Quantitation of total antibody (tAb) from antibody drug conjugate (ADC) PYX-201 in rat and monkey plasma using an enzyme-linked immunosorbent assay (ELISA) and its application in preclinical studies.

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA. This assay was validated with a dynamic range 250 - 10,000 ng/mL and 250 - 6000 ng/mL in rat and monkey K2EDTA plasma, respectively. PYX-201 tAb bioanalytical ELISA assay was reported for the first time in any biological matrix. This is the first time for a bioanalytical method to be validated for a tAb from an ADC drug targeting EDB + FN in any biological matrix.

Quantification of antibody-drug conjugate PYX-201 in rat and monkey plasma via ELISA and its application in preclinical studies.

Aim: PYX-201 is a novel antibody-drug conjugate targeting the extra domain B splice variant of fibronectin in the tumor microenvironment. Accurate quantification of PYX-201 is critical for PYX-201 pharmacokinetics profiling in preclinical studies. Materials & methods: ELISA was performed using reference standard PYX-201, mouse monoclonal anti-monomethyl auristatin E antibody, mouse IgG1, mouse monoclonal anti-human IgG horseradish peroxidase and donkey anti-human IgG horseradish peroxidase. Results: This assay was validated at 50.0-10,000 ng/ml in rat dipotassium EDTA plasma and 250-10,000 ng/ml in monkey dipotassium EDTA plasma. Conclusion: This is the first time for a PYX-201 bioanalytical assay in any matrix to be reported.

Recommendations on qPCR/ddPCR assay validation by GCC.

Gene therapy, cell therapy and vaccine research have led to an increased use of qPCR/ddPCR in bioanalytical laboratories. CROs are progressively undertaking the development and validation of qPCR and ddPCR assays. Currently, however, there is limited regulatory guidance for the use of qPCR and a complete lack of any regulatory guidelines for the use of the newer ddPCR to support regulated bioanalysis. Hence, the Global CRO Council in Bioanalysis (GCC) has issued this White Paper to provide; 1) a consensus on the different validation parameters required to support qPCR/ddPCR assays; 2) a harmonized approach to their validation and 3) a consistent development of standard operating procedures (SOPs) for all the bioanalytical laboratories using these techniques.

Biological Matrix Supply Chain Shortages: More Matrices Are Now Rare-the Case for Surrogate Matrices.

The COVID-19 pandemic has strained the biological matrix supply chain. An upsurge in demand driven by numerous COVID-19 therapeutic and vaccine development programs to combat the pandemic, along with logistical challenges sourcing and transporting matrix, has led to increased lead times for multiple matrices. Biological matrix shortages can potentially cause significant delays in drug development programs across the pharmaceutical and biotechnology industry. Given the current circumstances, discussion is warranted around what will likely be increased use of surrogate matrices in support of pharmacokinetic (PK), immunogenicity, and biomarker assays for regulatory filings. Regulatory authorities permit the use of surrogate matrix in bioanalytical methods in instances where matrix is rare or difficult to obtain, as long as the surrogate is appropriately selected and scientifically justified. Herein, the scientific justification and possible regulatory implications of employing surrogate matrix in PK, immunogenicity, and biomarker assays are discussed. In addition, the unique challenges that cell and gene therapy (C>) and other innovative therapeutic modalities place on matrix supply chains are outlined. Matrix suppliers and contract research organizations (CROs) are actively implementing mitigation strategies to alleviate the current strain on the matrix supply chain and better prepare the industry for any future unexpected strains. To maintain ethical standards, these mitigation strategies include projecting matrix needs with suppliers at least 6 months in advance and writing or updating study protocols to allow for additional matrix draws from study subjects and/or re-purposing of subject matrix from one drug development program to another.

Recommendations on ELISpot assay validation by the GCC.

Gene therapy, cell therapy and vaccine research have led to an increased need to perform cellular immunity testing in a regulated environment to ensure the safety and efficacy of these treatments. The most common method for the measurement of cellular immunity has been Enzyme-Linked Immunospot assays. However, there is a lack of regulatory guidance available discussing the recommendations for developing and validating these types of assays. Hence, the Global CRO Council has issued this white paper to provide a consensus on the different validation parameters required to support Enzyme-Linked Immunospot assays and a harmonized and consistent approach to Enzyme-Linked Immunospot validation among contract research organizations.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis. Similar challenges exist with critical reagents (e.g., capture and detection antibodies) used for assays supporting biologics. The recommendations provided in this publication are the minimum requirements for the content that GCC members believe should be included in Certificates of Analysis for reference standards obtained from commercial vendors, sponsors and compendial suppliers, for use in regulated bioanalytical studies. In addition, recommendations for internal standards, metabolites and critical reagents are discussed.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

Bis{2-amino-2-oxo-N-[(1E)-1-(pyridin-2-yl-κN)ethyl-idene]acetohydrazidato-κ(2)N',O(1)}nickel(II).

In the title compound, [Ni(C(9)H(9)N(4)O(2))(2)], the Ni(II) ion is situated on a twofold rotation axis and is coordinated by two O and four N atoms from two tridentate {2-amino-2-oxo-N-[(1E)-1-(pyridin-2-yl-κN)ethyl-idene]acetohydrazidate ligands in a distorted octa-hedral geometry. In the crystal, N-H⋯O and N-H⋯N hydrogen bonds link the mol-ecules into columns in [001]. The porous crystal packing is further stabilized via π-π inter-actions between the pyridine rings of neighbouring mol-ecules [centroid-centroid distance = 3.746 (3) Å] with voids of 270 Å(3).