Impurities & Degradants

Identification * Characterization * Assessment

September 28 - 29, 2020

One of the most important considerations in the drug discovery and development process is safety. Product-related and process-related impurities present in the drug substance and drug product need to be identified, characterized, and assessed to make sure no toxicity exists in the drugs administered to patients. Drug product degradation profiles should also be established to guide stable formulation and ensure suitable drug shelf life. Structural characterization of impurities and degradants is thus an integral part of biotherapeutic product development, of which the regulatory agency focuses much of its attention. CHI’s Impurities & Degradants conference aims to share best practices to assess and characterize these product-related and process-related impurities and degradation products to determine their risks to product stability and immunogenicity.

Monday, September 28

7:30 am Registration and Morning Coffee
8:30 Welcome by Conference Organizer
Mimi Langley, MBA, Senior Conference Director, Cambridge Healthtech Institute
8:35 Chairperson's Remarks
Krishnan Sampathkumar, PhD, Senior Director, Analytical and Drug Product Sciences, Development, Macrogenics

Joint Opening Plenary Session

8:40 Predicting Antibody Developability Profiles Through Early Stage Discovery Screening
Laurence Fayadat-Dilman, PhD, Senior Director, Protein Sciences, Merck Research Labs

The Impact of Excipients and Oxidative Degradation on Protein Product Stability

Satish K. Singh, PhD, Head, Sterile Product Technology, Moderna Therapeutics, Inc.

Excipients in drug products fulfill a range of functions. In parenteral protein biotherapeutics, excipients provide physical and chemical stability, while contributing to osmolality. Excipients can however display complex behavior and under certain circumstances, may even destabilize the active protein molecule. Additionally, excipients may not be pharmacologically inert. Excipients must therefore be selected with care, and their control considered during development of the control strategy for the product.

9:40 Current and Emerging Expectations for R&D CMC Data Integrity
Nadine M. Ritter, PhD, President & Analytical Advisor, Global Biotech Experts LLC
  • What kind of data integrity operational elements are appropriate for R&D vs. GxP labs?
  • How can it be confirmed that everyone in the lab understands the rationale and justification of quality practices for R&D CMC labs?
  • How can a regulatory affairs reviewer assure all of the R&D data included in a product dossier are in fact authentic, complete, accurate, etc.?
  • What should be done if errors or omissions are discovered in key R&D CMC study reports?
10:10 PANEL DISCUSSION: Challenges in Ensuring Data Integrity in R&D and GxP Labs: Emerging Regulatory Policies and Best Laboratory Practices
Panel Moderator:
Nadine M. Ritter, PhD, President & Analytical Advisor, Global Biotech Experts LLC
  • Which CMC data are at risk?
  • How to ensure the R&D data are authentic, complete and accurate?
  • What operational control mechanisms should be in place?
  • What have been good vs bad experiences with R&D data integrity?​

10:40 Networking Coffee Break

Characterizing Product-Related Impurities

11:10 Chairperson's Remarks
Bernice Yeung, PhD, Director, Protein Analytical Chemistry, Analytical Development, Biogen
11:15 Impurities and Potential Impact on Generating Immunogenicity Response to a Biotherapeutic
Boris Gorovits, PhD, Senior Director, Pharmacokinetics, Pharmacodynamics & Metabolism, Pfizer Inc.

All Biotherapeutics have the potential to induce an unwanted immunogenicity response with a significantly varying clinical sequelae.  Potential consequences of anti-drug antibody induction include loss of exposure, associated loss of efficacy as well as immediate or latent hypersensitivity reactions and other safety related concerns.  The degree of immune response may significantly contribute to the success or failure of the product.  Related risk factors are often described as intrinsic (drug substance specific) and extrinsic (associated with patient status, treatment and other product characteristics).  Intrinsic factors include protein aggregates, co-purified contaminants and impurities, variety of post-translational adducts. The origin and immunogenicity induction impact potential of various impurities and contaminants will be discussed.

11:45 In vitro Immunogenicity Risk Assessment of Aggregates & Impurities in Protein Formulations
Michael D. Swanson, PhD, Senior Scientist, Biologics & Vaccines Bioanalytics, Merck & Co., Inc.

