Monoclonal Antibodies Meeting the Challenges in
Manufacturing, Formulation, Delivery and Stability of Final
Drug Product
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Woodhead Publishing Series in Biomedicine:
Number 77
Monoclonal Antibodies
Meeting the Challenges in
Manufacturing, Formulation,
Delivery and Stability of Final
Drug Product
Steven J. Shire
AMSTERDAM • BOSTON • CAMBRIDGE • HEIDELBERG
LONDON • NEW YORK • OXFORD • PARIS • SAN DIEGO
SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO
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Contents
List of figures ix
List of tables xv
About the author xvii
Prefacexix
1 Introduction 1
Pharmaceutical development 1
Development of the API 2
mAbs as protein therapeutics 3
Brief review of mAb structure 12
References 14
2 Analytical tools used in the formulation and assessment of
stability of monoclonal antibodies (mAbs) 17
Analytical methods for evaluation of monoclonal antibody stability 17
References 39
3 Stability of monoclonal antibodies (mAbs) 45
Degradation routes in monoclonal antibodies 45
Chemical degradation 45
Mechanisms of oxidation 51
Nonenzymatic peptide fragmentation 62
Nonreducible cross-linking in mAbs 66
Physical degradation 67
Exposure to air/water interfaces due to agitation 77
Use of large-scale pumps in DP unit operations 78
Filtration 78
Filling 79
Adsorption to surfaces 80
References 81
4 Formulation of proteins and monoclonal antibodies (mAbs) 93
Formulation of monoclonal antibodies 93
Buffers for pH control 107
Ionic strength and tonicity modifiers 108
vi Contents
Surfactants and surface-active agents 110
Antioxidants 111
Protein Stabilizers 112
References 117
5 Challenges in the intravenous (IV) administration of
monoclonal antibodies (mAbs) 121
Extractables and leachables from IV bags and impact on
protein/mAb stability 121
References 127
6 Challenges in the subcutaneous (SC) administration of
monoclonal antibodies (mAbs) 131
The challenge of formulating at high concentration 131
Impact on delivery due to high viscosity at high
mAb concentrations 132
Impact on manufacturing of high-concentration SC formulations
due to high viscosity 134
Bioavailability of a high-concentration mAb formulation for SC delivery 135
Development of analytical tools for high-concentration formulation
development 136
References 137
7 Strategies to deal with challenges of developing high-concentration
subcutaneous (SC) formulations for monoclonal antibodies (mAbs) 139
Using existing manufacturing technologies through redesign of
equipment or modification of process variables to produce
high-concentration formulations 139
Development of alternative processes/formulations for manufacturing
of high-concentration dosage forms 140
Using formulation excipients to reduce viscosity 146
References 151
8 Development of delivery device technology to deal with the
challenges of highly viscous mAb formulations at high concentration 153
Using delivery devices to deliver large volume mAb formulations
by the subcutaneous route 153
Delivery of viscous solutions using a prefilled syringe 153
The technical challenges for device and formulation development 154
Primary container/closure systems for devices to be used with mAbs 154
Silicone oil interactions with proteins and mAbs in prefilled syringes 155
Impact of leachables from prefilled syringe components 157
Potential interactions with stainless steel needles 157
Potential problems with tungsten in prefilled syringes 159
Filling of highly concentrated mAbs into prefilled syringes 160
References 160
Contents vii
9 The molecular basis of high viscosity of monoclonal antibodies (mAbs)
at high concentration 163
What is viscosity? 163
How is viscosity measured experimentally? 165
Other methods for determination of viscosity 167
The dependence of viscosity on attractive protein–protein interactions 171
Specific interactions in mAb1 that result in increase of viscosity 175
Impact of net charge versus localized surface charge distribution
on protein–protein interactions and viscosity as a function of mAb
concentration 178
Linking amino acid sequence to self-association and viscoelastic
behavior of mAb1 and mAb2 182
Coarse-grained molecular dynamics computations 185
References 189
10 The future of monoclonal antibodies (mAbs) as therapeutics and
concluding remarks 193
References 194
Index197
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List of figures
Figure 1.1 Major contributors to successful pharmaceutical development.2
Figure 1.2 (a) Electron microscope picture of an IgG1 mAb. (b) Ribbon diagram of
an antibody showing main, secondary, and tertiary structural folding
