Post on 11-Aug-2020
ZURICH UNIVERSITY OF APPLIED SCIENCES
DEPARTMENT OF LIFE SCIENCES AND FACILITY MANAGEMENT
INSTITUTE OF BIOTECHNOLOGY
Specific enzymatic digestion and fragment isolation for the critical quality attribute assessment of IgG1
Master thesis
by
Rappo, Martin Alexander
Master of Life Sciences
20.10.2017
Pharmaceutical Biotechnology
Academic supervisor:
Prof. Dr. Jack Rohrer Head of the Cell Physiology and Cellular Engineering Group ZHAW Life Sciences und Facility Management, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland
Industrial supervisor:
Dr. Christoph Rösli Senior Fellow in the Physicochemical Analytics Group Novartis Pharma AG, Werk Klybeck, Postfach, 4002 Basel, Switzerland
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Imprint
Keywords
Critical Quality Attributes, CQA, Monoclonal Antibody, mab, Fab, Fc, F(ab’)2, Large hinge IgG
fragment, LHF, IdeS, IgdE, Lysine Gingipain, Kgp, EndoS2
Please cite this report as followed:
M. A. Rappo, Specific enzymatic digestion and fragment isolation for the critical quality attribute
assessment of IgG1, ZHAW LSFM (2017)
Contact information
Author
Martin Alexander Rappo Speiserstrasse 12 4600 Olten Switzerland Tel. +41 79 537 64 76 E-Mail: marappo@gmx.ch
Academic Supervisor
Prof. Dr. Jack Rohrer ZHAW Life Sciences und Facility Management Einsiedlerstrasse 31 8820 Wädenswil Switzerland Tel. +41 58 934 57 17 E-mail: jack.rohrer@zhaw.ch
Industrial Supervisor
Dr. Christoph Rösli Novartis Pharma AG Werk Klybeck Postfach 4002 Basel Switzerland Tel. +41 61 696 14 39 E-Mail: christoph.roesli@novartis.com Basel 20.10.2017
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Acknowledgement
I would like to express my very great appreciation to Christoph Rösli. I would like to thank him
for giving me the chance to work on this interesting project, the stimulating scientific
discussions and useful inputs given during the time I spend at Novartis.
I am particular grateful for the support and training given by Olivia Guerre. Also the interesting
exchange helped to approach problems with a different perspective.
I would like to offer my special thanks to Philippe Simeoni, who always offered this time
whenever a question or issue regarding CE came up.
Also Francois Griaud I would like to thank for giving me an introduction on antibody digestion.
A thanks also goes to Wolfram Kern for being so kind to let me use his work bench and all his
equipment.
The refreshing exchange with Manuel Diez inside and outside the office has been a great help
to keep motivated and stay open-minded.
I would like to thank Genovis for kindly providing me a free test sample of FabALACTICA™.
A special thanks also goes to Raphael Dreier for all the coffee and lunch breaks we had
together, the support during stressful times, extended brain-storming sessions and all the
laughs we shared.
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Abstract
Recent ICH quality guideline updates introduced the enhanced drug development which is
based on Quality by Design approach. For this a Quality Risk Management has to be
conducted based on scientific knowledge. Information gaps are closed through analysis of
molecules with domain-specific modifications. The aim of this thesis was to evaluate methods
to prepare a model IgG1 for such studies. The antibody fragments F(ab’)2, LHF, Fab and Fc
and the Fc-glycosylation cleaved IgG with residual N-acetylglucosamine were selected. A
representative IgG1 was digested with the recently discovered enzymes IdeS, Kgp, IgdE and
EndoS2 and the digestion products were purified with common biotechnological down-stream
methods. The F(ab’)2 was successfully isolated with a yield of 55% and a purity of 96%. After
an initial digestion parameter optimization the LHF, Fab and Fc could be generated and an
isolation method was established. A following scale-up was unsuccessful, due to
reproducibility issues. A promising alternative was identified. The enzymatic glycan cleavage
and product isolation was successful with a yield of 80% and a purity of 99%.
Zusammenfassung
Die kürzlich von der ICH aktualisierten Qualitätsrichtlinien führten den erweiterten
Wirkstoffentwicklungsvorgangs ein, welcher auf dem Quality-by-Design Prinzip basiert. Dazu
wird ein Risikomanagement, basierend wissenschaftlichen Fakten durchgeführt. Um
Informationslücken zu schliessen müssen proteindomänenspezifisch veränderte Moleküle
untersucht werden. Das Ziel dieser Arbeit war die Evaluation von Methoden zur Vorbereitung
eines Modell IgG1 für solche Studien mit minimaler unspezifischer Veränderungen.
Ausgewählt wurden die IgG-Fragmente F(ab‘)2, LHF, Fab und Fc und das Fc-Polysaccharid
gespaltene IgG mit restlichem N-Acetylglucosamin. Ein repräsentatives IgG1 wurde mit den
kürzlich entdeckten Enzymen IdeS, Kgp, IgdE und EndoS2 verdaut und mittels üblichen
biotechnologisch Reinigungsmethoden aufgereinigt. Das F(ab‘)2 Fragment konnte mit einer
Ausbeute von 55% und Reinheit von 96% isoliert werden. Nach einer initialen Optimierung der
Verdauungsparameter konnte das LHF, Fab und Fc erfolgreich hergestellt und eine Methode
zur Fragmentisolation gefunden werden. Die Herstellung einer grösseren Menge war erfolglos,
jedoch konnte eine alternative Lösung identifiziert werden. Die enzymatische Spaltung des
Polysaccharids und Isolation war erfolgreich mit einer Ausbeute von 80% und einer Reinheit
von 99%.
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Table of content
1 INTRODUCTION ............................................................................................................ 1
2 THEORETICAL BACKGROUND.................................................................................... 5
2.1 ANTIBODIES ............................................................................................................... 5 2.1.1 Structure ............................................................................................................ 5 2.1.2 Fragment nomenclature ..................................................................................... 7 2.1.3 Glycosylation ...................................................................................................... 8 2.1.4 Mechanism of action .......................................................................................... 8
2.2 ENZYMES ................................................................................................................... 8 2.2.1 Immunoglobulin-degrading enzyme of Streptococcus pyogenes ........................ 9 2.2.2 Lysine gingipain of Porphyromonas gingivalis .................................................. 11 2.2.3 Immunoglobulin G degrading enzyme of Streptococcus agalactiae .................. 13 2.2.4 Endo-beta-N-acetylglucosaminidase of Streptococcus pyogenes .................... 13
2.3 KINETICS MODEL FOR THE KGP DIGESTION ................................................................. 15
3 METHODS AND MATERIALS ...................................................................................... 17
3.1 INSTRUMENTS, CONSUMABLES, REAGENTS AND SOFTWARE. ....................................... 17 3.2 MODEL ANTIBODY ..................................................................................................... 17 3.3 BUFFER PREPARATION .............................................................................................. 17 3.4 PHYSICOCHEMICAL METHODS ................................................................................... 18
3.4.1 Cation exchange chromatography .................................................................... 18 3.4.2 Size exclusion chromatography ........................................................................ 19 3.4.3 Capillary electrophoresis – sodium dodecyl sulfate .......................................... 20
3.5 DIGESTION AND ISOLATION ........................................................................................ 22 3.5.1 Fab, Fc and LHF .............................................................................................. 22 3.5.2 F(ab’)2 .............................................................................................................. 27 3.5.3 Chitobiose cleavage ......................................................................................... 29
3.6 ECOLOGY, SAFETY AND DISPOSAL ............................................................................. 30
4 RESULTS AND DISCUSSION ..................................................................................... 31
4.1.1 Fab, Fc and LHF .............................................................................................. 31 4.1.2 F(ab’)2 .............................................................................................................. 44 4.1.3 Chitobiose cleavage ......................................................................................... 46
5 CONCLUSION .............................................................................................................. 48
6 OUTLOOK .................................................................................................................... 49
7 REFERENCES ............................................................................................................. 50
7.1 LIST OF ILLUSTRATIONS ............................................................................................ 55 7.2 LIST OF TABLES ........................................................................................................ 56 7.3 LIST OF EQUATIONS .................................................................................................. 57
ANNEX .................................................................................................................................. I
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Abbreviations
Table 1: Abbreviations.
Acronym Description
Ab Antibody
ADCC Antibody-dependent cellular cytotoxicity
CDR Complementary determining region
CE Capillary electrophoresis
CH1 First constant heavy chain domain
CH2 Second constant heavy chain domain
CH3 Third constant heavy chain domain
CL Constant light chain domain
DB Digestion buffer
DP Drug product
Fab Fragment antigen binding
Fc Fragment crystallizable
FcRn Neonatal Fc receptor
FcγR IgG-Fc receptor
Fuc Fucose
Gal Galactose
GAS Group A Streptococcus
GlcNAc N-acetylglucosamine
HC Heavy chain
HPLC High performance liquid chromatography
ICH International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use
Ig Immunoglobulin
IgdE Immunoglobulin G degrading enzyme of S. agalactiae
LC Light chain
Mab Monoclonal antibody
Man Mannose
NeuGc N-glycosylneuraminic acid
NR Non-reducing
PAGE Polyacrylamide gel electrophoresis
PD Pharmacodynamics
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PK Pharmacokinetics
QbD Quality by Design
QRM Quality risk management
Rgp Arginine gingipain
RMT Relative migration time
Rt Room temperature
SDS Sodium dodecyl sulfate
VH Variable heavy chain domain
VL Variable light chain domain
Amino acids
Figure 1: Amino acid properties Venn diagram [1].
Amino acid A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine
ZHAW LSFM, 2017 Introduction Martin Alexander Rappo
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1 Introduction
With the creation of the first monoclonal antibody (mab) in 1975 a new era of pharmaceutical
development was initiated [2]. The mab technology increased therapeutic options and opened
new treatment opportunities for diseases with an unmet medical need, such as cancer,
immunological disorders, chronic inflammatory disease, infectious disease and many more [3].
Because mabs have a higher target specificity and reduced systemic toxicity compared to prior
predominant small molecular drugs, focus of drug research and development slowly shifted
toward this technology [4]. In 1986, the first mab was licensed [2,5]. Sixteen years later, in
2002 the first human mab was approved by the U.S. Food and Drug Administration for
pharmaceutical use in humans [3]. In 2014 the annual global mab market was approximately
$20 billion, generated by roughly 30 products [2] and is estimated to grow to $125 billion with
around 70 products on the market by the year 2020 [5]. In parallel with the transition from small
to large molecular drug research and development, regulatory authorities introduced the new
quality guidelines Q8 – Q12 for drug development, listed in Table 2 [6–8].
Table 2: List of new ICH quality guidelines.
ICH Guideline Topic
Q8(R2) Pharmaceutical development
Q9 Quality Risk Management
Q10 Pharmaceutical Quality System
Q11 Development and Manufacture of Drug Substance
Q12 Technical and Regulatory Considerations for Pharmaceutical Product Lifecycle Management
The new guidelines propose an enhanced approach for drug development compared to the
traditional. Where the traditional way defines set values and operational ranges for process
parameters, based on tests and reproducibility studies to reach their acceptance criteria the
enhanced approach integrates quality directly into the process [8]. This is achieved by
elaborating a quality risk management (QRM) based on scientific evaluations throughout the
whole product lifecycle, a so called quality by design (QbD) approach. The QbD, illustrated in
Figure 2, includes a summary of characteristics of a drug product (DP) for its ideal quality, the
so called quality target product profile (QTPP). The QTPP contains for example information
regarding the indication, the mechanism of action, the dosage form or the dosage strength [9].
Based on the QTPP a critical quality attribute (CQA) assessment is performed, listing any
physical, chemical, biological, or microbiological property of a DP having an influence on the
quality with regard to activity/potency, pharmacokinetics/-dynamics (PK/PD), immunogenicity
and/or safety. Examples of CQAs are viral impurity, host cell proteins, glycosylation or high
molecular weight species. Afterwards operation parameters affecting CQAs, the critical
ZHAW LSFM, 2017 Introduction Martin Alexander Rappo
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process parameters (CPP), are defined and tested by a design of experiment (DoE). Based
on the results of these experiments, the criticality of the CQAs is adapted. Examples for CPPs
are: temperature, time and pH. Based on the findings a working area of each CPP is defined,
called the design space. At last a control strategy is introduced, ensuring the defined quality of
the final DP. The whole process is maintained and updated through the whole lifecycle and
should lead to an improved product understanding and ultimately higher patient safety [10–
14].
Figure 2: Illustration of the Quality by Design strategy [15,16].
To conduct a QRM potential risks are identified first, their likelihood is rated and the
consequences are assessed. Based on those points an impact factor for each risk is calculated
and an appropriated follow-up action is evaluated. Such can be “no action required”, “further
investigation required”, “close monitoring” or “risk prevention”. The basis for such a risk
identification for quality attributes emerges from four sources, illustrated in Figure 3: literature
& in-silico data, in-house knowledge and product comparison, project related experimental
data and specific clinical experience. The information availability is usually high for literature &
in-silico data and decreases for in-house knowledge & product comparison. Project related
experimental data and specific clinical expertise, which have both a high certainty score and
cost & time investment, are often only available in late development phases. Therefore, the
relation of effort to information gain has to be evaluated thoroughly.
Quality target product profiles (QTPP)
Critical process
parameters (CPP)
Critical quality attributes
(CQA)
Design
Space
Control
Strategy
Verification
Validation Iterative process throughout the lifecycle
ZHAW LSFM, 2017 Introduction Martin Alexander Rappo
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Figure 3: Potential information sources for the quality risk management [15].
The risks considered for impact assessment of quality attributes during drug development are
divided into four categories, listed in Figure 4.
Figure 4: The four quality categories for the risk assessment quality attributes [15,17–20].
This QbD approach slowly finds its way into pharma companies, where chemistry,
manufacturing and control strategies are adapted and updated accordingly. For an adequate
risk evaluation a fundamental molecule, mechanism and process understanding is necessary.
