The phenomena of protein aggregation is a complex conundrum that impacts biopharmaceutical development at virtually every stage. All mechanisms of aggregation are not conclusively known, but the industry must use every effort to characterize and control this condition – and to reflect these efforts in regulatory filings. A changing landscape of assays, instrumentation and processes support the need to collect, analyze and report this data. Requirements for understanding the correlation between aggregates and immunogenicity shape analytical and clinical studies. In addition, aggregation resulting from new higher concentration protein formulations yields new challenges in drug product development, delivery systems and manufacturing processes. The PEGS Protein Aggregation and Stability in Biopharmaceutical Products meeting offers important scientific updates and a forum for dialog among the stakeholders in this challenging arena.


12:00 pm Registration

12:35 Luncheon in the Exhibit Hall with Poster Viewing

1:40 Chairperson’s Remarks

Ankit Patel, Ph.D., Scientist, Genentech


New Aggregation Properties of Therapeutic Proteins Revealed by New Analytical Methods

Unpublished Data and Case Study Tudor Arvinte, Ph.D., Professor, Biopharmaceutics, University of Geneva; CEO, Therapeomic, Inc.

Proof and understanding of protein aggregation phenomena can be achieved only through a convergence of evidence from numerous lines of inquiry using classical and orthogonal analytical methods. Based on case studies, different orthogonal methods will be presented. No single result form these methods denotes a general, absolute proof of the aggregation state, but together they point to unmistakable conclusions and reveal a broad picture on the protein aggregation states.


2:20 Understanding Agitation-Induced Aggregation and Predicting Aggregation Propensity of Monoclonal Antibodies

Ankit Patel, Ph.D., Scientist, Genentech

While surfactants are commonly used to prevent agitation-induced aggregation, they are typically not present at earlier stages in the monoclonal antibody production process and may be significantly diluted during dose preparation in infusion systems. Several interfacial analytical techniques have been utilized to evaluate the dynamics of adsorption to the air-water interface and evaluate interfacial protein-protein interactions leading to aggregation. In addition, a simple method to predict the aggregation propensity of monoclonal antibodies will be presented.

2:50 Predicting Aggregation-Prone Sequences in Proteins

Case StudyJoost Schymkowitz, Ph.D., Principal Investigator, VIB Switch Lab, KULeuven, University of Leuven

As proteins have only evolved to be soluble at the concentration at which they naturally occur, they often tend to aggregate when employed in applications that require much higher concentrations. The propensity of proteins to aggregate is determined by their primary sequence and can be predicted with reasonable accuracy. Although computational assessment of protein quality is still in its infancy, it holds great promise to help shape the protein therapeutics of the future. I will discuss a number of key determinants and how to employ them to engineer polypeptide sequences to obtain superior proteins, with a particular focus on antibodies.

3:20 Fully Automated Antibody Structure Prediction from Sequence

Ken Butenhof, Ph.D., Advisory Field Applications Scientist, BIOVIA

We have captured the best practices developed during the Second Antibody Modeling Assessment in a single fully automated antibody structure prediction method implemented in Discovery Studio through a Pipeline Pilot protocol. In this assessment we predicted the structure of eleven unpublished antibody Fv fragments. The prediction method utilizes fully automated multiple framework and antibody CDR Loop template selection, alignment and modeling. The resulting models are compared to the revealed X-Ray crystal structures and manually derived models. The comparison demonstrates that Biovia Antibody Modeling Cascade is capable of generating quite accurate models without manual intervention for the framework and the canonical CDR regions, with RMSDs to the X-ray structure on average below 1 A˚ for most of these regions. Furthermore, local geometry assessment scores are similar to that of the target X-ray structures. This method is applicable to Fv, Fab, and scFv sequences and is suitable for submission of multiple antibody sequences.

