almost certainly

modifies a myriad of proteins of the host cells, thereby directly affecting a broad range of

162

biological activities which may impact the culture viability. With the advancement in protein

mass spectrometry and the increasing awareness of issues highlighted in this paper, similar

modifications and mechanisms will likely be revealed in other systems and proteins. Altogether,

better understanding and critical consideration of the latent reactivities of any addictives —and

particularly, deleterious consequences—would be prudent to ensure the quality of protein

products.

163

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168

Chapter 5. Perspectives and Future Directions

5.1 Perspectives and Future Directions

From a quote previously mentioned “analytical chemists are very good at finding what

they know”. Recombinant proteins are extremely complex molecules and in addition to changes

driven due to primary structure (deamidation, oxidation, N-terminal pyroglutamate, etc.), they

are also subject to modifications by enzymes, reactive metabolites, cell culture additives and

formulation excipients. The intracellular environment is in constant flux which is exacerbated by

the high demands of the protein drug expression, high cell densities and flux in redox. All of

these can induce changes to the environment where reactive molecules may appear.

As discussed in this dissertation, both advanced glycation end products and xylosone

have been implicated in protein crosslinking. More recently, it has been shown that protein

crosslinks may be detected using 18

O labeling with an algorithm which looks for a signature

crosslink pattern based on the presence of two C-termini1. Such an approach should be applied

to samples where the prevalence of AGEs or xylosone is apparent. In addition, such an approach

may uncover other reactive species in cell culture.

The targeted search for events known to be associated with ROS may prove fruitful. For

instance, racemization has been associated with protein aging and may be facilitated in

environments when the levels of reactive oxygen species increase. Recent reports present

analytical methodologies which may be applied to help detect and quantify the levels of

racemization which may be occurring2. Hydrogen-deuterium exchange of the alpha carbon

proton has proven a valuable approach in assessing which residues in the primary structure

underwent the formation of the racemization intermediate thereby providing regions worth

investigating further3. These occurrences along with changes in chromatographic behavior could

169

effectively probe the primary structure of the antibody for racemization as a result of a stressed

cellular environment.

As discussed previously, sulfenylation has emerged as another posttranslational

modification linked to the formation of ROS4. They should certainly be considered in

recombinant monoclonal antibody production. The recently reported trapping strategies may

prove quite useful. In addition, other variants associated with cysteine residues such as

trisulfides should be further investigated5. Like sulfenylated products, the trisulfides are also

labile therefore they may be more prevalent than currently thought.

The current work presented focused on the recombinant monoclonal antibody product.

However, modifications of other endogenous CHO cell proteins by reactive species such as

methylglyoxal could also have impactful consequences to the cell culture. Analytical studies to

focus of CHO proteins should be performed. Proteomics analysis of cell culture lysates from

varying degrees of stress could probe for proteins susceptible to this modification. The influence

such a modification could have on the cell viability or the expression levels should be

investigated.

The structure-function relationship of recombinant monoclonal antibodies exhibiting

chemical modification would prove prudent. Chemical modifications to the CDRs may likely

affect the ability of the antibody to bind its intended target. An investigation of antibody

function using surface plasmon resonance may provide clues to underlying chemical

modifications. If resolved regions of the ‘acidic species’ have reduced binding to the epitope, it

would suggest that a chemical modification is a likely cause. Additionally, modifications to the

FC region of the antibody could influence the Effector Functions. This could have further

reaching implications beyond the protein drug. These conserved regions share homology with

170

endogenous human IgG1 therefore sites susceptible be reactive species in CHO cell culture will

likely extrapolate to susceptible human IgG.

