The phenomenon 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. And 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 Biopharmaceuticals meeting offers important scientific updates and a forum for dialog among the stakeholders in this challenging arena.

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Recommended Pre-Conference Short Course*

SC10: Analyzing and Rationalizing Protein-Protein Interactions

*Separate registration required


12:35 pm Luncheon in the Exhibit Hall with Poster Viewing

1:40 Chairperson’s Remarks

Shantanu Sule, Ph.D., Senior Engineer, Parenteral Drug Product Development, Biogen


Subvisible Particle and Cell-Based Immunogenicity Characterization of Antibody Therapeutics Pumped Through Clinical Intravenous Infusion Systems: Current In-line Filters are not Adequate

John F. Carpenter, Ph.D., Professor, Pharmaceutical Sciences; Co-Director, Center for Pharmaceutical Biotechnology, University of Colorado Anschutz Medical Center

Numerous therapeutic proteins are administered by intravenous (IV) infusion and can cause immunogenicity and infusion reactions in patients. We found that IV saline contains > 20 million nano- and > 20,000 microparticles per ml; particle levels increased upon addition of therapeutic protein solution. The particles, even in solutions passed through in-line IV filters, stimulated several in vitro cellular functions associated with immunogenicity and/or infusion reactions including T cell proliferation, dendritic cell activation and cytokine secretion.


2:20 Predicting High-Concentration Protein-Protein Interactions, Aggregation, and Phase Behavior

Christopher J. Roberts, Ph.D., Associate Professor, Chemical & Biomolecular Engineering, University of Delaware

Proteins are increasingly formulated and delivered at high concentrations (~ 100 mg/mL or higher). This poses a number of challenges, including increased propensity for reversible and irreversible aggregation, elevated viscosity, and phase separation. This talk focuses on combined experimental and modeling approaches to predict the behavior of proteins at high concentrations using different levels of molecular and thermodynamic modeling. Examples include monoclonal antibodies and globular proteins.

2:50 Impact of Linker-Payload on the Physical Stability of Cysteine-Based Antibody-Drug Conjugates

Jianxin_GuoJianxin Guo, Ph.D., Principal Scientist, Pfizer

The impact of linker-payload on the physical stability of cysteine-based Antibody-Drug Conjugates (ADC) will be addressed with case studies. The application of biophysical techniques on the evaluation of structural stability and aggregation propensity will be elaborated. The implication of higher order structural characterization for process and formulation development will be illustrated.

3:20 Counting and Sizing Protein Aggregates Down to 0.15 um in sub-mL Volumes by New Focused-Beam Light Scattering Technology

David Nicoli, Ph.D., Vice-President, Research & Development, Particle Sizing Systems

A new single-particle optical sizing (SPOS) technique uses light scattering from a focused laser beam to count and size protein aggregates down to 0.15 um at high concentrations, inaccessible by conventional light obscuration and scattering. Addition of a hybrid light obscuration/scattering sensor extends the upper size limit to 150 um. Analysis can be made on sub-mL samples of high viscosity resulting from high protein concentrations, with conservation of the sample.

3:50 Refreshment Break

4:20 Protein Aggregation Kinetics; Monitoring and Mechanisms Under Applied Stressors

Wayne_ReedWayne F. Reed, Ph.D., Professor, Physics; Founding Director, PolyRMC, Tulane University

Time dependent light scattering signatures are used to measure aggregation rates and to make deductions about aggregation mechanisms. Simultaneous Multiple Sample Light Scattering (SMSLS) allows many samples under different stressors to be measured independently. Results focus on thermal and mechanical stressors and differentiating kinetics and mechanisms. Real time SMSLS data allow rational scheduling of complementary analyses, such as GPC and DSC. Relations between scattering and fluorescence based kinetics are also compared.

4:50 Rapid Characterization of Biotherapeutic Proteins by Size Exclusion Chromatography Coupled to Mative Mass Spectrometry

Michael_LeissMichael Leiss, Ph.D., Lab Manager, Development Analytics Roche Diagnostics GmbH

Dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats. An approach combining UHPLC size exclusion separation with native electrospray ionization mass spectrometry for the simultaneous identification and quantification of size variants in recombinant antibodies is presented. It enables us to study various bispecific antibody size variants and facilitates the detailed analysis of low-abundant and non-covalent size variants.

