2015 Archived Content
Cambridge Healthtech Institute’s Second Annual
Protein Aggregates and Particles
Tools and Techniques for Effective Prediction and Analysis of Aggregates and Particles
5-6 November 2015
Aggregation not only impacts a product’s developability, but can also affect its stability, formulation, and immunogenicity. Aggregation can occur at any stage from development to manufacturing, thus the detection, characterization and monitoring
of all aggregates, visible, sub-visible and submicron particles are becoming a major task in drug development. At the Protein Aggregates & Particles conference, we aim to uncover the underlying mechanism of aggregation, from which
to develop tools to detect and characterize aggregates and particles, and ultimately to design novel biotherapeutics with reduced aggregation.
Final Agenda
Day 1 | Day 2 | Speaker Biographies | Download Brochure
: Unpublished Data | 
: Case Study
Wednesday, 4 November
07:45 Registration and Morning Coffee
08:30 Chairperson’s Opening Remarks
Salvador Ventura, Ph.D., Professor, Biochemistry and Molecular Biology, Institute of Biotechnology and Biomedicine, University of Barcelona
KEYNOTE PRESENTATION
08:35 Understanding (and Controlling) Aggregation of Antibody-Drug Conjugates
Fred Jacobson, Ph.D., Staff Scientist, Kadcyla™ Technical Development Leader, Protein Analytical Chemistry,
Genentech, Inc.
09:20 Engineering and Screening for Antibodies with Favourable Developability Properties
Chris Lloyd, Ph.D., Senior Scientist, Antibody Discovery and Protein Engineering, MedImmune
Undesirable physicochemical properties, such as aggregation and chemical degradation may preclude the development of therapeutic antibodies, due to potential issues during manufacturing, storage and in vivo administration. Avoiding such properties early
on in drug discovery is therefore desirable. My presentation will highlight practical screening and antibody engineering strategies that are effective in identifying lead candidates suitable for drug development.
09:50 Stable Human Antibody Therapeutics and Phage Display Libraries through Engineering of Variable Domains
Daniel Christ, Ph.D., Associate Professor of Medicine; Head, Antibody Therapeutics, Immunology Program, Garvan Institute of
Medical Research
Human antibody variable domains often display poor biophysical properties and a propensity to aggregate. We have identified aggregation hotspots in the CDR regions of antibody variable VH and VL domains, and have developed generally applicable strategies
to overcome these limitations. Here we outline the application of the technology to human antibody therapeutics and antibody phage display libraries.
10:20 Innovative Technologies to Improve the Characterization of Protein Aggregates
Matthew Brown, Ph.D., Scientist, Malvern Instruments Ltd.
Understanding the process of protein aggregation is a key component of QbD approaches during biotherapeutic development or deviation resolution of legacy products. Advanced characterization technologies, now available to the biopharmaceutical industry,
offer detailed insights into protein behavior to improve product stability and process knowledge and understanding.
10:50 Coffee Break in the Exhibit Hall with Poster Viewing
11:45 Selected Poster Presentation: AF4 and SEC as Tools for Protein Aggregation Studies
Mats Leeman, Ph.D., Senior Research Scientist, Solve Research and Consultancy AB
Proteins with the potential to aggregate must be monitored in basic research as well as during product development. The conventional characterization technique, size exclusion chromatography (SEC), is the proper choice as long as the proteins are small
and stable. However, the technique is limited in the capacity to characterize large proteins and protein aggregates due to the possibility for shear or adsorption induced artefacts. This is one of the reasons why FDA requires validation with orthogonal
methods during development of protein based therapeutics. One of the recommended orthogonal methods to use is asymmetrical flow field-flow fractionation (AF4), where separation occurs in an open channel without packing material. This result in lower
shear forces which enables the behaviour of proteins and aggregates to be studied with minimal impact of the analytical method. However, the technique is still not that widely spread. Protein size and aggregation level has been characterized using
SEC and AF4 in combination with multi-angle light scattering (MALS) and UV detection. In the poster, the results from the two analytical techniques will be compared and the differences in instrument capabilities discussed to reveal when to use which
technique and why.
12:00 Formulation Development Based on Sub-Visible Particle Morphology Using Micro-Flow Imaging
Malin Persson, Ph.D., Senior Research Scientist, Biopharm Formulation Development, Novo Nordisk A/S
12:30 Simultaneous Detection
of Protein Aggregation & Affinity Measurements in a Single SPR Experiment
Aaron Martin, Senior Principal Scientist, Research & Development, SensiQ Technologies Inc
No SPR-biosensor has been capable of delivering information on drug affinity and detect protein aggregates simultaneously in the same experiment. SensiQ Technologies presents data from a current collaboration with Genentech describing how this limitation
can be eliminated via simultaneous detection of both protein aggregation and affinity determination in a single experiment as enabled by Pioneer FE SPR.