3:35 Sponsored Presentation (Opportunity Available)

3:50 Refreshment Break

4:20 Problem-Solving Breakout Discussions

5:20 End of Day

5:15 Registration for Dinner Short Courses


8:00 am Morning Coffee

Formulation and Process Strategies to Control Protein Aggregation

8:30 Chairperson’s Remarks

Jifeng Zhang, Ph.D., Head, Drug Product Analytics and Characterization, Drug Delivery and Delivery Development, MedImmune

8:35 Predicting Protein Aggregation in Lyophilized Solids Using Hydrogen Deuterium Exchange with Mass Spectrometric Analysis

Case Study
Elizabeth M. Topp, Ph.D., Dane O. Kildsig Chair and Head, Industrial and Physical Pharmacy, Purdue University

Our group has developed solid-state hydrogen deuterium exchange (ssHDX-MS) with mass spectrometric analysis to probe protein conformation and matrix interactions in lyophilized solids with peptide-level resolution. This case study presents ssHDX-MS results for equine myoglobin (Mb), demonstrating a strong correlation between Mb aggregation during one year of storage in the solid state and ssHDX-MS results immediately following lyophilization.

9:05 Thermal Unfolding and Aggregation of Human IgG1 Fc Fragment: the Relevance of Solution Composition

Case StudyJifeng Zhang, Ph.D., Head, Drug Product Analytics and Characterization, Drug Delivery and Delivery Development, MedImmune

This presentation highlights the importance of understanding the specific ion effect on the protein behavior with respect to the biophysical properties of proteins during formulation development.

9:35 Combined In Silico and Biophysical Approaches to Enhance Aggregation Resistance of Biologics

Unpublished DataMichaela Blech, Ph.D., Associate Director Protein Science, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany

To meet the requirements of current clinical indications with respect to drug delivery, highly concentrated therapeutic protein drugs up to 100-200 mg/ml are necessary. In order to achieve such concentrations, a number of protein properties like solubility, self-association, solution viscosity, and protein aggregation have to be controlled. Our current strategy uses a combination of in silico modeling approaches to identify critical amino acids or sequences, as well as biophysical tools to understand conformational and colloidal properties of the protein candidate at the molecular level and the impact on macroscopic solution properties.

10:05 Coffee Break

10:35 The Effect of Resin Pore Size, Particle Diameter, and Ionic Capacity on the Resolution of Monoclonal Antibody Monomer from Aggregate During Preparative Cation Exchange Chromatography

Unpublished Data
Benjamin Guzman, Senior Associate Scientist, Purification Process Development, Amgen

This work focuses on understanding the effect of resin properties on the removal of aggregates from a mAb feed stream utilizing a DOE approach. We will present results examining nine GE Healthcare Capto™ S Impact CEX prototype resins with variations in pore size, particle diameter, and ionic capacity within a designed space. In addition to the effect on resolution of aggregate and monomer, implications for other quality attributes and process integration will be discussed.

11:05 High Throughput Screening for Drug Like Properties

Unpublished Data
Patricia Lowden, Scientist, Protein Production and Analytics Department, Eleven Biotherapeutics

Eleven Biotherapeutics creates novel engineered proteins for ophthalmic indications.  As part of our drug discovery efforts, drug candidates are assessed for drug like properties to ensure manufacturability.   The analytical methods used to assess drug like properties include extrinsic fluorescence and light scattering techniques, which address thermal and colloidal stability.   Methods known to correlate to second virial coefficient, such as interaction parameter by DLS and self-interaction nanoparticle spectroscopy, are compared.  These methods are used to predict high concentration solution behavior, and may be useful in pre-formulation screens.

11:35 Targeted Mutagenesis of a Therapeutic Human Monoclonal IgG1 Antibody Prevents Gelation at High Concentrations

Paul Casaz, Ph.D., Research Scientist, University of Massachusetts Medical School

A human monoclonal antibody was found that forms an opaque white gel at concentrations >30 mg/mL and temperature <6°C. Gelation was prevented by a low pH or high sodium chloride concentrations but a protein engineering solution was also sought. The substitution of a glutamic acid residue in the heavy chain variable region framework with lysine was found to prevent gelation.