During the CQA assessment related to the active pharmaceutical ingredient in-depth
understanding of potential structural and molecular modifications and their impact are
evaluated. As literature and in-silico data are generally rated quiet uncertain and are often not
Literature & in-silico data
In-house knowledge & product comparison
Project related experimental
data
Specific clinical
experience
Cost and duration
Certainty
Availability
Biological activity/potency describes the ability of a molecule/drug to induce a certain response in a biological system at a defined concentration [17].
Pharmacokinetics (PK) is the study of progression of a drug during absorption, distribution, metabolism and excretion. Pharmacodynamics (PD) is the study of the biological effect caused by the drug at the site of
action [18].
Immunogenicity describes the ability of a substance inducing an immunological reaction in an organism This includes the formation of anti-drug antibodies [19].
Safety according to the World Health Organization are biological, chemical or physical agents or operations that are reasonably likely to cause illness or injury [20].
ZHAW LSFM, 2017 Introduction Martin Alexander Rappo
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even available for new biological entities, molecular entity-specific in-house knowledge has to
be expanded and tools to gain project-specific experimental data have to be established. So
far biopharmaceuticals have been exposed to controlled stressed conditions, like heat,
oxidation or shearing stress, for a defined period of time and degradation products were
analyzed. This approach may introduce multiple, uncontrolled modifications leading to vague
conclusions. Therefore, the development of new tools to modify a drug very specific (and
prevent undesired modifications) is necessary.
The topic of this study is to start establishing tools for the CQA assessment of
biopharmaceuticals. Potential modification are for the most part dependent on the molecular
structure of the drug substance and may vary from one project to another. As a starting point
the immunoglobulin G (IgG) 1 subclass was used in this thesis. IgG1 is currently the most used
subclass of mabs in drug development. A possibility of targeted modification is obtained
through enzymes. Especially in protein-related sciences enzymes offer a wide variety of
applications. Unfortunately, commonly used endoproteinase, such as trypsin, Lys-C, papain
or pepsin still tend to undesired over-digestion. In recent years new endoprotease were
discovered which promise to cleave IgGs sequence specific without the risk of over-digestion
[21–24]. The goal of this thesis is to screen and improve the enzymatic digestion of a model
IgG1 to obtain Fab, Fc, LHF and F(ab’)2 fragments, respectively the chitobiose cleaved IgG,
shown in Figure 5, and isolated the corresponding products. Undesired modifications have to
be prevented as far as possible. For a copy of the objective setting agreement please see
appendix page X.
Figure 5: Selected digestion products to be evaluated during the master thesis including their final purpose in the CQA assessment.
F(ab‘)2
Bivalent binding (potency) Monovalent binding (potency)
Monovalent binding (potency)
LHF
Fab Fc
(Fuc-)GlcNAc-IgG
Glycosylation pairing (PK/PD)
Fc methionine oxidation distribution
Change in higher order structure
FcRn, FcγR and C1q binding (PK/PD)
ZHAW LSFM, 2017 Theoretical background Martin Alexander Rappo
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2 Theoretical background
2.1 Antibodies
Antibodies (Ab), also called immunoglobulin (Ig), are glycoproteins and play an essential role
in the adaptive immune system. Abs are naturally either transmembrane proteins, as B-cell
receptors or soluble proteins released from plasma cells after an antigen recognition. Special
properties of such Abs are the high specificity and affinity to bind a certain antigen and initiate
an effector mechanism [25].
2.1.1 Structure
The basic structure of an Ab, illustrated in Figure 6, is Y-like shaped and consists of the stem,
the so called fragment crystallizable (Fc) responsible for the effector function (explained in
chapter 2.1.4) and the two arms, the so called fragment of antigen-binding (Fab) responsible
for the specific antigen binding [26]. Abs are axial symmetrical hetero-tetramers containing two
light chains (LC) and two heavy chains (HC). The LC consist of two domains of roughly
12.5 kDa, the variable (VL) and constant (CL) domain. The HC consists of four domains, also
one variable (VH) and three constant domains (CH1, CH2 and CH3). The two HC are interlinked
between CH1 and CH2, in the so called hinge-region via disulfide bridges [25,27]. Similarly, the
LC and the HC are liked via a disulfide bridge in the Fab region [25]. Additionally, each domain
contains an intra-chain disulfide bond [28]. The CH2 bears an N-glycosylation at position
Asn297, which plays an important role for the effector function, protein stability and the ability to
aggregate [29,30]. The highly specific binding ability of Igs origins from the hypervariable
regions within the variable region, the so called complement determining region (CDR) [25].
Each variable domain contains three CDRs and the CDR of the HC and LC lie close together
through the three-dimensional arrangement forming the so called antigen-binding pocket [31].
ZHAW LSFM, 2017 Theoretical background Martin Alexander Rappo
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Figure 6: Illustration of the IgG1 structure with light chain in apricot and heavy chain in blue.
Figure 7: Ig Fab with CDR in red and a hapten as epitope [32].
Figure 8: 3D surface structure of an IgG1 [33].
Fragment antigen-binding (Fab): antigen binding
Hinge region: flexibility and connection
Variable region
Constant region
IgG1 structure
Fragment crystallizable
(Fc): Effector function
Gly
co
syla
tion
Heavy chain
Light chain
Fab region with hapene in binding pocket
IgG1 3D structure
LC HC
Glycosylation
ZHAW LSFM, 2017 Theoretical background Martin Alexander Rappo
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In humans there are two different types of LCs, the κ and the λ chain and five different types
of HCs, the γ, δ, ε, α and μ chain. Depending on the HC type, antibodies are classified into five
classes or isotypes, namely IgG, IgD, IgE, IgA and IgM [25–27]. IgG have the highest half-life
of roughly three weeks and are therefore the most interesting class for therapeutic applications
[25]. The IgG class is further divided into four subclasses [25,27,34,35], depending on the
preferable antigen target, hinge region length, disulfide connection [26,28], serum
concentration, half-life, placental transfer potential, complement activation and Fc receptor
binding. Those subclasses are numbered ascending from highest to lowest relative abundance
(γ1, γ2, γ3, γ4) in human serum. IgG1 is usually found in the highest relative abundance in the
human serum compared to other subclasses and targets mainly soluble and membrane
proteins. Additionally, it has compared to other subclasses a unique disulfide connection
between LC and HC [34] and binds efficiently to the IgG-Fc receptor (FcγR) [36].
2.1.2 Fragment nomenclature
In Figure 9 common antibody fragments are listed.
Figure 9: Nomenclature of common IgG fragments [37–42].
Immunoglobulin G
IgG
Fragment antigen binding
Fab
F(ab‘)2
Fragment crystallizable
Fc
Fc/2
Large hinge fragment [40]
LHF
Fab‘ Reduced/half IgG rIgG/hIgG
Fd‘
The term Fabc is rarely used and can have two different meanings. Either as
equivalent for the intact IgG [37,42] or the IgG without CH3 domains [41].
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2.1.3 Glycosylation
Protein glycosylation is a highly complex post-translational modification but crucial for folding,
conformation, stability & half-live, solubility, pharmacokinetics, biological activity and
immunogenicity [29,43]. Ig of the subclass γ1 undergo glycosylation in the endoplasmic
reticulum and Golgi network and contain a conserved glycosylation at approximate position
Asn297 of both CH2 domains in the Fc region. Roughly 20% of mabs contain an additional N-
glycosylation in the variable region. Usually glycosylation of Abs consists of a complex bi-
antennary glycan with core-fucosylation [44]. An illustration of transgenic animal respectively
Chinese hamster ovary cell N-glycosylation structures is shown in Figure 10. Minor size and
structural differences of the glycan from one protein to another lead to an unequal mixture,
which is called glycoform heterogeneity [44,45]. Specifically for IgG the glycosylation has an
effect on thermo-, unfolding- and protease-stability, impacts the aggregation tendency [46] and
influences the effector functions, especially the antibody-dependent cell cell-mediated
cytotoxicity (ADCC) and the complement dependent cytotoxicity (CDC) [29,46,47].
Figure 10: Fc glycosylation structure of transgenic animal/Chinese hamster ovary cells.
2.1.4 Mechanism of action
Abs can have different effects on an antigen. Important for a functional Ab is the ability to bind
a certain epitope with the binding pocket and initiate an effector function from the Fc region.
With both of these properties Abs act as adapter and bring antigen and the immune cell
together. Especially IgG1 and IgG3 are known to initiate such effector mechanisms [34].
Therapeutic mabs on the other hand usually do not depend on an effector function and in some
cases those are even undesired. A summary of the most important effector functions are listed
in the appendix on p. II.
2.2 Enzymes
Enzymes are proteins with a wide function of catalyzing bio-chemical reactions in an organism.
Some enzymes catalyze a certain reactions high specific and under physiological conditions.
Fc glycosylation of Chinese hamster ovary cells
Chitobiose core
ZHAW LSFM, 2017 Theoretical background Martin Alexander Rappo
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Enzymes also have a high relevance in industrial and diagnostic applications. A summary of
commonly used enzymes for the mab analytic is listed in the appendix on p. III.
2.2.1 Immunoglobulin-degrading enzyme of Streptococcus pyogenes
Name Immunoglobulin-degrading enzyme of Streptococcus pyogenes
Abbreviation IdeS, streptococcal Mac-1
Catalytic type Cysteine [48]
Family Protease C66 [49]; CA clan [50]
Organism Streptococcus pyogenes
Molecular weight 36.2 kDa [50]
Molar absorbance 1.362 (calculated) [51]
Figure 11: Illustration of IdeS.
Streptococcus pyogenes is a bacteria causing strep throat and impetigo in humans [52].
Recent studies revealed its secreted cysteine endopeptidase (IdeS or streptococcal Mac-1) to
naturally protect the bacteria from the host’s immune system by cleaving surrounding Ig at the
lower hinge region.
Figure 12: IgG fragmentation of IdeS.
IdeS contains similar structural features as papain but shows only minor sequence homology.
Both can structurally be divided into a left-hand (L) and right-hand (R) domain and the catalytic
site is located at the interface in between. High specificity of substrate recognition towards the
hinge region of IgG is a unique feature of IdeS. So far only few accepted sequences of the Ig
hinge region have been identified, shown in Figure 13 [50]. This high specificity, which prevents
over-digestion is exploited for bioanalytical applications.
Catalytic domain
L R
ZHAW LSFM, 2017 Theoretical background Martin Alexander Rappo
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Figure 13: Accepted sequence for the IdeS cleavage. Identical amino acid in black, closely chemically similar in blue, weakly chemically similar in sienna and non-similar in apricot.
The catalytic mechanism proposed for the IdeS digestion is similar to the one of the papain
cysteine protease superfamily, forming a thiolate-imidazolium ion with the catalytic dyad Cys94
and His262, shown in Figure 14 [50]. Through proton transfer from His262 to Cys94 the enzyme
is activated. The thiolate of Cys94 attacks the carbonyl of the substrate and forms an
enzyme-substrate complex. The scissile peptide bond of the substrate is broken and the C-
terminal fragment is released, where the remaining chain forms together with the enzyme a
thioester, the so called acyl-enzyme intermediate. The thioester is hydrolyzed in a
subsequent step and the N-terminal chain is also released [53].
Figure 14: Proposed catalytic cycle of the IdeS cleavage.
Asp284 and Asp286, close to the catalytic pocket, have been identified to have a positive impact
on the catalytic activity, by facilitating the correct three-dimensional orientation resp. creating
240 250
....|............|....
IgG1 ...CPAPELLG | GPSVF...
IgG2 ...---APPVA | GPSVF...
IgG3 ...CPAPELLG | GPSVF...
IgG4 ...CPAPEFLG | GPSVF...
Accepted IdeS cleavage sequence
ZHAW LSFM, 2017 Theoretical background Martin Alexander Rappo
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a suitable electrostatic milieu. Additionally, Lys84, which is stabilized through the hydrogen
bond-salt link of Asp286, forms together with the active site Cys94 a stabilizing pocket for the
substrate, the so called oxyanion hole [50]. This is illustrated in Figure 15.
Figure 15: Catalytic pocket and stabilizing effects.
Initial kinetic studies showed a non-Michaelis-Menten behavior, but a curve of positive
cooperativity [54]. Later studies revealed an underlying two-step Michaelis-Menten kinetics,
where the first reaction is 100 times faster than the subsequent [55].
Genovis®, a life science company, offers recombinant IdeS in various forms, e.g. as lyophilisate
(FabriCATOR®), immobilized on agarose (FragIT™) or in combination with a CaptureSelect™
column (FragIT™ kit) including digestion procedure.
2.2.2 Lysine gingipain of Porphyromonas gingivalis
Name Lysine gingipain
Abbreviation Kgp
Enzyme class EC 3.4.22.47 [54]
Catalytic type Cysteine
Family Protease C25; CD clan [53]
Organism Porphyromonas gingivalis
Molecular weight 187 kDa (native) [56] 48 kDa (recombinant; catalytic and immunoglobulin superfamily-like domain) [53,57]
Molar absorbance 1.971 (recombinant, calculated) [58]
Figure 16: Illustration of recombinant Kgp.
The periodontal disease is a wide spread bacterial infection of the gums and the bacteria
supposedly to be the main cause is Porphyromonas gingivalis. P. gingivalis expresses two
Hole with catalytic pocket
Immunoglobulin superfamily- like domain
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types of cysteine peptidase gingipain, the lysine (Kgp) and the arginine gingipain (RgpA and
RgpB). These two enzymes contribute to a large part to the extracellular proteolytic activity
responsible for its high virulence [57]. Structurally Kgp consists of five domains; the signal
peptide, a pro-domain, the Lys-specific catalytic subdomain, an immunoglobulin superfamily-
like domain, and the C-terminal domain [53,57]. Kgp cleaves multiple substrates after Lys [53]
and also plays an important role in evading the host’s immune system by degrading Ig at the
upper hinge region, illustrated in Figure 17 [54].
Figure 17: IgG fragmentation of Kgp and IgdE.
