Optimizing Signal Peptides for Expression of Recombinant Antibodies in HEK293 Cells

Link¨opingUniversity | Department of Physics, Chemistry and Biology Master’s Thesis, 30 hp | M.Sc. in Chemical Biology: Protein Science and Technology Spring term 2020 | LITH-IFM-A-EX−20/3776−SE

Optimizing signal peptides for expression of recombinant antibodies in HEK293 cells

Gustav Myhrinder Science for Life Laboratory

Examiner and internal supervisor: Lars-G¨oranM˚artensson External supervisors: Leif Dahllund & Anders Olsson

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2020-06-11 Department of Physics, Chemistry and Biology Linköping University

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Svenska/Swedish Licentiatavhandling ISRN: LITH-IFM-A-EX--20/3776--SE Engelska/English Examensarbete ______C-uppsats D-uppsats Serietitel och serienummer ISSN ______Övrig rapport Title of series, numbering ______

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Titel Title

Optimizing signal peptides for expression of recombinant antibodies in HEK293 cells

Författare Author

Gustav Myhrinder

Sammanfattning Abstract Monoclonal antibodies are well-established as a therapeutic in the biopharmaceutical market, targeting a variety of diseases and with 79 approved products by the United States Food and Drug Administration in December 2019. Therapeutic monoclonal antibodies are commonly produced as recombinant proteins in mammalian cell lines, due to their capacity of post-translational modifications, most notably glycosylation. Furthermore, an identified bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the endoplasmic reticulum. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion. Several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems. The aim of this study was to evaluate the expression of three human recombinant antibodies in Human Embryonic Kidney 293 (HEK293) cells by evaluating 16 different signal peptide combinations, consisting of eight heavy chain (HC) and two light chain (LC) signal peptides. The impact goal was an efficient secretion of recombinant antibodies, and thus lower production cost of recombinant antibodies in HEK293 cells. First, 16 HC and LC signal peptide plasmid constructs were generated for each of the three recombinant antibodies. Thereafter, transient gene expression in HEK293 cells were performed at three independent experiments. Finally, the antibody titers were quantified using Biacore concentration analysis. The produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, the evaluated HC and LC signal peptide combinations resulted in 3 times higher and 2 times higher antibody titers compared to the original signal peptides used by the Drug Discovery and Development platform at Science for Life Laboratory, for two of the studied antibodies respectively. The results presented in this report further demonstrates the necessity to evaluate signal peptides in order to achieve increased expression of recombinant antibodies in mammalian systems.

Nyckelord Keyword

Signal peptide, antibody, human immunoglobulin G, mammalian cell, HEK293, protein expression, secretory pathway, transient transfection, Biacore, Biacore concentration analysis, SDS-PAGE, kinetic screening Abstract

Monoclonal antibodies are well-established as a therapeutic in the biopharmaceutical market, targeting a variety of diseases and with 79 approved products by the United States Food and Drug Administration in December 2019. Therapeutic monoclonal antibodies are commonly produced as recombinant proteins in mammalian cell lines, due to their capacity of post-translational modiﬁcations, most notably glycosylation. Furthermore, an identiﬁed bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the endoplasmic reticulum. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion. Several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems.

The aim of this study was to evaluate the expression of three human recombinant antibodies in Human Embryonic Kidney 293 (HEK293) cells by evaluating 16 different signal peptide combinations, consisting of eight heavy chain (HC) and two light chain (LC) signal peptides. The impact goal was an efficient secretion of recombinant antibodies, and thus lower production cost of recombinant antibodies in HEK293 cells. First, 16 HC and LC signal peptide plasmid constructs were generated for each of the three recombinant antibodies. Thereafter, transient gene expression in HEK293 cells were performed at three independent experiments. Finally, the antibody titers were quantified using Biacore concentration analysis.

The produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, the evaluated HC and LC signal peptide combinations resulted in 3 times higher and 2 times higher antibody titers compared to the reference HC and LC signal peptides used by the Drug Discovery and Development platform at Science for Life Laboratory, for two of the studied antibodies respectively. The results presented in this report further demonstrates the necessity to evaluate signal peptides in order to achieve increased expression of recombinant antibodies in mammalian systems.

III Acronyms and Abbreviations

Ab Antibody ANOVA Analysis of variance CHO cells Chinese Hamster Ovary cells CH Constant heavy chain domain CL Constant light chain domain

dH2O Sterile water DNA Deoxyribonucleic acid ER Endoplasmic reticulum E. coli Escherichia coli Fab region Fragment antigen-binding region Fc region Fragment crystallizable region FDA United States Food and Drug Administration GFP Green ﬂuorescent protein HC Heavy chain HEK293 cells Human Embryonic Kidney 293 cells Ig Immunoglobulin IgG Immunoglobulin G IgG1 Immunoglobulin G subclass 1 LC Light chain mAb Monoclonal antibody mRNA Messenger RNA PCR Polymerase chain reaction PTM Post-translational modiﬁcation RNA Ribonucleic acid RNC Ribosome-nascent chain complex rpm Revolutions per minute SciLifeLab Science for Life Laboratory SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM Standard error of the mean SP Signal peptide

SPHC Heavy chain signal peptides SPLC Light chain signal peptides SPase Signal peptidase SRP Signal recognition particle SR Signal recognition particle receptor SPR Surface plasmon resonance

Tm Melting temperature TGE Transient gene expression VH Heavy chain variable domain VL Light chain variable domain

IV Table of Content

Abstract III

Acronyms and Abbreviations IV

1 Introduction 1 1.1 Background ...... 1 1.2 Aim ...... 1 1.3 Motivation ...... 1 1.4 Work plan ...... 2 1.5 Limitations ...... 2

2 Theory 5 2.1 Scientiﬁc background ...... 5 2.1.1 Antibody function and structure ...... 5 2.1.2 Secretory pathway ...... 6 2.1.3 Signal peptides ...... 7 2.1.4 Surface plasmon resonance ...... 8 2.1.5 Biacore biosensors ...... 9 2.2 Methodology ...... 11 2.2.1 In-Fusion cloning ...... 11 2.2.2 Cloning approach ...... 11 2.2.3 Colony PCR screening ...... 13 2.2.4 Restriction digest screening ...... 13 2.2.5 Transient transfection ...... 13 2.2.6 Transfection eﬃciency ...... 13 2.2.7 Biacore concentration analysis ...... 14 2.2.8 Biacore kinetic screening ...... 14

3 Methods 15 3.1 Preparation of variable domains ...... 15 3.1.1 PCR amplification of variable domains ...... 15 3.1.2 Purification of LC variable domains ...... 15 3.1.3 Purification of HC variable domains ...... 15 3.2 Preparation of signal peptides ...... 16 3.3 Linearization of plasmid backbone ...... 16 3.4 Construction of LC plasmid constructs ...... 17 3.4.1 In-Fusion cloning of LC variable domains ...... 17 3.4.2 Plasmid transformation into competent E. coli ...... 17 3.4.3 Colony PCR screening of LC plasmid constructs ...... 17 3.4.4 Minipreparations of LC plasmid constructs ...... 18 3.4.5 Restriction digest screening of LC plasmid constructs ...... 18 3.4.6 Sequencing of LC plasmid constructs ...... 18 3.5 Linearization of LC plasmid constructs ...... 19 3.6 Construction of HC and LC plasmid constructs ...... 19 3.6.1 In-Fusion cloning of HC variable domains ...... 19 3.6.2 Plasmid transformation into competent E. coli ...... 19 3.6.3 Colony PCR screening of HC and LC plasmid constructs ...... 19

V 3.6.4 Minipreparations of HC and LC plasmid constructs ...... 20 3.6.5 Restriction digest screening of HC and LC plasmid constructs . . . 20 3.6.6 Sequencing of HC and LC plasmid constructs ...... 21 3.7 Sterile ﬁltration of plasmid constructs ...... 21 3.8 Maintenance of HEK293 cells ...... 21 3.9 Transient gene expression of recombinant antibodies ...... 21 3.9.1 Transfection of plasmid constructs into HEK293 cells ...... 21 3.9.2 Measurement of transfection eﬃciency ...... 22 3.9.3 Harvest of expressed recombinant antibodies ...... 22 3.10 Biacore concentration analysis of recombinant antibodies ...... 23 3.11 Statistics ...... 23 3.12 SDS-PAGE analysis of recombinant antibodies ...... 23 3.13 Kinetic screening of recombinant antibodies ...... 24

4 Results 26 4.1 Cloning of HC and LC plasmid constructs ...... 26 4.2 Evaluation of HC and LC signal peptides for recombinant antibody expression in HEK293 cells ...... 26 4.3 Comparisons between the evaluated HC and LC signal peptides and the reference signal peptides ...... 30 4.4 Control of full-length recombinant antibodies using SDS-PAGE ...... 31 4.5 Kinetic activity of recombinant antibodies ...... 33 4.6 Control of HEK293 cell maintenance culture ...... 34 4.7 Control of transfection eﬃciency ...... 34 4.8 Sequence alignment of HC signal peptides ...... 35 4.9 Sequence alignment of LC signal peptides ...... 36 4.10 Sequence alignment of the evaluated HC and LC signal peptides and the reference signal peptides ...... 37 4.11 Process analysis ...... 38

5 Discussion 39

6 Conclusions 42

Acknowledgements 43

References 44

Appendix A. Materials 47

Appendix B. Cloning of plasmid constructs 51 B.1 Preparation of cloning fragments ...... 51 B.2 LC plasmid constructs ...... 52 B.3 HC and LC plasmid constructs ...... 53

Appendix C. Biacore concentration analysis of recombinant antibodies 54

VI 1. Introduction

1 Introduction

1.1 Background Therapeutic monoclonal antibodies are well-established in the biopharmaceutical market with 79 approved products by the United States Food and Drug Administration (FDA) in December 2019 [1]. Interestingly, there is a variety of diseases that can be targeted with monoclonal antibodies (mAb): acute myeloid leukemia, multiple myeloma, multiple sclerosis, rheumatoid arthritis and severe asthma, just to mention a few [2][3]. In 2018, the global therapeutic monoclonal antibody market was valued at approximately US$ 115.2 billion and the market is still expanding [1]. Thus, the future for therapeutic antibodies shines bright with new monoclonal antibodies that are being commercialized and approved as a therapeutic every year [3]. The major production system for a monoclonal antibody is the expression as a recombinant protein in a mammalian cell line [4], including Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney 293 (HEK293) cells [5]. Mammalian expression systems are usually preferred for production of biopharmaceuticals due to their capacity for correct folding, protein secretion and post-translational modifications (PTMs), most notably glycosylation [6][7]. Nevertheless, mammalian cell cultures have a number of in- trinsic disadvantages causing their cultivation to be time-consuming and cost-intensive [8]. Therefore, research and development is crucial for improved production cell lines and cell culture processes [9]. Optimization work is an instrumental part in process development activities and different factors regarding the expression of recombinant antibodies can be studied to increase the product titer. An identified bottleneck within the secretory pathway of recombinant proteins is the translocation of secretory proteins from the cytosol into the lumen of the endoplasmic reticulum (ER) [10]. Moreover, in the process of protein secretion a central role is played by the signal peptide, which is located at the N-terminal of nascent proteins [11]. Previous studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems [10][12][13]. Furthermore, a previous study analyzed all naturally occurring human antibody signal peptides and clustered them based on sequence similarity. Based on clustering, eight HC signal peptides and two LC signal peptides were generated [10].

1.2 Aim The aim of this project was to evaluate the expression of three recombinant antibodies in HEK293 cells by evaluating different combinations of eight HC and two LC signal peptides. The evaluation of signal peptides were performed using three human immunoglobulin G subclass 1 (IgG1) recombinant antibodies with different variable domains, aimed for two different therapeutic areas: infectious disease, oncology and one control. The impact goal was an efficient secretion of recombinant antibodies, and thus an increased expression and lower production cost of recombinant antibodies in HEK293 cells.

1.3 Motivation This master’s thesis project was performed at Science for Life Laboratory (SciLifeLab) in Stockholm, which is a national center for molecular life science research in Sweden. The

1 1. Introduction evaluation of signal peptides for the expression of recombinant antibodies in HEK293 cells is of great interest for SciLifeLab, especially for their Protein Expression and Char- acterization facility. The three human recombinant antibodies that were evaluated with diﬀerent signal peptides were provided by the Drug Discovery and Development platform at SciLifeLab.

1.4 Work plan The project was divided into several objectives:

• Preparation of cloning fragments: signal peptides, the human IgG1-backbone plasmid and the variable domains for each of the three recombinant antibodies.

• Construction of LC plasmid constructs with diﬀerent signal peptides. Transforma- tion of plasmid constructs into Escherichia coli (E. coli) and thereafter plasmid preparation, puriﬁcation and sequencing for quality control.

• Selection of successful LC plasmid constructs for further cloning with the HC variable domains and signal peptides.

• Construction of HC and LC plasmid constructs with diﬀerent combinations of signal peptides. Transformation of plasmid constructs into E. coli and thereafter plasmid preparation, puriﬁcation and sequencing for quality control.

• Selection of successful HC and LC plasmid constructs for transient transfection into HEK293 cells.

• Transient gene expression (TGE) of the three recombinant antibodies with 16 different HC and LC signal peptide combinations in HEK293 cells.

• Quantiﬁcation of expressed recombinant antibodies using Biacore concentration analysis.

• Analyzation of expressed recombinant antibody titers with signal peptides under investigation.

The objectives were divided into diﬀerent activities and milestones. The milestones of the project are shown in Table 1. The diﬀerent activities with their estimated time, actual consumed time as well as the milestones of the project are illustrated in a Gantt chart in Figure 1.

1.5 Limitations Some materials, methods and results are not presented in full detail due to conﬁdential- ity commitment to the Drug Discovery and Development platform at Science for Life Laboratory.

2 1. Introduction

Table 1: Milestones for the master’s thesis project ”Optimizing signal peptides for expression of recombinant antibodies in HEK293 cells”.