(directional arrows are β sheet structures). (c) Schematic of an IgG mAb
showing key structural features.12
Figure 2.1 Schematic of tentacle ion exchangers compared to conventional ion exchanger
(shown for an anion exchange resin).19
Figure 2.2 Isoelectric focusing of TMVP and r-TMVP in 8 M urea. Lanes 1–3
correspond to r-TMVP, pl markers (Pharmacia), and wild type TMVP,
respectively.23
Figure 2.3 The circular dichroism (CD) for various secondary structures.26
Figure 2.4 Far UV (a) and near UV (b) CD spectra of an IgG (0.2 mg/ml) in a 10-mM
phosphate buffer, pH 8.1.27
Figure 2.5 Size distribution of a full-length mAb determined by size
exclusion chromatography and analytical ultracentrifugation (AUC)
sedimentation velocity.31
Figure 2.6 Active concentration of 4D5 HER2 murine mAb determined by ELISA
binding to the extracellular domain (ECD) of p185HER2, RIA binding to
ECD and the bioassay as a function of aggregate content.37
Figure 2.7 Format of the ELISA-based receptor inhibition assay for anti-IgE mAb
and correlation of the determined potencies using the plate binding assay,
receptor inhibition assay, and the rat mast cell bioassay of stability samples
of rhu anti-IgE mAb.38
Figure 3.1 Schematic of chemical and physical protein degradation.46
Figure 3.2 General acid and base catalysis of deamidation.47
Figure 3.3 The half-lives and first-order rate constants of deamidation for GlyLeuGln
AlaGly and Gly ArgGlnAlaGly as a function of pH.48
Figure 3.4 Formation of a cyclic imide as a result of nucleophilic attack by main chain
amide nitrogen on the carboxyl carbon of either the side chain amide or
carboxylic acid groups and formation of either Asp or isoAsp as a result of
hydrolysis of either of the carbonyl nitrogen bonds in the cyclic imide.49
Figure 3.5 The differences between the primary sequences of two closely related
mAbs (mAbI and mAbII), and the pH rate of Asp isomerization in two
closely related mAbs and respective model peptides.50
Figure 3.6 Acid–base catalysis of Met to Met sulfoxide by hydrogen peroxide
(HA = acid), and photooxidation of Met to Met sulfoxide by singlet oxygen.52
x List of figures
Figure 3.7 The structures of Trp, hydroxyTrp, kynurenine, N-formyl kynurenine,
and 3-hydroxy kynurenine and their absorption spectrum of the major
oxidation products of Trp.55
Figure 3.8 Photooxidation of surface-exposed Trp in a mAb, which catalyzes the
generation of H2O2. The H2O2 produced can in turn oxidize other susceptible
residues such as Met, and Liquid chromatography–mass spectrometry/mass
spectrometry tryptic peptide map analysis of a mAb at 50 mg/mL in
formulation buffer exposed to light.57
Figure 3.9 Main degradation products resulting from oxidation of Tyr.58
Figure 3.10 Generation of disulfide cross-links (cysteine), sulfenic, sulfonic, and
cysteic acids from H2O2 oxidation of Cys, and proposed mechanism for
metal-catalyzed oxidation of Cys.60
Figure 3.11 Generation of new disulfide bonds in the presence of free thiol, and altered
intramolecular and intermolecular disulfides in a protein as a result of
disulfide reduction and reoxidation.61
Figure 3.12 Generation of a free thiol from a β-elimination reaction of a disulfide bond. 61
Figure 3.13 (a) Pseudo first-order reaction kinetics for formation of aggregate in freeze-
dried mAb (left panel). Size exclusion chromatograph (SEC) after storage at
30 °C for 1 year (lower right panel). Online weight average molecular weight
(MW) using light scattering detection (upper right panel) and lower table, (b)
Nonreduced and reduced SDS PAGE (using β-mercaptoethanol). Lane 1: MW
standards; lane 2: mAb freeze-dried without excipients after 1 year at 30 °C.63
Figure 3.14 Possible pathways for cleavage of peptide bonds at Asp sites.64
Figure 3.15 Size exclusion chromatography of four humanized IgG1 mAbs after
incubation at pH 5.2 for 1 month at 40 °C.65
Figure 3.16 Generation of a 92 kDa thioester cross-link between the heavy and light
chain in a humanized mAb. Panel A: Reducing Capillary Gel �Electrophoresis,
Panel B: SDS PAGE, lane 1—molecular weight standards, lanes 2 and 3
reduced. Panel C: Schematic of the heavy chain, light chain, and 92 kDa
cross-linked species.67
Figure 3.17 Levels of structural organization in proteins: (a) primary structure,
(b) secondary structure showing α helix and β sheet structures, (c) tertiary
structure (folding of the polypeptide chains with secondary structure into an
organized spatial structure), and (d) quaternary, association different tertiary
structures.68
Figure 3.18 (a) Sizing chromatography of human relaxin. 50 μL at x mg/mL
loaded onto a TSK G2000 SWXL column (300 × 7.5 mm I.D.)