For Fc glycosylated IgG1 only one cleavage site, above the hinge region, has been identified,
shown in Figure 18. For IgG1 without glycosylation a second cleavage site in the Fc region
was observed [22]. Also IgG3 have been reported to be cleaved by Kgp but not as specific as
IgG1. For IgG2 and IgG4 no cleavage sites have been identified [54].
Figure 18: Accepted Kgp cleavage sequence of IgG1 [22].
The proposed mechanism is similar to IdeS Cys/His dyad involving Cys477 and His444, but
experiments have shown the importance of Asp388 for the catalytic cleavage [57]. Typical for
cysteine proteases is an activity increase in a mild reducing environment, like glutathione or
cysteine [53]. Kinetic studies revealed that the digestion does not follow the Michaelis-Menten
kinetic, but rather a mechanism of positive cooperativity [54], usually meaning the first ligand
binding to the enzyme simplifies a subsequent binding [59]. Similar to IdeS, Kgp kinetics could
also underlie a consecutive two-step Michaelis-Menten kinetic, but this theory was not tested
yet. It is reported that the digestion can be stopped by drastically decrease pH or addition of
iodoacetic acid resp. Nα-Tosyl-L-lysine chloromethyl ketone [54]. The mayor advantage of Kgp
over commonly used papain or Lys-C is the high specificity without the risk of over-digestion.
Genovis® offers a recombinant Kgp in a kit including a cysteine solution as reducing agent,
called GingisKHAN®.
230 240
.....|............|...
...KSCDK | THTCPPCP...
Accepted Kgp cleavage sequence of IgG1
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2.2.3 Immunoglobulin G degrading enzyme of Streptococcus agalactiae
Name Immunoglobulin G-degrading Enzyme
Abbreviation IgdE
Catalytic type Cysteine
Family Protease C113, CA clan [60]
Organism Streptococcus agalactiae
Molecular weight 70 kDa (recombinant)
Molar absorbance 0.584 (calculated) [61]
Figure 19: Illustration of IgdE.
The immunoglobulin G degrading enzymes (IgdE) of the Streptococcus species, known for
their virulence in several species, were recently discovered and have been identified to help
the bacteria evade the host’s immune system by degrading surrounding Ig. Exceptional for
those enzymes is their high specificity towards IgG of the streptococci’s main host and distinct
cleavage site. One IgdE, degrading human IgG1, origins from S. agalactiae and cleaves the
Ab similar to papain, above the hinge region, shown in Figure 20, but without further cleavage.
The involved mechanism is the same as for Kgp and is formed by the catalytic triad Cys302
His333 and Asp348. Several applicable cysteine protease inhibitors like, Z-LVG-CHN2 and
iodoacetamide have been reported [36,49,55].
Figure 20: Accepted IgdEagalactiae cleavage sequence of human IgG1 [36].
2.2.4 Endo-beta-N-acetylglucosaminidase of Streptococcus pyogenes
Name Endo-beta-N-acetylglucosaminidase
Abbreviation EndoS2
Family Glycoside hydrolases 18
Organism Streptococcus pyogenes group A (GAS) (Serotype M49) [62]
Molecular weight 95 kDa (native) [63] 92 kDa (recombinant) [24]
Molar absorbance 1.044 (calculated) [64]
Figure 21: Illustration of EndoS2.
Streptococcus pyogenes, as explained in chapter 2.2.1, possesses another way to evade the
human immune system. The bacteria produces, next to IdeS, the endoglycosidase EndoS.
Catalytic pocket
230 240
.....|............|...
...KSCDKT | HTCPPCP...
Accepted IgdE cleavage sequence of IgG1
Catalytic site
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This enzyme is hydrolyzing the glycosidic bond between the two acetylglucosamine (GlcNAc)
in the chitobiose core structure of IgG impairing an effector function and increasing bacterial
survival [62].
Figure 22: Chemical structure of the chitobiose core.
The EndoS is conserved in almost all identified S. pyogenes serotypes with the exception of
serotype M49 GAS strain. The serotype M49 GAS expresses instead the enzyme EndoS2, with
similar function but a sequence homology of only 37% [63]. Both enzymes cleave complex
type glycans. However EndoS2 cleaves in addition also high-mannose and hybrid type glycans
[65]. All human IgG subclasses and Abs of several species, such as mouse, rat, monkey,
sheep, goat, cow and horse have been identified as substrate. Importantly, the enzyme works
only on native proteins, which indicates a protein-protein interaction. Through a sequence
alignment and later active-site mutation the catalytic Glu186 and several Trp have been
identified to be of importance for the digestion [62]. Next to IgG only one other protein, the α1-
acid glycoprotein, has been identified to act as substrate and therefore EndoS and EndoS2 do
not show general chitinase activity.
Figure 23: Chitobiose cleavage of EndoS2.
Genovis® offers an EndoS (IgGZero™) and an EndoS2 (GlycINATOR®) ready-to-use kit,
containing the corresponding enzyme with a poly-his tag as lyophilisate or immobilized on
agarose.
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2.3 Kinetics model for the Kgp digestion
To simplify result comparison and reduce enzyme consumption a model was applied for
studies on Kgp. This model is based on the reaction kinetics of a consecutive first-order
reaction as described below [66]. The kinetic order was not experimentally determined but
assumed as a first order reaction.
Figure 24: Proposed consecutive reaction scheme of an IgG and Kgp.
The IgG reacts in a first reaction step with the kinetic constant k1 to the LHF and a Fab and in
a subsequent step with k2 to two Fabs and one Fc. k1 is calculated using Equation 1 and kinetic
constants for different digestion parameter are compared. The higher the kinetic constant the
faster the digestion.
Equation 1: Determination of the kinetic constant k1 [66].
𝑘1 =1
𝑡ln[𝐴]0[𝐴]𝑡
It is assumed that k1 and k2 are almost the same, as neither the enzyme nor the substrate
change by a lot. Only the accessibility of the substrate of the second reaction is presumably
higher compared to the first and k1 is therefore slightly smaller than k2.
𝑘1 ≈ 𝑘2 𝑘1 < 𝑘2
The molar concentration of the intermediate product is calculated according to Equation 2.
Equation 2: Calculation of the intermediate product concentration [I].
[𝐼] =𝑘1
𝑘2 − 𝑘1(𝑒−𝑘1𝑡 − 𝑒−𝑘2𝑡)[𝐴]0
The molar concentration of the product is calculated according to Equation 3.
Equation 3: Calculation of the product concentration [P].
[𝑃] = (1 +𝑘1𝑒
−𝑘2𝑡 − 𝑘2𝑒−𝑘1𝑡
𝑘2 − 𝑘1) [𝐴]0
The time point where the intermediate product is highest is calculated according to Equation
4.
k1 k2
A I P
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Equation 4: Calculation of the time point of the maximal intermediate concentration is reached.
𝑡[𝐼]𝑚𝑎𝑥 =1
𝑘1 − 𝑘2ln (
𝑘1𝑘2)
Transformed back to purity the digestion progress is expected to behave as shown in Figure
25.
Figure 25: Simulated Kgp digestion progress based on a consecutive first-order kinetic.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Purity
in A
%
Time in min
Simulated Kgp digestion progress for a consecutive first order reaction kinetic
IgG1
LHF
Fc
Fab
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3 Methods and materials
3.1 Instruments, consumables, reagents and software.
High performance liquid chromatography (HPLC) was performed on instruments from Agilent,
the capillary electrophorese (CE) on BECKMAN-COULTER instruments and the content was
measured on a Nanodrop™ by Thermo Scientific. All enzyme were purchased from Genovis®.
Analytical data evaluation was performed on Chromeleon™ 6.8. A detailed list of all
instruments, consumables, reagents and software can be found in the appendix on p. IV - VI.
3.2 Model antibody
During this studies a model IgG1 was used. The used mab is developed by Novartis and
blinded in this report for the protection of the company’s intellectual property. It is simply
referred as mIgG1 standing for model IgG1. The mIgG1 belongs to the IgG1 subclass with a κ
LC and the standard mab solution is formulated at approximately 200 mg/mL in 5 mM L-
histidine/L-histidine hydrochloride at pH 5.8.
3.3 Buffer preparation
Table 3: List of used buffer.
Name Composition
CaptureSelect™ binding buffer 10 mM sodium phosphate, 150 mM sodium chloride pH 7.4
CaptureSelect™ elution buffer 100 mM glycine pH 3.0
CE-SDS NR gel buffer Beckman Coulter gel buffer, Cat. no. 391163
CE-SDS NR sample buffer 100 mM tris base, 10 g/L SDS, pH 7.00
CE-SDS red gel buffer Beckman Coulter gel buffer, Cat. no. 391163 + water (1:1.2)
CE-SDS red sample buffer 50 mM tris base, 5 g/L SDS, pH 8.5
cOmplete™ solution 1 Tablet in 50 mL Kgp DB
EndoS2 digestion buffer 10 mM sodium phosphate, 150 mM NaCl, pH 7.4
Formulation buffer 5 mM L-histidine/L-histidine-HCl pH 5.8
IdeS digestion buffer 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH 6.0
IgdE digestion buffer 150 mM sodium phosphate pH 7.0
Kgp digestion buffer 100 mM tris pH 8.0/7.3/6.0
Protein L binding buffer 20 mM sodium phosphate, 150 mM NaCl, pH 7.2
Protein L elution buffer 100 mM sodium citrate, pH 2.0 – 3.5
Reducing solution 20 mM cysteine
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3.4 Physicochemical methods
Several physicochemical techniques were used for sample analysis and fragment isolation.
Detection was always by UV and the digestion progress was evaluated according to purity by
capillary electrophoresis (CE).
3.4.1 Cation exchange chromatography
Cation exchange chromatography (CEX) is a liquid chromatography technique and belongs to
the ion-exchange chromatography. It is used to separate species with a different net surface
charge on an ionic stationary phase. It was used to detect the rate of deamidation, shown in
Figure 26, by monitoring purity of the acidic species.
Figure 26: Deamidation of asparagine.
During this study a salt based separation gradient was applied with parameter listed in Table
4.
Table 4: Applied CEX parameter.
Parameter Setting
Sample injection concentration 1 mg/mL
Sample injection volume 50 μL
Sample temperature 5°C
Mobile phase A 25 mM sodium phosphate, pH 6.0
Mobile phase B 25 mM sodium phosphate, 250 mM sodium chloride, pH 6.0
Flow rate 1.0 mL/min.
Column ProPac™ WCX-10
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Column temperature 25°C
Detection 220 nm
Gradient Time in min %B
0 10
30.0 37
30.1 100
32.0 100
32.1 10
40.0 10
An example chromatogram is displayed in Figure 27.
Figure 27: Example cation exchange chromatogram.
3.4.2 Size exclusion chromatography
Size exclusion chromatography (SEC) separates species according to their hydrodynamic
radius. This technique was used to separate and collect the native IgG1, LHF and Fab fractions
using the parameters listed in Table 5.
Table 5: Applied SEC parameter for fraction collection.
Parameter Setting
Sample injection amount 40 – 400 μg
Sample injection volume 10 – 100 μL
Sample temperature 5°C
Mobile phase 150 mM potassium phosphate, pH 6.5
Flow rate 0.4 mL/min.
12 13 14 15 16 17 18 19 20 21 22
Absorb
ance in m
AU
Time in min
Example CEX chromatogram of mIgG1
Acidic variants Main Basic variants
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Column TSKgel G3000SWXL; 2 – 3 columns in series
Column temperature 30°C
Detection 210 nm
Run time 55 – 85 min
3.4.3 Capillary electrophoresis – sodium dodecyl sulfate
CE is, like the polyacrylamide gel electrophoresis (PAGE), a physicochemical separation
technique but instead of a planar gel the separation occurs in a thin capillary [67]. Sodium
dodecyl sulfate (SDS) denatures the protein and ensures a similar charge to mass ratio of all
proteins, thus migration time increases with higher molecular weight. The technique is superior
to SEC in regard to peak resolution of fragments but has the disadvantage that sample
fractionation is not possible.
Non-reducing conditions
For non-reducing (NR) CE-SDS, the protein sample was pre-diluted to 6 mg/mL in water, 20 μL
of this solution were further diluted in 75 μL sample buffer and 5 μL 250 mM iodoacetamide
solution were added for alkylation. Afterwards the protein was denatured at 70°C for 10 min,
the solution was cooled down to room temperature (rt), centrifuged and analyzed by CE-SDS
with parameters listed in Table 6.
Table 6: List of CE-SDS NR parameter.
Parameter Setting
Auto sampler temperature 20°C
Injection type Outlet
Injection voltage 5 kV
Capillary length 30 cm
Capillary diameter 50 µm
Capillary temperature 25°C
Polarity Positive
Voltage 15 kV
Detection 214 nm
An example CE-SDS NR electropherogram of the Kgp digestion, with assigned peaks is shown
in Figure 28.
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Figure 28: Example CE-SDS NR electropherogram of the Kgp digestion.
Reducing conditions
For reducing (red) CE-SDS the protein sample was pre-diluted to 6 mg/mL in water, 20 μL of
this solution were further diluted in 75 μL sample buffer and 5 μL 2-mercaptoethanol were
added for reduction. Afterwards the protein was denatured at 70°C for 10 min, the solution was
cooled down to rt, centrifuged and analyzed by CE-SDS with the parameters listed in Table 7.
Table 7: List of CE-SDS red parameter.
Parameter Setting
Auto sampler temperature 20°C
Injection type Inlet
Injection voltage 5 kV
Capillary length 30 cm
Capillary diameter 50 µm
Capillary temperature 25°C
Polarity Reverse
Voltage 14.2 kV
Detection 214 nm
An example CE-SDS red electropherogram of the EndoS2 digestion, with assigned peaks is
shown in Figure 29.
15 30 45 60 75 90 105 120 135 150 165 180 195
Absorb
ance in m
AU
MW in kDa
Example CE-SDS NR electropherogram of the Kgp digestion
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Figure 29: Example CE-SDS red electropherogram of the EndoS2 digestion.