Milestone Description Calendar Week M1 Planning report finished 6 M2 LC plasmid constructs are finished 8 M3 HC and LC plasmid constructs are finished 12 M4 Half-time presentation 14 M5 First quantification of expressed antibodies 15 M6 Finished quantification of expressed antibodies 19 M7 Final report draft submitted 21 M8 Project presentation and review 23 M9 Corrected final report 25

3 1. Introduction ”Optimizing signal peptides for . Gantt chart with actual consumed time, planned time and milestones for the master’s thesis project Figure 1: expression of recombinant antibodies in HEK293 cells”

4 2. Theory

2 Theory

2.1 Scientiﬁc background 2.1.1 Antibody function and structure An antibody, also referred to as an immunoglobulin (Ig), is a protein that recognize foreign material (e.g. pathogenic bacteria and viruses) through a unique molecular structure, called an antigen. Antibodies are part of the immune system and can be membrane- bound, functioning as an antigen receptor at the surface of B cells, or secreted in the blood plasma by plasma cells. The antibody recognize and bind to the antigen through its fragment antigen-binding (Fab) region. Thus, the antibody can tag the antigen and stimulate an immune response mediated via its fragment crystallizable (Fc) region, which interacts with receptors at the cell surface. Moreover, an antibody can also directly neutralize an antigen by inhibiting an important structure (e.g. blocking an active site at the pathogen). However, there are a variety of antibodies, specialized in various biological activities and with diﬀerent structural properties [14].

Immunoglobulin G (IgG) is the most common isotype of antibodies in the blood and extracellular ﬂuid and consists of two identical light chains (LCs) and two identical heavy chains (HCs) [14]. The LC is divided into the variable domain (VL) and the constant domain (CL). The HC is divided into the variable domain (VH), the constant domains (CH1-CH3) and a hinge region between the CH1 and CH2. Furthermore, the diﬀerent chains are linked together with disulphide bridges. Moreover, the IgG molecule consists of two Fab regions and one Fc region. The Fc region comprise of the paired CH2 and CH3 domains and the Fab region consists of one variable and one constant domain from both the HC and LC [15]. A schematic structure of an IgG antibody can be seen in Figure 2.

Fab region

CH2 CH2

Fc region

CH3 CH3

Figure 2: Schematic structure of an immunoglobulin G (IgG) antibody. The light chain (LC) (colored in blue), the heavy chain (HC) (colored in yellow), the hinge region (colored in black) and the disulphide bridges (colored in red). The LC is divided into the variable domain (VL) and the constant domain (CL). The HC is divided into the variable domain (VH) and the constant domains (CH1-CH3). The Fab region is composed of one variable and one constant domain from both the HC and LC. The Fc region consists of the paired CH2 and CH3 domains. Hence, an antibody consists of two Fab regions and one Fc region.

5 2. Theory

2.1.2 Secretory pathway Recombinant antibodies are secreted from the mammalian host cell via the co-translational translocation pathway. First, each chain of the recombinant antibody is translated on the ribosome with a signal peptide at the N-terminal. The signal peptide is recognized by the signal recognition particle (SRP) while the protein still is being synthesized on the ribosome. The SRP transports the ribosome-nascent chain complex (RNC), consisting of the ribosome and the growing protein, to the endoplasmic reticulum (ER) membrane. Thereafter, at the ER membrane, SRP binds to the SRP receptor (SR) and delivers the RNC to the ER membrane-bound translocon which transports the synthesized protein into the ER lumen. Then, the signal peptide is cleaved oﬀ by a recruited signal peptidase (SPase) and the newly synthesized protein is released into the ER lumen [16]. The co-translational translocation pathway is illustrated in Figure 3.

1. Ribosome 2. mRNA RNC Translated protein

Signal peptide Cytosol

SRP SRP receptor ER lumen Translocon 3. 4.

Cytosol Cytosol

SRP receptor ER lumen ER lumen Translocon SPase

Figure 3: The co-translational translocation pathway. 1. The protein is translated on the ribosome with a signal peptide at the N-terminal, which is detected by the signal recognition particle (SRP). 2. SRP binds to the signal peptide and transports the ribosome-nascent chain complex (RNC), containing the ribosome and the growing protein, to the endoplasmic reticulum (ER) membrane. 3. SRP binds to the SRP receptor and the RNC is transferred to the membrane-bound translocon, which transports the translated protein into the ER lumen. 4. The signal peptide is cleaved oﬀ by a recruited signal peptidase (SPase) and the synthesized protein is released from the ribosome into the ER lumen.

In the rough ER, the newly synthesized proteins are incorporated into transport vesicles. The transport vesicles fuse together with the cis-Golgi vesicle and release the synthesized proteins into the Golgi lumen. Then, the cis-Golgi vesicle moves from the cis- position (nearest the ER) to the trans-position (farthest from the ER), in a process called cisternal migration. When the Golgi vesicle reaches the trans-position the secretory pro-

6 2. Theory teins are moved into the trans-Golgi reticulum. The secretory proteins are incorporated into transport vesicles which move towards the plasma membrane and release the secretory proteins by exocytosis [17].

2.1.3 Signal peptides Signal peptides are located at the N-terminal of nascent proteins and are on average 15−30 amino acids in length for eukaryotes. Comparisons of signal peptide sequences indicated that they typically comprise of three distinct domains: a positively charged amino-terminal region (n-region), a central hydrophobic region (h-region) and a polar carboxy-terminal region (c-region). Apart from this three-domain structure with a ”positive-hydrophobic-polar” design, the signal peptide sequence is highly variable [11]. In order to address the diﬀerent residue positions in the signal peptide, the last position is referred to as −1, the second last position as −2 and so on. The ﬁrst residue position in the mature protein is referred to as +1. A schematic structure of a signal peptide can be seen in Figure 4.

The positively charged n-region is responsible for interactions with the phosphate backbone of the SRP and the phosphate group of lipid bilayers, which is crucial for an efficient translocation [18]. The n-region varies strongly with the overall length of the signal peptide, but usually consists of five residues [19]. Furthermore, the hydrophobic h-region determines the conformation of the signal peptide and is essential in protein pro- cessing and translocation [18]. Interestingly, previous studies suggested that hydrophobic h-regions stabilize the interactions between the signal peptide and SRP [20][21]. The h-region has some variability in length, though the most important hydrophobic residues consist of position −6 to −13 in eukaryotes [11]. Further, the c-region is independent of the total length of the signal peptide and usually comprise of residue −1 to −5 in eukaryotes [11]. The cleavage site for the SPase is located in the c-region, between residue −1 and +1. Interestingly, critical points in the c-region are located at position −1 and −3, well-known as the ”(−3, −1)-design” of signal peptides [22]. Furthermore, a previous study presented conserved residues at position −1 and −3 in the signal peptide sequence through a rigorous analysis of 1877 eukaryotic signal peptide sequences. The results showed that small residues are conserved at position −1; alanine (A) and glycine (G). Position −3 favoured small aliphatic residues; alanine (A) and valine (V). Moreover, serine (S), threonine (T) and cysteine (C) are also noticeable at position −1 and −3 [23]. The conserved ”(−3, −1)-design” fits within a pocket in the catalytic domain of the SPase and thereby defines the cleavage site between position −1 and +1 [18].

n-region h-region c-region mature protein

NH2 –

cleavage site

Figure 4: Schematic structure of a signal peptide with the three distinct domains: a positively charged n-region, a hydrophobic h-region and a polar c-region. The cleavage site between the signal peptide and the mature protein is also visualized.

Interestingly, previous studies have shown that the signal peptide play a key role in the

7 2. Theory efficiency of the co-translational translocation pathway, which have been identified as a crucial step in the secretory pathway. An optimal secretion path for a recombinant protein would increase the product yield in a mammalian host system [12]. Nonetheless, there is a variety of signal peptides with various sequences that show completely different impact on the protein secretion [10][12][13]. However, a previous study analyzed all naturally occurring human antibody signal peptides (172 HC signal peptides and 62 LC signal peptides) and clustered them based on their sequence similarity. This resulted in eight HC signal peptides (H1-H8) and two LC signal peptides (L1-L2) [10]. These eight HC and two LC signal peptides were used in this project for the evaluation of different combinations of HC and LC signal peptides. The amino acid sequence for the eight HC signal peptides (H1-H8) and two LC signal peptides (L1-L2) are shown in Table 2.

Table 2: The amino acid sequence for the eight heavy chain (HC) signal peptides (H1-H8) and the two light chain (LC) signal peptides (L1-L2) that were used in this study.

HC signal peptide Amino acid sequence H1 MELGLSWIFLLAILKGVQC H2 MELGLRWVFLVAILEGVQC H3 MKHLWFFLLLVAAPRWVLS H4 MDWTWRILFLVAAATGAHS H5 MDWTWRFLFVVAAATGVQS H6 MEFGLSWLFLVAILKGVQC H7 MEFGLSWVFLVALFRGVQC H8 MDLLHKNMKHLWFFLLLVAAPRWVLS LC signal peptide Amino acid sequence L1 MDMRVPAQLLGLLLLWLSGARC L2 MKYLLPTAAAGLLLLAAQPAMA

2.1.4 Surface plasmon resonance Surface plasmon resonance (SPR) is a phenomenon that occurs in thin conducting films, often a gold layer, at the interface of materials with different refractive indices. First, incident light passes through a prism causing an internal reflection at the metal-prism interface. As a result, an electromagnetic evanescent wave occurs in the metal that prop- agates along the metal-ambient interface. At a certain energy and angle of the incident light, surface plasmons in the metal layer are excited which can be seen as a drop in the intensity of the reflected light [24][25], which is illustrated in Figure 5.

8 2. Theory

Reflected light intensity

SPR angle Angle of reflection

Figure 5: The surface plasmon resonance (SPR) detection principle. A speciﬁc angle of the incident light (referred to as the SPR angle) excite surface plasmons in the metal layer which result in a drop in the intensity of the reﬂected light [25].

2.1.5 Biacore biosensors Biacore biosensor systems utilizes SPR to monitor molecular interactions in real-time and can be used to study specificity, kinetics, affinity and concentration analysis. Biacore systems measures the change in the refractive index, which is due to molecular interactions at the sensor surface. In Biacore systems, the conducting film is a gold layer and the materials with different refractive indices are a glass slide and the sample solution that flows through the flow cell. A schematic illustration of a Biacore system can be seen in Figure 6. The gold film is coated with a carboxymethylated dextran matrix to which ligand molecules are immobilized. In direct binding assays, analytes in the sample solution binds directly to the immobilized ligands, which alters the refractive index. Thus, the response is directly proportional to the analyte concentration at the sensor surface. The SPR signal is recorded in a sensorgram which plots response against time, and hence showing the progress of the interaction between the analyte and the ligand [25], which is illustrated in Figure 7.

9 2. Theory

Incident Reflected light light SPR angle

Glass slide Sensor chip Gold layer

Flow cell

Figure 6: Schematic illustration of a Biacore system. The conducting film is the gold layer and the media with different refractive indices are the glass slide of the sensor chip along with the sample solution that flows through the flow cell. The SPR angle refers to the angle of the incident light that result in a drop in the intensity of the reflected light [25].

Response Analyte signal (RU) Ligand

Dissociation

Baseline Association

Buffer Sample Buffer Regeneration Buffer Time

Figure 7: Schematic illustration of a sensorgram with the different phases and the detection of binding events in a direct binding assay. The bars below the sensorgram curve indicate the solution that flows over the sensor surface. First, buffer flows over the sensor surface with immobilized ligands, which results in a baseline response signal. Thereafter, as the analyte begins to bind to the immobilized ligand, the refractive index on the sensor surface change and the response signal increases. After sample injection, buffer flows over the sensor surface and the analyte starts to dissociate from the ligand, resulting in a decrease of the response signal. Then, regeneration solution removes bound analyte from the ligand at the sensor surface and the signal response returns back to baseline [25].

10 2. Theory

2.2 Methodology 2.2.1 In-Fusion cloning A key step in the expression of a recombinant protein is the construction of a vector containing the target protein. Traditionally, restriction enzymes and DNA ligase are used to manipulate the plasmid vector, which is quite time consuming and relies on unique sites for the restriction enzymes. In contrast, In-Fusion cloning uses a recombinant exonuclease that exposes a complementary sequence, containing 15 bases, at the vector and insert. The 15 bases homologous overlap fuses the vector and insert together. The design of primers to create these overlaps, at both ends, for the insert are therefore key for a successful In-Fusion cloning [26]. In-Fusion cloning can join multiple DNA fragments into a vector during a single reaction, as long as they share 15 bases of overlap at each end [27]. An illustration of an In-Fusion cloning can be seen in Figure 8.

1. 2. 3. Insert Insert Insert

Linearized plasmid Linearized plasmid Plasmid

Figure 8: In-Fusion cloning with two fragments: insert and plasmid. Before the In-Fusion cloning the plasmid is linearized with restriction enzymes. 1. Thereafter, the In-Fusion cloning starts by mixing the two fragments and a recombinant exonuclease. 2. The exonuclease exposes the 15 bases overlaps at each fragment. 3. The homologous overlaps fuses the two fragments together, resulting in a plasmid which contains the desired insert.

2.2.2 Cloning approach The plasmid constructs in this project were based on an existing vector provided by the facility that contained the constant domains (CL and CH) for a human IgG1 antibody along with VL and VH stuffer fragments. Also, all of the plasmid constructs in this project had the same promoter sequence and regulatory elements. Before cloning, the plasmid was linearized with restriction enzymes that cleaved the VL stuffer fragment. Thereafter, each VL was fused to the two LC signal peptides along with the CL in the plasmid by In-Fusion cloning. Three LC In-Fusion reactions were carried out, one for each antibody. The LC In-Fusion cloning resulted in two different constructs for each antibody: one with L1 as a signal peptide and one with L2. An illustration of the LC In-Fusion cloning can be seen in Figure 9. Thereafter, the fused LC plasmid constructs were transformed into competent E. coli cells for amplification. The colonies were screened for a successful cloning with colony PCR. After plasmid purification from successful colonies, restriction digest screening was used to determine which colonies that contained which LC signal peptide, L1 or L2. Then, the purified plasmid constructs were sequenced for quality control.