The mass spectrometry search algorithms are lacking. These tools are capable of

reporting chemical modifications which are considered by the application. They are not capable

of reporting mystery mass shifts however due to the algorithms de-coupling of MS and MS/MS

data. The MS/MS spectral data used to identify a peptide should be linked with the observed

parent mass. Should the fragmentation profile be in good agreement with neutral losses

corresponding to a region of the fasta sequence but the parent mass deviate from the theoretical,

it should flag it along with the mass shift. In this way, the target peptide, the mass change and

potentially the target residue would be reported. Seldom, do modifications happen at only a

single site therefore the presence of multiple flags for the same variant would increase the

likelihood of its validity. Such information would be a major breakthrough for the identification

of unknown chemical modification. Communicating with groups like Protein Metrics or Peaks

on needs for future versions of their data analysis packages could make this approach a reality.

Mass spectrometry is constantly evolving to meet the demands of drug discovery.

Accordingly, instruments are achieving greater sensitivity, faster scan rates and higher

resolution. In addition, fragmentation technologies are also improving to where Top Down and

Middle Down approaches are becoming more and more robust6-7

. Such approaches may be able

to detect lower abundance mass shifts enabling easier identification of variants. In addition

ETD, electron transfer dissociation, has matured into a valuable tool for measuring more labile

chemical modification. ETD has been used to successfully perform Top Down analysis on the

heavy and light chains of a recombinant monoclonal antibody8. Using this approach to carefully

171

dissect and discern isolated regions of the weak cation exchange profile may provide a molecular

mapping of the antibody elution profile and help to uncover what is influencing it.

Alternative approaches to evaluating charge heterogeneity should be investigated. The

Agilent OffGel Fractionator may be applied to the separation of charge variants in a recombinant

monoclonal antibody. The separation mechanism is equivalent to isoelectric focusing, however,

discrete bands separated by differences in pI can be recovered to measurable amounts and

subjected to analysis by mass spectrometry9. This approach would differ from weak cation

exchange chromatography in that charge differences not associated with surface residues could

be elucidated.

Alternatively, chromatofocusing is becoming more widely used for the analysis of charge

heterogeneity. It is a hybrid technique which utilizes ampholytes to produce a pH gradient thus

influencing the charge as the pH approaches the isoelectric point10

. Additionally, the interaction

between the protein and the weak cation exchange column still relies on surface charge. The

intriguing aspect of chromatofocusing is the possibility of coupling this technique directly to a

mass spectrometer. Salt based separations are not compatible with mass spectrometry but a

separation which uses MS friendly ampholytes may be able to bridge the gap between isolation

and analysis. This approach deserves further investigation.

In conclusion, the most critical aspects to discover an unknown variant in recombinant

monoclonal antibody is staying well informed of the science. A continued pursuit in

understanding chemical biology, biochemistry, advances in analytics and the current reports in

the literature pertaining to antibody heterogeneity are essential. Being well informed of possible

reactive species increases the likelihood that an unassuming peak in a mass spectrum is not

missed. It is of course through my training over the past five years that I have come this point

172

where I have contributed to what is known about recombinant monoclonal antibody

heterogeneity and as I look towards the future, it is the continued pursuit of the science which

will lead to the yet unwritten chapters.

173

5.2 References



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status report. Proteomics 2014, 14. 1130-40.

8. L. Fornelli, E. Damoc, P. M. Thomas, N. L. Kelleher, K. Aizikov, E. Denisov, A.

Makarov, Y. O. Tsybin, Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer

Dissociation Orbitrap FTMS. Molecular & Cellular Proteomics : MCP 2012, 11. 1758-1767,

DOI: 10.1074/mcp.M112.019620.

9. E. Mostovenko, C. Hassan, J. Rattke, A. M. Deelder, P. A. van Veelen, M. Palmblad,

Comparison of peptide and protein fractionation methods in proteomics. EuPA Open Proteomics

2013, 1. 30-37, DOI: http://dx.doi.org/10.1016/j.euprot.2013.09.001.

10. A. Jungbauer, C. Tauer, E. Wenisch, K. Uhl, J. Brunner, M. Purtscher, F. Steindl, A.

Buchacher, Isolation of isoproteins from monoclonal antibodies and recombinant proteins by

chromatofocusing. J Chromatogr 1990, 512. 157-63.

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