High-Throughput Developability Assessment of Therapeutic Antibody Candidates in Discovery Stage
The stability of a candidate drug-protein is critical to the success or failure of its development as a viable drug. Low stability could adversely impact production consistency, shipping and handling excursions, and long-term storage. Developability assessments are becoming an increasingly important criterion to mitigate this risk. The conventional approach is to perform the developability assessment on the final few shortlisted candidates at the mid-to-late stage of drug development.
At Aragen, we have moved the developability assessment into the discovery phase during antibody screening, accelerating the candidate selection process. Integration of the Uncle platform into our process was essential for early assessments due to its small sample requirements and rapid analysis time. In this study, we show that humanized antibody candidates of a murine monoclonal antibody differ in their thermal melting and thermal aggregation profiles due to differences in the stability of the Fab domain (that carries the target binding residues) because of the humanization process. Destabilization of the Fab domain or lower aggregation onset can lead to problems later during production, shipment, or storage. Assessment of thermal stability and aggregation potential can be an important tool for early selection of viable drug candidates. Analysis during antibody screening (discovery stage) and clone selection (early process development stage) can significantly reduce the time and cost of downstream manufacturing.
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An Integrated Stepwise Approach to Developability Assessment of Therapeutic Antibody Candidates from Discovery to Development
The developability of a candidate drug protein is a strong indicator whether it can successfully be developed into a viable drug, since developability challenges can adversely impact production consistency, shipping and handling excursions, and long-term storage. Therefore, it is increasingly important to perform developability assessments to mitigate this risk and the risk of costly late-stage failures.
In this poster, we propose an integrated approach to developability assessment starting from early discovery through late process development. We present few example data to visualize the concept. First step is to integrate ForteBio’s high throughput Octet® and Unchained Lab’s high throughput Uncle during early stage discovery for developability screening using potency, self-interaction (technique CSI-BLI) or cross-interaction (CI-BLI), thermal stability, and aggregation propensity data. Hundreds of candidates can be screened in just a few hours with minimal sample consumption using this approach. Promising candidates can then be evaluated using relatively lower throughput methods like Biacore, standup monolayer adsorption chromatography (SMAC), size exclusion chromatography (SEC), and capillary electrophoresis (CE) for developability assessment using molecule properties including binding kinetics, colloidal stability, aggregation, fragmentation, charge variants. This approach can lead to the early identification of developable candidates, significantly reducing the time and cost of downstream manufacturing.
Resolving Precipitation Issues of an Antibody-Fusion During Protein A Purification Through Flocculation
Two distinct antibody-fusion proteins that share a common fusion partner have the propensity to precipitate upon elution during Protein A purification. Further precipitation occurs upon pH neutralization of the pH 3 eluted antibody-fusion protein. After removing the precipitate, the remaining soluble antibodyfusion was highly aggregated when assessed by analytical SEC. A flocculation strategy was employed on clarified cell culture harvests to resolve these precipitation issues.
Biological and Functional Characterization of a Bleomycin-Induced Pulmonary Fibrosis Model in Mice
Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease that causes lung tissue scarring and thickening, leading to respiratory failure. Despite the approval of Pirfenidone and Nintedanib for IPF treatment in multiple countries, lung fibrosis remains a major unmet medical need. Bleomycin-induced pulmonary fibrosis has been a useful pre-clinical model in several species and is most prevalent in rodent models to evaluate potential prophylactic and therapeutic drugs for IPF. Spanning more than a decade, Aragen Bioscience has completed more than three hundred studies for our numerous clients, which have resulted in pre-clinical data for multiple INDs. Our extensive experience with this model has led to a proven track record of highly reproducible and successful preclinical studies. Biological and functional analysis further demonstrates that this model is a very reliable and relevant model for the evaluation of anti-fibrotic agents. This poster details a selection of assays, including gene expression analysis using Aragen’s FibroPanel™.