11 2. Theory

1. 2. 3. VL VL SPLC SPLC SPLC VL

Linearized plasmid Linearized plasmid LC plasmid

Figure 9: In-Fusion cloning with LC variable domain (VL), LC signal peptides (SPLC) and plasmid.2.1 First, the plasmid is linearized2.2 with restriction enzymes. 2.31. The In-Fusion cloning VH VH starts by mixing the three fragments and a recombinant exonuclease. 2. The exonuclease exposes SPHC SPHC the 15 bases overlap at the VL, SPLC and plasmid. 3. The homologousSP overlapsHC VH fuses the three fragments together, resulting in a LC plasmid construct.

After the LC In-Fusion cloning six different LC plasmid constructs were generated in SPLC VL SPLC VL SPLC VL total: one construct with L1 as a signal peptide and one construct with L2, for each of the LC + HC plasmid threeLinearized antibodies.LC These plasmid LC plasmidLinearized constructsLC were plasmid linearized with restriction enzymes that cleaved the VH stuffer fragment. Six HC In-Fusion reactions were carried out, one for each LC plasmid construct. In the HC In-Fusion cloning the VH was fused to the eight HC signal peptides along with the CH in the human IgG1-backbone plasmid. The HC In-Fusion cloning resulted in plasmid constructs that contained both the LC and the HC with their respective signal peptide, which is illustrated in Figure 10. These plasmid constructs were transformed into competent E. coli cells for amplification. Thereafter, the plasmids were purified and screened for a successful cloning with restriction digest 1. 2. 3. screening. Then,VL the plasmid constructs with a successful cloning were sequenced for VL qualitySP controlLC and most importantly to determine which colonies that contained which SPLC combination of the HC and LC signal peptides. SPLC VL

Finally, after a successful two-step In-Fusion cloning, 16 diﬀerent plasmid constructs were generated for each antibody: all combinations of the two LC signal peptides and the eight HC signal peptides. Hence, 48 diﬀerent plasmid constructs in total. Linearized plasmid Linearized plasmid LC plasmid

1. 2. 3. VH VH SPHC SPHC SPHC VH

SPLC VL SPLC VL SPLC VL Linearized LC plasmid Linearized LC plasmid HC and LC plasmid

Figure 10: In-Fusion cloning with HC variable domain (VH), HC signal peptides (SPHC) and LC plasmid construct. First, the LC plasmid is linearized with restriction enzymes. 1. The In-Fusion cloning starts by mixing the three fragments and a recombinant exonuclease. 2. The exonuclease exposes the 15 bases overlap at the VH, SPHC and LC plasmid. 3. The homologous overlaps fuses the three fragments together, resulting in a HC and LC plasmid construct.

12 2. Theory

2.2.3 Colony PCR screening Colony PCR is a rapid screening method to determine which colonies, following transformation, that carries the desired genetic insert. First, PCR primers are designed that generate a product of known size only if the desired insert is present. Thereafter, the bacteria are thermally lysed and their DNA are used as template in the PCR. Following the ampliﬁcation, the PCR products are analysed using gel electrophoresis. Colonies with gel bands of the correct size indicates a successful transformation. Hence, colony PCR can be used to distinguish between a successful and an unsuccessful transformation [28].

2.2.4 Restriction digest screening Restriction digest can be used as a screening method for puriﬁed plasmid constructs to determine if the cloning was successful, i.e. if the plasmid construct contains the desired insert. The plasmid constructs are cleaved with restriction enzymes that generate diﬀerent digestion patterns depending on if the desired insert is present or not. Thereafter, the digested plasmid constructs are analysed using gel electrophoresis. Restriction mapping and in silico digestions are performed to determine which restriction enzymes that can be used to identify the presence of the desired insert [29].

2.2.5 Transient transfection Transfection is a method that introduces foreign DNA into eukaryotic cells and can either be used to yield transient or stable expression of the gene of interest. For stable transfection, the foreign DNA is integrated into the host genome, stably expressed and passed on to daughter cells. In contrast, transiently transfected cells express the introduced DNA for a limited amount of time and the foreign DNA is not integrated into the host genome. Thus, transiently transfected DNA is not passed on to daughter cells [30]. Neverthe- less, transient transfection is a rapid method for the expression of recombinant proteins in mammalian cells without having to establish a stable cell line [31]. However, foreign DNA can be introduced into eukaryotic cells with a variety of methods and transfection reagents: calcium phosphate, cationic polymers, cationic lipids and electroporation, just to mention a few [32]. An excess of cationic transfection reagent interacts with the negatively charged DNA and forms positively charged complexes, which is endocytosed by the transfected cell [33]. For plasmid DNA, eﬃcient transfection reagents delivers the gene into the nucleus of the host cell, where it is expressed [34]. In this project, FectoPRO® was used, which is an optimized transfection reagent for transient gene expression (TGE) of recombinant proteins and mAb in suspension CHO cells and HEK293 cells [35].

2.2.6 Transfection efficiency The transfection efficiency is a crucial parameter to determine in gene expression experiments and describes the proportion of cells that have been successfully transfected. Commonly, transfection efficiency is often monitored through fluorescent reporter genes, including the expression of green fluorescent protein (GFP) [36][37]. In this project, GFP was used as an internal control for transfection efficiency and was transfected in a sepa- rate well for each cell culture plate. The transfection efficiency was determined using a Countess™ II FL Automated Cell Counter.

13 2. Theory

2.2.7 Biacore concentration analysis Biacore systems can be utilized for many purposes, including concentration analysis. In direct binding concentration assays, responses from samples with known analyte concentrations are monitored and used to calculate a standard curve. Thereafter, the standard curve can be utilized to determine the concentration of samples with unknown concentrations [25]. In this project, Biacore T200 and Series S Sensor Chip Protein A were used for the concentration assay. The sensor surface is precoated with recombinant Protein A, which predominantly binds human antibodies of the subclasses IgG1, IgG2 and IgG4. The recombinant Protein A only binds to the HC within the Fc region, thus ensuring an orientation-speciﬁc binding of the antibody [38]. Hence, in the concentration assay the immobilized ligand was the recombinant Protein A and the analytes were the expressed recombinant antibodies. Moreover, the standard curve was calculated from samples with known concentrations of Herceptin® (Trastuzumab), which is a recombinant humanized IgG1 mAb [39].

2.2.8 Biacore kinetic screening Determination of real-time interaction kinetics with high resolution can be studied using Biacore systems. Further, antibody screening can be performed to identify antibody constructs with suitable kinetic and affinity properties against an antigen. The affinity for the binding interaction, such as the equilibrium dissociation constant (KD), can be derived from the kinetic rate constants for the interaction (ka for association rate constant and kd for dissociation rate constant) [40]. In this project, a Human Fab Capture Kit was used for immobilization at the sensor surface on a Sensor Chip CM5. The sensor surface was coated with capturing molecules, monoclonal antibodies that bind specifically to the LC of human Fab regions [41]. Thereafter, the expressed recombinant antibodies, referred to as the ligand, was flowed across the sensor surface and captured through interactions with the immobilized capturing molecules. Then, the antigen, referred to as the analyte, was injected and the binding interaction was monitored [40].

14 3. Methods

3 Methods

The materials and instruments used in this project are found in Appendix A, except for general laboratory equipment. Computer software used in the project are cited in the text.

3.1 Preparation of variable domains 3.1.1 PCR amplification of variable domains The two variable domains (VH and VL) for each of the three recombinant antibodies (referred to as Antibody A, Antibody B and Antibody C) were amplified respectively with CloneAmp™ HiFi PCR Premix (2X). 1 µL of each variable domain (10 ng/µL) were added to PCR tubes along with 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 25 µL ™ CloneAmp HiFi PCR Premix (2X) and 22 µL dH2O, resulting in a total volume of 50 µL. The melting temperatures (Tm) for the forward and reverse primer were calculated to be 65°C and 72°C respectively, using Thermo Fisher Scientific Tm Calculator [42]. Therefore, a two-step PCR was feasible since the primers annealing temperature were within 3°C of the extension temperature at 68°C [43]. Thus, the amplifications were performed with a two-step PCR with the following program: a denaturation step at 98°C for 10 seconds, followed by a combined annealing and extension step at 68°C for 30 seconds. The thermal cycle was repeated 30 times and thereafter the temperature decreased to 4°C.

3.1.2 Purification of LC variable domains After the amplification, the LC variable domains were purified using GeneJET PCR Purification Kit. 50 µL dH2O, 100 µL Binding buffer and 100 µL 2-propanol were added to each PCR mixture, resulting in a total volume of 300 µL. Then, the PCR mixtures were transferred to GeneJET purification columns and washed according to the manufacturer’s protocol. The VL were eluted in 30 µL Elution buffer. The concentration of the VL were determined using an Implen NanoPhotometer® NP80.

Then, the VL were analysed with gel electrophoresis for quality control. 1 µL of each ™ VL was mixed with 1 µL FlashGel Loading Dye (5X) and 3 µL dH2O and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder for size comparisons. Thereafter, the VL were further purified with gel electrophoresis. 5 µL FlashGel™ Loading Dye (5X) was added to 20 µL of each VL and applied to the FlashGel™ Recovery Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder. The identified gel bands for the VL were extracted from the FlashGel™ Recovery Cassette (1.2% agarose). The concentration of the extracted VL were determined using an Implen NanoPhotometer® NP80. To control that the gel electrophoresis purification had worked 4 µL of each VL was mixed with 1 µL FlashGel™ Loading Dye (5X) and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder.

3.1.3 Purification of HC variable domains After the amplification, the HC variable domains (VH) were purified using GeneJET PCR Purification Kit. 50 µL dH2O, 100 µL Binding buffer and 100 µL 2-propanol were added to each PCR mixture, resulting in a total volume of 300 µL. Then, the PCR mixtures were

15 3. Methods transferred to GeneJET puriﬁcation columns and washed according to the manufacturer’s protocol. The VH were eluted in 30 µL Elution buﬀer. The concentration of the VH were determined using an Implen NanoPhotometer® NP80.

Then, the VH were analysed with gel electrophoresis for quality control. 1 µL of each ™ VH was mixed with 1 µL FlashGel Loading Dye (5X) and 3 µL dH2O and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder for size comparisons. Thereafter, the VH were further purified with gel electrophoresis. 7.5 µL FlashGel™ Loading Dye (5X) was added to 30 µL of each VH and applied to the FlashGel™ Recovery Cassette (1.2% agarose). The identified gel bands for the VH were extracted from the FlashGel™ Recovery Cassette (1.2% agarose). The concentration of the extracted VH were determined using an Implen NanoPhotometer® NP80. Then, the VH were concentrated using GeneJET PCR Purification Kit. 100 µL Binding buffer and 100 µL 2-propanol were added to each VH. The VH were washed and eluted in 30 µL Elution buffer according to the manufacturer’s protocol. The concentration of the VH were determined using an Implen NanoPhotometer® NP80. For control, 1 ™ µL of each VH was mixed with 1 µL FlashGel Loading Dye (5X), 3 µL dH2O and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder.

3.2 Preparation of signal peptides A vector containing all of the signal peptides (L1-L2 and H1-H8) with their respective 15 bases In-Fusion overlap was ordered from GeneArt™ (Thermo Fisher Scientific). The vector was designed with cleavage sites for the restriction enzyme BveI FastDigest™ between every signal peptide construct. Therefore, the vector was cleaved to generate a mixture containing all of the signal peptides. 5 µL vector (3 µg) was added to a PCR tube along with 6 µL FastDigest™ buffer (10X), 3 µL BveI FastDigest™ restriction enzyme, 3 µL oligonucleotide (20X) and 43 µL dH2O, resulting in a total volume of 60 µL. The PCR tube was incubated at 37°C for three hours, and then the enzymes were inactivated by heating for five minutes at 80°C.

Thereafter, the signal peptides were purified using gel electrophoresis. 15 µL FlashGel™ Loading Dye (5X) was added to 60 µL solution containing the signal peptides and applied to the FlashGel™ Recovery Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder. The identified gel bands for the signal peptides were extracted from the FlashGel™ Recovery Cassette (1.2% agarose). Then, the concentration of the signal peptides were determined using an Implen NanoPhotometer® NP80. To control that the gel electrophoresis purification had worked 4 µL solution containing the signal peptides was mixed with 1 µL FlashGel™ Loading Dye (5X) and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder.

3.3 Linearization of plasmid backbone A human IgG1-backbone plasmid with a VL and VH stuﬀer fragment was provided by the facility. Before the LC In-Fusion cloning the plasmid was linearized with restriction enzymes that cleaved the VL stuﬀer fragment. Restriction mapping were performed to determine which restriction enzymes that could be used, using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). 6 µL plasmid (3 µg) was added to a PCR tube along with 6 µL

16 3. Methods

FastDigest™ buffer (10X), 3 µL of each restriction enzyme (Pfl23II FastDigest™ and SgsI ™ FastDigest ) and 42 µL dH2O, resulting in a total volume of 60 µL. The PCR tube was incubated at 37°C for three hours and thereafter the enzymes were inactivated by heating for five minutes at 80°C.

The plasmid was purified using GeneJET PCR Purification Kit. 40 µL dH2O and 100 µL Binding buffer were added to the plasmid solution, resulting in a total volume of 200 µL. Then, the solution was transferred to a GeneJET purification column and the plasmid was purified and eluted in 30 µL Elution buffer according to the manufacturer’s protocol. The concentration of the plasmid was determined using an Implen NanoPhotometer® ™ NP80. For control, 1 µL FlashGel Loading Dye (5X) and 3 µL dH2O were added to 1 µL plasmid and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with a control (non-cleaved) plasmid and 5 µL FastRuler Middle Range DNA Ladder for size comparisons.