12:45 Enjoy Lunch on Your Own
14:00 Chairperson’s Remarks
Jennifer McManus, Ph.D., Lecturer, Department of Chemistry, National University of Ireland Maynooth
14:05 Understanding Protein Aggregation in Pharmaceutical Products
Ankit Patel, Ph.D., Scientist, Late Stage Pharmaceutical Development, Genentech, Inc.
Protein aggregates are common degradation products for therapeutic proteins. Due to the complex nature of protein aggregation, the underline mechanisms and their potential biological impacts are not always well understood. In this presentation, we will
give an overview of protein aggregation phenomenon for a few pharmaceutical products. The mechanism and implication of these protein aggregates will be reviewed and discussed.
14:35 Aggregation Analysis at High and Low Protein Concentrations
Jennifer McManus, Ph.D., Lecturer, Department of Chemistry, National University of Ireland Maynooth
Aggregation of proteins may occur by a number of different mechanisms, which can lead to a range of aggregate types. Using a range of analytical techniques the formation of protein aggregates by various mechanisms has been assessed at low and where possible,
at moderate to high protein concentrations. The effect of sugars on protein stability will also be discussed.
15:05 Effects of Protein Aggregation on
Uptake, Processing and Presentation by Dendritic Cells – A Case Study
Anja Langenkamp, Ph.D., Principal Scientist, Immunopathology, Pharmaceutical Sciences, Roche Pharmaceutical Research & Early Development, Roche Innovation Center Basel
Studies show that tolerance can be broken in transgenic mouse models by harshly stressed protein therapeutics. However, the underlying mechanisms and relevance for humans remain unclear. Thus, we studied the influence of aggregation on the uptake,
presentation and activation of dendritic cells - the key regulators for adaptive immunity. First results will be presented that provide mechanistic insights into the properties of monomeric and aggregated variants of a therapeutic monoclonal antibody.
15:35 Refreshment Break in the Exhibit Hall with Poster Viewing
16:15 Tuning the Aggregation Propensity of Protein Structures
Salvador Ventura, Ph.D., Professor, Biochemistry and Molecular Biology, Institute of Biotechnology
and Biomedicine, University of Barcelona
This talk presents a method to overcome the current limitations of predicting aggregation by sequencing. The AGGRESCAN3D (A3D) server overcomes the limitations by taking into account the protein structure and the experimental aggregation propensity
scale. The identified aggregation-prone residues can be virtually mutated to design variants with increased solubility. Additionally, the A3D server takes into account the dynamic fluctuations of protein structure in solution, which may influence
aggregation propensity.
16:45 Rational Design of Protein Solubility
Michele Vendruscolo, Ph.D., Professor, Department of Chemistry, University of Cambridge
I will discuss the extent to which the solubility and aggregation of proteins are related to the physico-chemical properties of their amino acid sequences. Based on these properties, I will present methods for the prediction of the solubility
and aggregation of proteins and illustrate how these methods can be of practical interest and importance.
17:15 Standards, Measurements, and Analysis for Protein Particle Characterization
Richard Cavicchi, Ph.D., Scientist, Bioprocess Measurements Group, National Institute of
Standards and Technology
Concentration measurements of protein aggregates obtained on orthogonal instrument types often differ significantly. NIST is developing a protein aggregate standard that simulates aggregate properties for sizes from 1 µm to visible particles.
In addition, we are using a custom microfluidic device that compares orthogonal measurements on single particles to analyze microfabricated particles of defined dimensions and protein aggregates to discern the effects of shape and porosity.
17:45 Oral Poster Presentation
High Resolution Antibody Modeling Identifies "Design-able" Antibody
Hiroki Shirai, Ph.D., Executive Fellow, Bioscience Research Laboratories, Astellas Pharma Inc.
“Antibody informatics” can be considered as a set of technologies to select and/or design antibodies to remove various obstacles for drug discovery, and antibody modeling is the basis of all above(ref.1). In the course of drug
discovery, researchers should select a limited numbers of lead antibodies to be optimized among many candidates, but the difficulty in antibody engineering sometimes depends on antibody sequence. If one can identify “design-able”
antibodies which are easy to build models for design with high accuracy, it would be helpful for rational selection. We set up high resolution antibody modeling technology especially focused on CDR-H3 modeling by integrated approach
by informatics and simulation(ref.2). It proved competitive at a world wide assessment(ref.2,3). We can predict “degree of accuracy” of model for the particular sequences. Based on the “accuracy prediction”,
we can identify “design-able” antibodies. We are seeking some collaborators to maximize the benefit.