5:20 End of Day

5:15 Registration for Dinner Short Courses

Recommended Dinner Short Course*

SC12: Strategic Bioassay Design and Analysis

*Separate registration required

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8:00 am Registration and Morning Coffee

Understanding Aggregation

8:30 Chairperson’s Remarks

Dana Filoti, Ph.D., Senior Scientist, Protein Analytics, AbbVie

8:35 Understanding the Role of Aggregation in the Immunogenicity of Biotherapeutic Proteins

Jeremy_DerrickJeremy Derrick, Ph.D., Professor, Molecular Microbiology, University of Manchester

We have compared the immunological responses to immunization with an scFv fragment in monomeric and aggregated forms in a mouse model; the results were indicative of a Th1-type response. In addition, our data also indicate that the heat shock protein DnaK, a common HCP, could play a role in modulating the immune response; the implications for our understanding of the immunogenicity of biotherapeutic proteins will be discussed.

9:05 Functional and Structural Characterization of Process-Related Aggregate Species of an IgG4 Monoclonal Antibody

Flaviu_GruiaFlaviu Gruia, Ph.D., Scientist, Analytical Biotechnology, MedImmune

This study details the characterization of aggregate species of an IgG4 monoclonal antibody. Monomer, dimer and high-molecular-weight (HMW) species were chromatographically separated. A comparative analysis of the three species was performed. Progressive oxidation and conformational differences were detected for aggregate species. The activity of the HMW species was higher than dimer but lower than monomer species. Further characterization determined that non-covalent mechanisms provided the main path for protein aggregation.

9:35 The Role of Surfactants in Managing Protein Aggregation

Hanns-Christian_MahlerHanns-Christian Mahler, Ph.D., Head, Drug Product Services, Lonza AG

Non-Ionic surfactants are commonly used in protein formulations to protect the protein from aggregation and particle formation. Polysorbates can undergo autooxidation, cleavage at ethylene oxide subunits and hydrolysis of fatty acid ester bonds. Possibly, the cascade of degradation may impact product quality, e.g., formation of hydroperoxides and degradation products that may impact protein stability. This talk aims to discuss benefits and concerns related to the use of surfactants in formulations.

10:05 Coffee Break

10:35  (SPEAKER HAS CANCELLED: SESSION WILL BEGIN AT 11:05) New Insight into the Stability of Concentrated Protein Solutions from a Combination of Light, Neutron and X-Ray Scattering, Viscometry and Computer Simulations

 Anna_StradnerAnna Stradner, Ph.D., Associate Professor, Physical Chemistry, Lund University

Many biopharmaceuticals require a single dose delivery at high concentration, where the stability and the resulting viscosity are very difficult to control. We show how we can use a combination of advanced characterization techniques such as small-angle neutron (SANS) and x-ray scattering (SAXS) or 3D cross correlation light scattering (3DDLS), combined with state-of-the-art computer simulations to assess and predict interparticle interactions, stability and flow behavior of concentrated solutions of biopharmaceuticals.

11:05 Aggregate Recursors and Aggregation Kinetics

Dana_FilotiDana Filoti, Ph.D., Senior Scientist, Drug Product Preformulations, AbbVie

Due to the complex nature of protein aggregation kinetics, the underline mechanistic truths and physiological consequences of the solubility of proteins are not always well understood. In this presentation, we will discuss the time-dependent protein aggregation mechanism, particle formation and the nature of electrostatic intermolecular interactions governing protein aggregation kinetics at low and high concentrations for a monoclonal antibody.


11:35 Profiling Product Quality & Stability for Process Development

Christine_ChanChristine P. Chan, Ph.D., Principal Scientist and Technical Lead, Global Manufacturing Science and Technology, Genzyme - a SANOFI Company

Protein therapeutics encounters many external factors that influence aggregation and stability during the manufacturing process stages. Due to the complexity of biopharmaceuticals, a combination of orthogonal analytical methods is often required to facilitate evaluation of process impact and define relevant operational controls. This presentation will discuss strategies in product testing and review case studies on characterization of different proteins in support of process development.