3.5 Digestion and isolation
3.5.1 Fab, Fc and LHF
Figure 30: Flow chart of the Fab, Fc and LHF generation and isolation.
Stability evaluation in digestion buffer with and without reducing solution
Increased pH and temperature are usually used to enforce asparagine deamidation
in proteins [68] and reducing agents lead to breaking of disulfide bridges and loss of
tertiary structure. As all three conditions are present in the Kgp digestion, specified
in the Genovis protocol the rate of deamidation and reduction was evaluated.
15 17 19 21 23 25 27 29 31 33 35
Absorb
ance in m
AU
Time in min
Example CE-SDS red. electropherogram of the EndoS2
digestion
Undigested Digested
Dig.
AC
BE
SEC
BE
Control
Digestion
IPC
Protein L affinity chromatography
Buffer exchange
Buffer exchange
Size exclusion chromatography
Purity analysis
IPC
IPC
In-process control
In-process control
In-process control
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Deamidation stability: 20.0 µL mIgG1 solution were diluted in 788 µL digestion
buffer (DB) to 5.00 mg/mL (final pH 7.87), split in two equal portions, kept at rt resp.
at 37°C/300 rpm for 1, 2 and 24 h and were analyzed by CEX and CE-SDS NR.
Reduction stability: 20.0 µL mIgG1 solution were pre-diluted in 60.0 µL DB to
50.5 mg/mL and 24.8 µL of this pre-dilution solution were further diluted in 50 µL
Genovis reducing solution (RS) and 175.2 µL DB, kept at 37°C/300 rpm for 2 h and
analyzed by CE-SDS NR.
Digestion progress of Kgp and influence factors
Enzyme ratio: To assess the initial digestion rate and test the applicability of the
model, the initial digestion was performed according to the provided protocol from
Genovis [22]. 20.0 µL mIgG1 solution were diluted in 60.0 µL DB and 24.8 µL
(1’250 µg) of this solution were further diluted in 50.0 µL DB, 125.8 µL Kgp 10 U/µL
(1’258 U) and 50.0 µL fresh RS to 5.00 mg/mL mIgG1. The digestion solution was
incubated at 37°C/300 rpm for 5, 15, 30, 60 and 120 min and tested by CE-SDS NR.
The 5 min sample was injected twice to prove that digestion stops in presence of 1%
SDS.
In the next experiment, the enzyme amount was reduced by a factor of ten. 20.0 µL
mIgG1 were diluted in 60.0 µL DB and 24.8 µL (1’250 µg) of this stock solution were
further diluted in 162.7 µL DB, 12.5 µL Kgp 10 U/µL (125 U) and 50.0 µL fresh
reducing agent to 5.00 mg/mL. The digestion solution was incubated at
37°C/300 rpm for 5, 15, 30, 60 and 120 min and tested by CE-SDS NR. After first
data evaluation the experiment was repeated with pull points from 6 to 15 min every
minute.
Enzyme inhibition: As the intermediate product LHF is of interest slowing down or
stopping the digestion progress would simplify the procedure. In this experiment the
influence of cooling the digestion mixture to rt resp. 5°C, acidifying to pH 5.5 and/or
addition of cOmplete™ protease inhibitor was evaluated. As extreme pH changes
(e.g. by spiking TFA) might lead to undesired modifications, the maximum pH drop
was set to pH 5.5, where most IgG1 are stable.
20.0 µL mIgG1 were diluted in 60.0 µL DB and 5.00 µL of this pre-dilution, 2.50 µL
Kgp 10 U/µL and 10.0 µL fresh reducing agent were diluted in 32.5 µL corresponding
DB, digested for 15 min at 37°C/300 rpm, 22°C or 5°C and analyzed by CE-SDS NR.
Cysteine influence: To evaluate the influence of cysteine on the enzyme activity
several digestions at different concentration levels, origins and age were tested and
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reaction constants compared. As a rate of reduction also the LC peak, at a relative
migration time (RMT) of 0.54, was observed through the digestion.
20.0 µL mIgG1 were diluted in 60.0 µL DB to 50.5 mg/mL and the digestion solution
were prepared as described in Table 8 with the corresponding RS, incubated for
15 min at 37°C/300 rmp and analyzed by CE-SDS NR.
Table 8: Dilution table of the cysteine influencing digestion.
µL
Genovis Self-made/fresh Self-made/7d-aged
2.0 mM 0.4 mM 2.0 mM 1.0 mM 0.2 mM 1.0 mM 0.2 mM
DB pH 8.0 37.5 41.5 37.5 40.0 42.0 40.0 42.0
mIgG1 50.5 mg/mL
5.0 5.0 5.0 5.0 5.0 5.0 5.0
Kgp 10 U/mL
2.5 2.5 2.5 2.5 2.5 2.5 2.5
RS 20 mM 5.0 1.0 5.0 2.5 0.5 2.5 0.5
Genovis 2.0 and 0.4 mM and fresh/self-made 2.0 mM were then repeated in a three
times larger volume and analyzed after 5, 15, 30, 60 and 120 min.
To evaluate the reaction kinetics of the Kgp digestion in absence of cysteine 9.9 µL
mIgG1 (50.5 mg/mL) were diluted in 85.1 µL DB and 5.0 µL Kgp (10 U/µL) were
added. Afterwards, the digestion mixture was incubated at 37°C/300 rpm for 15, 30
and 60 min and analyzed by CE-SDS NR.
pH influence: To be able to evaluate the impact of the pH and better control the
digestion rate mIgG1 was digested at pH 7.3 (slightly basic) and 6.0 (slightly acidic).
20.0 µL mIgG1 were diluted in 60.0 µL DB to 50.5 mg/mL and 9.9 μL of this pre-
dilution solution were further diluted in 75.1 μL DB pH 7.3 resp. 6.0, 5.0 μL Kgp
(10 U/μL) and 10.0 μL RS were added. Afterwards, the digestion mixture was
incubated at 37°C/300 rpm for 15, 30 and 60 min and analyzed by CE-SDS NR.
Digestion parameter optimization: To optimally use the reagents included in the
kit and digest to 20 – 30% LHF within a reasonable time frame the enzyme content
was set ten times lower compared to the standard protocol and the cysteine
concentration was reduced to 1.25 mM. pH 6.0 and 8.0 were tested on those
conditions. 20.0 µL mIgG1 solution were diluted in 60.0 µL DB to 50 mg/mL and
19.8 μL of this pre-dilution solution were diluted in 157.7 μL DB at pH 6.0 resp. 8.0,
10 μL Kgp solution (10 U/μL) and 12.5 μL RS were added, incubated at
37°C/300 rpm for 5, 15, 30, 60 and 120 min and analyzed by CE-SDS NR.
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Fc isolation: To confirm the identity of the Fc peak (RMT = 0.73) a Protein L affinity
chromatography (AC) was performed. 20.0 µL mIgG1 were diluted in 60.0 µL DB to
50.5 mg/mL, 24.8 μL of this pre-dilution solution were further diluted in 187.7 μL DB,
12.5 μL Kgp (10 U/μL) and 25 μL RS were added and incubated for 2 h at
37°C/300 rpm. After full digestion a sample of 24 µL was taken for CE-SDS NR, the
remaining volume was loaded on a Protein L AC pre-equilibrated in binding buffer
(BB) and eluted with elution buffer (EB) according to Table 9. Acidic elution fractions
were neutralized with 60 µL 1 M tris pH 8.0 per milliliter fraction.
Table 9: Elution of Fc isolation.
Number of steps
Volume in mL Buffer Fraction volume in mL
Fraction name
1 1 BB 1.2 Flow through
4 1 BB 4 x 1 Wash 1 – 4
5 1 EB pH 3.5 5 x 1 Elution 1 – 5
The content of each fraction was determined by Nanodrop. The rinsing fraction was
concentrated using a 0.5 mL 10K-centrifugal filter, the concentration was determined
and the purity analyzed by CE-SDS NR.
SEC screening
To generate material for the preliminary trials 4.00 mg mIgG1 were digested
according to the optimized conditions and quenched after 30 min with 84 μL
trifluoroacetic acid 0.5%.
Number of columns: To evaluate the optimal number of serially connected columns
required to reach the needed resolution, 40 μg mIgG1 were injected on two and three
columns in series.
Column load: To evaluate the maximal injection amount without losing resolution
40, 120, 200, 300 and 400 µg mIgG1 were loaded on three columns in series.
Fractionation range: To evaluate an appropriate range to collect desired fractions
20 runs of 40 µg mIgG1 each were injected on three columns in series and collected
according to Table 10. Collected fractions were pooled, concentrated with 15 and
0.5 mL 10K-centrifugal filters and analyzed by Nanodrop and CE-SDS NR.
Table 10: SEC fractionation range.
Fragment name Fraction range in min.
IgG 59.0 – 61.0
LHF 63.5 – 66.0
Fab (+ Fc) 71.5 – 75.0
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Protein L AC and SEC loading buffer evaluation: To generate fragments free of
Fc and to stop further fragmentation the digestion was repeated with 8.00 mg mIgG1.
After 30 min digestion, the mixture was loaded on a Protein L AC column and eluted
as described in Table 11. Acidic elution fractions were neutralized with 60 µL 1 M tris
pH 8.0 per milliliter fraction.
Table 11: Elution of Protein L AC.
Number of steps
Volume in mL Buffer Fraction volume in mL
Fraction name
1 1 BB 1.8 Flow through
4 1 BB 4 Wash
8 1 EB pH 2.0 8 Elution
The concentration of each fraction was measured. Elution fraction 2 – 8 were pooled,
concentrated with a 15 mL 10K-centrifugal filter to approximately 1 mL and analyzed
by Nanodrop. 60 µL (413 µg) were analyzed by SEC.
The sample was then buffer exchanged to SEC mobile phase with a 0.5 mL 10K-
centrifugal filter, rinsed and diluted with 2 x 500 µL mobile phase. Afterwards, the
content was determined. 8 fraction collection runs of 85 µL (8 x 406 µg) were
performed. The collected fractions were pooled, concentrated and buffer exchanged
into formulation buffer in a 4 mL 10K-centrifugal filter to approximately 150 µL
sample. The fractions were analyzed by Nanodrop and CE-SDS NR.
Sample stability: To evaluate the sample stability in mobile phase the injection
sample was analyzed before and after fraction collection (T1d) by CE-SDS NR.
Digestion reproducibility and fragment isolation: To evaluate the reproducibility
the digestion was repeated with 4.00 mg mIgG1. After 30 min a sample was
analyzed by CE-SDS NR. The remaining solution was loaded on a Protein L AC
column and eluted as described in Table 12. Acidic elution fractions were neutralized
with 60 µL 1 M tris pH 8.0 per milliliter fraction.
Table 12: Elution of Protein L AC for reproducibility.
Number of steps
Volume in mL Buffer Fraction volume in mL
Fraction name
1 1 BB 1.4 Flow through
4 1 BB 4 Wash
10 1 EB pH 2.0 10 Elution
The eluate was concentrated and buffer exchanged to approximately 150 µL and
rinsed with 250 µL formulation buffer. The concentration was measured and the
samples diluted with additional 800 µL formulation buffer to 3.8 mg/mL. 7 fraction
collection runs of 100 µL (7 x 380 µg) were performed. The fractions were pooled,
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concentrated and buffer exchanged into formulation buffer in a 4 and 0.5 mL 10K-
centrifugal filter to approximately 42 µL and rinsed with 2 x 10 µL formulation buffer.
The fractions were analyzed by Nanodrop and CE-SDS NR.
Digestion progress of IgdE
During the time of this master’s thesis Genovis launched their IgdE product, called
FabALACTICA™. Its cleavage pattern is the same as Kgps, apart from the cleavage
site shift of one amino acid. Compared the GingisKHAN®, FabALACTICA™ is
superior in regard to digestion at neutral pH and absence of reducing agent. However
it has a lower digestion rate. In this experiment it was evaluated if IgdE is suitable for
LHF generation.
10.0 µL mIgG1 solution was pre-diluted in 40.5 µL IgdE DB to 40.0 mg/mL, 5.0 µL of
this pre-dilution solution (200 µg) were further diluted in 10.0 µL DB to 10 mg/mL and
5.0 µL IgdE 40 U/µL (200 U) were added, incubated at 37°C/300 rpm for 3 h and
analyzed by CE-SDS NR.
This procedure was repeated with 800 µg mIgG1 and analyzed after 30, 50, 60 and
90 min and a third time for time points at 5, 10, 15, 20 and 25 min.
3.5.2 F(ab’)2
Figure 31: Flow chart of the F(ab)2 generation and isolation.
IdeS digestion and Protein L AC pH 3.5 elution
The IdeS digestion was performed according to the Genovis protocol. 4.95 µL mIgG1
were diluted with 80.0 µL DB and 15.0 µL IdeS (66.7 U/mL) were added. The solution
was incubated at 37°C/300 rpm for 30 min. After the digestion a sample of 18.0 µL
was taken and analyzed by CE-SDS NR.
Afterwards the Protein L AC was performed according to GE Healthcare Instructions
29-0078-77 AC [69]. The digestion mixture was loaded on the column and eluted as
Dig.
AC
BE
Control
Digestion
IPC
Affinity chromatography
Buffer exchange
Purity analysis
In-process control
ZHAW LSFM, 2017 Methods and materials Martin Alexander Rappo
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listed in Table 13. Acidic elution fractions were neutralized with 60 µL 1 M tris pH 8
per milliliter buffer.
Table 13: Elution of Protein L AC after IdeS digestion.
Number of steps
Volume in mL Buffer Fraction volume in mL
Fraction name
1 0.6 BB 0.68 Flow through
5 1.0 BB 5 x 1 Wash
5 1.0 EB 5 x 1 Elution
The content of each fraction was determined by Nanodrop. Flow through and wash
fraction 1 were pooled, concentrated with 4 and 0.5 mL 10K-centrifugal filter to
approximately 20 µL, rinsed with 20 µL formulation buffer and analyzed by CE-SDS
NR. All elution fraction were pooled, concentrated with 4 and 0.5 mL 30K-centrifugal
filter and rinsed with 2 x 10 µL formulation buffer. Concentration of both samples
were determined.