18:15 Networking Reception in the Exhibit Hall with Poster Viewing
19:15 End of Day
Day 1 | Day 2 | Speaker Biographies | Download Brochure
Thursday, 5 November
08:00 Morning Coffee
08:30 Chairperson’s Remarks
Antonio Ribeiro, Ph.D., Professor, Pharmaceutical Technology, University of Coimbra Azinhaga de Santa Comba
08:35
Nanoparticle Tracking Analysis for Studying Aggregation Profile of Particles
Antonio Ribeiro, Ph.D., Professor, Pharmaceutical Technology, University of Coimbra Azinhaga de Santa Comba
Subvisible particles do not constitute a mass fraction to be quantified by using size exclusion chromatography (SEC). Moreover, SEC requires high dilution of the sample, which itself can change the aggregation profile. Nanoparticle tracking
analysis (NTA) can count and measure size individual species in undiluted particles and may be more appropriate than SEC for studying particles aggregation. Moreover, using fluorescent labeled particles, NTA may allow distinguishing
individual from aggregated particles.
09:05 Hydrodynamic Diameter (By DLS) and Molecular Mass Measurement (By SLS After FFF) Can Characterise
Aggregation Level of A HMW Protein Not Characterisable by SE-HPLC
Peter Matthiessen, Ph.D., Senior Manager, Formulation, Fill/Finish, Baxter Innovations GmbH
The aggregation level of a HMW Protein cannot be quantified by SE-HPLC. Field flow fractionation can partially separate potential aggregates and SLS was used for quantitation of molecular mass increase. Also DLS was used to monitor hydrodynamic
diameter increase by potential aggregates. Various temperature stress conditions in liquid and lyophilized form and mechanical stress by shaking or stirring, both known to induce aggregation, were investigated by DLS and SLS. The correlation
of aggregation and subvisible particle levels was investigated.
09:35
New, Orthogonal Methods to Detect Protein Aggregation at High and Low Concentration
Tudor Arvinte, Ph.D., Chairman & CEO, Therapeomic, Inc.; Titular Professor of Biopharmaceutics, School of Pharmacy, University of Geneva
Based on case studies, different orthogonal methods will be presented that permit detection and characterisation of protein aggregates and particulate matter in liquid formulations of biopharmaceuticals at high and low a protein concentrations.
A method alone cannot provide absolute information on the aggregates present in a sample. By using different methods to analyse a sample, we can obtain strong conclusions and a broad picture on the protein aggregation states and particulates
present in the solutions.
10:05 Protein-Protein Interactions, in Multi Protein-Albumin or Peptide-Albumin Co-Formulations Using Recombinant Human Serum Albumin (rHSA) to Prevent Aggregation
Darrell Sleep, Director, R&D, Novozymes Biopharma
It is well-known that rHSA has the ability to prevent protein and peptide aggregation. At drug concentrations <1mg/mL coating of hydrophobic and hydrophilic surfaces of the primary packaging material and process equipment is expected
to prevent depletion and surface induced aggregation. When aggregation is independent of the surfaces (>1mg/mL) the mechanism of aggregation prevention is less well understood and is likely to depend on the aggregation pathway of
the drug. Some case studies suggest that hydrophobic patches on rHSA interact with hydrophobic patches on the drugs forming an rHSA-drug complex where such surface areas are shielded and hereby preventing self-association. Other case
studies suggest an excluded volume effect with no rHSA-drug complex formation.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 Detection and Characterisation of Visible, Sub Visible Particles and Other Aggregates: Achievements and Challenges
Anacelia Rios Quiroz, MSc, Late Stage Pharmaceutical and Process Development,
Pharmaceutical Development & Supplies, PTD Biologics Europe, (PTDE-PF), F. Hoffmann-La Roche, Ltd.
The talk will give an overview of required and commercially available counting methodologies for detection of protein aggregates and visible and sub visible particles (SbVP); species ubiquitously present in protein formulations. Focus
will be SbVP as they are gaining attention regarding immunogenicity and quality attributes. Lack of a well-defined methodology for SbVP makes it important to increase our knowledge of emerging instruments’ performance. Applicability
towards the assessment of a meaningful array of particle counting techniques will be discussed.
11:45 Chemical Kinetics and Microfluidic Sizing for the Analysis of the Aggregation of Therapeutic Proteins
Paolo Arosio, Ph.D., Marie Curie Postdoc Fellow, Department of Chemistry, University of Cambridge
In this presentation, we show how chemical kinetic analysis can identify the protein aggregation path-ways at the molecular level. We demonstrate the potential of this approach by analyzing the aggregation mechanisms of different IgGs
and of human insulin under conditions that are relevant for downstream and formulation. Finally, we show a novel microfluidic technique that enables the sizing of polydisperse protein samples under native conditions on a second timescale.
12:15 Enjoy Lunch on Your Own
13:00 Dessert Break in the Exhibit Hall with Poster Viewing
13:30 End of Protein Aggregates & Particles
: Unpublished Data | : Case Study
Day 1 | Day 2 | Speaker Biographies | Download Brochure