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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 these conditions, applying a rapidly changing landscape of assays, instrumentation, formulation strategies and process steps. The PEGS Protein Aggregation and Stability in Biopharmaceuticals conference offers important scientific updates and a forum for dialog among the stakeholders in this challenging arena.

Final Agenda

Recommended Short Course*

SC14: Overcoming the Challenges of Immunogenicity Assays, Risk Assessment and Regulatory Requirements - Detailed Agenda

*Separate registration required

THURSDAY, MAY 4

12:00 pm Registration

12:35 Luncheon in the Exhibit Hall with Poster Viewing

1:40 Chairperson’s Remarks

Gayatri Ganeshan, Scientist, Biologics and Vaccines, Merck

1:50 KEYNOTE PRESENTATION: Measuring Changes in Protein Structure in Lyophilized Solids and Impacts on Aggregation

Elizabeth_ToppElizabeth M. Topp, Ph.D., Dane O. Kildsig Chair and Department Head, Department of Industrial and Physical Pharmacy, Perdue University

The lack of adequate methods to measure protein structure in the solid state has slowed the development of protein drug products. Here, we present solid-state hydrogen deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a high resolution method to assess protein structure in lyophilized solids. We also show that ssHDX-MS results are highly correlated with the aggregation of a MAb and of myoglobin during storage.

Understanding Aggregation Phenomena

2:20 Chaperonin-Based Biolayer Interferometry to Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins

Mark_FisherMark T. Fisher, Ph.D., Professor, Biochemistry and Molecular Biology, University of Kansas Medical School

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. This presentation describes an automated method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for metastable proteins using a GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform.

2:50 Understanding the Causes of Immunogenicity in Biotherapeutic Proteins

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

Understanding the origins and factors contributing to the immunogenicity of biotherapeutic proteins remains a challenge. Here I will discuss our recent work in which we seek to investigate the roles of aggregation, chemical modification and host cell protein impurities on the immune response to selected proteins. I will also show how aggregation, in particular, affects the quality as well as the amplitude of the immune response.

Albumedix3:20 Reviving Otherwise Hard-to-Stabilize Biopharmaceuticals through Protein Co-Formulation

Eleonora_CerasoliEleonora Cerasoli, Ph.D., Senior Research Scientist, Bioprocessing and Characterisation, Albumedix Ltd.

Aggregation and depletion of biopharmaceuticals is a source of both dosage form and storage instability, compromising safety and efficacy. Human serum albumin is well known to stabilize proteins preventing adsorption, aggregation and oxidation due to its natural roles and inherent properties. Recombinant human albumin is therefore a promising stabilizer for hard-to-formulate biopharmaceuticals. We will present data on the use of Albumedix™ Recombumin® as a stabilizing agent for model biopharmaceuticals and elucidate on potential mechanisms.

3:50 Refreshment Break

4:20 Understanding What Could Make Subvisible Protein Particles Immunogenic

Anacel_Rios_QuirozAnacelia Ríos Quiroz, Ph.D., Group Leader, Particle Lab, Pharma Technical Development Europe (Biologics) Analytics, F. Hoffmann-La Roche Ltd.

The likely immunogenic reactions that have been attributed to subvisible protein particles constitute a safety concern around the use of biotherapeutic proteins. The talk will give a comprehensive overview of the current status of the related literature together with the results of a study which evaluated the particle characteristics needed to break immune tolerance in an IgG1 transgenic mouse. This talk will help increase our understanding on the biological consequences of particulate matter.

4:50 Speaker has cancelled - delegates may attend concurrent sessions. Precipitation Methods to Assess Colloidal Stability

Ramil_LatypovRamil Latypov, Ph.D., Associate Director, Research & Development, Sanofi Genzyme

The use of precipitation methods to assess colloidal stability of antibodies has become widespread. Concentrated antibodies can lose their colloidal stability through various phase transitions, including aggregation, gelation and crystallization. Colloidal stability assessments are done to characterize the propensity of antibodies and their formulations to undergo such phase transformations. PEG-induced liquid-liquid phase separation method has evolved into a fast and convenient way to quantitatively evaluate colloidal stability of antibody solutions.

5:20 End of Day

5:20 Registration for Dinner Short Courses

Recommended Dinner Short Course*

SC15: Critical Considerations for the Design and Development of Antibody-Drug Conjugates

*Separate registration required

FRIDAY, MAY 5

8:00 am Morning Coffee

Particle Characterization

8:30 Chairperson’s Remarks

Reema Raghavendra, Scientist, AbbVie

8:35 Protein Particle Standards: Use and Guidelines for Analyses

Dean_RippleDean Ripple, Ph.D., Leader, Bioprocess Measurements Group, National Institute of Standards and Technology

New particle standards, spanning the subvisible and visible size range, are being developed using ethylene tetrafluoroethylene (ETFE) polymer and a photoresist because they have desirable properties that make them better than currently available standards. These particles can be used to 1) standardize subvisible particle measurements, primarily through correcting instrument bias and 2) develop a semi-quantitative method for monitoring visible proteinaceous particles.

9:05 Flow Cytometry: A Promising Tool for Sub-Visible Particle Characterization

Reema_RaghavendraReema Raghavendra, Scientist, AbbVie

Immunogenicity concerns of therapeutic protein aggregates have necessitated detailed particle characterization not only in the sub-visible range, but also into the sub-micron range. Flow cytometry has potential to bridge particle characterization across these regimes. Here, we present quantitative particle measurements of novel protein-like standards and therapeutic proteins by flow cytometry. Particle responses were compared to micro-flow imaging and corrected for refractive index biasing, allowing accurate particle quantitation.