Over-digestion screening and Protein L AC pH 2.0 – 3.0 elution
To check whether the digestion is complete after 30 min and exclude over-digestion,
the digestion time was extended to 60 min. To evaluate the correct pH value to elute
the F(ab’)2 fragment from the column a stepwise elution gradient of pH 3.0, 2.5 and
2.0 was applied.
The digestion conditions are identical to the digestion before. After 60 min digestion
a sample of 18.0 µL was taken for CE-SDS NR and the remaining volume was
loaded on the Protein L column, washed and eluted according to Table 14.
Table 14: Protein L AC purification table.
Number of steps
Volume in mL Buffer Fraction volume in mL
Fraction name
2 (vial rinse) 0.45 BB 1 Flow through
4 1.0 BB 4 Wash
8 0.25 EB pH 3.0 5 pH 3.0 elution
3 1.0
8 0.25 EB pH 2.5 5 pH 2.5 elution
3 1.0
8 0.25 EB pH 2.0 5 pH 2.0 elution
3 1.0
The protein content of each fraction was determined. The flow through, wash and all
fractions of the identical EB (pH 2.5 and 2.0) were pooled individually, concentrated
with a 4 and 0.5 mL 10K-centrifugal filter and each sample was analyzed by
Nanodrop and CE-SDS NR.
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FragIT™ kit digestion and fragment isolation
For simple enzyme removal and F(ab’)2 isolation Genovis offers the FragIT™ kit,
containing agarose-immobilized IdeS and a CaptureSelect™ column with an
immobilized 13 kDa lama antibody fragment [70]. The digestion and fragment
isolation were performed according to the FragIT™ kit instructions [70] including Fc/2
elution and all steps for maximal recovery. To evaluate if the columns could be
reused the whole procedure was repeated at the same day and after 17 days.
49.5 µL mIgG1 solution (10 mg IgG) were diluted with 450 µL DB to 20 mg/mL,
loaded on the digestion column and rotated end-over-end for 30 min at rt. Afterwards
the solution was collected through centrifugation at 100 g for 1 min and the column
was washed with 2 x 1 mL BB. The content was determined and a sample for CE-
SDS NR was taken. The solution was loaded on the CaptureSelect™ column, rotated
end-over-end for 30 min at rt, collected by centrifugation at 200 g for 1 min and
washed with 2 x 1 mL BB. The Fc/2 was collected through re-suspending with
2 x 1 mL CaptureSelect™ EB and collecting at 200 g resp. 1000 g at the final step.
Afterwards, the fractions (4.5 mL F(ab’)2 and 2.5 mL Fc/2-fraction) were
concentrated and buffer exchanged to formulation buffer with 4 and 0.5 mL 10K-
centrifugal filter to approximately 42 µL and rinsed with 3 x 20 µL. Samples were
analyzed by Nanodrop and CE-SDS NR. This procedure was repeated after 150 min
and 17 days.
3.5.3 Chitobiose cleavage
Figure 32: Flow chart of the EndoS2 digestion and fragment isolation.
EndoS2 digestion
The EndoS2 digestion was performed according to the Genovis protocol [24] at
20 mg/mL. For stability reasons the pH of the DB was set to 6.5.
Dig.
AC
BE
Control
Digestion
IPC
Affinity chromatography
Buffer exchange
Purity analysis
In-process control
ZHAW LSFM, 2017 Methods and materials Martin Alexander Rappo
30
5.90 µL mIgG1 (1.19 mg) were diluted in 24.1 µL DB and 30.0 µL EndoS2 40 U/µL
(1’200 U) added. The solution was incubated at 37°C/300 rpm for 30 min. After the
digestion a sample of 9.0 µL analyzed by CE-SDS red. The remaining digestion
mixture was buffer exchanged with a 100 K-spin filter, the membrane was rinsed with
2 x 20 µL fresh formulation buffer and the sample was analyzed by Nanodrop and
CE-SDS red.
Immobilized GlycINATOR®
To ensure complete enzyme removal immobilized EndoS2 (immobilized
GlycINATOR® MidiSpin) is used for digestion. The digestion was performed
according the Genovis standard protocol for A0-GL6-100 Version 17.1.1 [71]
including steps for maximum recovery.
49.5 µL mIgG1 (10 mg) were diluted in 450.5 µL DB, loaded on the equilibrated
column and rotated end-over-end for 30 min at rt. Afterwards, the solution was
collected through centrifugation at 100 g for 1 min and the column was washed with
2 x 1 mL BB. All fractions were pooled, concentrated and buffer exchanged into
formulation buffer with 4 and 0.5 mL 10K-centrifugal filters to approximately 42 µL.
The filter was rinsed with 2 x 20 µL formulation buffer and the sample was analyzed
by Nanodrop and CE-SDS red.
3.6 Ecology, safety and disposal
Standard laboratory safety equipment consisting of lab coat and protective goggles were used
during all tasks. During acid or base handling regular nitrile and during handling of organic
solvents butyl gloves were used. Contaminated solutions and consumables were discarded in
the chemical waste container. Powdered SDS and 2-mercaptoethanol were only handled
under the hood, as SDS creates irritating dust and 2-mercaptoethanol creates toxic fumes and
may be fatal in contact with skin [72].
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
31
4 Results and discussion
4.1.1 Fab, Fc and LHF
Stability evaluation in digestion buffer with and without reducing solution
Deamidation stability: A negligible deamidation rate of < 0.5% was detected within
the two hours in DB for both temperatures. However after 24 h an increase of 0.7
and 2.6% was detected for rt and 37°C, respectively. The chromatogram is shown in
the appendix on p. VII. Neither fragmentation nor aggregation was detected by CE-
SDS NR during the measured time frame.
Stability in digestion buffer
Figure 33: Purity trend of the stability in digestion buffer by CEX (deamidation).
Figure 34: Purity trend of the stability in digestion buffer by and CE-SDS NR (fragmentation/aggregation).
Reduction stability: The relative area increase of the sum of reduced species (L,
H, HL, HH, and HHL) after 2 h at 37°C is below 0.1% and therefore consider non-
critical. The electropherogram overlay is displayed in Figure 35.
7.5
8
8.5
9
9.5
10
10.5
11
11.5
12
12.5
0 4 8 12 16 20 24
Purity
(Acid
ic)
in A
%
Time in h
Purity of acidic variants by CEX
95
95.5
96
96.5
97
97.5
98
98.5
99
99.5
100
0 4 8 12 16 20 24
Purity
(Main
) in
A%
Time in h
Purity of main by CE-SDS NR
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
32
Figure 35: CE-SDS NR electropherogram of the mIgG1 stability in digestion buffer with reducing agent.
Digestion progress of Kgp and influence factors
Enzyme ratio: The consistency of the CE-SDS NR double injection of the digestion
sample after 5 min proves, that the digestion stops in presence of 1% SDS. The
digestion according to the Genovis protocol is almost complete after 5 min (appendix
p. VII) and is therefore not suitable for the generation of LHF. With the adjusted
enzyme-protein ratio of 1:10 a maximum LHF concentration of 24 area% is reached
after 14 min and the digestion is complete after 2 h. Linear regression of the kinetic
ln([A]/[A]0) VS. time-plot, displayed in the appendix on p. VIII shows a coefficient of
determination (R2) of 0.9683. This high value is an indication of first order-like
behavior [66]. A comparison of the measured and the simulated values, presented
in Figure 36, shows a good overlap of the IgG1 and LHF curve. Fc and Fab
concentrations are slightly overestimated by the simulation. As the generation of LHF
has priority over Fc and Fab this model is considered fit for purpose.
7 9 11 13 15 17 19
Absorb
ance in m
Ab
Time in min.
CE-SDS NR electropherogram - Digestion buffer stability with reducing agent
T0 T120 min
LH HL HH
HHL
Ma
in
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
33
Figure 36: CE-SDS NR purity progress of the 1:10 Kgp-to-IgG digestion and comparison of the simulated values.
Enzyme inhibition: An evaluation of the reaction constant k1 (see Figure 37) and
tmax(LHF) (see Figure 38) revealed following inhibition potential:
Cooling > Acidify to pH 5.5 > cOmplete™
Figure 37: Reaction constant k1 in dependency of temperature for tested inhibitory factors (standard digestion, cOmplete™ and pH 5.5).
Figure 38: Calculated time point of maximal LHF concentration in dependency of temperature for tested inhibitory factors (standard digestion, cOmplete™ and pH 5.5).
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 15 30 45 60 75 90 105 120
Purity
in A
%
Time in min
1:10 enzyme:protein digestion progress - measured VS. calculated
IgG1(measured)
IgG1(calculated)
LHF (measured)
LHF (calculated)
Fc (measured)
Fc (calculated)
Fab (measured)
Fab (calculated)
R² = 0.9946
R² = 0.9999
R² = 0.9654
0.00
0.02
0.04
0.06
0.08
0.10
0.12
5 15 25 35
Reaction c
onsta
nt
k1
Temperature / °C
Kinetic constant
0
20
40
60
80
100
120
5 15 25 35
t ma
x(L
HF
) in
min
Temperature / °C
Time of maximal LHF conentration
Inhibitory factor influence
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
34
The cOmplete™ protease inhibitor cocktail will be omitted for further studies, as the
inhibition potential is low. Additionally, as the composition is not clearly defined
tracking during fragment isolation and full removal cannot be ensured. For further
studies cooling to 5°C potentially in combination with acidifying and enzyme removal
for minimal molecule impact will be applied.
Cysteine influence: The comparison between digestions a different cysteine
concentrations, shown in Figure 39 and Figure 40, revealed a high influence of
cysteine on the Kgp digestion rate. The Kgp digestion rate is drastically reduced resp.
the digestion time is increased in absence of cysteine.
Figure 39: Kinetic constants of the Kgp digestion for different cysteine concentration of Genovis, self-made/fresh and self-made/aged RS.
Figure 40: Calculated time point of maximal LHF concentration of the Kgp digestion for different cysteine concentration of Genovis, self-made/fresh and self-made/aged RS.
The self-made cysteine solution had the highest digestion rate resp. lowest digestion
time for all concentrations compared to other conditions. The digestion using aged
self-made cysteine solution was slower than freshly made RS for all concentrations.
The highest digestion rate and lowest digestion time was determined at a
concentration of 1 mM, even though similar results were obtained for a concentration
of 2 mM cysteine. A possible explanation for the lower kinetic constant of Genovis’
RS compared to the self-made might be small differences regarding final
concentration. The cysteine in aged RS is most probably partly oxidized and not able
to provide the optimal reducing environment anymore.
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Kin
etic c
onsta
nt
k1
Cysteine concentration in mM
Kinetic constant
0
50
100
150
200
250
Tim
e o
f [L
HF
] ma
xin
min
Cysteine concentration in mM
Time point of the maximal LHF concentration
Cysteine concentration influence
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
35
The purity progress of the peak at RMT 0.54 through the digestion time, displayed in
Figure 41, shows a comparable pattern for all tested cysteine concentrations. The
purity initially increases and later decreases again. As expected, the rate of reduction
increases with increasing cysteine content, but is throughout low. All experiments
using the RS of Genovis show a purity maximum at 30 min, whereas the self-made
RS shows its maximum after 60 min, which is significant higher compared to the
Genovis RS at equivalent concentration. This is an indication that this peak does not
only contain LC but also another unidentified species, which is formed and degraded
during the digestion.
Figure 41: Progress of the 25 kDa-peak formation during the IgG1 Kgp digestion at different cysteine concentrations.
Therefore the cysteine concentration is considered crucial for the digestion progress
and Genovis and self-made RS do not deliver equal results.
pH influence: The experiment showed that the pH has a large impact on the
digestion kinetic. At pH 8.0 the highest and at pH 6.0 lowest kinetic constant was
measured. This is visualized in Figure 42 and Figure 43.
0%
1%
2%
3%
4%
5%
6%
7%
8%
9%
0 15 30 45 60 75 90 105 120
Purity
in A
%
Time in min
Purity of the peak at a RMT = 0.54 through the digestion course at different cysteine conentrations
Genovis 4 mM
Genovis 2 mM
Genovis 1 mM
Self-made 2 mM
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
36
pH influence
Figure 42: Kinetic constants of the Kgp digestion at different pH.
Figure 43: Calculated time point of maximal LHF concentration of the Kgp digestion at different pH.
Digestion parameter adjustment: It was observed that upon reconstitution of the
cysteine according to the Genovis protocol, a suspension was obtained. The cysteine
only fully dissolved after further dilution for digestion. The purity of IgG, LHF, Fc and
Fab at pH 6.0 and 8.0 are visualized in Figure 44 and Figure 45, respectively. The
digestion at pH 6.0 is relatively slow and the maximum concentration of LHF is not
reached until the upper time limitation of 120 min. The digestion at pH 8.0 on the
other hand shows a maximum LHF concentration after 30 min followed by a shallow
decrease, allowing the scientist to perform the digestion in a reasonable time frame.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
6 6.5 7 7.5 8
Kin
etic c
onsta
nt
k1
pH
Kinetic constant
0
5
10
15
20
25
30
35
6 6.5 7 7.5 8
Tim
e o
f [L
HF
] ma
xin
min
pH
Time point of the maximal LHF concentration
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
37
Digestion parameter adjustment
Figure 44: Digestion course of the parameter adjusted Kgp digestion at pH 6.0.
Figure 45: Digestion course of the parameter adjusted Kgp digestion at pH 8.0.
Fc isolation: The Protein L AC successfully bound the Fab and the Fc could be
collected with a recovery of 47%. Later Fab elution, displayed in Figure 46, was
minimal with a recovery of 20%. A possible explanation is, that the pH of the EB was
not low enough to reverse the binding.