equilibrated with 10 mM sodium citrate, pH 5.0 in 0.25 M sodium
chloride. Flow rate at 0.5 mL/min with detection at 214 nm. (b) Weight
average molecular weight (Mw) determined by sedimentation equilibrium
analytical ultracentrifugation divided by the monomer molecular weight
(M1) determined from amino acid composition of human relaxin.
Human relaxin at concentrations ranging from 0.05 to 0.2 mg/mL in
10 mM sodium citrate, 0.15 M sodium chloride at pH 5.0 was centrifuged
at 22 or 32 k rpm at 19–21 °C for 18 h and concentration gradients detected
at 280 nm.72
Figure 3.19 Generation of aggregates during bioprocessing.73
Figure 3.20 Protein unfolding at air–water interfaces.77
Figure 3.21 Particulate formation in protein solutions filled using rotary piston pumps
when filled into vials.80
List of figures xi
Figure 4.1 Stability of a mAb after 1 year of storage at −70, 5, 30, and 40 °C as
assessed by size exclusion chromatography and hydrophobic interaction
chromatography after papain digestion.104
Figure 4.2 Blind men investigating the appearance of an elephant.104
Figure 4.3 The pseudo first order rate constants for deamidation and isomerization
in an IgG1 mAb as a function of pH.107
Figure 4.4 The pseudo first order aggregation rate constant for an IgG1 mAb stored
at 40 °C as a function of buffer species.108
Figure 4.5 IgG1 mAb solubility/gel formation at pH 6 at 15 and 150 mM NaCl and
B22 determined by SLS and kD determined by DLS at pH 6 at 15 and
150 mM NaCl.109
Figure 4.6 Schematic of protein stabilization due to increased surface area of the
denatured state and ensuing preferential hydration.112
Figure 4.7 Depictions of an unfolded protein with hydrogen bonded clathrate water
structure surrounding hydrophobic residues and of a folded protein with
exposed hydrophilic residues hydrogen bonding to specific water molecules.113
Figure 4.8 Generation of aggregate as determined by size exclusion chromatography
for an IgG1 anti-IgE mAb formulated in 10 mM succinate buffer at pH 5
with different sugars at 275 mM.115
Figure 4.9 Effect of sugars at 275 mM on aggregate formation of a lyophilized
IgG1 mAb at 5 mg/mL in 5 mM sodium succinate, pH 5.0 during 1 year
storage at 40 °C, and effect of increased pH to 6.0 on prevention of aggregate
formation of a lyophilized IgG1 mAb at 5 mg/ mL in 5 mM His-HCl
during 24 weeks storage at 40 °C.116
Figure 5.1 (a) Comparison of the absorbance of saline solutions from different
intravenous (IV) administration infusion bags. (b) Stability of dulanermin
in 100 mL IV infusion bags and the effect of freezing and thawing.
