Predicting Long-Term Storage Stability of Protein Therapeutics

Current methods for predicting long-term storage stability of protein therapeutics are troubled with the empirical problems which become apparent when extrapolating across accelerated stress conditions like rising temperature or agitation. Researchers are struggling to develop a sufficient understanding of the kinetics of the formation of aggregates from dimers to sizable protein particles

The PIPPI consortium members and fellows, gathered at MedImmune in Cambridge, UK.

Image Credit: Wyatt Technology

The Challenge

The formation of aggregates requires multiple pathways. Rate controlling steps include partially folded intermediates that arise at relatively low populations. They are also hard to isolate and study. A full spectrum of protein aggregate sizes thus emerges from these intermediates. To understand individual kinetics, the final size spectrum and the intermediates are then studied over time.

The Goal

Focusing on small numbers of partially folded intermediates and the full spectrum of aggregate sizes, the goal is to develop a set of tools for forecasting aggregation.

Overview of the PIPPI research and training program.

Lorenzo Gentiluomo was given the above problem, challenge and goal when he accepted the PIPPI fellowship. PIPPI is an academic-industrial association funded by the European Horizon 2020 program, it stands for Protein-excipient Interactions and Protein-Protein Interactions. PIPPI is an Innovative Training Network (ITN) which addresses the challenges in the creation of protein-based drugs.

PIPPI’s mandate is to merge investigations of the physico-chemical qualities of several proteins with a detailed insight into the molecular interactions beyond their macroscopic behavior. The overall objective is to establish tools, methodologies, and databases to advise the rational formulation of protein-based therapeutics.

PIPPI Fellow Lorenzo Gentiluomo, hard at work in the lab!”

Lorenzo, a Ph.D. student supervised by Prof. Dr. Wolfgang Friess of the Ludwig Maximilians University (LMU), undertakes much of his work under Dr. Dierk Roessner from Wyatt Technology Europe GmbH (WTE) in Dernbach, Germany. As an important partner in the PIPPI fellowship, WTE supplies lab and office space, as well as the unique instrumentation required for understanding protein aggregation pathways.

Explaining his research to attendees of Wyatt’s workshop on protein characterization in Dernbach.

With the experience working as a formulation scientist at Henkel and researching at Jyväskylä University, and a M.Sc. cum laude in organic and biomolecular chemistry from La Sapienza University of Rome and the University of Jyväskylä, Lorenzo is thoroughly ready to take on the challenge from PIPPI.

The Novelty: Lorenzo will have to overcome the challenges with the following methods:

1. Using high-throughput static and dynamic light scattering (HT-DLS) and special size-exclusion chromatography-multi angle light scattering (SEC-MALS) setups along with modified sampling to quantifying aggregation kinetics.
2. Creating a large-scale database of proteins developed in various conditions using the first systematic studies of aggregation kinetics.
3. Using predictive models like the initial unfolding step, the reversible association, and the interactions between partially folded proteins that integrate knowledge of individual steps in aggregation pathways.
4. Taking advantage of informative techniques like asymmetric-flow field-flow fractionation (AF4) or raster image correlation spectroscopy, to provide greater detail about the time evolution of monomer and oligomer species, thus refining aggregation models.

As of July 16^th, 2018, Lorenzo has been part of the program for two years, he has familiarized himself with SEC-MALS, µSEC-MALS , CG-MALS, HT-DLS and differential viscometry. Some of the tools he has made use of are:

• miniDAWN TREOS II  and Optilab T-rEX with Agilent HPLC for SEC-MALS analysis of proteins, aggregates and other degradants;
• µDAWN^® and Optilab^® UT-rEX with Waters Acquity UPLC for fast, low-volume µSEC-MALS analyses;
• DynaPro^® Plate Reader III for HT-DLS/SLS;
• Calypso^® II and DAWN^® HELEOS^® II for characterization of interactions by CG-MALS;
• Eclipse^® DualTec^® for advanced separations of large and small aggregates by AF4;
• DynaPro Nanostar^® for quasi-equilibrium studies by DLS and SLS;
• Möbius^® for electrophoretic mobility measurements to determine protein charge;
• Viscostar^® III for a new application of differential viscometry in biotechnology.

Including Wyatt Technology in the consortium has been instrumental for the success of the project. The access to best-in-class Wyatt instruments and their scientific expertise has been really important to be able to characterize all the proteins in an efficient way. Also the possibility for the PhD students to go to Wyatt and be part of the highly specialized, industrial environment has made it possible for the students to gain knowledge they would otherwise not have obtained.