Figure 46: Protein content of the Protein L affinity chromatography by Nanodrop.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 15 30 45 60 75 90 105 120
Purity
in A
%
Time in min
pH 6.0
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 15 30 45 60 75 90 105 120P
urity
in A
%
Time in min
pH 8.0
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Concentr
ation in m
g/m
L
Step
Protein content of the Protein L affinity column fractions
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
38
The Fc could successfully be isolated with a final concentration of 3.43 mg/mL by
Protein L AC and the peak at RMT 0.73 in CE-SDS NR could be assigned. The
corresponding electropherogram is shown in Figure 47.
Figure 47: CE-SDS NR electropherogram of the Kgp digestion and concentrated Protein L flow through.
SEC screening
Number of columns: Two SEC columns in series are capable of separating IgG,
LHF and Fab. Three columns in series prolong the run time from 55 to 85 min,
increase the resolution and an additional peak between LHF and Fab could be
separated. Baseline separated is not reached in both cases. Corresponding
chromatograms are shown in Figure 48.
6 7 8 9 10 11 12 13 14 15 16
Absorb
ance in m
AU
Time in min
CE-SDS NR electropherogram before and after Protein L AC
Kgp digestion
Protein L flow through
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
39
Figure 48: SEC chromatogram overlay of 2 and 3 columns in series.
Column volume: Testing column loads from 40 to 400 μg mIgG1 showed no
significant loss of peak resolution. The SEC chromatogram overlay is displayed in
Figure 49.
Figure 49: SEC chromatogram of the injection amount evaluation.
Fractionation range: The chosen fractionation range was sufficient to isolate the
desired fragments. Importantly, the chromatographic overlays (Figure 50) revealed,
that the TFA spiking did not fully quench the digestion and the sample did
fragmentation continued at 5°C (shown by decreasing peaks at retention time 59.6
0 10 20 30 40 50 60 70 80
Absorb
ance in m
AU
Time in min
SEC chromatogram of the Kgp digest - 2 VS. 3 columns
2 columns 3 columns
IgG
1
LH
F
Fab
IgG
1
LH
F
Fab
55 60 65 70 75 80 85
Absorb
ance in m
AU
Time in min
SEC chromatogram - Increasing column load
40 µg 120 µg 200 µg 300 µg 400 µg
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
40
(IgG) and 64.5 min (LHF) as well as an increase in the Fab peak at retention time
72 min).
Figure 50: SEC chromatogram overlay of the fraction collection trial of 20 times 40 μg injection.
A summary of the isolated fragment is listed in Table 15.
Table 15: Summary of isolated fragments during SEC trials.
Fragment Volume in µL
Conc. In mg/mL
Mass in µg Recovery in %
Purity in A%
IgG1 120 0.10 11 4 86
LHF 120 0.18 22 14 75
Fab+Fc 120 0.81 64+32 45+30 98
Figure 51: CE-SDS NR overlay of the isolated IgG, LHF and Fab+Fc fractions.
9 10 11 12 13 14 15 16
Absorb
ance in m
AU
Time in min.
CE-SDS NR electropherogram - Purity of isolated fragments
IgG LHF Fab+Fc
71%
27%
75% 86%
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
41
Protein L AC and SEC loading buffer evaluation: The Protein L AC could
successfully remove the Fc part and the enzyme (see appendix p. VIII) and stop
further digestion. As a trade-off, a small amount of IgG, LHF and Fab was lost during
the wash steps. The intact IgG purity by CE-SDS NR was higher than expected.
Reason for this could possibly be a slower digestion rate and imply issues regarding
reproducibility. Telquel injection on the SEC showed a different chromatographic
pattern, displayed in Figure 52, than previous injections without prior Protein L AC.
A possible explanation for this behavior is the different buffer composition influencing
the hydrodynamic radius.
Figure 52: SEC chromatogram of the Kgp digestion fragment isolation with and without Protein L AC.
Prior buffer exchange into SEC mobile phase improved pattern resemblance on the
SEC column, shown in Figure 53. Peaks before the IgG peak indicate sample
instability of mixed sample but also isolated fragments. The lower UV trace of one of
the sample injections can be explained by a smaller injection volume. A summary of
isolated fragments is listed in Table 16. Reason for the low LHF purity is the relatively
high IgG content in the initial sample tailing into the LHF fraction.
Table 16: Summary of isolated fragments during SEC injection buffer evaluation.
Fragment Volume in µL
Conc. In mg/mL
Mass in µg Recovery in %
Purity in A%
IgG1 130 4.55 591 89 98
LHF 110 1.29 142 21 45
Fab 110 1.24 136 15 95
43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83
Absorb
ance in m
AU
Time in min
Comparison: Fraction collection after digest and after Protein L AC
Without Protein L AC With Protein L AC IgG fraction
LHF fractio Fab fraction
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
42
Figure 53: SEC chromatogram overlay of the fragment isolation after Kgp digestion, Protein L AC and buffer exchange in mobile phase.
Sample stability: The CE-SDS NR electropherogram of the SEC injection sample
before and after fraction collection, displayed in Figure 54, shows no significant
changes. A possible explanation are reversible aggregates, which dissociate in CE-
SDS NR sample buffer.
Figure 54: CE-SDS NR electropherogram of initial and one-day aged injection sample.
Digestion reproducibility and fragment isolation: CE-SDS NR comparison of two
digestions with identical parameters but different GingisKHAN® batches (see Figure
35 40 45 50 55 60 65 70 75 80 85
Absorb
ance in m
AU
Time in min
Kgp digestion after Protein L AC and desalting: Fraction collection
Injection 1 Injection 2 Injection 3 Injection 4 Injection 5
Injection 6 Injection 7 IgG fraction LHF fraction Fab fraction
9 10 11 12 13 14 15 16 17 18 19
Absorb
ance in m
AU
Time in min
CE-SDS NR electropherogram - Injection sample stability in SEC mobile phase at 5°C
Before fraction collection After fraction collection
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
43
55) showed inconsistent digestion progress. A possible explanation for this are
differences of the RS, as a different solubility was overserved during reconstitution.
Figure 55: CE-SDS NR electropherogram overlay of two Kgp digestions with identical parameter apart from different GingisKHAN® batches.
Buffer exchange into formulation buffer improved the sample stability and less peaks
before IgG were observed.
Figure 56: SEC chromatogram overlay of the fragment isolation after Kgp digestion, Protein L AC and buffer exchange in formulation buffer.
9 10 11 12 13 14 15 16 17
Absorb
ance in m
AU
Time in min
CE-SDS NR electropherogram - Comparison of two 30 min Kgp digestions
Digest 1 Digest 2
35 40 45 50 55 60 65 70 75 80 85
Absorb
ance in m
AU
Time in min
SEC chromatogram - Kgp digestion after Protein L AC and buffer exchange in formulation buffer: Fraction collection
Injection 1 Injection 2 Injection 3 Injection 4 Injection 5
Injection 6 IgG fraction LHF fraction Fab fraction
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
44
A summary of isolated fragments is listed below.
Table 17: Summary of isolated fragments during SEC fraction collection.
Fragment Volume in µL
Conc. In mg/mL
Mass in µg Recovery in %
Purity in A%
IgG1 20 6.21 124 4 97
LHF 20 2.24 45 9 41
Fab 20 2.32 46 15 82
Digestion progress of IgdE
The IgdE digestion progress, displayed in Figure 57, showed a much lower digestion
time required than the 16 –18 h suggested in the Genovis protocol. The maximum
LHF concentration of 29% was detected after 10 min and would even allow reduction
of the enzyme-to-protein ratio to slow down the digestion. The higher intermediate
concentration, the poly-his tag for simple enzyme removal and mild digestion
conditions make IgdE superior to Kgp for the LHF generation.
Figure 57: Digestion progress of IgdE by CE-SDS NR.
4.1.2 F(ab’)2
IdeS digestion and Protein L AC pH 3.5 elution
The IdeS digestion was complete after 30 min. Fc/2, IdeS and some other, minor
impurities could successfully be removed by the Protein L AC. The F(ab’)2 could not
be eluted with EB at pH 3.5.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 15 30 45 60 75 90 105 120 135 150 165 180
Purity
in A
%
Time in min
Digestion progress of the IgdE digestion
IgG
LHF
Fc
Fab
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
45
Over digestion screening and Protein L AC pH 2.0 – 3.0 elution
After 1 h of digestion no sign of over-digestion could be detected by CE-SDS NR.
Elution at pH 3.0 was very slow, at pH 2.5 moderate and at pH 2.0 acceptable. The
electropherogram is displayed in the appendix on p. IX.
Figure 58: Elution profile of Protein L AC at pH 3.0, 2.5 and 2.0.
FragIT™ kit digestion and fragment isolation
The on-column IdeS digestion was complete after 30 min and the Fc/2 could
successfully be removed with the CaptureSelect™ column, as shown in Figure 59.
Figure 59: CE-SDS NR electropherogram of the IdeS digestion and isolated products.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
Concentr
ation in m
g/m
L
Elution buffer volume in mL
Protein L AC elution at pH 3.0 - 2.0
pH 3.0
pH 2.5
pH 2.0
7 8 9 10 11 12 13 14 15 16
Absorb
ance in m
AU
Time in min
CE-SDS NR electropherogram - FabIT kit digestion and purification
Undigested
Digested
F(ab)2
Fc/2
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
46
Initial and second digestion at the same day show the same digestion rate. The
digestion after 17 days showed a loss in activity and is after 30 min not complete. No
differences of digestion products was observed. A summary of the isolated fragments
is listed in Table 18.
Table 18: Summary of isolated fragments for FragIT™ kit digestion and fragment isolation.
Run Volume in
µL Conc. in mg/mL
Mass in µg Recovery in %
Purity in A%
1 100 31.65 3’165 48 91
2 100 36.12 3’612 55 96
3 100 12.71 1271 19 97
Figure 60: CE-SDS NR electropherogram overlay of the three FragIT™ digestion.
4.1.3 Chitobiose cleavage
EndoS2 digestion
The digestion according to the Genovis® protocol and the subsequent buffer
exchange were successful. The enzyme could not be removed but reduced to below
0.5%. 40 µL chitobiose-cleaved mIgG1 27.06 mg/mL (1’082 µg = 94% yield) could
be isolated.
7 8 9 10 11 12 13 14 15
Absorb
ance in m
AU
Time in min
CE-SDS NR electropherogram - Overlay of FragIT™ digestion at different time points
1st run (T0) 2nd run (T150min.) 3rd run (T17d)
ZHAW LSFM, 2017 Results and discussion Martin Alexander Rappo
47
Figure 61: CE-SDS red. electropherogram of the EndoS2 digestion and buffer exchange.
Immobilized GlycINATOR®
The Genovis® digestion procedure for the immobilized GlycINATOR® was
successfully applied to mIgG1. The digestion product was be concentrated and
buffer exchanged into formulation buffer. 62 µL chitobiose-cleaved mIgG1 at
125 mg/mL (7’750 µg = 80% yield) were isolated.
15 17 19 21 23 25 27 29 31 33 35
Absorb
ance in m
AU
Time in min.
CE-SDS red. electropherogram of the soluble EndoS2
digestion and buffer exchange
Reference Digestion T30min Buffer exchange
ZHAW LSFM, 2017 Conclusion Martin Alexander Rappo
48
5 Conclusion
The influence of the undesired modifications, deamidation and reduction during digestion
where assessed and are considered minimal (> 0.5%) within 2 The Kgp digestion of the mIgG1
using GingisKHAN® was successful and the LHF could be detected as intermediate by CE-
SDS NR after reducing the enzyme-to-protein ratio to 1:10. Cysteine concentration and pH of
the DB was classified as a crucial influence parameter for the digestion kinetic. Through
digestion parameter optimization for LHF isolation the time for the maximal LHF was adjusted
to 30 min at 37°C, which is considered an appropriate time frame to handle. Of the evaluated
digestion inhibition parameter (temperature, pH and cOmplete™ inhibitor cocktail) nothing fully
inhibited but slowed down further fragmentation. Inhibition potential is in decreasing order:
cooling to 5°C, acidify to pH 5.5 and adding cOmplete™. Therefore enzyme removal by AC is
proposed to stop further digestion. Through Protein L AC the Fc could be isolated and following
elution at pH 2.0 the IgG, LHF and Fab mixture could be eluted. Small quantities could be
separated by SEC with a purity of > 75%. Scale-up and reproducibility experiments revealed
issues regarding inconsistent digestion progresses for different batches. Slow digestion led
finally to large IgG contamination of the LHF fraction. This digestion kinetic variations probably
origin from RS differences. Experiments on the recently launched FabALACTICA™ (IgdE)
showed promising results with an acceptable digestion time, mild digestion conditions, less
influencing factors and a poly-his tag on the enzyme for simple removal and digestion stop.
Also the outlook on the immobilized IgdE, which is according to Genovis® supposed to be
launched in fall 2017 makes IgdE and better alternative for LHF generation than Kgp.
Digestion with FabRICATOR® (soluble IdeS) was complete after 30 min and Fc/2 was removed
by Protein L AC. The F(ab‘)2 was successfully generated by applying standard FragIT™
(immobilized IdeS) procedure and the desired fragment was isolated by CaptureSelect™
without exposure to acidic solution. The maximal recovery was 55% and purity 96% and no
undesired modifications were detected. The procedure was repeated with identical outcome
at the same day using the same mab and digestion kit.
The chitobiose cleavage by GlyCINATOR® (soluble EndoS2) was complete after 30 min at
37°C but a simple 100K-centrifugal filtration was not sufficient to fully remove the enzyme.
Immobilized GlyCINATOR® provided a full digestion after 30 min at rt and full enzyme removal
with a yield of 80%.
ZHAW LSFM, 2017 Outlook Martin Alexander Rappo
49
6 Outlook
Following experiments should include identification and in-depth analysis of undesired
modification by mass spectrometry.
Also other types of IgG1, mutants, other applicable subclasses or proteins should be tested on
the implemented methods.
As soon as the immobilized FabALACTICA™ (IgdE) is launched, digestion progress and
applicability for the LHF generation should be evaluated. Important is also a reproducibility and
batch-to-batch comparison.