Dulanermin was diluted into 100 mL IV bags to a final concentration
of 0.08 mg/mL and then removed for analysis immediately or after 16 h.123
Figure 5.2 Comparison of disassembled components of 100 and 250 mL polyolefin
(PO) bags from the same manufacturer.124
Figure 6.1 The effect of protein concentration on viscosity (closed triangles) and time
to draw 1 cc through a syringe with a ½″ 27 g needle (open triangles).133
Figure 6.2 Glide forces as a function of viscosity determined at 2 × 103/s (25 °C)
compared to glide forces calculated based on the Hagen–Poiseuille’s
equation.133
Figure 6.3 The non-Newtonian behavior of an IgG1 mAb as shown by dependence of
viscosity on shear rate at 10 mg/mL and 200 mg/mL.134
Figure 6.4 Bioavailability of seven different mAbs after subcutaneous delivery.135
Figure 7.1 (a) Pressure drop in a tangential flow filtration (TFF) system as a function
of temperature, (b) Viscosity of an IgG1 mAb at 150 mg/mL as a function
of temperature, and (c) The TFF flux (L/M2/h) versus transmembrane
pressure (psi) at different temperatures for an IgG1MAb1, initially at
30 mg/mL.141
Figure 7.2 Using lyophilization to manufacture a high concentration subcutaneous
(SC) formulation.142
Figure 7.3 Stability and final tonicity after reconstitution as a function of
lyoprotectant:mAb molar ratio.143
Figure 7.4 Stability of an IgG1 mAb as a function of lyoprotectant:mAb molar ratio
and temperature of storage.143
xii List of figures
Figure 7.5 Three humanized IgG1 mAbs with the same IgG1 human Fc construct
but different complementarity determining regions (CDRs) formulated
identically and lyophilized using the same lyophilization process. (a) Time
to reconstitute the lyophilized cake resulting in a final mAb concentration
of 100 mg/mL. (b) Viscosity at 25 °C after reconstitution to a final mAb
concentration of 85 mg/mL.144
Figure 7.6 Lyophilization of an mAb as a function of loading concentration, scanning
electron microscopy of lyophilized solid for the 40 and 110 mg/mL mAb
loading concentrations.145
Figure 7.7 Viscosity measurements of infliximab as a crystalline suspension vs. aqueous
formulation.146
Figure 7.8 Solution viscosity of 125 mg/mL IgG1 mAbmAb1 in 30 mM histidine
buffer at pH 6.0 with different addition of salts as a function of ionic
strength.147
Figure 7.9 The pH-dependence of the viscosity of a 300 mg/mL γ-globulin solution
with no excipient added or in the presence of 0.5 M NaI or 0.5 M
trimethylphenylammonium iodide.148
Figure 7.10 (a) The pseudo first-order rate constants for deamidation and isomerization
in an IgG1 mAb as a function of pH. (b) The kinematic viscosity as a function
of pH at 125 mg/mL IgG1 mAb. (c) Viscosity of the IgG1 mAb with no
excipients and addition of 200 mM ArgHCl.149
Figure 7.11 Effect of arginine compounds on (a) viscosity and (b) aggregation as measured
by decrease in monomer by SEC for an IgG4 mAb. SEC, Size-exclusion
chromatography.150
Figure 8.1 Injectors used for subcutaneous (SC) delivery.155
Figure 8.2 Particle released for polysorbate 80 solutions in phosphate buffered saline
for (a) sprayed-on silicone (regular), baked silicone, and cross-linked
silicone on glass (XSi) (b) Submicron counts measured by Archimedes,
Occhio, and Nanosight instruments.156
Figure 9.1 A volume element moving relative to a lower volume element with relative
velocity equal to du.164
Figure 9.2 G′(a), and kD (b) for an IgG2 mAb as a function of pH and ionic strength
at 25 °C.170
Figure 9.3 Viscosity at 25 °C as a function of mAb concentration for lyophilized and
TFF concentrated mAb1.171
Figure 9.4 Viscosity at 25 °C as a function of mAb concentration for (a) reconstituted
lyophilized and nonlyophilized mAb2 ± NaCl and (b) lyophilized and
reconstituted and nonlyophilized mAb1 at 150 mM NaCl.173
Figure 9.5 Sedimentation equilibrium measurements using preparative
centrifuges.174
Figure 9.6 The corrected weight average molecular weight as a function of
concentration for mAb1 ± NaCl.175
Figure 9.7 (a) Viscosity of mAb1, mAb2 F(ab′)2 fragments compared with mAb1
F(ab′)2 in buffer with 200 mM NaSCN, (b) Viscosity of full-length
MAb1 mixed with Fab fragments (c) Viscosity of MAb1 and MAb2 Fab
fragments in 30 mM histidine, pH 6.0. (d) Viscosity of full-length MAb2
mixed with Fab fragments.176
Figure 9.8 Viscosity and net charge for four mAbs at pH 6, 15 mM ionic strength.179
Figure 9.9 Schematic differentiating the effect on solution viscosity of long-range network