12:05 pm A Novel Screening Method to Assess Developability of Antibody-Like Molecules

Melissa_GeddieMelissa Geddie, Ph.D., Principal Scientist, Merrimack Pharmaceuticals

The discovery of antibodies that bind to particular targets with high affinity is now a routine exercise. However, it is still challenging to identify antibodies that in addition to having the desired biological effect also express well, remain soluble at different pH levels, remain stable at high concentrations, can withstand high shear stress, and have minimal non-specific interactions. Here, we present a simple HPLC based screening method to assess these developability factors earlier in discovery process.

Albumedix12:35 Luncheon Presentation:  Recombinant Human Albumin an Effective Approach for Stabilization of Hard-to-Formulate Biopharmaceuticals

Darrell Sleep, Ph.D., CSO, Albumedix Ltd.

The expanding field of biotherapeutics gives promise for improvement of several treatment options. Many of the biopharmaceuticals found to be efficacious, however continue to face ex vivo instability challenges. Recombinant human albumin, however, can potentially alleviate these shortcomings. The mechanisms by which albumin help stabilize biopharmaceuticals are multiple and dependent on the specific drug. Data is presented here that exemplifies these different mechanisms.

1:05 Refreshment Break


1:35 Chairperson’s Remarks

Camellia Zamiri, Ph.D., Scientist, Late Stage Pharmaceutical Development, Genentech

1:40 Polysorbate 20 Hydrolysis and Exploring Options for Mitigating Particulate Formation

Camellia_ZamiriCamellia Zamiri, Ph.D., Scientist, Late Stage Pharmaceutical Development, Genentech

Visible particles in mAb Drug Products have been identified upon storage at 2-8°C. Studies indicated hydrolytic (enzymatic) polysorbate 20 degradation in mAb formulations resulted in accumulation of fatty acids. At low temperatures (e.g. 2-8°C), fatty acids can exceed their solubility limit and form visible particles. Effect of polysorbate 20 source on particle formation will be presented. In addition, for screening studies selection of enzyme and method conditions that mimic polysorbate 20 degradation profile in mAb products will be discussed.

2:10 Efficient Membrane Chromatography Devices for Monoclonal Antibody Separation

Raja_GhoshRaja Ghosh, Ph.D., Professor, Chemical Engineering, McMaster University

Column chromatography, which is widely used for monoclonal antibody aggregate separation is slow and poorly scalable. Membrane chromatography is a fast alternative that is increasingly being used in the biopharmaceutical industry. However, resolution of separation obtained with currently available devices tends to be poor. Novel membrane chromatography devices suitable for high-resolution protein purification, and their used for monoclonal antibody aggregate separation will be discussed in this presentation.

2:40 Impact of Uncontrolled Drug Substance Attributes on Downstream Biologic Product Quality

Shantanu_SuleShantanu Sule, Ph.D., Senior Engineer, Parenteral Drug Product Development, Biogen

This presentation will discuss how uncontrolled chemical modifications and impurities during upstream drug substance process influence protein stability in the downstream drug product. Formulation and process control strategies will be presented to address such challenges along with opportunities to improve drug product quality by impurity removal during purification. Further, novel stability study design approaches will be examined specific to high titer concentrated biopharmaceuticals where such situations can commonly arise.

3:10 Stabilization of Antibody Solutions Using a Novel Excipient

Iris Batalha, Ph.D., Postdoctoral Research Associate, Chemical Engineering and Biotechnology, University of Cambridge

We report a novel excipient that at concentrations of only 10 mM can reduce the viscosity of high concentration protein liquid formulations (150 mg/mL). The excipient also reduced the phase separation of the same antibody formulations during heating from 4 to 40ᵒ C. A salt screen identified various counterions to improve the solubility of excipient, which did not cause protein conformational instability as measured by differential scanning calorimetry.

3:40 End of Protein Aggregation & Stability

Add these programs to your agenda for a full week of comprehensive coverage!

Characterization of Biotherapeutics

Biophysical Analysis of Biotherapeutics

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