Isolated digestion products have to be tested for regarding CQA. This could include: cell
bioassay for fragments and higher order structure analysis by differential scanning calorimetry
and circular dichroism in the far UV for the chitobiose chelated product.
ZHAW LSFM, 2017 References Martin Alexander Rappo
50
7 References
[1] R.O. Esquivel, M. Molina-Espiritu, F. Salas, C. Soriano, C. Barrientos, J.S. Dehesa, J.A. Dobado, Decoding the building bloks of life from the perspective of quantum information, Licens. InTech. (2013) 641–669. doi:http://dx.doi.org/10.5772/55160 1.
[2] J.K.H. Liu, The history of monoclonal antibody development - Progress, remaining challenges and future innovations, Ann. Med. Surg. 3 (2014) 113–116. doi:10.1016/j.amsu.2014.09.001.
[3] A.L. Nelson, E. Dhimolea, Development trends for human monoclonal antibody therapeutics, Nat. Rev. Drug Discov. 9 (2010) 767–774. doi:10.1038/nrd3229.
[4] H.M. Shepard, G.L. Phillips, C.D. Thanos, M. Feldmann, Developments in therapy with monoclonal antibodies and related proteins, Clin. Med. J. R. Coll. Physicians London. 17 (2017) 220–232. doi:10.7861/clinmedicine.17-3-220.
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7.1 List of illustrations
FIGURE 1: AMINO ACID PROPERTIES VENN DIAGRAM [1]. .......................................................... VIII FIGURE 2: ILLUSTRATION OF THE QUALITY BY DESIGN STRATEGY [15,16]. .................................. 2 FIGURE 3: POTENTIAL INFORMATION SOURCES FOR THE QUALITY RISK MANAGEMENT [15]. .......... 3 FIGURE 4: THE FOUR QUALITY CATEGORIES FOR THE RISK ASSESSMENT QUALITY ATTRIBUTES
[15,17–20]. ...................................................................................................................... 3 FIGURE 5: SELECTED DIGESTION PRODUCTS TO BE EVALUATED DURING THE MASTER THESIS
INCLUDING THEIR FINAL PURPOSE IN THE CQA ASSESSMENT. .............................................. 4 FIGURE 6: ILLUSTRATION OF THE IGG1 STRUCTURE WITH LIGHT CHAIN IN APRICOT AND HEAVY
CHAIN IN BLUE. ................................................................................................................. 6 FIGURE 7: IG FAB WITH CDR IN RED AND A HAPTEN AS EPITOPE [32]. ......................................... 6 FIGURE 8: 3D SURFACE STRUCTURE OF AN IGG1 [33]. .............................................................. 6 FIGURE 9: NOMENCLATURE OF COMMON IGG FRAGMENTS [37–42]. ........................................... 7 FIGURE 10: FC GLYCOSYLATION STRUCTURE OF TRANSGENIC ANIMAL/CHINESE HAMSTER OVARY
CELLS. ............................................................................................................................. 8 FIGURE 11: ILLUSTRATION OF IDES. ......................................................................................... 9 FIGURE 12: IGG FRAGMENTATION OF IDES. .............................................................................. 9 FIGURE 13: ACCEPTED SEQUENCE FOR THE IDES CLEAVAGE. IDENTICAL AMINO ACID IN BLACK,
CLOSELY CHEMICALLY SIMILAR IN BLUE, WEAKLY CHEMICALLY SIMILAR IN SIENNA AND NON-SIMILAR IN APRICOT. ........................................................................................................ 10
FIGURE 14: PROPOSED CATALYTIC CYCLE OF THE IDES CLEAVAGE. ......................................... 10 FIGURE 15: CATALYTIC POCKET AND STABILIZING EFFECTS. ..................................................... 11 FIGURE 16: ILLUSTRATION OF RECOMBINANT KGP. .................................................................. 11 FIGURE 17: IGG FRAGMENTATION OF KGP AND IGDE. .............................................................. 12 FIGURE 18: ACCEPTED KGP CLEAVAGE SEQUENCE OF IGG1 [22]. ............................................ 12 FIGURE 19: ILLUSTRATION OF IGDE. ....................................................................................... 13 FIGURE 20: ACCEPTED IGDEAGALACTIAE CLEAVAGE SEQUENCE OF HUMAN IGG1 [36]. ..................... 13 FIGURE 21: ILLUSTRATION OF ENDOS2................................................................................... 13 FIGURE 22: CHEMICAL STRUCTURE OF THE CHITOBIOSE CORE. ................................................ 14 FIGURE 23: CHITOBIOSE CLEAVAGE OF ENDOS2. .................................................................... 14 FIGURE 24: PROPOSED CONSECUTIVE REACTION SCHEME OF AN IGG AND KGP. ....................... 15 FIGURE 25: SIMULATED KGP DIGESTION PROGRESS BASED ON A CONSECUTIVE FIRST-ORDER
KINETIC. ......................................................................................................................... 16 FIGURE 26: DEAMIDATION OF ASPARAGINE. ............................................................................ 18 FIGURE 27: EXAMPLE CATION EXCHANGE CHROMATOGRAM. .................................................... 19 FIGURE 28: EXAMPLE CE-SDS NR ELECTROPHEROGRAM OF THE KGP DIGESTION. .................. 21 FIGURE 29: EXAMPLE CE-SDS RED ELECTROPHEROGRAM OF THE ENDOS2 DIGESTION. ........... 22 FIGURE 30: FLOW CHART OF THE FAB, FC AND LHF GENERATION AND ISOLATION. .................... 22 FIGURE 31: FLOW CHART OF THE F(AB)2 GENERATION AND ISOLATION. ..................................... 27 FIGURE 32: FLOW CHART OF THE ENDOS2 DIGESTION AND FRAGMENT ISOLATION. .................... 29 FIGURE 33: PURITY TREND OF THE STABILITY IN DIGESTION BUFFER BY CEX (DEAMIDATION). .... 31 FIGURE 34: PURITY TREND OF THE STABILITY IN DIGESTION BUFFER BY AND CE-SDS NR
(FRAGMENTATION/AGGREGATION). ................................................................................... 31 FIGURE 35: CE-SDS NR ELECTROPHEROGRAM OF THE MIGG1 STABILITY IN DIGESTION BUFFER
WITH REDUCING AGENT. .................................................................................................. 32 FIGURE 36: CE-SDS NR PURITY PROGRESS OF THE 1:10 KGP-TO-IGG DIGESTION AND
COMPARISON OF THE SIMULATED VALUES. ........................................................................ 33 FIGURE 37: REACTION CONSTANT K1 IN DEPENDENCY OF TEMPERATURE FOR TESTED INHIBITORY
FACTORS (STANDARD DIGESTION, COMPLETE™ AND PH 5.5). ........................................... 33 FIGURE 38: CALCULATED TIME POINT OF MAXIMAL LHF CONCENTRATION IN DEPENDENCY OF
TEMPERATURE FOR TESTED INHIBITORY FACTORS (STANDARD DIGESTION, COMPLETE™ AND
PH 5.5). ......................................................................................................................... 33
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FIGURE 39: KINETIC CONSTANTS OF THE KGP DIGESTION FOR DIFFERENT CYSTEINE
CONCENTRATION OF GENOVIS, SELF-MADE/FRESH AND SELF-MADE/AGED RS. ................... 34 FIGURE 40: CALCULATED TIME POINT OF MAXIMAL LHF CONCENTRATION OF THE KGP DIGESTION
FOR DIFFERENT CYSTEINE CONCENTRATION OF GENOVIS, SELF-MADE/FRESH AND SELF-MADE/AGED RS. ............................................................................................................. 34
FIGURE 41: PROGRESS OF THE 25 KDA-PEAK FORMATION DURING THE IGG1 KGP DIGESTION AT
DIFFERENT CYSTEINE CONCENTRATIONS. ......................................................................... 35 FIGURE 42: KINETIC CONSTANTS OF THE KGP DIGESTION AT DIFFERENT PH. ............................. 36 FIGURE 43: CALCULATED TIME POINT OF MAXIMAL LHF CONCENTRATION OF THE KGP DIGESTION
AT DIFFERENT PH. .......................................................................................................... 36 FIGURE 44: DIGESTION COURSE OF THE PARAMETER ADJUSTED KGP DIGESTION AT PH 6.0. ...... 37 FIGURE 45: DIGESTION COURSE OF THE PARAMETER ADJUSTED KGP DIGESTION AT PH 8.0. ...... 37 FIGURE 46: PROTEIN CONTENT OF THE PROTEIN L AFFINITY CHROMATOGRAPHY BY NANODROP. 37 FIGURE 47: CE-SDS NR ELECTROPHEROGRAM OF THE KGP DIGESTION AND CONCENTRATED
PROTEIN L FLOW THROUGH. ............................................................................................ 38 FIGURE 48: SEC CHROMATOGRAM OVERLAY OF 2 AND 3 COLUMNS IN SERIES. .......................... 39 FIGURE 49: SEC CHROMATOGRAM OF THE INJECTION AMOUNT EVALUATION. ............................ 39 FIGURE 50: SEC CHROMATOGRAM OVERLAY OF THE FRACTION COLLECTION TRIAL OF 20 TIMES
40 ΜG INJECTION. ........................................................................................................... 40 FIGURE 51: CE-SDS NR OVERLAY OF THE ISOLATED IGG, LHF AND FAB+FC FRACTIONS. ........ 40 FIGURE 52: SEC CHROMATOGRAM OF THE KGP DIGESTION FRAGMENT ISOLATION WITH AND
WITHOUT PROTEIN L AC. ................................................................................................ 41 FIGURE 53: SEC CHROMATOGRAM OVERLAY OF THE FRAGMENT ISOLATION AFTER KGP
DIGESTION, PROTEIN L AC AND BUFFER EXCHANGE IN MOBILE PHASE. ............................... 42 FIGURE 54: CE-SDS NR ELECTROPHEROGRAM OF INITIAL AND ONE-DAY AGED INJECTION
SAMPLE. ......................................................................................................................... 42 FIGURE 55: CE-SDS NR ELECTROPHEROGRAM OVERLAY OF TWO KGP DIGESTIONS WITH
IDENTICAL PARAMETER APART FROM DIFFERENT GINGISKHAN® BATCHES. ......................... 43 FIGURE 56: SEC CHROMATOGRAM OVERLAY OF THE FRAGMENT ISOLATION AFTER KGP
DIGESTION, PROTEIN L AC AND BUFFER EXCHANGE IN FORMULATION BUFFER. ................... 43 FIGURE 57: DIGESTION PROGRESS OF IGDE BY CE-SDS NR. .................................................. 44 FIGURE 58: ELUTION PROFILE OF PROTEIN L AC AT PH 3.0, 2.5 AND 2.0. ................................. 45 FIGURE 59: CE-SDS NR ELECTROPHEROGRAM OF THE IDES DIGESTION AND ISOLATED
PRODUCTS. .................................................................................................................... 45 FIGURE 60: CE-SDS NR ELECTROPHEROGRAM OVERLAY OF THE THREE FRAGIT™ DIGESTION. 46 FIGURE 61: CE-SDS RED. ELECTROPHEROGRAM OF THE ENDOS2 DIGESTION AND BUFFER
EXCHANGE. .................................................................................................................... 47 FIGURE 62: CEX CHROMATOGRAM OF THE DIGESTION BUFFER STABILITY AT 37°C. ................... VII FIGURE 63: DIGESTION PROGRESS OF THE DIGESTION ACCORDING TO GENOVIS® STANDARD
PROTOCOL. .................................................................................................................... VII FIGURE 64: KINETIC MODEL PLOT LN([A]/[A]0) IN DEPENDENCY OF TIME TO JUSTIFY A FIRST ORDER
KINETIC MODEL. ............................................................................................................. VIII FIGURE 65: CE-SDS NR ELECTROPHEROGRAM OF THE PROTEIN L AC FLOW THROUGH AND
ELUTION. ....................................................................................................................... VIII FIGURE 66: CE-SDS NR ELECTROPHEROGRAM OF THE IDES DIGESTION AND PROTEIN L AC. .... IX FIGURE 67: CE-SDS RED ELECTROPHEROGRAM OF THE DIGESTION BY IMMOBILIZED ENDOS2 AND
BUFFER EXCAHNGED SAMPLE. .......................................................................................... IX
7.2 List of tables
TABLE 1: ABBREVIATIONS. ...................................................................................................... VII TABLE 2: LIST OF NEW ICH QUALITY GUIDELINES. ...................................................................... 1 TABLE 3: LIST OF USED BUFFER. ............................................................................................ 17 TABLE 4: APPLIED CEX PARAMETER. ..................................................................................... 18 TABLE 5: APPLIED SEC PARAMETER FOR FRACTION COLLECTION. ............................................ 19 TABLE 6: LIST OF CE-SDS NR PARAMETER. ........................................................................... 20
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TABLE 7: LIST OF CE-SDS RED PARAMETER. .......................................................................... 21 TABLE 8: DILUTION TABLE OF THE CYSTEINE INFLUENCING DIGESTION. ..................................... 24 TABLE 9: ELUTION OF FC ISOLATION. ...................................................................................... 25 TABLE 10: SEC FRACTIONATION RANGE. ................................................................................ 25 TABLE 11: ELUTION OF PROTEIN L AC. ................................................................................... 26 TABLE 12: ELUTION OF PROTEIN L AC FOR REPRODUCIBILITY. ................................................. 26 TABLE 13: ELUTION OF PROTEIN L AC AFTER IDES DIGESTION................................................. 28 TABLE 14: PROTEIN L AC PURIFICATION TABLE. ...................................................................... 28 TABLE 15: SUMMARY OF ISOLATED FRAGMENTS DURING SEC TRIALS. ...................................... 40 TABLE 16: SUMMARY OF ISOLATED FRAGMENTS DURING SEC INJECTION BUFFER EVALUATION. . 41 TABLE 17: SUMMARY OF ISOLATED FRAGMENTS DURING SEC FRACTION COLLECTION. .............. 44 TABLE 18: SUMMARY OF ISOLATED FRAGMENTS FOR FRAGIT™ KIT DIGESTION AND FRAGMENT
ISOLATION. ..................................................................................................................... 46 TABLE 19: MOST IMPORTANT EFFECTOR FUNCTIONS OF IGG. ..................................................... II TABLE 20: LIST OF IG FRAGMENTING ENZYMES. ........................................................................ III TABLE 21: LIST OF IG DEGLYCOSYLATING ENZYMES. ................................................................. III TABLE 22: LIST OF USED INSTRUMENTS. .................................................................................. IV TABLE 23: LIST OF USED CONSUMABLES. ................................................................................. IV TABLE 24: LIST OF USED REAGENTS. ........................................................................................ V TABLE 25: LIST OF USED SOFTWARE. ....................................................................................... VI
7.3 List of equations
EQUATION 1: DETERMINATION OF THE KINETIC CONSTANT K1 [66]. ............................................ 15 EQUATION 2: CALCULATION OF THE INTERMEDIATE PRODUCT CONCENTRATION [I]. .................... 15 EQUATION 3: CALCULATION OF THE PRODUCT CONCENTRATION [P]. ......................................... 15 EQUATION 4: CALCULATION OF THE TIME POINT OF THE MAXIMAL INTERMEDIATE CONCENTRATION
IS REACHED. ................................................................................................................... 16
Specific enzymatic digestion and fragment isolation for the critical quality attribute assessment of IgG1
Annex
Novartis 20.10.2017
I
Table of content
I. MECHANISM OF ACTION ............................................................................................. II
II. ENZYMES IN MAB ANALYTICS ................................................................................... III
I.1. FRAGMENTATION ....................................................................................................... III I.2. DEGLYCOSYLATION ................................................................................................... III
III. INSTRUMENTS .......................................................................................................... IV
IV. CONSUMABLES ........................................................................................................ IV
V. REAGENTS ................................................................................................................. V
VI. ENZYMES .................................................................................................................. VI
VII. SOFTWARE ............................................................................................................... VI
VIII. CHROMATOGRAM AND ELECTROPHEROGRAM ................................................. VII
I.3. FAB, FC AND LHF ..................................................................................................... VII I.4. F(AB’)2 ...................................................................................................................... IX I.5. CHITOBIOSE CLEAVAGE ............................................................................................. IX
IX. COPY OF THE OBJECTIVE SETTING AGREEMENT ................................................ X
X. STATEMENT OF AUTHORSHIP .............................................................................. XII
XI. REPORT IN DIGITAL FORMAT ............................................................................... XIII
XII. POSTER ................................................................................................................... XIV
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II
I. Mechanism of action
Table 19: Most important effector functions of IgG.