 Partical Sizing Systems9:35 Counting and Sizing Protein Aggregates down to 0.15um by Focused-Beam Light Scattering Technology

David_NicoliDavid Nicoli, Ph.D., Vice President, Research & Development, Particle Sizing Systems LLC

A novel single-particle optical sizing (SPOS) technique using a focused laser beam collects scattered light from individual particles in a flowing suspension, allowing protein aggregates to be counted/sized down to 0.15 um, at concentrations much higher than possible using normal light scattering technology. Combining this "FX-Nano" sensor with a second conventional sensor extends the upper size limit to 200 um, with analysis possible on sub-mL high-viscosity samples, with conservation of the sample.

10:05 Coffee Break

Formulation and Process Control

10:35 Improvements in Downstream Processes to Minimize and Mitigate Aggregation

Steve_CramerSteven Cramer, Ph.D., Professor, Rensselaer Polytechnic Institute

The propensity of downstream processing conditions for forming aggregates will be discussed along with strategies for avoiding these scenarios via high throughput screening, protein property analyses and appropriate use of mobile phase modifiers. Techniques for removal of aggregates will then be discussed including how best to operate various chromatographic resin systems for these separations and how to properly screen and develop optimal modes of column operation using various strategies and modeling approaches.

11:05 The Role of Surfactants in Phase I Vaccine Formulations

Sashi_BanappagariSashikanth Banappagari, Ph.D., Scientist, Formulation Development, Vaccine Research Center, National Institutes of Health

Non-ionic surfactants are commonly used in vaccine/protein formulations to minimize instability induced by self-interaction and/or interfacial interactions occurring during manufacturing and long term storage. These interactions may result in conformational changes leading to aggregation or material loss via container adsorption, which may be further enhanced by the low doses common to vaccine formulations. Two case studies, involving Chikungunya Virus Virus-Like Particles and Hemagglutinin-Ferritin Nanoparticles, will be discussed.

11:35 Novel Biophysical Tools for the Study of Insulin Aggregates and Their Ability to Distinguish between Fibrillar and Amorphous Aggregates

Gayatri_GaneshanGayatri Ganeshan, Scientist, Biologics and Vaccines, Merck

Fluorescent dyes including Thioflavin-T are typically used to measure fibrils in insulins. However, some of the newer unfibrillated insulin analogs (Tresiba®, Levemir®, etc.) show high background ThT fluorescence making it difficult to screen for fibrils. We have developed a Flow Cytometry method using ThT that can distinguish between amorphous aggregates and fibrils in different stressed commercial insulins. Its sensitivity and turnover time are valuable as a design tool to de-risk key attributes.

12:05 pm Optimization Strategy to Generate Antibody with Low Viscosity for High Concentration Subcutaneous Formulation

Masaru_MuraokaMasaru Muraoka, Ph.D., Research Scientist, Chugai Pharmabody Research PTE. LTD.

Developing high concentration formulations brings forth several challenges due to solubility limitations and increased viscosity. High solubility and low viscosity are desired. However, in some cases, protein engineering has adverse effects on physicochemical properties of antibody, even with a single mutation. This presentation will provide case studies covering these issues and discuss the optimization strategies for protein engineering.

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

1:05 Refreshment Break

Controlling Stability in High Concentration Protein Formulations

1:35 Chairperson’s Remarks

Shantanu Sule, Ph.D., Senior Research Scientist, Eli Lilly & Company

1:40 Profiling Molecular Feasibility for High Dose Biological Therapeutics

Shantanu_SuleShantanu Sule, Ph.D., Senior Research Scientist, Eli Lilly & Company

Developing biological therapeutics often faces challenges from the high efficacious doses while maintain safety. Here we outline state-of-the-art approaches to develop high dose/high concentration biologics, molecular engineering and excipient technologies that address such challenges. New findings on the impact of manufacturing operations on biologic quality will be shared. The implications of high concentration processing and delivery devices will then be summarized for the current clinical and commercial landscape.

2:10 Affinity Capture Self-Interaction Nanoparticle Spectroscopy (AC-SINS) Measurements of Weak Interactions in Dilute Solution Support Early Assessment of Antibody Manufacturability

Marissa Mock, Ph.D., Senior Scientist, Therapeutic Discovery; Biologics, Amgen

Methods to identify molecules with potential manufacturability liabilities are desired during the preclinical selection of large molecule therapeutics. Current techniques are often impractical in early pipelines because they require large amounts of highly pure material. We evaluated and implemented the high-throughput AC-SINS assay, developed by Prof. Peter Tessier at RPI, and will present a test case demonstrating that AC-SINS successfully predicted high viscosity variants from an antibody engineering panel.

Long Term Stability Prediction

2:40 Evaluation of Early Stage Formulation Development Strategies for Monoclonal Antibodies

Hardeep_SamraHardeep Samra, Ph.D., Senior Scientist, Formulation Sciences, MedImmune

Selection of optimal formulation conditions for monoclonal antibodies can be challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long term stability. High-throughput screening (HTS) techniques can be used to screen solution conditions for early formulation development. The utility of using accelerated stability, differential scanning light scattering (DSLS), and differential scanning fluorescence (DSF) were evaluated as early formulation screening techniques. This talk highlights the correlation between data from these techniques and predictability to real time stability.

3:10 Using Viscosity-Derived Parameters and Thermal Analysis to Evaluate the Solution Properties of Bispecific Dual Variable Domain Antibodies

Ralf_CarilloRalf Joe Carrillo, Ph.D., Senior Scientist, Physical Chemistry, AbbVie

Changes in Simha shape, maximum packing fraction, intrinsic viscosity and DSC thermal stability for mAbs and bi-specific Dual Variable Domain Immunoglobulins DVD-Ig proteins have been studied and analyzed to ascertain how the viscoelastic properties for mAbs and DVD-Igs differ and how these properties can predict long term stability.

3:40 End of Conference


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