3.4 Construction of LC plasmid constructs 3.4.1 In-Fusion cloning of LC variable domains 2 µL of each VL (≈ 14 ng) were added to a PCR tube along with 4 µL signal peptides (≈ 13 ng), 1 µL linearized plasmid (≈ 65 ng), 2 µL In-Fusion® HD Enzyme Premix (5X) and 1 µL dH2O, resulting in a total volume of 10 µL. For control, 1 µL linearized plasmid ® (≈ 65 ng), 2 µL In-Fusion HD Enzyme Premix (5X) and 7 µL dH2O were added to a PCR tube. The four PCR tubes (three diﬀerent VL and one control) were incubated at 50°C for 15 minutes according to the manufacturer’s protocol.

3.4.2 Plasmid transformation into competent E. coli 3 µL In-Fusion product and 50 µL Stellar™ Competent Cells were added to a 1.5 mL microcentrifuge tube and incubated on ice for 30 minutes. Thereafter, the cells were heat shocked at 42°C for 45 seconds and placed back on ice for 2 minutes. 450 µL Invitrogen™ S.O.C. Medium was added to the transformed cells followed by shake-incubation at 37°C and 240 rpm for 1 hour. Then, the cells were spread onto agar plates with 100 µg/mL Carbenicillin and incubated overnight at 37°C.

3.4.3 Colony PCR screening of LC plasmid constructs Initially, colony PCR screening was not performed and plasmid DNA were directly isolated with minipreparation. However, due to a high transformation background for three of the LC plasmid constructs, colony PCR was used to select colonies for these constructs. Colonies for colony PCR were picked from agar plates using pipette tips and put in wells of a PCR plate. 1 µL (1 ng) plasmid with a successful insert was added to a well for positive control. 1 µL (1 ng) plasmid with no insert was added to a well for negative control along with a well with no added colony or plasmid. Thereafter, the pipette tips were moved to a 24 well cell culture plate and the colonies were inoculated in 2.5 mL LB medium containing 100 µg/mL Carbenicillin. The samples were shake-incubated overnight at 37°C and 240 rpm. 12.5 µL DreamTaq™ Green PCR Master Mix (2X) was added to each PCR well along with 0.5 µL (10 µM) forward primer, 0.5 µL (10 µM) reverse primer and 11.5 µL dH2O,

17 3. Methods resulting in a total volume of 25 µL. The colony PCR was performed with the following program: an initial step at 95°C for 7 minutes, followed by a thermal cycle of 95°C for one minute and 68°C for one minute, which was repeated 25 times. After the thermal cycle, the temperature was kept at 68°C for 7 minutes and thereafter the temperature decreased to 4°C. 3 µL of each PCR reaction was directly applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder for size comparisons. Colonies with identiﬁed gel bands of the correct size, indicating a successful transformation, were used for minipreparation of plasmid DNA.

3.4.4 Minipreparations of LC plasmid constructs Colonies that were not screened with colony PCR were picked from agar plates using sterile culture loops and inoculated in 5 mL LB medium containing 100 µg/mL Carbenicillin in 14 mL Falcon tubes. The samples were shake-incubated overnight at 37°C and 240 rpm. Thereafter, 1.8 mL overnight cell culture, either screened with colony PCR or not, was transferred to a 2 mL microcentrifuge tube and centrifuged at 17,000 × g for 2 minutes at room temperature. The supernatants were thrown to waste. The cell pellets were resuspended and lysed followed by plasmid DNA puriﬁcation using GeneJET Plasmid Miniprep Kit. The plasmid constructs were eluted in 50 µL Elution buﬀer according to the manufacturer’s protocol. Then, the concentration of the plasmid constructs were determined using an Implen NanoPhotometer® NP80.

3.4.5 Restriction digest screening of LC plasmid constructs In silico digestions with the restriction enzymes NcoI and XbaI were performed on LC plasmid constructs, for both L1 and L2, and one control plasmid with no inserts using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). 1 µL plasmid construct was added to a PCR tube along with 0.5 µL FastDigest™ buﬀer (10X), 0.25 µL of each restriction enzyme ™ ™ (NcoI FastDigest and XbaI FastDigest ) and 3 µL dH2O, resulting in a total volume of 5 µL. The PCR tubes were incubated at 37°C for 20 minutes, and then the enzymes were inactivated by heating for ﬁve minutes at 80°C. 1.25 µL FlashGel™ Loading Dye (5X) was added to each PCR tube and thereafter 5 µL was applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder for size comparisons. The in silico digestions were used as references for digestion patterns in the gels. Plasmid constructs that generated gel bands with digestion patterns that indicated a successful cloning were sequenced for further quality control.

3.4.6 Sequencing of LC plasmid constructs LC plasmid constructs were sequenced at KIGene, Karolinska Institutet. The obtained DNA sequence electropherogram for each LC plasmid construct was analysed using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). LC plasmid constructs that showed a correct DNA sequence when mapped to the reference plasmids were accepted as candidates for further cloning. Two candidates for each antibody, one with L1 as a signal peptide and one with L2, were chosen for cloning with the VH. Hence, six LC plasmid constructs in total.

18 3. Methods

3.5 Linearization of LC plasmid constructs After sequencing, the six chosen LC plasmid constructs were linearized with restriction enzymes that cleaved the VH stuffer fragment. Restriction mapping were performed to determine which restriction enzymes that could be used, using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). 2 µg LC plasmid construct was added to a PCR tube along with 6 µL FastDigest™ buffer (10X), 1.5 µL of each of the four restriction enzymes (BamHI ™ ™ ™ ™ FastDigest , BspTI FastDigest , Eco91I FastDigest and HindIII FastDigest ) and dH2O, resulting in a total volume of 60 µL. All of the LC plasmid constructs had different concentrations from the minipreparations, and thus the volume of dH2O differed from each reaction so that the total volume was 60 µL. The PCR tubes were incubated at 37°C for three hours and thereafter the enzymes were inactivated by heating for five minutes at 80°C.

Thereafter, the LC plasmid constructs were purified using GeneJET PCR Purification Kit. 40 µL dH2O and 100 µL Binding buffer were added to the LC plasmid constructs, resulting in a total volume of 200 µL. The solutions were transferred to a GeneJET purification columns and the LC plasmid constructs were purified and eluted in 30 µL Elution buffer according to the manufacturer’s protocol. The concentration of the LC plasmid constructs were determined using an Implen NanoPhotometer® NP80. For control, 1 ™ µL FlashGel Loading Dye (5X) and 3 µL dH2O were added to 1 µL LC plasmid construct and applied to the FlashGel™ DNA Cassette (1.2% agarose) along with two control plasmids (one non-cleaved plasmid and one cleaved, but non-purified plasmid) and 5 µL FastRuler Middle Range DNA Ladder for size comparisons.

3.6 Construction of HC and LC plasmid constructs 3.6.1 In-Fusion cloning of HC variable domains 2 µL of each VH (≈ 16 ng) were added to a PCR tube along with 4 µL signal peptides (≈ 13 ng), 2 µL linearized LC plasmid construct (≈ 66 ng), 4 µL In-Fusion® HD Enzyme Premix (5X) and 8 µL dH2O, resulting in a total volume of 20 µL. For control, 1 µL linearized LC plasmid construct (≈ 66 ng), 2 µL In-Fusion® HD Enzyme Premix (5X) and 7 µL dH2O were added to a PCR tube. The PCR tubes were incubated at 50°C for 15 minutes according to the manufacturer’s protocol.

3.6.2 Plasmid transformation into competent E. coli 5 µL In-Fusion product and 50 µL Stellar™ Competent Cells were added to a 1.5 mL microcentrifuge tube and incubated on ice for 30 minutes. Thereafter, the cells were heat shocked at 42°C for 45 seconds and placed back on ice for 2 minutes. 450 µL Invitrogen™ S.O.C. Medium was added to the transformed cells followed by shake-incubation at 37°C and 240 rpm for 1 hour. Then, the cells were spread onto agar plates with 100 µg/mL Carbenicillin and incubated overnight at 37°C.

3.6.3 Colony PCR screening of HC and LC plasmid constructs Initially, colony PCR screening was performed for one of the In-Fusion products to rapidly determine the success rate of the transformation. Thereafter, because of a successful

19 3. Methods transformation, colony PCR screening was not performed and the HC and LC plasmid constructs were directly puriﬁed with minipreparations.

Colonies for the initial screening were picked from agar plates using pipette tips and put in wells of a PCR plate. 1 ng plasmid with a successful insert was added to a well for positive control. 1 ng plasmid with no insert was added to a well for negative control along with a well with no added colony or plasmid. Thereafter, the pipette tips were moved to a 24 well cell culture plate and the colonies were inoculated in 2.5 mL LB medium containing 100 µg/mL Carbenicillin. The samples were shake-incubated overnight at 37°C and 240 rpm.

12.5 µL DreamTaq™ Green PCR Master Mix (2X) was added to each PCR well along with 0.5 µL (10 µM) forward primer, 0.5 µL (10 µM) reverse primer and 11.5 µL dH2O, resulting in a total volume of 25 µL. The colony PCR was performed with the following program: an initial step at 95°C for 7 minutes, followed by a thermal cycle of 95°C for one minute and 68°C for one minute, which was repeated 25 times. After the thermal cycle, the temperature was kept at 68°C for 7 minutes and thereafter the temperature decreased to 4°C. 3 µL of each PCR reaction was directly applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder for size comparisons.

3.6.4 Minipreparations of HC and LC plasmid constructs Colonies were picked from agar plates using pipette tips and inoculated in 3 mL LB medium containing 100 µg/mL Carbenicillin in a 24 well cell culture plate. The cultures were shake-incubated overnight at 37°C and 240 rpm. Thereafter, 100 µL of each cell culture was transferred into a PCR plate and stored at 4°C. The 24 well cell culture plates were centrifuged at 2,500 × g for 10 minutes at 4°C. The supernatants were thrown to waste. The cell pellets were resuspended and lysed, followed by plasmid DNA puriﬁcation using QIAGEN® Plasmid Plus 96 Miniprep Kit and QIAvac 96 Vacuum Manifold. The plasmid constructs were eluted in 80 µL Elution buﬀer according to the manufacturer’s protocol.

3.6.5 Restriction digest screening of HC and LC plasmid constructs In silico digestions were performed on HC and LC plasmid constructs and control plasmids with no inserts, either with the restriction enzymes NcoI and XbaI or ApaI and HindIII, using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). 200 ng plasmid construct was added to a PCR tube along with 0.5 µL FastDigest™ buﬀer (10X), 0.25 µL of each restriction enzyme (either NcoI FastDigest™ and XbaI FastDigest™ or ApaI FastDigest™ ™ and HindIII FastDigest ) and dH2O, resulting in a total volume of 5 µL. The PCR tubes were incubated at 37°C for 20 minutes, and then the enzymes were inactivated by heating for ﬁve minutes at 80°C. 1.25 µL FlashGel™ Loading Dye (5X) was added to each PCR tube and thereafter 5 µL was applied to the FlashGel™ DNA Cassette (1.2% agarose) along with 5 µL FastRuler Middle Range DNA Ladder for size comparisons. The in silico digestions were used as references for digestion patterns in the gels. Plasmid constructs that generated gel bands with digestion patterns that indicated a HC and LC plasmid construct were sequenced for further quality control.

20 3. Methods

3.6.6 Sequencing of HC and LC plasmid constructs HC and LC plasmid constructs were sequenced at KIGene, Karolinska Institutet. The obtained DNA sequence electropherograms were analysed and the HC signal peptide was determined for each HC and LC plasmid construct using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). HC and LC plasmid constructs that showed a correct DNA sequence when mapped to the reference plasmids were accepted as candidates for transfection. 16 plasmid constructs for each antibody, with all the HC and LC signal peptide combinations, were chosen for transient transfection into HEK293 cells.

3.7 Sterile filtration of plasmid constructs Before transfection, the plasmid constructs were sterile filtered using Corning® Costar® Spin-X® centrifuge tube filter with 0.22 µm pore size cellulose acetate membrane. The plasmid constructs from the minipreparations were transferred to the centrifuge tube filters and centrifuged at 17,000 × g for 5 minutes at room temperature. The concentration of the sterile plasmid constructs were determined using an Implen NanoPhotometer® NP80.

3.8 Maintenance of HEK293 cells A maintenance culture of Expi293F™ cells were cultivated and split twice a week, either a 3-day split or a 4-day split. Total cell count and percent viability for the maintenance culture was determined before each split using a Countess™ II FL Automated Cell Counter. 50 µL cell culture was mixed with 50 µL Trypan Blue Stain (0.4%). 10 µL sample mixture was loaded to each chamber in the sample slide and analysed according to the manufacturer’s protocol. At each split, Expi293F™ cells were transferred to fresh Expi293™ Expression Medium, pre-warmed to 37°C, in an Erlenmeyer cell culture ﬂask. The seeding density was either 0.75 × 106 cells/mL or 0.6 × 106 cells/mL, for a 3-day split or a 4-day split respectively. The maintenance culture was shake-incubated at 37°C, 125 rpm, 50 mm shaking diameter, 8% CO2 and 70% relative humidity.