formation versus soluble irreversible aggregate and suspension formation.180
List of figures xiii
Figure 9.10 Measured overall dipole moment for mAb1 versus mAb2 as a function of pH.182
Figure 9.11 Computed electrostatic potential surfaces for mAb1 and mAb2.182
Figure 9.12 (a) Viscosity as a function of mAb concentration of mAb1 and mAb2
mutants with (b) schematics showing amino acid substitutions for the
mutants.185
Figure 9.13 The corrected weight average molecular weights for mAb1 and mutants,
at low and high ionic strength.186
Figure 9.14 Coarse grained (CG) computations for mAb1 versus mAb2 using a 12 site
compact configuration model at 125 mg/mL and pH 6. (a) The 12 site model
overlaid on the atomistic model (ribbon diagram) and structures formed of
mAb1 versus mAb2 (b) Distribution of mAb clusters.188
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List of tables
Table 1.1 Therapeutic monoclonal antibodies, Fc fusion and Fab conjugates approved
or in review in the European Union or United States4
Table 2.1 Secondary structure of an IgG2 determined by far UV circular dichroism
measurements as a function of temperature28
Table 2.2 Comparison of rate constants for pulmozyme deamidation at 5 °C obtained
from Arrhenius kinetics and real-time stability data after 88 days at
2–8 °C storage33
Table 3.1 Electrospray fragments of mAb B collected from size exclusion
chromatography66
Table 4.1 Formulations for antibodies, Fc fusion, and Fab conjugates approved
in the US94
Table 5.1a Summary of four mAb preparations used in intravenous (IV)
administration bag dilution study126
Table 5.1b Summary of percentage of aggregates for mAb1 overtime after agitation
in 250 mL polyolefin intravenous (IV) administration bags with controlled
60 mL headspace and headspace removed126
Table 5.1c Summary of percentage of aggregates for mAb2 overtime after agitation
in 250 mL PO IV bags with controlled 60 mL headspace and headspace
removed127
Table 8.1 Some drugs formulated in chloride-based formulations and filled into staked
needle syringes158
Table 9.1 Viscosities of different fluids in mPas164
Table 9.2 Summary of nondissociable soluble aggregates of mAb1 in different
formulations181
Table 9.3 Summary of mutants as a result of performing mutations in the
complementary determining region (CDR) sequence of mAb1 and
mAb2. Bold amino acid residues are charged residues in the CDR
sequences of the mAbs184
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About the author
Dr Shire has over 30â•›years experience in the pharmaceutical biotechnology indus-
try. He received his PhD from Indiana University Chemistry Department and after
postdoctoral training at the University of Connecticut began his career at Genentech
as research scientist in the Department of Protein Chemistry. He was involved in the
early work to isolate heterologous recombinant proteins expressed in bacterial sys-
tems. This work led to the granting of a patent and served as the basis for further
product development of proteins expressed in bacterial systems. During his tenure in
the Protein Chemistry Department, he used numerous physicochemical techniques to
characterize Genentech proteins at various stages of development. Shortly after the
creation of the Pharmaceutical Research and Development Department at Genentech,
he joined the department where he made numerous contributions to development of
protein formulation and delivery. In addition, he set up one of the first analytical ultra-
centrifugation laboratories in the biotechnology industry. He had been responsible
for directing research and development of formulations for a variety of recombinant
human proteins including Pulmozyme® and Xolair®. After 32â•›years at Genentech,
Inc. he retired from his position as a staff scientist in the Late Stage Pharmaceutical
Development Department at Genentech. Currently he is an adjunct faculty member of
the USC School of Pharmacy and University of Connecticut School of Pharmaceuti-
cal Sciences, and serves as a consultant for the biotechnology industry. Dr Shire has
served as the chair of the American Association of Pharmaceutical Scientists (AAPS)
Biotechnology Section, and was elected as a fellow of AAPS in 1998 and member at
large to the AAPS Executive Council in 2001. He has published over 90 reviews and
papers dealing with various aspects of formulation and pharmaceutical development
of therapeutic proteins.
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