Function Mechanism
Complement activation After epitope binding on a cell an IgG undergoes a conformational change. Two bound Igs next to each other allow for binding of C1q, a protein complex, at the CH2 (Glu318, Lys320, Lys322). Subsequently, the complement cascade is initiated, which leads to pore formation and finally the destruction of the cell [25,73].
Ab-dependent cellular cytotoxicity (ADCC)
Cell bound IgGs allow natural killer-cells to bind the Fc region with a Fc receptor (FcR), the FcRs are cross-linked and initiate the release of granzymes and leading to apoptosis of the cell [25,74].
Opsonization Igs bound to bacteria present their Fc part to phagocytes and simplify the internalization [25].
Blocking Ig binding to a receptor may prevent the access for ligands and their physiological function [25]. A special form of blocking is the binding of an Ig to the CDR of another Ig, so called anti idiotypic Ab. This leads to the elimination of this respective Ab [25].
Masking Binding of non-cytotoxic Ig may prevent cytotoxic T-cells resp. Igs to recognize and bind an antigen [25].
Neutralization Small molecular toxins might act as an antigen and through binding by an Ig the binding to a receptor and the toxic effect is prevented [25].
Receptor binding Ab binding to a receptor can have three possible effects:
No reaction The receptor is not effected by the Ab [25].
Agonistic Ab The Ig binding itself imitates a ligand binding and triggers a response. This effect is only relevant for auto-immune disease [25].
Antagonistic Ab The Ig blocks the access of the receptor for ligands and prevents a physiological reaction [25].
Inhibition Some specific soluble immune complexes have the ability to suppress the activation of B-cells. This is important to reduce the antigen specific B-cell response [25].
Penetration Igs are able to cross polarized epithelial cells through cell-mediated transcytosis. For IgG the neonatal Fc receptor (FcRn) is of importance and even allows passing of the placenta [25,75].
Precipitation Binding of polyclonal Ab on multiple epitopes first forms soluble complexes and leads later to precipitation [25].
Agglutination Linking multiple particular antigens, e.g. on the surface of cells, leads to clump [25].
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II. Enzymes in mab analytics
I.1. Fragmentation
Table 20: List of Ig fragmenting enzymes.
Application Name Trade name Information
Fab + Fc Papain Papain Cleaves hydrophilic-R/K ↓ not V [76]. Over-digestion possible.
Lys-C Lys-C Cleaves C-terminal of K. Over-digestion possible. [77]
SpeB FabULOUS™ Cleaves human IgG1: KTHT ↓ CPPCPAPEL under reducing conditions. [78]
Kgp GingisKHAN® Cleaves human IgG1: KSCDK ↓ THTCPPCP under mild reducing conditions.
IgdE FabALACTICA™ Cleaves IgG1: KSCDKT ↓ HTCPPCP [23]
F(ab’)2 Pepsin Pepsin Cleaves the peptide bond of hydrophobic and aromatic amino acids [79]. Risk of oxidation.
IdeS FabRICATOR® Cleaves human IgG1, 3 and 4: CPAPELLG ↓ GPSVF and human IgG2: CPAPPVA ↓ GPSVF. [21]
Peptide mapping Trypsin Trypsin Cleaves C-terminal of K/R-not P [80].
Rgp GingisREX™ Cleaves C-terminal of R [81].
Removal of C-terminal K
Carboxypeptidase B
Carboxypeptidase B
Cleaves K and R at the C-terminus [80].
I.2. Deglycosylation
Table 21: List of Ig deglycosylating enzymes.
Cleavage site Name Trade name Information
-GlcNAc | Asn- PNGase F PNGase F Complex, high-mannose and hybrid type oligosaccharides of N-linked glycoproteins [80].
-GlcNAc | GlcNAc- EndoS IgGZERO® Only complex oligosaccharides of IgG and the α1-acid glycoprotein [82].
-GlcNAc | GlcNAc- EndoS2 GlycINATOR® Complex, high-mannose and hybrid type oligosaccharides of IgG and the α1-acid glycoprotein [82].
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Removal of α2,3-, α2,6- and α2,8-linked sialic acids
Two sialidase of Akkermansia muciniphila
SialEXO™ Cleaves N- and O-glycans[82].
O-glycosylated proteins of core 1 and core 3
endo-α-N-acetylgalactos-aminidase
OglyZOR™ Required prior sialic aids removal [82].
O-glycosylated proteins N-terminally of the S/T glycosylation site
Endoprotease of Akkermansia muciniphila
OpeRATOR™ Improved enzyme activity with removed sialic aids [82].
III. Instruments
Table 22: List of used instruments.
Type Model Manufacturer
Centrifuge 0.5 mL tubes 1-14 SIGMA
Centrifuge 4 & 15 mL tubes 3-18KS SIGMA
CE-SDS NR/Red. PA800plus Pharmaceutical Analysis System
BECKMAN COULTER
HPLC (CEX and SEC) 1100 Series Agilent Technology
HPLC fraction collector 1260 Infinity Agilent Technology
Thermo shaker Thermomixer comfort EPPENDORF
Tube rotator plate Tube rotator BOEKEL
Water bath Eco Silver Lauda
Nanodrop™ NanoDrop1000 Spectrophotometer
Thermo Scientific™
IV. Consumables
Table 23: List of used consumables.
Description Manufacturer Item number
Amicon® Ultra – 0.5mL Centrifugal Filters Ultracel® - 10K
Merk UFC501096
Amicon ® Ultra – 4 Centrifugal Filters Ultracel® - 10K
Merk UFC801024
Amicon ® Ultra – 15 Centrifugal Filters Ultracel® - 10K
Merk UFC901096
Stericup® 150ml Millipore® Express PLUS
Millipore SCGPU01RE
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0.22µm PES
Protein L affinity chromatography HiScreen™ Capto™ L HiTrap™ Protein L, 1 mL
GE Healthcare 29048665
Crimp vials 6 mL + 20 mm crimp caps with septum
Agilent Technology 9301-1419 + 9301-1425
V. Reagents
Table 24: List of used reagents.
Name Supplier Part number Purity / Conc. MW in g/mol
2-mercaptoethanol
SIGMA-ALDRICH 63689 ≥ 99.0% 78.13
cOmplete™ protease inhibitor cocktail
Roche 11 697 498 001 n.a. n.a.
Cysteine SIGMA-ALDRICH 30089 98.5% 121.16
Hydrochloric acid Merk 1.09060 1 M 36.46
Iodoacetamide SIGMA-ALDRICH I1149 ≥ 99% 184.96
L-Histidine SIGMA-ALDRICH H8000 ≥ 99% 155.15
L-Histidine hydrochloride monohydride
SIGMA-ALDRICH H8125 ≥ 98% 209.63
ortho-phosphoric acid
Merk 1.00552 85% 97.99
Potassium chloride
SIGMA-ALDRICH P9541 ≥ 99.0% 74.55
SDS-MW Gel buffer
BECKMAN COULTER
A30341 n.a. n.a.
Sodium chloride Honeywell | Fluka 71380 ≥ 99.5% 58.44
Sodium hydroxide solution
Merk 1.09137 1 M 40.00
Sodium phosphate dibasic anhydrous
SIGMA-ALDRICH S7907 ≥ 99.0% 141.96
Sodium phosphate monobasic monohydrate
SIGMA-ALDRICH 71507 ≥ 99.5 137.99
Trifluoroacetic acid
Thermo Scientific 28903 Sequencing grade 114.02
Tris-HCl Promega H5123 ≥ 99.0% 157.56
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Trisodium citrate dihydrate
SIGMA-ALDRICH S1804 100% 294.10
Trizma® base SIGMA-ALDRICH T1503 99.9% 121.14
UltraPure™ Tris-HCl pH 8.0
Gibco 15568025 1 M 157.60
VI. Enzymes
Name Supplier Part number Amount in units MW in kDa
FabALACTICA™ Genovis A0-AG1-020 2000 70
FabRICATOR® Genovis A0-FR1-020 2000 38
FragIT™ kit midispin
Genovis A0-FR6-100 n.a. 38
GingisKHAN® Genovis B0-GKH-020 2000 50
GlyCINATOR® Genovis A0-GL1-020 2000 92
Immobilized GlycINATOR® MidiSpin
Genovis A0-GL6-100 n.a. 92
VII. Software
Table 25: List of used software.
Name Version Developer
ChemDraw Ultra 14.0 PerkinElmer
Chromeleon 6.8 Thermo Scientific™
GPMAW 10.0 Lighthouse data
Open Lab CDS Chemstation Edition for LC & LC/MS Systems
C.01.05 [41] Agilent Technologies
PyMOL Molecular Graphics System
1.5.0.5 SCHRÖDINGER
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VIII. Chromatogram and electropherogram
I.3. Fab, Fc and LHF
Stability evaluation in digestion buffer with and without reducing solution
Figure 62: CEX chromatogram of the digestion buffer stability at 37°C.
Figure 63: Digestion progress of the digestion according to Genovis® standard protocol.
12 13 14 15 16 17 18 19 20 21 22
Absorb
ance in m
AU
Time in min.
CEX chromatogram - Digestion buffer stability at 37°C
T0@37°C T1h@37°C T2h@37°C T24h@37°C
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0 15 30 45 60 75 90 105 120
Purity
in A
%
Time in min
Genovis protocol digestion procedure
IgG
LHF
Fc
Fab
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Figure 64: Kinetic model plot ln([A]/[A]0) in dependency of time to justify a first order kinetic model.
Figure 65: CE-SDS NR electropherogram of the Protein L AC flow through and elution.
y = -0.0838x - 0.2684R² = 0.9683
-5.5
-4.5
-3.5
-2.5
-1.5
-0.5
0.5
0 10 20 30 40 50 60
ln([
A]/[A
] 0
Time in min.
Kinetic plot: ln([A]/[A]0) VS. time
ln([A]/[A]0)
Linear (ln([A]/[A]0))
9 10 11 12 13 14 15 16 17
Absorb
ance in m
AU
Time in min.
CE-SDS NR: Pooled and conentrated Protein L AC fractions
Protein L Wash Protein L elution
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I.4. F(ab’)2
Figure 66: CE-SDS NR electropherogram of the IdeS digestion and Protein L AC.
I.5. Chitobiose cleavage
Figure 67: CE-SDS red electropherogram of the digestion by immobilized EndoS2 and buffer excahnged sample.
7 8 9 10 11 12 13 14 15 16 17
Absorb
ance in m
AU
Time in min.
CE-SDS NR electropherogram of the Protein L affinity chromatography and F(ab')2 isolation
Reference 1h digestion Flow through
Wash Elute pH 2.5 Elute pH 2.0
15 20 25 30 35 40 45 50
Absorb
ance in m
AU
Time in min.
CE-SDS red. electropherogram of the immob. EndoS2 chitobiose cleavage and buffer exchange
Reference EndoS2 digestion Buffer exchange
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IX. Copy of the objective setting agreement
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X. Statement of authorship
I certify hereby, that I have written this report autonomous, without outside assistance and I
have used only allowed resources. I affirm particularly, that I have mentioned all literal and
paraphrase transfers as such and assigned the original author and document.
Any violation will lead to actions according to §39 and §40 of the General Academic
Regulations for Bachelor’s and Master’s degree programs at the Zurich University of Applied
Sciences of 29.01.2008.
Location Date Signature
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XI. Report in digital format
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XII. Poster