3.9 Transient gene expression of recombinant antibodies 3.9.1 Transfection of plasmid constructs into HEK293 cells On the day prior to transfection (Day −1), total cell count and percent viability was determined using a Countess™ II FL Automated Cell Counter, according to the manufacturer’s protocol. Then, Expi293F™ cells were diluted in fresh Expi293™ Expression Medium, pre-warmed to 37°C, to a ﬁnal density of 2.35 × 106 cells/mL. Thereafter, the cells were shake-incubated overnight at 37°C, 125 rpm, 50 mm shaking diameter, 8% CO2 and 70% relative humidity. On the next day (Day 0), total cell count and percent viability was determined using a Countess™ II FL Automated Cell Counter, according to the manufacturer’s protocol. The Expi293F™ cells were diluted in fresh Expi293™ Expression Medium, pre-warmed to 37°C, to a ﬁnal density of 2.5 × 106 cells/mL. The total transient gene expression (TGE) volume for each plasmid construct was 4 mL. 3.6 mL Expi293F™ cells (2.5 × 106 cells/mL) (0.9 mL × total TGE volume) were added to a round bottom 24 well cell culture plate. 3.2 µg plasmid DNA (0.8 µg × total TGE volume) was added to a 96 deep well plate. 3.2 µL FectoPRO® (FectoPRO® to DNA ratio was 1 µL : 1 µg) was

21 3. Methods mixed with 400 µL Opti-MEM™ I Reduced Serum Medium (0.1 mL × total TGE volume) and transferred to the plasmid DNA in the 96 deep well plate. The transfection mixture containing plasmid DNA, FectoPRO® and Opti-MEM™ I Reduced Serum Medium was homogenized and incubated at room temperature for 20 minutes. After incubation, the transfection mixture was added to the Expi293F™ cells. Thereafter, the transfected cells ® were incubated at 37°C, 8% CO2, 80% relative humidity and placed on a MixMate shaker at 400 rpm.

In each round bottom 24 well cell culture plate, one well was transfected with pmaxGFP™ vector, which was used as an internal control for transfection eﬃciency. Moreover, a human IgG1-backbone plasmid with the reference HC and LC signal peptides used by the facility for each recombinant antibody were transfected in one well in each cell culture plate. The antibody titer for the reference HC and LC signal peptides for each recombinant antibody was well-documented at the facility, and therefore used as a control in the TGE. The amino acid sequence for the reference HC and LC signal peptides used by the facility are shown in Table 3. The TGE experiment described above was repeated three times for each HC and LC signal peptide construct. Hence, three independent transfections for the 16 diﬀerent combinations of the HC and LC signal peptides for each antibody were performed. Thus, 144 transfections were carried out in total, excluding controls (3 repeats × 3 antibodies × 16 HC and LC plasmid constructs). Therefore, the TGE experiments were carried out in seven independent batches, one batch consisted of a 24 well round bottom cell culture plate.

Table 3: The reference heavy chain (HC) and light chain (LC) signal peptides used by the facility and their respective amino acid sequence.

Reference signal peptide Amino acid sequence HC signal peptide MSVSFLIFLPVLGLPWGVLS LC signal peptide MEAPAQLLFLLLLWLPDTTG

3.9.2 Measurement of transfection efficiency After 48 hours post-transfection (Day 2), the transfection efficiency was determined. The percentage of Expi293F™ cells that were transfected with GFP was used as an internal control for transfection efficiency. 1 mL cell culture that had been transfected with pmaxGFP™ vector was collected. 50 µL cell culture was mixed with 50 µL Trypan Blue Stain (0.4%). 10 µL sample mixture was loaded to each chamber in the sample slide and analysed using a Countess™ II FL Automated Cell Counter, according to the manufacturer’s protocol.

3.9.3 Harvest of expressed recombinant antibodies After 120 hours post-transfection (Day 5), the expressed recombinant antibodies were harvested. The round bottom 24 well cell culture plates were centrifuged at 2,500 × g for 5 minutes at 4°C. Thereafter, the culture supernatants were collected and stored at −20°C until further analysis.

22 3. Methods

3.10 Biacore concentration analysis of recombinant antibodies First, HBS-EP+ buffer (pH 7.4) was prepared, containing 10 mM HEPES, 150 mM sodium chloride, 3mM EDTA and 0.05% TWEEN® 20, and used as running buffer. 10 mM Glycine (pH 1.5) was prepared and used as regeneration buffer. Then, standard curve samples were prepared from Herceptin® stock solution (1 mg/mL), diluted in Expi293F™ Expression Medium to a final concentration according to Table 4. Thereafter, the culture supernatants from the TGE of recombinant antibodies were diluted in Expi293F™ Expression Medium. First, dilution factors for each recombinant antibody were based on expected antibody titers, according to previous data from the facility. After quantification, the dilution factors were evaluated and selected for each HC and LC signal peptide construct in order for the antibody titers to lie within the linear range of the standard curve. After an appropriate dilution, the culture supernatants were filtered using MultiScreenHTS HV Filter 96 well plate, with 0.45 µm pore size membrane. The culture supernatans were transferred to the 96 well filter plate and centrifuged at 700 × g for 2 minutes at room temperature. Series S Sensor Chip Protein A was docked to the Biacore T200, the samples were loaded and the system was primed with running buffer. Thereafter, the Biacore analysis was performed with the following settings: two startup cycles with running buffer. Sample injection with 135 seconds sample contact time, 10 µL/minute sample flow rate. Regeneration with 30 seconds regeneration buffer contact time, 30 µL/minute regeneration buffer flow rate. The standard curve was measured before the first sample and after every 24th sample.

Table 4: The ten diﬀerent concentrations of Herceptin® that were used as standard samples for the standard curve in the Biacore concentration analysis.

Standard curve Analyte 1 2 3 4 5 6 7 8 9 10 Herceptin® (µg/mL) 0.5 1 3 5 10 15 20 30 40 50

3.11 Statistics The quantified titer for each recombinant antibody was analyzed using a two-way analysis of variance (ANOVA) with a full factorial model, with the LC signal peptide and HC signal peptide as independent variables. Moreover, post hoc Tukey’s honestly significant difference (HSD) test for multiple comparisons were also performed. Statistical data analysis was performed with IBM SPSS Statistics 26 (IBM Corporation). A P value ≤ .05 was used as level of significance in all statistical analysis.

3.12 SDS-PAGE analysis of recombinant antibodies First, evaluation of antibody titers for each of the three recombinant antibodies with different signal peptides were performed. Thereafter, the reference signal peptide constructs and the HC and LC signal peptide constructs that resulted in the signiﬁcantly highest antibody titers for each antibody were chosen for further control using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The evaluated HC and LC signal peptide constructs H6/L1, H7/L1 for Antibody A, H6/L1 for Antibody B and H2/L1,

23 3. Methods

H6/L1 for Antibody C were selected along with the reference signal peptide constructs for each antibody.

Due to a low antibody titer, the three Antibody C constructs were concentrated. First, 1 mL sample solution from the transient gene expression of Antibody C constructs were sterile filtered using Corning® Costar® Spin-X® centrifuge tube filter with 0.22 µm pore size cellulose acetate membrane. The sample solutions were transferred to the centrifuge tube filters and centrifuged at 17,000 × g for 10 minutes at 4°C. Thereafter, the sample solutions from Antibody C constructs were transferred to Amicon® Ultra-4 Centrifugal filters (10 kDa cutoff) and centrifuged at 2,500 × g for 15 minutes at 4°C. The sample solutions from the transient gene expression for the three Antibody C constructs were concentrated from 1 mL to 250 µL. Thereafter, the different antibody constructs were prepared for separation under reducing conditions. 15.6 µL sample solution was added to a PCR tube along with 6 µL NuPAGE™ LDS Sample Buffer (4X) and 2.4 µL NuPAGE™ Reducing Agent (10X), resulting in a total volume of 24 µL. The sample solutions were heated at 95°C for 5 minutes. Then, the NuPAGE™ 4−12% gradient Bis-Tris Gel was mounted in the XCell SureLock® Mini-Cell. Running buffer was prepared with 50 mL NuPAGE™ MES SDS Running Buffer (20X) and 950 mL dH2O. The upper and lower buffer chambers were filled with the prepared 1X running buffer. Moreover, 500 µL NuPAGE™ Antioxidant was applied in the upper buffer chamber. Thereafter, 20 µL of each sample solution were loaded to the NuPAGE™ 4−12% gradient Bis-Tris Gel along with 10 µL Novex™ Sharp Pre-stained Pro- tein Standard for size comparisons. Then, 200 V was applied for 35 minutes. The gel was removed from the XCell SureLock® Mini-Cell and submersed in InstantBlue™ Protein Gel Stain for 1 hour. The gel was imaged using LI-COR Odyssey® Fc Imaging System.

The different antibody constructs were also separated under non-reducing conditions. 15.6 µL sample solution was added to a PCR tube along with 6 µL NuPAGE™ LDS Sample ™ Buffer (4X) and 2.4 dH2O, resulting in a total volume of 24 µL. The NuPAGE 4−12% gradient Bis-Tris Gel was mounted in the XCell SureLock® Mini-Cell. The prepared 1X running buffer containing NuPAGE™ MES SDS Running Buffer was used. Thereafter, 20 µL of each sample solution were loaded to the NuPAGE™ 4−12% gradient Bis-Tris Gel along with 10 µL Novex™ Sharp Pre-stained Protein Standard for size comparisons. Then, 200 V was applied for 35 minutes. The gel was removed from the XCell SureLock® Mini- Cell and submersed in InstantBlue™ Protein Gel Stain for 1 hour. The gel was imaged using LI-COR Odyssey® Fc Imaging System.

3.13 Kinetic screening of recombinant antibodies The HC and LC signal peptide constructs that resulted in the signiﬁcantly highest antibody titer together with the reference signal peptide constructs for each antibody were selected for a kinetic screen. The signal peptide constructs H6/L1, H7/L1 for Antibody A, H6/L1 for Antibody B and H2/L1, H6/L1 for Antibody C were selected along with the reference signal peptide constructs for each antibody. The three recombinant antibodies (Antibody A, Antibody B and Antibody C) were screened against their respective antigen, referred to as Antigen A, Antigen B and Antigen C.

First, a Human Fab Capture Kit was used for immobilization at the sensor surface on a Sensor Chip CM5 according to the manufacturer’s protocol. Second, a test capture

24 3. Methods experiment was performed where each antibody construct flowed across the sensor surface and was test captured at the Fab capture surface. Then, the antibody constructs were diluted in HBS-EP+ buffer to yield capture levels around 400 RU. Samples containing 50 nM antigen were prepared for each antibody. HBS-EP+ buffer was used as running buffer and 10 mM Glycine (pH 1.5) was used as regeneration buffer. Thereafter, the kinetic screen was performed with the following settings: three startup cycles with running buffer, 60 seconds contact time, 30 µL/minute flow rate. Antibody constructs were captured, 60 seconds contact time, 10 µL/minute flow rate. Antigen was injected, 120 seconds contact time, 300 seconds dissociation time, 30 µL/minute flow rate. Two regeneration steps, 60 seconds contact time, 30 µL/minute flow rate and ending with 300 seconds stabilization. Data from the reference flow cell was subtracted from the flow cells where the antigen binding activity was monitored.

25 4. Results

4 Results

4.1 Cloning of HC and LC plasmid constructs 16 plasmid constructs, every combination of the eight HC and two LC signal peptides, were generated for each of the three antibodies (Antibody A, Antibody B and Antibody C). Sequencing results from KIGene (Karolinska Institutet) for the 16 HC and LC plasmid constructs for each antibody showed a correct DNA sequence when mapped to the reference plasmids using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). Detailed results regarding the cloning of the 48 diﬀerent plasmid constructs are found in Appendix B.

4.2 Evaluation of HC and LC signal peptides for recombinant antibody expression in HEK293 cells To evaluate the impact of signal peptides on the expression of recombinant antibodies, 16 HC and LC signal peptide constructs for each antibody was transiently transfected into HEK293 cells. Three independent transfections for the 16 diﬀerent combinations of the HC and LC signal peptide for each antibody were performed. Furthermore, a vector expressing GFP was also included in each cell culture plate (referred to as each transfection batch) and used as an internal control for transfection eﬃciency, which are shown in Fig- ure 17. The recombinant antibodies were harvested 5 days post-transfection. Thereafter, the recombinant antibody titers were determined using Biacore concentration analysis. A standard curve was calculated from samples with known concentration of Herceptin®, according to Table 4, and utilized to determine the concentration of the expressed recombinant antibodies. The standard curve as well as the sensorgram of the standard samples with Herceptin® used in the Biacore concentration analysis are presented in Appendix C.

The recombinant antibody titers for the 16 HC and LC signal peptide constructs for each recombinant antibody are shown in Figure 11. The expression of Antibody C with L2 as the LC signal peptide were lower than 0.5 µg/mL for all of the eight HC signal peptides, due to a response lower than the lowest calibration standard in the standard curve. The presented data are the mean and the error bars are the standard error of the mean (SEM) of three independent transfections for each HC and LC signal peptide construct. As clearly shown in Figure 11, the produced antibody titers were highly dependent on the used signal peptides. For Antibody A, the mean antibody titer of all the eight HC signal peptides were 16 times higher for LC signal peptide L1 compared to L2. For Antibody B, the mean antibody titer of all the eight HC signal peptides were 36 times higher for LC signal peptide L1 compared to L2.

The quantified recombinant antibody titers were analyzed using a two-way ANOVA with a full factorial model, with the LC signal peptide and HC signal peptide as independent variables. However, for Antibody C, the recombinant antibody titers with L1 LC signal peptide were analyzed using a one-way ANOVA with the HC signal peptide as an independent variable. Since the L2 LC signal peptide resulted in antibody titers that were lower than 0.5 µg/mL for all of the eight HC signal peptides for Antibody C, a two-way ANOVA was not feasible for Antibody C. There were significant interactions in the two-way ANOVA between the HC and LC signal peptides for Antibody A and Anti- body B, and thus simple main effects analysis was performed. Furthermore, there were

26 4. Results significant difference between the HC signal peptides with L1 as the LC signal peptide for Antibody A and Antibody B. However, there were no significant difference between the HC signal peptides with L2 as the LC signal peptide for Antibody A and Antibody B. As for Antibody C, there were significant difference between the HC signal peptides with L1 as the LC signal peptide. Therefore, the presented recombinant antibody titers in Figure 12 were divided based on their LC signal peptide (L1 or L2) for each antibody. The presented antibody titers in Figure 12 show the same data as in Figure 11, however the graphs were divided based on the LC signal peptide (L1 or L2) for each antibody. Thus, the eight HC signal peptides (H1-H8) were compared for each LC signal peptide (L1 or L2) and for each antibody in Figure 12.