A Novel Approach to Real Time Monitoring of Antibody Titer in a Fed-Batch Bioreactor Run
In most cases, the titer of an antibody being expressed in a stirred bioreactor is measured offline. The process involves removing an aliquot aseptically from the bioreactor, centrifuging the aliquot and analyzing the resulting supernatant. The supernatant is analyzed either by bio-layer interferometry (BLI), Protein A HPLC or ELISA. Such an approach may limit throughput, workflow and efficiency. Aragen is evaluating a prototype chromatography-based device that is directly connected to a bioreactor. The compact device automatically measures the titer of an antibody being expressed from a stable DG44 CHO clone for 19 days, starting from day 3, post-inoculation. Results of this novel approach to real-time monitoring of antibody titer in a fed-batch stirred bioreactor run are presented.
A Highly Reproducible In Vivo Model for Bleomycin-Induced Lung Fibrosis in Mice to Evaluate Drugs for the Treatment of Idiopathic Pulmonary Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease that causes scarring and thickening of the lung tissue leading to respiratory failure. Despite the approval of Pirfenidone and Nintedanib in multiple countries for the treatment of IPF, lung fibrosis remains a major unmet medical need. Bleomycin-induced pulmonary fibrosis has been a useful pre-clinical model in several species and is most prevalent in rodent models to evaluate potential prophylactic and therapeutic drugs for IPF. As a tissue injury and repair model of fibrosis, bleomycin (BLM) has contributed significantly to studies of the pathobiology of pulmonary fibrosis. The induction and progression of the disease in rodents is of a short duration, making it a practical model for evaluating test compounds in preclinical research. Major drawbacks for this model have been its high mortality rate and inconsistency in the induction of disease. At Aragen Bioscience, our extensive experience with this model has led to a proven track record of highly reproducible and successful preclinical studies. Aragen has run more than a hundred studies for our numerous clients which have resulted in pre-clinical data for multiple INDs.
In Vivo Model and Ex Vivo Analysis for Murine Cytomegalovirus in BALB/c Mice
Human CMV (hCMV), like other herpes viruses, establishes life-long persistent and latent infections in their host (ie cold sores, chicken pox/shingles). Most people become infected with hCMV during early childhood and adolescence with few to no symptoms in the immunocompetent. In immunocompromised individuals such as organ transplants and AIDS patients hCMV infection (primary or reactivation) can cause serious disease and mortality. In utero infections with hCMV are also problematic with congenital CMV infections occurring at a frequency of 1-2% of all live births, resulting in central nervous system birth defects such as hearing loss and learning disabilities. (Handbook of Animal Models of infection, 1999; chapter 111).
FDA approved drugs are currently available for the treatment/prevention of CMV infections in immunocompromised patients (ganciclovir, valganciclovir, foscarnet, maribavir and cidofovir) and have dramatically improved outcomes for immunocompromised hosts. However, the clinical utility of most of these drugs is limited by poor oral bioavailability, associated toxicities, and the potential for development of resistance with extended use. (Infect Disord Drug Targets. 2011 Oct;11(5):475-503.) Therefore novel therapeutic agents are needed to address limitations of FDA approved drugs.
Due to strict species specificity it is not possible to test hCMV viral infections in rodent models. However, infection of mice with Mouse CMV (mCMV) sets up an infection model with many similarities to the human disease and the mCMV model has proven to be an excellent pre-clinical model for predicting anti-viral drug efficacy in hCMV disease.