Protein aggregates and other impurities found in drug products have the potential to stimulate immune responses. Aggregation of proteins can result in increased innate and adaptive phase responses. Contaminating host cell proteins (HCP) can also be immunogenic and could potentially impact drug efficacy and safety. Here describe in silico and in vitro tools for immunogenicity risk assessment of aggregates and impurities. 

12:15 pm Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own
12:40 Session Break

Characterizing Product-Related Impurities (CONT.)

1:30 Chairperson's Remarks
Bernice Yeung, PhD, Director, Protein Analytical Chemistry, Analytical Development, Biogen
1:35 Identification, Characterization and Criticality Assessment of Product-Related Quality Attributes
Romina Hofele, PhD, Scientist II, AstraZeneca

Assessing criticality of product-related impurities is a key step in characterizing quality attributes. When establishing the structure-function relationship of quality attributes in biotherapeutics, product-specific and conserved attributes warrant different considerations. We studied the formation and characterization of both product-specific and conserved quality attributes in monoclonal antibodies (mAbs) using mass spectrometry and molecular modelling. In the first example, we studied the kinetics of isomerization of two CDR sites at high and low pH, including the formation of a stable succinimide intermediate. In the second example, we investigated oxidation rates of amino acid residues in conserved regions across mAbs with various Fc formats. Last, implications to molecule understanding, platform mAb knowledge, criticality assessment and control strategy will be discussed.

2:05 Detection, Characterization and Control of Impurities in Biologics
Mark A. Skasko, PhD, Science & Standards Liaison, Global Biologics, USP

A critical aspect of ensuring the product quality of biologic pharmaceuticals is identifying, monitoring, and controlling impurities. Biologic pharmaceutical impurities run the gamut of process-related impurities, such as host cell DNA, proteins, and particulates, to product-related impurities, such as degradants, by-products, precursors, and aggregates. This presentation will provide an overview of approaches available through USP to monitor and control both process-related and product-related impurities.

2:35 Sponsored Presentation (Opportunity Available)
3:05 Networking Refreshment Break and Breakout Discussions
4:30 Results of an Interlaboratory Comparison for Counting of Sub-Micrometer Particles
Kurt D. Benkstein, Research Chemist, Biomolecular Measurement Division, NIST

NIST has conducted a recent interlaboratory comparison of counting and sizing particles in the sub-micrometer size regime to study varied approaches (including particle tracking analysis, resonant mass measurement, electrical sensing zone, and others). We will discuss the study design, including the polydisperse particle dispersion that was circulated in the study, and initial observations from the anonymized dataset, which features responses from 20 outside laboratories.

Protein Interactions and Degradation at Interfaces

5:00 Protein Adsorption and Degradation at Surfaces
Cavan Kalonia, PhD, Scientist II, Formulation, AstraZeneca Biologics

Physical degradation and aggregation of proteins at interfaces (e.g., solid-liquid, liquid-liquid, and air-liquid) can negatively impact the manufacturability, shelf-life stability, and administration of protein therapeutics.  Despite the critical impact of surfaces on protein stability, the mechanisms of interfacial degradations remain poorly understood and highly speculative in the pharmaceutical literature.  In this work, we have collaborated with the National Institute of Standards and Technology (NIST) and University of Manchester to implement and develop state of the art metrology and modeling tools to investigate protein interfacial degradation at pharmaceutically relevant surfaces (e.g., stainless steel, glass, and silicone oil).  Our improved understanding of protein interactions and degradation at interfaces could lead to establishing novel and prospective mitigation strategies, spanning from protein engineering, formulation development and manufacturing process controls. 

5:30 Close of Day One and Dinner Short Course Registration
6:00 Dinner Short Courses
SC1: Particles in Biotherapeutics: Characterization & Impact
SC2: Advanced Analytical Technologies for Developability & Formulation Assessments

Tuesday, September 29

8:00 am Morning Coffee

Characterizing HCPs and Residual DNA

8:30 Chairperson's Remarks
Jennifer Hu, PhD, Scientist, Analytical Development, Juno Therapeutics
Veronika Reisinger, PhD, Lab Head, Physico Chemical Characterization, Novartis AG

Residual HCPs can impact product quality in different ways. To date, the standard method for HCP detection is ELISA which is usually not capable to identify and quantify single HCPs. Therefore, orthogonal methods, where individual HCPs can be detected, identified and quanitified, are evolving. So far, the most popular orthogonal method for identification and quantification of individual HCPs is LCMS. Here, we present workflows to support the identification and quantification of HCPs impacting product stability and their aggregation propensity.