Thereafter, post hoc Tukey’s honestly significant difference (HSD) multiple compari- son were performed for the eight HC constructs with L1 as the LC signal peptide for each of the three recombinant antibodies. For Antibody A, signal peptides H6/L1 resulted in significantly higher recombinant antibody titer than all the other studied signal peptides, except for H7/L1 where no significant difference was found. For Antibody B, signal peptides H6/L1 resulted in significantly higher recombinant antibody titer than all the other studied signal peptides. For Antibody C, signal peptides H6/L1 resulted in significantly higher recombinant antibody titer than all the other studied signal peptides, except for H2/L1 where no significant difference was found. To summarize, the evaluated HC and LC signal peptides that resulted in the significantly highest recombinant antibody titers for each antibody were:

• Antibody A: signal peptides H6/L1 and H7/L1

• Antibody B: signal peptides H6/L1

• Antibody C: signal peptides H2/L1 and H6/L1

27 4. Results

Figure 11: Recombinant antibody titers from triplicate transient gene expression in HEK293 cells. For each antibody, eight HC signal peptides (H1-H8) were paired with two LC signal peptides (L1-L2). The recombinant antibodies were harvested 5 days post-transfection and antibody titers were quantiﬁed using Biacore concentration analysis. The presented data are the mean and the error bars are the standard error of the mean (SEM) of three independent transfections for each HC and LC signal peptide construct. The expression of Antibody C with L2 as the LC signal peptide were lower than 0.5 µg/mL for all of the eight HC signal peptides.

28 4. Results

Figure 12: Recombinant antibody titers from triplicate transient gene expression in HEK293 cells. For each antibody, eight HC signal peptides (H1-H8) were paired with two LC signal peptides (L1: left panel, L2: right panel). The recombinant antibodies were harvested 5 days post-transfection and antibody titers were quantiﬁed using Biacore concentration analysis. The presented data are the mean and the error bars are the standard error of the mean (SEM) of three independent transfections for each HC and LC signal peptide construct. The expression of Antibody C with L2 as the LC signal peptide were lower than 0.5 µg/mL for all of the eight HC signal peptides. The presented antibody titers in this ﬁgure (Figure 12) show the same data as in Figure 11, however the graphs were divided based on the LC signal peptide (L1 or L2) for each antibody.

29 4. Results

4.3 Comparisons between the evaluated HC and LC signal peptides and the reference signal peptides In addition to the evaluated HC and LC signal peptide constructs, a vector with the reference HC and LC signal peptides used by the facility for each antibody were also transiently transfected into HEK293 cells at three independent experiments. The amino acid sequence for the reference HC and LC signal peptides used by the facility are shown in Table 3. It is therefore crucial to investigate whether the evaluated HC and LC signal peptides resulted in a more efficient secretion of the studied recombinant antibodies than the reference signal peptides. Thus, in Figure 13 the recombinant antibody titer for the reference signal peptides used by the facility were compared to the evaluated HC and LC signal peptides that resulted in the significantly highest antibody titer for each antibody. For Antibody A, no significant difference was found in antibody titer between the reference signal peptides and the evaluated HC and LC signal peptide combinations H6/L1 and H7/L1. For Antibody B the signal peptides H6/L1 resulted in 2 times higher recombinant antibody titer compared to the reference signal peptides. Interestingly, for Antibody C the signal peptide combinations H6/L1 and H2/L1 resulted in 3 times higher and 2 times higher recombinant antibody titer compared to the reference signal peptides respectively.

30 4. Results

Figure 13: Recombinant antibody titers from triplicate transient gene expression in HEK293 cells. Comparisons between the reference HC and LC signal peptides used by the facility and the evaluated HC and LC signal peptides that resulted in the signiﬁcantly highest recombinant antibody titer for each antibody (Antibody A: H6/L1 and H7/L1, Antibody B: H6/L1, Antibody C: H2/L1 and H6/L1). The recombinant antibodies were harvested 5 days post-transfection and antibody titers were quantiﬁed using Biacore concentration analysis. The presented data are the mean and the error bars are the standard error of the mean (SEM) of three independent transfections for each HC and LC signal peptide construct.

4.4 Control of full-length recombinant antibodies using SDS- PAGE To further verify the presented recombinant antibody titers, determined by Biacore concentration analysis, SDS-PAGE analysis of sample solutions from the transient gene expression of recombinant antibodies in HEK293 cells was performed. The SDS-PAGE analysis was carried out using both reduced and non-reduced sample solutions. The HC and LC signal peptide constructs that resulted in the signiﬁcantly highest antibody titer together with the reference signal peptide constructs for each antibody, shown in Figure 13, were selected for SDS-PAGE analysis. Hence, the signal peptide constructs H6/L1, H7/L1 for Antibody A, H6/L1 for Antibody B and H2/L1, H6/L1 for Antibody C were selected along with the reference signal peptide constructs for each antibody.

The two SDS-PAGE gels are shown in Figure 14. The sample solutions for the three Antibody C constructs (reference, H2/L1 and H6/L1 signal peptides; lanes 7, 8 and 9 respectively) were concentrated 4X (10 kDa cutoﬀ), due to a low antibody titer, before

31 4. Results

SDS-PAGE analysis. All the eight studied non-reduced sample solutions showed a clear band around 150 kDa, indicating a full-length IgG antibody with no or very low amounts of unpaired or partially paired molecules. Moreover, all the eight studied reduced sample solutions showed clear bands around 25 kDa and 50 kDa, indicating free LC and free HC respectively. Since the sample solutions for Antibody C were concentrated, in addition to recombinant antibodies, artifacts in the sample solution were also concentrated, shown as more distinct bands around 70 kDa.

(a) Non-reduced sample solutions

(b) Reduced sample solutions

Figure 14: SDS-PAGE analysis of sample solutions from the transient gene expression in HEK293 cells for the reference signal peptide used by the facility and the evaluated HC and LC signal peptides that resulted in the signiﬁcantly highest recombinant antibody titer for each antibody (Antibody A: H6/L1 and H7/L1, Antibody B: H6/L1, Antibody C: H2/L1 and H6/L1). Expected molecular weights of full-length antibody, free heavy chain and free light chain are indicated. (a) Non-reduced sample solutions. (b) Reduced sample solutions. 1. Novex™ Sharp Pre-stained Protein Standard. 2. Antibody A reference. 3. Antibody A H6/L1. 4. Antibody A H7/L1. 5. Antibody B reference. 6. Antibody B H6/L1. 7. Antibody C reference. 8. Antibody C H2/L1. 9. Antibody C H6/L1. The sample solutions for the three Antibody C constructs (reference, H2/L1 and H6/L1 signal peptides) were concentrated 4X (10 kDa cutoﬀ), due to a low antibody titer, before SDS-PAGE analysis.

32 4. Results

4.5 Kinetic activity of recombinant antibodies To investigate whether the different signal peptides affected the activity of the expressed recombinant antibodies, the three recombinant antibodies (Antibody A, Antibody B and Antibody C) were selected for a kinetic screen against their respective antigen, referred to as Antigen A, Antigen B and Antigen C. The HC and LC signal peptide constructs that resulted in the significantly highest antibody titer together with the reference signal peptide constructs for each antibody, shown in Figure 13, were selected for a kinetic screen. Hence, the signal peptide constructs H6/L1, H7/L1 for Antibody A, H6/L1 for Antibody B and H2/L1, H6/L1 for Antibody C were selected along with the reference signal peptide constructs for each antibody.

Each antigen was injected with a concentration of 50 nM and binding kinetics were monitored for each captured antibody construct. All of the antibody constructs bound to their respective antigen. However, Antigen A and Antigen B were also captured on the reference flow cell surface, coated with capturing molecules. The unspecific reference flow cell binding of Antigen A and Antigen B resulted in inaccurate sensorgrams for the binding kinetics of Antibody A and Antibody B. Thus, only the kinetics and affinities for Antibody C constructs were evaluated, which are shown in Figure 15. The determined binding constants for Antibody C constructs are shown in Table 5. For Antibody C, the evaluated HC and LC signal peptide constructs (H2/L1 and H6/L1) bound to Antigen C and showed no difference in affinity and kinetics compared to the reference signal peptide construct.

Figure 15: Antibody screening data for the reference signal peptide used by the facility, H2/L1 and H6/L1 signal peptides for Antibody C. Antigen C were injected with a concentration of 50 nM. Antigen responses are colored in red and their 1 : 1 binding interaction models are colored in black.

Table 5: Kinetic and equilibrium dissociation constants for Antibody C and Antigen C complex formation, for the reference signal peptide used by the facility, H2/L1 and H6/L1 signal peptides.

Antibody C construct ka (1/Ms) kd (1/s) KD (nM) Reference signal peptide 7.73 × 105 9.22 × 10-4 1.19 H2/L1 signal peptide 8.01 × 105 9.68 × 10-4 1.21 H6/L1 signal peptide 7.11 × 105 8.60 × 10-4 1.21

ka: association rate constant, kd: dissociation rate constant, KD: equilibrium dissociation constant.

33 4. Results

4.6 Control of HEK293 cell maintenance culture Quality control of the HEK293 cell maintenance culture was crucial for subculturing and transfection. Total cell count and viability for the maintenance culture, measured at each split, are shown in Figure 16. Total cell count was within 3−4.5 × 106 cells/mL and cell viability was between 93% to 94% at each split, except for a decrease to 91% viability at the fourth split.

Figure 16: Total cell count and viability for the HEK293 cell maintenance culture, measured at each split.

4.7 Control of transfection efficiency Transfection efficiency was monitored with GFP as an internal control for each independent transfection batch. The percentage of cells that were transfected with GFP was determined 48 hours post-transfection. The transfection efficiency for the seven transfection batches are shown in Figure 17. The transfection efficiency was between 88% to 92% for all the transfection batches.

34 4. Results

Figure 17: Transfection eﬃciency for the seven independent transfection batches, monitored as the percentage of cells that were transfected with GFP 48 hours post-transfection.

4.8 Sequence alignment of HC signal peptides A sequence identity alignment of the eight HC signal peptides is presented in Figure 18. All the HC signal peptides contained 19 amino acids, except for H8 which contained 26 amino acids. H8 and H3 had identical sequences, except for the fact that H8 had seven more amino acids prior to H3 in the n-region. Signal peptides H1, H2, H6 and H7 had similar sequences with 68.4% to 84.2% sequence identity. H4 and H5 had similar sequences with 78.9% sequence identity. Furthermore, residue −1 to −5 formed the polar c-region (−1: the last residue of the signal peptide) and residue −6 to −13 comprised the hydrophobic h-region in the signal peptides. Four of the signal peptides (H1, H2, H6, H7) terminated with a cysteine (C), while the other four (H3, H4, H5, H8) terminated with a serine (S). In all the signal peptides residue −3 was a valine (V), except for H4 where it was an alanine (A). Moreover, in three of the n-regions in the signal peptides (H1, H6, H7), there were no positively charged amino acids, however one negatively charged amino acid (glutamic acid, E).

35 4. Results

1 10 20 26 Consensus Identity H1 H2 H3 H4 H5 H6 H7 H8

(a) 1 10 20 22 Consensus Identity L1 L2

(b)

Figure 18: (a) Sequence identity alignment of the eight HC signal peptides (H1-H8). The alignment was colored relative to the sequences percentage identity (black: 100% identity, dark grey: 80% to 100% identity, light grey: 60% to 80% identity, white: less than 60% identity). The HC signal peptides were also divided into the n-region, h-region and c-region. (b) Percentage of residues that were identical between the eight HC signal peptides (H1-H8) [44]. 1 10 20 26 Consensus Identity 4.9 SequenceH1 alignment of LC signal peptides H2 A sequenceH3 identity alignment of the two LC signal peptides is shown in Figure 19. H4 The two LCH5 signal peptides contained 22 amino acids and had 36.4% sequence identity. Residue −1H6 to −5 formed the polar c-region and residue −6 to −13 comprised the hy- H7 drophobic h-regionH8 in the signal peptides. In both the LC signal peptides (L1 and L2), residue −3 was an alanine (A). Signal peptide L1 terminated with a cysteine (C), while L2 terminated with an alanine (A).

1 10 20 22 Consensus Identity L1 L2

H1 78.9% 21.1% 26.3% 31.6% 84.2% 68.4% 21.1% Figure 19:H2Sequence78.9% identity alignment26.3% of36.8% the two42.1% LC signal78.9% peptides73.7% (L1-L2).26.3% The alignment was coloredH3 relative21.1% to the26.3% sequences identity42.1% (black:47.4% identical,31.6% white:31.6% non-identical).100% The LC signal peptidesH4 26.3% L1 and L236.8% had a42.1% 36.4% sequence78.9% identity.36.8% The LC31.6% signal42.1% peptides were also H5 31.6% 42.1% 47.4% 78.9% 42.1% 36.8% 47.4% divided intoH6 the84.2% n-region,78.9% h-region31.6% and c-region36.8% [44].42.1% 78.9% 31.6% H7 68.4% 73.7% 31.6% 31.6% 36.8% 78.9% 31.6% H8 21.1% 26.3% 100% 42.1% 47.4% 31.6% 31.6%

36 4. Results

4.10 Sequence alignment of the evaluated HC and LC signal peptides and the reference signal peptides A sequence identity alignment of the evaluated HC signal peptides that resulted in the signiﬁcantly highest recombinant antibody titer for the studied antibodies (H2, H6 and H7) and the reference HC signal peptide used by the facility is shown in Figure 20. The reference HC signal peptide used by the facility contained 20 amino acids, compared 1 10 20 26 to the evaluated HC signal peptides which contained 19 amino acids. Furthermore, the Consensus reference HC signal peptide used by the facility had a dissimilar sequence compared to Identity the evaluated HC signal peptides (H2, H6 and H7) with 15.8% to 21.1% sequence identity. H1 H2 H3 1 10 20 H4 Consensus H5 Identity H6 H7 H2 H8 H6 H7 Reference

1 10 20 22 Consensus (a) Identity 1 10 20 22 L1 L2 Consensus Identity L1 Reference (b)

78.9% 21.1% 26.3% 31.6% 84.2% 68.4% 21.1% 78.9% 26.3% 36.8% 42.1% 78.9% 73.7% 26.3% 21.1% 26.3% 42.1% 47.4% 31.6% 31.6% 100% Figure 20: (a) Sequence identity alignment of the reference HC signal peptide used by the 26.3% 36.8% 42.1% 78.9% 36.8% 31.6% 42.1% facility and the evaluated HC signal peptides that resulted in the signiﬁcantly highest recom- 31.6% 42.1% 47.4% 78.9% 42.1% 36.8% 47.4% binant antibody titer for the studied antibodies (H2, H6 and H7). The alignment was colored 84.2% 78.9% 31.6% 36.8% 42.1% 78.9% 31.6% 78.9% 73.7% 15.8% 68.4% 73.7% 31.6% 31.6% 36.8% 78.9% 31.6% relative to the sequences percentage78.9% identity (black:78.9% 100%21.1% identity, dark grey: 80% to 100% 21.1% 26.3% 100% 42.1% 47.4% 31.6% 31.6% identity, light grey: 60% to73.7% 80% identity,78.9% white: less than 60%21.1% identity). The HC signal peptides were also divided into the n-region,15.8% h-region21.1% and21.1% c-region. (b) Percentage of residues that were identical between the reference signal peptide used by the facility and the evaluated HC signal peptides (H2, H6 and H7) [44].