The UNcle System for Thermostability and Aggregation Profiling of Antibodies and Fc-Fusions in a CRO Setting
The UNcle system from Unchained Laboratories is an integrated analytics platform for fast turnaround thermodynamic and aggregation profiling of proteins. We have assessed two biosimilar antibodies and one Fc fusion for thermostability and aggregation profiling for formulation studies. The Tm values obtained for the antibodies and Fc-fusion using the UNcle system were similar to values obtained by DSF using a SyproOrange detecting flourophore for all buffer conditions tested. Aggregation was measured using both the UNcle and SEC-HPLC prior to the thermal ramp and no aggregation of antibody or Fc-fusion was observed using either platform. The UNcle detected aggregation (Tagg) of both antibodies and Fc-fusion between the first and second melting transitions (Tm1 and Tm2). We were able to confirm the Tagg measured by the UNcle by performing a thermal ramp on un-labelled protein to the reported Tagg and analyzing the protein by SEC-HPLC. We conclude that the UNcle system provides a quick and robust assessment of formulation buffers for antibodies, Fc-fusions, and other proteins enabling out clients to robustly characterize their products.
A Platform to Rapidly Purify and Formulate Immunoglobulins from Mammalian Expression Systems
At Aragen, our typical steps to purify and formulate human IgG from transient system cultures up to 2 L involve clarification of harvest, affinity purification using Protein A and a combination of dialysis and concentration using centrifugal filter units for formulation. This platform can take from 5 to 7 days to complete, including assessment of the formulated antibody for purity. Recently introduced technologies such as Protein A membrane devices and TFF systems capable of handling ultrafiltration and diafiltration of samples as low as 1 mL have tremendously decreased the turnaround time down to 3 days. The typical and improved platforms will be compared.
In Vivo Models and In Vitro Assays for Human RSV Infection – Pre-Clinical Antibody, Small Molecule and Vaccine Development
Respiratory syncytial virus (RSV) infection is the major cause of severe respiratory illness in infants and young children, as well as immune compromised individuals and the elderly. It causes a range of illnesses varying from mild infection to life-threatening bronchiolitis and respiratory failure. Despite over 50 years of research, disease due to RSV infection remains an unmet medical need. Rodent models for testing efficacy and safety in preclinical studies provides a critical component to the development of anti-RSV antibodies, small molecules and vaccines. Aragen Bioscience offers both mouse and cotton rat RSV infection models as well as a wide range of functional readouts to assess the efficacy of anti-RSV biologics and small molecules. These models enable the development of customized study designs that are performed by trained personnel in dedicated work areas specifically designed for infectious disease models. We have now conducted more than 60 pre-clinical studies in rodent models of RSV infection (30-130 animals per study). While BALB/c mice (which are semi-permissive to RSV infection) provide quick information on preliminary proof of concept studies, the cotton rat (Sigmodon hispidus) model represents a clinically relevant model of RSV infection that can be used to develop next generation RSV therapeutics.
Custom Monoclonal Antibodies for Therapeutic and Diagnostic Targets – A One-Stop-Shop for Pre-Clinical Antibody Research
A Comprehensive Portfolio of In Vitro and In Vivo Assays for the Characterization and Development of Novel Anti-TNFa Biologics
Optimizing Effector Functions of Therapeutic Antibodies & Biosimilars Using a Diverse Portfolio of Biophysical and Functional Cell-Based Assays
Summary of ADCC and CDC data presented at the American Society of Hematology conference
Allosteric inhibition of lysyl oxidase–like-2 impedes the development of a pathologic microenvironment. Nature Medicine 16, 1009–1017 (2010). The in-life portion of this study was performed at Aragen Bioscience.
Potent High-Affinity Antibodies for Treatment and Prophylaxis of Respiratory Syncytial Virus Derived from B Cells of Infected Patients The Journal of Immunology, November 15, 2009 vol. 183 no. 10 6338-6345
Strategies for Rapid Production of Therapeutic Proteins in Mammalian Cells
A High Yielding, CHO-K1-Based Transient Transfection System: Rapid Production for Therapeutic Protein Development
Comprehensive Solutions for the Biopharmaceutical Industry
An introduction to our services from concept to IND. 17 minutes
Using Activity Measurements to Drive Clone Selection
A case study highlighting the creation of a stable CHO cell line as well as the development of a scalable purification scheme suitable for cGMP production. The challenge of identifying and separating an inactive form of the protein of interest will be discussed. 15 minutes