12:05 pm Detection, Analysis, Purification and Processing of Monoclonal Antibody Aggregates

Unpublished Data and Case Study
Raja Ghosh, Ph.D., Professor, Canada Research Chair in Bioseparations Engineering, McMaster University

Monoclonal antibodies (mAbs) which are an important category of biopharmaceuticals are particularly prone to aggregation. The presence of aggregates in formulations is undesirable. Recent studies have shown that the hydrophobicity of a mAb increases with the degree of aggregation and aggregates can be fractionated using hydrophobic interaction membrane chromatography (HIMC). Different case studies where HIMC is used to detect, analyse, separate and process mAb aggregates will be discussed in this presentation.

12:35 Luncheon Presentation (Sponsorship Opportunity Available)
or Enjoy Lunch on Your Own

1:05 Refreshment Break

Developability Evaluation and Optimization

1:35 Chairperson’s Remarks

Peter Ihnat, Ph.D., Principal Research Scientist, Drug Product Development Pre-Formulation, AbbVie

1:40 Defining Antibody Developability Rule of Thumb by It’s Self- and Cross-Interactions

Unpublished Data
Yingda Xu, Ph.D., Associate Director, Protein Analytics, Adimab

Developability issues, such as aggregation, low solubility, high viscosity and poor pK can usually be linked to antibody self- or cross-interactions. Ideally, high-throughput screening assays can be applied to catch candidates possessing these undesirable properties to minimize downstream risks early in the process. Here we report the application of such assays on a large panel of clinical stage mAbs to develop a simple model for antibody developability rule of thumb.

2:10 Anion-Protein Interactions and Relevance to Solution Properties and Stability

Unpublished Data
Peter Ihnat, Ph.D., Principal Research Scientist, Drug Product Development Pre-Formulation, AbbVie

Cosolutes stabilize thermodynamic and colloidal properties of the protein to prevent aggregation in solution and improve hydration of the protein. We used light scattering techniques, thermal analysis and viscometry to probe the interactions between a series of anions and model proteins in solution. This information supports better insight into the stabilizing roles of common excipients and an improved rational approach to developing stable protein solution formulations.

2:40 Mutational Approaches to Improve the Biophysical Properties of Human Single-Domain Antibodies

Case Study
Jamshid Tanha, Ph.D., Senior Research Officer, National Research Council, Canada

Various mutational approaches for improving the biophysical properties of VH and VL single-domain antibodies have been described. Here we zoom in on one particular approach, namely disulfide engineering approach, which improves the stability VHs and VLs. The approach appears to be universally applicable across all VHs and VLs.

3:10 Concentration Dependent Viscosity of Monoclonal Antibody Solutions: Explaining Experimental Behavior in Terms of Molecular Properties

LiLiLi Li, Ph.D., Senior Principal Scientist, Pfizer

Antibody variable regions (Fv) were analyzed to explain concentration dependent viscosity behaviors of 11 mAbs. Net charge, x-potential and pI of the Fv regions correlate with viscosities of the mAbs at high concentrations. Coarse-grained molecular dynamics simulations of two problematic mAbs suggest that negative net charge on their Fv regions facilitates attractive inter-molecular interactions, leading to formation of transient antibody networks. These networks cause the problematic mAbs to diffuse slowly.

3:40 PEG-Induced Liquid-Liquid Phase Separation in Protein Solutions

YingWangYing Wang, Ph.D., Research Associate, Physics and Biological Physics, Massachusetts Institute of Technology

Globular proteins, e.g. antibodies, can lose their solubility without unfolding, both in-vivo and in pharmaceutical formulations. Proteins with low colloidal stability are associated with some diseases and can cause problems in the development of protein drugs. In this talk, I will present a universal method for quantifying colloidal stability of protein solutions using PEG-induced liquid-liquid phase separation. The physical basis of liquid-liquid phase separation and depletion interaction will be discussed.

4:10 End of Conference

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