A sequence identity alignment of the evaluated LC signal peptide L1 that resulted in the signiﬁcantly highest recombinant antibody titer for the studied antibodies and the reference LC signal peptide used by the facility is presented in Figure 21. The reference LC signal peptide used by the facility contained 20 amino acids, compared to the evaluated LC signal peptide L1 which contained 22 amino acids. Furthermore, the reference LC signal peptide used by the facility and the evaluated LC signal peptide L1 had similar sequences with 60% sequence identity.

37 1 10 20 26 Consensus Identity H1 H2 H3 1 10 20 H4 Consensus H5 Identity H6 H7 H2 H8 H6 H7 Reference

4. Results 1 10 20 22 Consensus Identity 1 10 20 22 L1 L2 Consensus Identity L1 Reference

78.9% 21.1% 26.3% 31.6% 84.2% 68.4% 21.1% 78.9% 26.3% 36.8% 42.1% 78.9% 73.7% 26.3% 21.1% 26.3% 42.1% 47.4% 31.6% 31.6% 100% 26.3% 36.8% 42.1% 78.9% 36.8% 31.6% 42.1% Figure 21: Sequence identity alignment of the reference LC signal peptide used by the facility 31.6% 42.1% 47.4% 78.9% 42.1% 36.8% 47.4% 84.2% 78.9% 31.6% 36.8% 42.1% 78.9% 31.6% and the evaluated LC signal peptide L178.9% that resulted73.7% in the15.8% signiﬁcantly highest recombinant 68.4% 73.7% 31.6% 31.6% 36.8% 78.9% 31.6% antibody titer for the studied78.9% antibodies. The alignment78.9% was21.1% colored relative to the sequences 21.1% 26.3% 100% 42.1% 47.4% 31.6% 31.6% identity (black: identical, white:73.7% non-identical).78.9% The reference21.1% LC signal peptide and the L1 LC signal peptide had a 60% sequence15.8% identity.21.1% The21.1% LC signal peptides were also divided into the n-region, h-region and c-region [44].

4.11 Process analysis The initial weeks of the project were going according to plan and the planning report was finished on time. Preparation of cloning fragments and In-Fusion cloning of the LC plasmid constructs were performed in two weeks instead of three, which freed up time for screening and sequencing of the LC plasmid constructs. Due to a lower transformation efficiency than expected, more colonies had to be screened to obtain the six different LC plasmid constructs. Nevertheless, successful LC plasmid constructs were still achieved on time. However, the work plan for the HC In-Fusion cloning and plasmid purification was changed and the procedure was carried out in two batches, which can be seen as two iterations of the HC and LC plasmid construct procedure in Figure 1. First, only one LC plasmid construct was cloned with the HC to investigate the transformation efficiency with colony PCR screening. The transformation efficiency was acceptable and no further purification of the cloning fragments were therefore needed for the remaining HC In- Fusion cloning. Then, the HC and LC plasmid constructs from the first batch were used to evaluate the QIAGEN® Plasmid Plus 96 Miniprep Kit. First, colonies were inoculated in 1.3 mL LB medium containing 100 µg/mL Carbenicillin in a 96 well cell culture plate. However, plasmid construct that were purified from cultures in 1.3 mL LB medium generated a lower yield of plasmid DNA than required for a triplicate transient transfection into HEK293 cells. Therefore, the volume was increased to 3 mL LB medium containing 100 µg/mL Carbenicillin in a 24 well cell culture plate for the remaining minipreparations. Despite the change in work plan for the HC and LC plasmid constructs, successful plasmid constructs were still obtained on time. The half-time presentation was postponed one week to better suit the weekly schedule.

The transient gene expression (TGE) experiments started on time and were performed during four weeks. Since all the transfections were successful no additional reruns of the experiments were necessary. Therefore, the triplicate TGE of recombinant antibodies were ﬁnished two weeks earlier than planned. The Biacore concentration analysis was performed continuously during the TGE experiments. Additional control experiments, including separation using SDS-PAGE and kinetic screening of the expressed recombinant antibodies were performed during project week 16 and 17.

38 5. Discussion

5 Discussion

An identiﬁed bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the ER [10]. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion [11]. Further, several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems [10][12][13].

The aim of this study was to evaluate the expression of three human IgG1 recombinant antibodies in HEK293 cells by evaluating different combinations of eight HC signal peptides and two LC signal peptides, shown in Table 2. First, 16 human IgG1-backbone plasmid constructs with different combinations of the HC and LC signal peptides were generated and sequenced for quality control for each of the three antibodies (referred to as Antibody A, Antibody B and Antibody C). All of the plasmid constructs in this project were based on an existing vector provided by the facility and contained the same promoter sequence and regulatory elements. Transient gene expression of the different recombinant antibodies in HEK293 cells were performed at three independent experiments. The antibody titers were quantified using Biacore concentration analysis. Further, total cell count and viability for the HEK293 cell maintenance culture were determined at each split, shown in Figure 16, and were crucial for successful and uniform transfection experiments. Moreover, transfection efficiency was monitored with GFP as an internal control and was between 88% to 92% for all transfection experiments, shown in Figure 17. Thus, the consistent cell viability and transfection efficiency between all transfection experiments made it possible to make direct comparisons between the different signal peptides and their impact on recombinant antibody expression.

As clearly shown in Figure 11, the produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, LC signal peptide L2 resulted in dramatically lower antibody titers than LC signal peptide L1 for all of the eight HC signal peptides, across all the studied antibodies. Moreover, the mean antibody titer of all the eight HC signal peptides were 16 times lower for LC signal peptide L2 compared to L1 for Antibody A and 36 times lower for Antibody B. The two LC signal peptides (L1 and L2) had 36.4% sequence identity, shown in Figure 19. Furthermore, the expression of Antibody C with L2 as the LC signal peptide were lower than 0.5 µg/mL for all of the eight HC signal peptides. Thus, LC signal peptide L2 resulted in an inefficient antibody secretion for the three studied recombinant antibodies, regardless of the HC signal peptide. Therefore, the different HC signal peptides were evaluated for the LC signal peptide L1, for each recombinant antibody, shown in Figure 12. Interestingly, the signal peptides H6/L1 resulted in the significantly highest recombinant antibody titer for all of the three recombinant antibodies, except for H7/L1 for Antibody A and H2/L1 for Antibody C where no significant difference was found. The signal peptide H6 have a similar sequence compared to signal peptides H2 and H7, with a 78.9% sequence identity, shown in Figure 18. Furthermore, the presented data in Fig- ure 12 clearly shows that two signal peptides (e.g. H2 and H7) had different impact on the antibody titer depending on which antibody (e.g. Antibody A and Antibody C) they were linked to. The antibody titer for H7/L1-Antibody A was significantly higher than H2/L1-Antibody A, however the antibody titer for H7/L1-Antibody C was significantly lower than H2/L1-Antibody C. These results indicated that the variable domain of the

39 5. Discussion antibody, in addition to the signal peptide, also affected the secretion efficiency. Thus, signal peptides need to be evaluated for individual recombinant antibodies. Thereafter, comparisons between the evaluated HC and LC signal peptides and the reference HC and LC signal peptides used by the facility, shown in Table 3, were performed for each antibody and the antibody titers are presented in Figure 13. For Antibody A, signal peptides H6/L1 and H7/L1 resulted in no significant difference in recombinant antibody titer compared to the reference signal peptides. For Antibody B, signal peptides H6/L1 resulted in 2 times higher recombinant antibody titer compared to the reference signal peptides. And for Antibody C the signal peptide combinations H6/L1 and H2/L1 resulted in 3 times higher and 2 times higher recombinant antibody titer compared to the reference signal peptides respectively. Thus, the evaluated signal peptides H6/L1 were more efficient, or at least as efficient, in expressing all of the three recombinant antibodies compared to the reference signal peptides used by the facility. Therefore, the use of signal peptides H6/L1 for Antibody B and Antibody C would lower the production cost for these recombinant antibodies in HEK293 cells. Interestingly, the reference HC signal peptide used by the facility have a dissimilar sequence compared to the evaluated HC signal peptides (H2, H6 and H7) with 15.8% to 21.1% sequence identity, shown in Figure 20. In contrast, the reference LC signal peptide used by the facility and the evaluated LC signal peptide L1 have similar sequences with 60% sequence identity, presented in Figure 21.

Sample solutions from the transient gene expression of recombinant antibodies in HEK293 cells were analysed using SDS-PAGE to further verify the presented antibody titers, determined by Biacore concentration analysis. The SDS-PAGE analysis was carried out using both reduced and non-reduced sample solutions and the gels are shown in Figure 14. All the non-reduced sample solutions showed a clear band around 150 kDa, indicating a full-length IgG antibody. Hence, the transient gene expression in HEK293 cells resulted in intact full-length IgG antibodies. These results veriﬁed that the presented antibody titers were for intact full-length IgG antibodies and none mispaired antibody molecules. Additionally, the reduced sample solutions showed clear bands around 25 kDa and 50 kDa, indicating free LC and free HC respectively, further supporting these results.

Furthermore, kinetic screening was performed for each antibody against their respective antigen to investigate whether the different HC and LC signal peptides affected the kinetics and affinity compared to the reference signal peptide. All of the antibody constructs bound to their respective antigen. However, due to unspecific reference flow cell binding of Antigen A and Antigen B only the antigen kinetics and affinities for Antibody C constructs were evaluated, shown in Figure 15 and Table 5. For Antibody C, the evaluated signal peptide constructs H2/L1 and H6/L1 showed no difference in affinity and kinetics towards Antigen C compared to the reference signal peptide construct, indicating that the signal peptides did not affect the conformation of the antigen binding Fab region.

It has been suggested by previous studies that the signal peptide comprise of a three- domain structure, with a ”positive-hydrophobic-polar” design, referred to as the n-region, h-region and c-region [11]. All the studied HC signal peptides contained 19 amino acids, except for H8 which contained 26 amino acids. Most of the n-regions contained six amino acids, followed by eight amino acids in the h-region and thereafter ﬁve amino acids in the c-region. Interestingly, three of the signal peptides (H1, H6, H7) contained one negatively charged amino acid and no positively charged amino acid in the n-region. Thus, not all

40 5. Discussion n-regions of the signal peptides are positively charged. Further, a critical ”(−3, −1)- design” in the c-region of the signal peptide have previously been determined [22]. Small residues were conserved at position −1; alanine (A), glycine (G), serine (S), threonine (T) and cysteine (C) (−1: the last residue of the signal peptide). Position −3 favoured small aliphatic residues; alanine (A) and valine (V) [18]. Interestingly, four of the HC signal peptides (H1, H2, H6, H7) terminated with a cysteine (C), while the other four (H3, H4, H5, H8) terminated with a serine (S). Also, all the studied HC signal peptides had a valine (V) at position −3, except for H4 where it was an alanine (A). Further, the two LC signal peptides both had an alanine (A) at position −3 and either a cysteine (C) or an alanine (A) at position −1. Hence, all the eight HC and two LC signal peptides had a conserved ”(−3, −1)-design”.

To further understand the difference in the secretion, the intracellular levels of expressed HC and LC could be analyzed as well as the effect that the different signal peptides have on the transcriptional and translational machinery. In this project, the signal peptides and their effect on antibody secretion was evaluated in transiently transfected cells. To further evaluate and verify the HC and LC signal peptides and their effect on antibody secretion, the expression of antibodies in stably transfected cell lines could be analyzed.

41 6. Conclusions

6 Conclusions

The produced antibody titers for the studied recombinant antibodies were highly dependent on the used signal peptides. The evaluated signal peptides H6/L1 resulted in the significantly highest recombinant antibody titer for all of the three studied antibodies, except for H7/L1 for Antibody A and H2/L1 for Antibody C where no significant difference was found. Interestingly, signal peptides H6/L1 resulted in 3 times higher antibody titer compared to the reference signal peptide for Antibody C and 2 times higher antibody titer for Antibody B. Thus, the optimal use of signal peptides H6/L1 for Antibody B and Antibody C would lower the production cost for these recombinant antibodies in HEK293 cells.

