Protein Aggregation and Host Cell Protein

INVESTIGATING THE IMMUNOGENICITY OF THERAPEUTIC PROTEINS: PROTEIN AGGREGATION AND HOST CELL PROTEIN IMPURITIES A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2016 Kirsty D Ratanji Table of Contents 1 Introduction 15 1.1 Review article: Immunogenicity of therapeutic proteins: Influence of aggregation 15 1.1.1 Abstract 17 1.1.2 Introduction 18 1.1.3 Protein aggregation 19 1.1.4 Methods to reduce aggregation 24 1.1.5 Immunogenicity of biotherapeutics 26 1.1.6 Clinical examples of aggregation associated immunogenicity 32 1.1.7 Screening for aggregation and immunogenicity 38 1.1.8 Concluding remarks 44 1.2 Introduction (cont’d) 45 1.2.1 Immunogenicity testing 45 1.2.2 Biophysical methods for aggregate analysis 52 1.2.3 Thesis aims 59 1.3 Alternative format 63 2 Paper 1: Producing and characterising subvisible protein aggregates for immunogenicity studies 65 2.1 Abbreviations 66 2.2 Abstract 67 2.3 Introduction 68 2.4 Methods 71 2.5 Results 76 2.6 Discussion 86 2 2.7 Acknowledgements 92 2.8 Appendix 93 2.9 References 93 3 Paper 2: Subvisible aggregates of immunogenic proteins promote a Th1-type response 97 3.1 Abstract 99 3.2 Introduction 100 3.3 Materials and Methods 103 3.4 Results 108 3.5 Discussion 124 3.6 Acknowledgements 129 3.7 Supplementary data 130 3.8 References 131 4 Paper 3: Influence of E.coli chaperone DnaK on protein immunogenicity 136 4.1 Abbreviations 138 4.2 Abstract 139 4.3 Introduction 140 4.4 Methods 143 4.5 Results 148 4.6 Discussion 163 4.7 Acknowledgements 168 4.8 References 169 5 Supplementary chapter 174 5.1 Abbreviations 175 3 5.2 Introduction 176 5.3 Methods 177 5.4 Results 182 5.5 Discussion 193 5.6 References 196 6 Discussion 197 6.1 General discussion 198 6.1.1 Revisiting the background and initial aims 198 6.1.2 Biophysical analyses of protein preparations 200 6.1.3 Characterising the immunogenicity of subvisible protein aggregates 201 6.1.4 Impact of aggregation and HCP impurities on protein immunogenicity 206 6.1.5 Predicting immunogenicity 208 6.2 Future work 211 6.3 Concluding remarks 214 7 Bibliography 216 Word count = 60, 456 4 Table of figures 1 Introduction Figure 1.1. Schematic model of protein aggregation 20 Figure 1.2. B-cell activation mechanisms 28 Figure 1.3. Factors that may influence biotherapeutic immunogenicity 32 2 Paper 1: Producing and characterising subvisible protein aggregates for immunogenicity studies Figure 2.1. scFv SEC-MALLS 76 Figure 2.2. SLS temperature ramp analysis of scFv 77 Figure 2.3. Mean protein particle diameter analysis by DLS 79 Figure 2.4. Size analysis of scFv, OVA and mouse albumin aggregates by RICS 81 Figure 2.5. Secondary structure analysis by CD 82 Figure 2.6. TEM analysis of OVA stir aggregates 84 Figure 2.7. Aggregate imaging by AFM 85 3 Paper 2: Subvisible aggregates of immunogenic proteins promote a Th1-type response Figure 3.1. Characterisation of immune responses to scFv: comparisons of monomer and heat stressed aggregates 110 Figure 3.2. Antibody response to scFv: Influence of dose and aggregation status 112 Figure 3.3. Antibody responses to scFv monomer, heat stressed and stir stressed aggregates 114 5 Figure 3.4. Splenocyte culture 3H-thymidine incorporation following ex vivo scFv challenge 116 Figure 3.5 Splenocyte culture IFN-γ and IL-13 cytokine secretion ex vivo following scFv challenge 118 Figure 3.6. Characterisation of immune responses to OVA: comparisons of monomer and stir stressed aggregates 120 Figure 3.7. Splenocyte culture 3H-Thymidine incorporation following ex vivo OVA challenge 122 Figure 3.8. Splenocyte culture IFN-γ, IL-4 and IL-13 cytokine secretion following ex vivo OVA challenge 123 Supplementary figure 3.1. Characterisation of immune responses to scFv following ip or sc routes of administration 130 4 Paper 3: Subvisible aggregates of immunogenic proteins promote a Th1-type response Figure 4.1. scFv purification and aggregation 149 Figure 4.2. A comparison of scFv Fraction M and Fraction A aggregate immunogenicity 150 Figure 4.3. Naïve mouse sera IgG antibody binding to scFv substrates 152 Figure 4.4. DnaK detection in scFv pellets and supernatants, and antibody responses to scFv monomer aggregate spiked with DnaK 155 Figure 4.5. DLS and circular dichroism spectra of native and heat treated proteins 158 Figure 4.6 DnaK detection