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.

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. 

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.

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.

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.

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. 

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.

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.  

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.