All of the plasmid constructs in this project were based on an existing vector provided by the facility and contained the same promoter sequence and regulatory element. Also, transfection efficiency and cell viability were consistent between the transfection experiments. Therefore, it was possible to make direct comparisons between the different signal peptides and their impact on recombinant antibody titer. Further, the SDS-PAGE analysis for sample solutions from the transient gene expression in HEK293 cells clearly indicated full-length IgG antibodies, verifying a successful expression of full-length recombinant antibodies. Hence, the presented antibody titers were for intact full-length IgG antibodies and none mispaired antibody molecules. The Biacore kinetic screening for the antibody constructs revealed that all antibody constructs bound to their respective antigen, indicating that the signal peptides did not affect the conformation of the antigen binding Fab region. Thus, the presented results further supports the signal peptide as a good optimization candidate and the necessity to evaluate signal peptides for attempting to achieve increased expression of recombinant antibodies in mammalian systems.

42 Acknowledgements

This master’s thesis project at Science for Life Laboratory in Stockholm concludes my time as a student at Link¨opingUniversity. I am deeply grateful towards Science for Life Laboratory and especially the Protein Expression and Characterization facility where I have been performing my master’s thesis project. Thank you to all the fantastic people at SciLifeLab that I have met during this project, with special thanks to:

• My supervisors Leif Dahllund and Anders Olsson at SciLifeLab deserves my deepest thanks for guiding me through the project. You have helped me organize, think and discuss diﬀerent ideas throughout the project. Your engagement and curiosity towards this study has been inspiring.

• Esmeralda Woestenenk and Yasmin Andersson at SciLifeLab are to be thanked for their generosity and incredible knowledge in molecular biology. Also, thanks to Esmeralda Woestenenk for your input regarding colony PCR.

• Camilla Hofstr¨omat SciLifeLab are to be thanked for Biacore introduction and expertise.

• All the fantastic people at the Protein Expression and Characterization facility and the Human Antibody Therapeutics facility at the Drug Discovery and Development platform at SciLifeLab for welcoming me with open arms.

I would also like to acknowledge my examiner Lars-G¨oranM˚artenssonat Link¨oping University for his interest in my thesis and for providing valuable feedback. Finally, I would like to thank my family and friends for their support and encouragement during my thesis.

Stockholm, June 2020 Gustav Myhrinder

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46 Appendix A. Materials

This appendix will list the materials and instruments used in this project, except for general laboratory equipment. Computer software used in the project are cited in the paper.

Table A-1: General materials.

Material Supplier 2-Propanol EMSURE® Merck Ethanol absolute ≥ 99.8% AnalaR NORMAPUR® VWR Chemicals Glycine, Analytical reagent grade Fisher Scientiﬁc HEPES ≥ 99.5% for biochemistry VWR Chemicals Sodium chloride, GPR RECTAPUR® VWR Chemicals Sterile water Merck Titriplex® III for analysis (EDTA) Merck TWEEN® 20, BioXtra, viscous liquid Sigma-Aldrich

Table A-2: Polymerase chain reaction (PCR).

Material Supplier CloneAmp™ HiFi PCR Premix (2X) Clontech® Laboratories DreamTaq™ Green PCR Master Mix (2X) Thermo Fisher Scientiﬁc

Table A-3: Restriction enzymes.

Material Supplier ApaI FastDigest™ Thermo Fisher Scientific BamHI FastDigest™ Thermo Fisher Scientific BveI FastDigest™ Thermo Fisher Scientific BveI FastDigest™ Oligonucleotide (20X) Thermo Fisher Scientific BspTI FastDigest™ Thermo Fisher Scientific Eco91I FastDigest™ Thermo Fisher Scientific HindIII FastDigest™ Thermo Fisher Scientific NcoI FastDigest™ Thermo Fisher Scientific Pfl23II FastDigest™ Thermo Fisher Scientific SgsI FastDigest™ Thermo Fisher Scientific XbaI FastDigest™ Thermo Fisher Scientific FastDigest™ Buffer (10X) Thermo Fisher Scientific

47 Table A-4: Agarose gel electrophoresis.

Material Supplier FastRuler Middle Range DNA Ladder Thermo Fisher Scientiﬁc FlashGel™ Loading Dye (5X) Lonza FlashGel™ DNA Cassette (1.2% agarose) Lonza FlashGel™ Recovery Cassette (1.2% agarose) Lonza

Table A-5: In-Fusion cloning.

Material Supplier In-Fusion® HD Enzyme Premix (5X) Clontech® Laboratories

Table A-6: Transformation.

Material Supplier Carbenicillin (100 mg/mL) Sigma-Aldrich LB Broth (Lennox) Sigma-Aldrich LB Broth with agar (Lennox) tablet Sigma-Aldrich S.O.C. Medium (Invitrogen™) Thermo Fisher Scientiﬁc Stellar™ Competent Cells Clontech® Laboratories

Table A-7: Plasmid preparation.

Material Supplier GeneJET PCR Purification Kit Thermo Fisher Scientific GeneJET Plasmid MiniPrep Kit Thermo Fisher Scientific QIAGEN® Plasmid Plus 96 Miniprep Kit QIAGEN®

Table A-8: Filtration.

Material Supplier Amicon® Ultra-4 Centrifugal filters (10 kDa cutoff) Merck Corning® Costar® Spin-X® centrifuge tube filter (0.22 Sigma-Aldrich µm pore size) MultiScreenHTS HV Filter 96 well plate (0.45 µm pore Merck size)

48 Table A-9: Transfection and transient gene expression.

Material Supplier 24-well Blocks Round-Bottom (RB) QIAGEN® Expi293F™ Cells Thermo Fisher Scientific Expi293F™ Expression Medium Thermo Fisher Scientific FectoPRO® Polyplus-transfection® Opti-MEM™ I Reduced Serum Medium Thermo Fisher Scientific pmaxGFP™ vector Lonza Trypan Blue Stain (0.4%) Thermo Fisher Scientific

Table A-10: Biacore concentration analysis.

Material Supplier Herceptin® stock (1 mg/mL) Roche Series S Sensor Chip Protein A Cytiva

Table A-11: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Material Supplier InstantBlue™ Protein Gel Stain Expedeon Novex™ Sharp Pre-stained Protein Standard Thermo Fisher Scientific NuPAGE™ 4−12% gradient Bis-Tris Gel Thermo Fisher Scientific NuPAGE™ Antioxidant Thermo Fisher Scientific NuPAGE™ LDS Sample Buffer (4X) Thermo Fisher Scientific NuPAGE™ MES SDS Running Buffer (20X) Thermo Fisher Scientific NuPAGE™ Reducing Agent (10X) Thermo Fisher Scientific

Table A-12: Biacore kinetic screen.

Material Supplier Human Fab Capture Kit Cytiva Sensor Chip CM5 Cytiva

49 Table A-13: Instruments.

Instrument Supplier Applied Biosystems® 2720 Thermal Cycler Applied Biosystems Biacore T200 Cytiva Centrifuge 5810 R Eppendorf Countess™ II FL Automated Cell Counter Thermo Fisher Scientific FlashGel™ Dock Lonza ™ ™ Forma Steri-Cult CO2 Incubator Thermo Fisher Scientific Heraeus™ Fresco™ 17 Microcentrifuge Thermo Fisher Scientific MixMate® Eppendorf Multitron Pro Incubation Shaker Infors HT NanoPhotometer® NP80 Implen Synergy® UV Water Purification System Merck Odyssey® Fc Imaging System LI-COR Biosciences QIAvac 96 Vacuum Manifold QIAGEN® XCell SureLock® Mini-Cell Thermo Fisher Scientific

50 Appendix B. Cloning of plasmid constructs

This appendix will disclosure the detailed results from the cloning of the 48 diﬀerent HC and LC plasmid constructs.

B.1 Preparation of cloning fragments The concentration of the purified variable domains (VL and VH) for the three different antibodies (referred to as Antibody A, Antibody B and Antibody C) can be seen in Table B-1. The concentration of the solution containing all of the signal peptides (L1-L2 and H1-H8) was 3.2 ng/µL. After linearization and purification the concentration of the human IgG1-backbone plasmid was 65.7 ng/µL. For control, the variable domains (VL and VH), signal peptides and linearized plasmid were analysed using gel electrophoresis, which can be seen in Figure B-1 and Figure B-2.

Table B-1: The concentration (ng/µL) of the puriﬁed LC variable domain (VL) and HC variable domain (VH) for each antibody: Antibody A, Antibody B and Antibody C.

Antibody A Antibody B Antibody C VL 7.4 4.2 9.2 VH 11.6 7.8 4.8

5000 bp 2000 bp 850 bp 400 bp

100 bp

1. 2. 3. 4. 5. 6. 7. 8.

Figure B-1: Gel electrophoresis of puriﬁed fragments: signal peptides, linearized human IgG1- backbone plasmid50 and00 LCbp variable domains (VL) for each antibody: Antibody A, Antibody B and Antibody C. 1. FastRuler Middle Range DNA Ladder. 2. Solution containing the signal peptides (L1-L2 and200 H1-H8).0 bp 3. Antibody A VL. 4. Antibody B VL. 5. Antibody C VL. 6. Linearized human IgG1-backbone plasmid. 7. Control, non-linearized human IgG1-backbone plasmid. 8. FastRuler Middle Range DNA Ladder. 850 bp

400 bp

100 bp 51 1. 2. 3. 4. 5. 5000 bp 2000 bp 850 bp 400 bp

100 bp

1. 2. 3. 4. 5. 6. 7. 8.

5000 bp

2000 bp

850 bp

400 bp

100 bp

1. 2. 3. 4. 5.

Figure B-2: Gel electrophoresis of puriﬁed fragments: signal peptides and HC variable domains (VH) for each antibody: Antibody A, Antibody B and Antibody C. 1. FastRuler Middle Range DNA Ladder. 2. Antibody A VH. 3. Antibody B VH. 4. Antibody C VH. 5. Solution containing the signal peptides (L1-L2 and H1-H8).

B.2 LC plasmid constructs Six LC plasmid constructs were generated using In-Fusion cloning, one with L1 as a signal peptide and one with L2, for each antibody. The concentration of the LC plasmid constructs after minipreparations can be seen in Table B-2. Sequencing results from KI- Gene (Karolinska Institutet) for the six LC plasmid constructs showed a correct sequence when mapped to the reference plasmids using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). Thereafter, the six LC plasmid constructs were linearized and puriﬁed before the HC In-Fusion cloning. For control, the linearized LC plasmid constructs were analyzed using gel electrophoresis, which can be seen in Figure B-3.

Table B-2: The concentration (ng/µL) of the six diﬀerent LC plasmid constructs, after minipreparations, with their respective signal peptide (L1 or L2) for each antibody (Antibody A, Antibody B and Antibody C).

Antibody A Antibody B Antibody C L1 272 373 429 L2 258 218 281

52 5000 bp 5000 bp 2000 bp 2000 bp 850 bp 850 bp 400 bp 400 bp 100 bp 100 bp 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 2. 3. 4. 5. 6. 7. 8.

Figure B-3: Gel electrophoresis of the LC plasmid constructs after linearization and purification. LC plasmid constructs with their respective signal peptide (L1 or L2) for each antibody 5000 bp (Antibody A, Antibody B and Antibody C). 1. FastRuler Middle Range DNA Ladder. 2. Antibody A L1 plasmid construct. 3. Antibody A L2 plasmid construct. 4. Antibody B L1 2000 bp plasmid construct. 5. Antibody B L2 plasmid construct. 6. Antibody C L1 plasmid construct. 7. Antibody C L2 plasmid construct. 8. Control, non-purified, linearized LC plasmid construct. 9. Control, non-linearized LC plasmid construct. 850 bp B.3 HC and LC plasmid constructs 400 bp 16 plasmid constructs were generated for each antibody, every combination of the eight HC signal peptides and two LC signal peptides. Sequencing results from KIGene (Karolinska 100 bp Institutet) for the 16 HC and LC plasmid constructs for each antibody showed a correct sequence when mapped to the reference plasmids using Geneious Prime® v. 9.1.8 (Biomatters Ltd.). After sterile filtration, the concentration of the HC and LC plasmid 1. 2. 3. 4. 5. constructs were determined, which can be seen in Table B-3.

Table B-3: The concentration (ng/µL) of the 16 diﬀerent HC and LC plasmid constructs, after sterile ﬁltration, with their respective signal peptide (H1-H8 and L1-L2) for each antibody (Antibody A, Antibody B and Antibody C).

Antibody A Antibody B Antibody C L1 L2 L1 L2 L1 L2 H1 330 370 329 233 198 245 H2 418 302 340 212 222 233 H3 300 350 310 212 139 260 H4 341 305 354 218 212 238 H5 348 407 370 226 206 216 H6 329 279 331 218 120 330 H7 373 334 447 258 153 226 H8 350 324 337 254 173 226

53 Appendix C. Biacore concentration analysis of recombinant antibodies

This appendix will disclosure supplementary data from the Biacore concentration analysis of the recombinant antibodies.

A standard curve was utilized to determine the concentration of the expressed recombinant antibodies using Biacore T200 and Series S Sensor Chip Protein A. The standard curve was prepared with known concentrations of Herceptin®, diluted in Expi293F™ Ex- pression Medium, according to Table 4. The sensorgrams of the ten diﬀerent standard samples with known concentration of Herceptin® are shown in Figure C-1. Thereafter, a standard curve was calculated from the samples with known concentration of Herceptin®, which is shown in Figure C-2. Before the Biacore concentration analysis, the samples with expressed recombinant antibodies were diluted in Expi293F™ Expression Medium to lie within the linear range of the standard curve (0.5−20 µg/mL).

RU Sensorgram 42000

40000

38000 0.5

1 36000 3

5 34000 10

15 32000 Response 20

30 30000 40

50 28000

26000

24000 0 50 100 150 200 250 300 350 Time s

Figure C-1: Sensorgram of the ten diﬀerent standard samples that were used in the Biacore concentration analysis, colored by concentration of Herceptin® (µg/mL).

54 RU Herceptin in Expi293 12000

10000

8000

6000

Relative Response Relative 4000

2000

0 -10 0 10 20 30 40 50 60 Concentration µg/ml

Figure C-2: Standard curve for the Biacore concentration analysis with ten diﬀerent concentrations of Herceptin®, diluted in Expi293F™ Expression Medium.

55 End of report