in mouse albumin pellets and supernatants 159 Figure 4.7 Characterization of immune responses to mouse albumin: comparisons of monomer and heat stressed aggregates 161 6 5 Supplementary chapter Figure 5.1. scFv purification optimisation 182 Figure 5.2. Splenocyte culture 3H-thymidine incorporation following ex vivo scFv challenge 184 Figure 5.3. Splenocyte 3H-thymidine incorporation: dose response to scFv 186 Figure 5.4. The effect of FCS concentration on splenocyte proliferation 187 Figure 5.5. Characterisation of bone marrow-derived dendritic cell membrane marker expression 188 Figure 5.6. The effect of BMDC co-culture with splenocytes on antigen specific proliferation and IFNγ secretion 189 Figure 5.7. Antibody responses to scFv Fraction M, Fraction A and Fraction A aggregate 191 Figure 5.8. Characterisation of immune responses to OVA: comparison of monomer and heat stressed aggregates 192 6 Discussion Figure 6.1. Proposed mechanisms for enhanced IgG2a/IgM production with subvisible protein aggregates 206 7 List of Tables 1 Introduction Table 1.1. Methods for aggregate analysis in therapeutic protein development. 42 4 Paper 3: Subvisible aggregates of immunogenic proteins promote a Th1-type response Table 4.1. Statistical analysis of antibody quantification by serum dilution 162 8 Abstract Institution: The University of Manchester Name: Kirsty D Ratanji Degree title: PhD Immunology Thesis title: Investigating the immunogenicity of therapeutic proteins: protein aggregation and host cell protein impurities Date: 2016 The development of anti-drug antibodies (ADA) against therapeutic proteins can impact upon drug safety and efficacy. This is a major challenge in the development of biotherapeutics. Various factors have the potential to contribute to protein immunogenicity and the production of ADA. Protein aggregation is one of these factors, though the mechanisms underlying aggregate immunogenicity are poorly understood. In this thesis the effect of protein aggregation on immunogenicity has been investigated. The thermal and/or mechanical stresses required in order to achieve subvisible aggregates of three test proteins were determined. Stressed preparations of proteins were characterised using a suite of biophysical techniques, including dynamic light scattering and circular dichroism. The immunogenic potential of subvisible aggregates of a humanised single chain variable fragment (scFv) and ovalbumin (OVA) was studied following intraperitoneal exposure in BALB/c strain mice. Monomeric proteins induced a T helper (Th) 2 dominant immune response, but when aggregated, the responses gained a Th1 phenotype, with a significant increase in the antigen- specific IgG2a antibody response. Cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were also consistent with aggregated preparations of OVA inducing a Th1-type response. Host cell protein (HCP) impurities can also contribute to immunogenicity. Mass spectrometry analysis of an scFv preparation identified the presence of the Escherichia coli (E.coli) heat shock protein DnaK, amongst other HCP, as an impurity. Protein preparations free from DnaK were spiked with recombinant E.coli DnaK to mimic the HCP impurity. The effect of DnaK on the immunogenicity of aggregated and monomeric scFv preparations was then investigated. BALB/c mice were immunised with monomeric and aggregated preparations, with and without E.coli DnaK at 0.1% by mass. Aggregation alone resulted in an enhanced IgG2a antibody response, and the presence of DnaK increased this further. Comparable investigations were also conducted using mouse albumin; here an increase in immunogenicity was observed with protein aggregation, and the presence of DnaK was found to increase the IgG2a response. Collectively, the evidence presented in this thesis shows that aggregation can impact upon the magnitude and characterof induced immune responses, and that subvisible aggregation promotes a Th1 immune skewing. Additionally, E.coli HCP DnaK enhances protein aggregate immunogenicity, which indicates that heat shock proteins, as a class of HCP, could have an adjuvant-like effect on biotherapeutic aggregates. 9 Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. Copyright Statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions.

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