Prof. Pernille Harris, Head of the PIPPI consortium and Professor, Technical University of Denmark

Accomplishments

In Lorenzo’s first two years, he completed the first screening of a sequence of protein provided by Roche, MedImmune and Novozyme, which assessed conformational and colloidal stability across numerous formulations. Lorenzo’s research, along with WTE’s support, amounted to about 80% of the total data set to be used by the 15 PIPPI fellows, this was an important first step in putting together the PIPPI database.

There has been a lack of high-value knowledge regarding the protein formulation conditions that result in stable drug products – but this database will provide a solution. Lorenzo will be foremost author on the manuscript reporting these results.

Lorenzo trained an artificial neural network to forecast colloidal and conformational stability of mAbs entirely based on amino acid composition from this dataset. By predicted their melting temperature Tm, aggregation onset temperature Tagg, diffusion interaction parameter kD and monomer loss over thermal stress, this approach could optimize the selection of candidate proteins with the highest potential for successful product development, even before their expression, harvesting and purification.

Moreover, the neural network model is believed to diminish the number of formulation conditions (i.e. pH and NaCl concentration) that need to be physically tested throughout early formulation development.

In-depth studies of native/reversible aggregates are sparse, and most of the aggregation kinetic literature centers on non-native/irreversible aggregation. Using a series of methods ranging from static and dynamic light scattering to analytical ultracentrifugation and small angle x-ray scattering, Lorenzo completed a study of the reversible, native aggregation of one protein. This work, in parallel to the work mentioned above, will also be submitted for publication.

Additional Achievements During the Two Year Period

• Lorenzo studied the physical stability of monoclonal antibodies by denaturant dilution, using the DynaPro Plate Reader, with PIPPI fellow Hristo Svilenov and Prof. Gerhard Winter’s group at LMU. The research is currently under review and will be published in J. Pharm. Sci.
• With PIPPI fellow Aisling Roche and Prof. Robin Curtis of the University of Manchester, he established a strategy to anticipate protein viscosity at high concentration using the ViscoStar and differential viscometry.
• He is collecting further data to use in the predicting the second viral coefficient, utilizing the high-throughput B22 screening abilities of the DynaPro Plate Reader III to generate datasets across many proteins and formulations with just a few µL per sample and within few days. For the a priori prediction of B22 of small proteins based on amino acid composition, these data will be used by PIPPI fellow Marco Polimeni and Dr. Mikael Lund of Lund University to fit Monte Carlo simulations.
• He is using CG-MALS to correlate B22 to the structure factor at angle 0° obtain by SAXS to study protein-protein interactions in collaboration with PIPPI fellow Sujata Mahapatra, Prof. Pernille Harris, Prof. Peter Günther of the Technical University of Denmark, and Dr. Werner Streicher of Novozyme.
• He is helping to study peptide gel formation by the use of AF4 in collaboration with PIPPI fellow Christin Pohl, Prof. Pernille Harris, Prof. Peter Günther of the Technical University of Denmark, and Dr. Allan Nørgaard of Novozyme.
• He is studying the relative bias between SEC, AF4 and AUC aggregation analyses for a series of proteins in collaboration with Dr. Christoph Johann of WTE as well as Sujata Mahapatra, Dr. Werner Streicher, Christin Pohl, Dr. Allan Nørgaard, Prof. Peter Günther and Prof. Pernille Harris.
• He is characterizing an antibody-drug conjugate in collaboration with Dr. Christopher van der Walle of Medimmune and PIPPI fellow Maria Laura Greco.

Prospects

In the final year of Lorenzo’s partnership with PIPPI, he will prioritize the screening of stressed formulations to improve understanding of the effect of excipients on protein aggregation. To study protein aggregation, he will use a new high-throughput approach, and to create models capable of predicting protein aggregation, he will use a machine learning algorithm.

Additionally, he expects to develop new applications for the Wyatt instruments. Finally, with help from the PIPPI network, he is developing a series of projects in collaboration with the other members.

We look forward to the publication of these stimulating studies.

As part of the company mission to “… delight its customers by providing outstanding analytical tools, as well as unparalleled levels of personal service, to support life-enhancing macromolecular and nanoparticle science”, Wyatt Technology supports and interacts with the PIPPI consortium.

From the insights produced by Lorenzo’s research, the development of life-saving medications that are more available, efficacious and lower-cost with fewer side-effects than traditional drugs will be enabled - benefit humanity as a whole. Furthermore, his work will advise Wyatt’s invention of the instruments and software needed by the biopharmaceutical industry to meet these goals.

This information has been sourced, reviewed and adapted from materials provided by Wyatt Technology.

For more information on this source, please visit Wyatt Technology.