9:05 Characterization of HEK 293 Host Cell DNA in Cell Therapy
Jennifer Hu, PhD, Bioanalytical Scientist, Cell Therapy Development & Operations, Bristol-Myers Squibb

The generation of chimeric antigen receptor (CAR)-expressing T cells from patient lymphocytes commonly relies on viral vectors for transgene delivery. Viral vector production occurs in a producer cell line and the manufacturing process requires partitioning of intact viral particles away from host cell impurities. Although a nuclease treatment step aids in size reduction and improves the clearance of residual DNA species, there is still a potential for nucleic acid impurities to exist in the vector product, to be transferred into the CAR T drug product manufacturing process, and subsequently to be present in the CAR T cell infusion. Residual DNA is a key critical quality attribute. As such, a risk-based analytical strategy for the characterization of residual DNA impurities will be presented.

9:35 Sponsored Presentation (Opportunity Available)
10:05 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
10:45 Developing Next-Generation Sequencing Approaches to Quantify Residual DNA and Characterize AAV Vector Genomes
Magalie Penaud-Budloo, PhD, Research Scientist, INSERM UMR1089, University of Nantes

The increasing number of successful clinical trials using adeno-associated virus (AAV) augurs a bright future for these small viral vectors. However, the development of new drugs goes hand-in-hand with higher quantity and quality requirements. In particular, new methods to assess the level of residual DNA in AAV stocks are welcome, considering the potential risk of co-transferring oncogenic or immunogenic sequences with the therapeutic vectors. Our laboratory has developed an assay based on next-generation sequencing to exhaustively identify and quantify DNA species in recombinant AAV batches. Compiled with a computational analysis of the single nucleotide variants, Single-Stranded Virus Sequencing (SSV-Seq) also provides information regarding AAV genome identity. In parallel, single-molecule sequencing technologies show great promise to analyze genome truncation events and consequently, could be used as a tool to improve the safety and potency of AAV viral vectors.  

11:15 A Novel Method for Removing PEI from Biopharmaceutical Samples: Improving Assay Sensitivity of Residual DNA qPCR
Shu Min Zhang, Associate Fellow & Investigator, BPD Analytical Sciences & Biopharm R&D, GlaxoSmithKline

Polyethyleneimine (PEI) is a flocculent and widely used in Mab downstream purification processes. PEI is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative polymerase chain reaction (qPCR) assay and other assays, used in product quality and release testing for biopharmaceutical products. Very high sample dilutions (e.g., 1:10,000) can overcome the interference of PEI, but at a cost of DNA assay sensitivity. Diluting samples poses a significant risk to the assay sensitivity needed to satisfy regulatory requirements on the amount of residual genomic DNA present per parenteral dose (i.e., 10 ng/dose). Removing PEI while retaining DNA can overcome the interference of PEI on the residual host cell DNA qPCR assay and other assays used for process development and for drug substance release testing. We have devised a new sample preparation protocol that has demonstrated the ability to overcome the interfering effect on qPCR due to PEI. The PEI sample preparation method utilizes a novel mixture of anionic peptide and detergent, added to the samples prior to heat denaturation and centrifugation. Spike recovery results have demonstrated that DNA recovery, which is totally inhibited in the presence of ≥20 ppm PEI, is overcome in most in-process sample matrices. The use of sodium dodecyl sulfate (SDS) and NaHO effectively removes 20 – 200 ppm PEI from samples and increased assay sensitivity 10 fold for bulk drug substance (BDS). For higher concentrations of PEI, in the range 1000 ppm, addition of Heparin and Sarkosyl increased qPCR assay sensitivity for in-process samples by 5-100 fold. Overcoming residual DNA assay interference is essential to achieve the assay sensitivity necessary to demonstrate DNA clearance for biopharmaceutical BDS.

11:45 Presentation to be Announced
12:15 pm Close of Impurities & Degradants

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