Albert Torri

Antidrug Antibodies in Patients Treated with Alirocumab

In 10 clinical trials involving 4747 patients, among those who received the PCSK9 inhibitor alirocumab, antidrug antibodies developed in 5.1%, although no significant effect was seen on the reduction in LDL cholesterol levels.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 3 – LBA, biomarkers and immunogenicity)

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.

Minimizing target interference in PK immunoassays: new approaches for low-pH-sample treatment

BACKGROUND Quantitating total levels of monoclonal antibody (mAb) biotherapeutics in serum using ELISA may be hindered by soluble targets. RESULTS We developed two low-pH-sample-pretreatment techniques to minimize target interference. The first procedure involves sample pretreatment at pH <3.0 before neutralization and analysis in a target capture ELISA. Careful monitoring of acidification time is required to minimize potential impact on mAb detection. The second approach involves sample dilution into mild acid (pH ∼4.5) before transferring to an anti-human capture-antibody-coated plate without neutralization. Analysis of target-drug and drug-capture antibody interactions at pH 4.5 indicated that the capture antibody binds to the drug, while the drug and the target were dissociated. Using these procedures, total biotherapeutic levels were accurately measured when soluble target was >30-fold molar excess. CONCLUSION These techniques provide alternatives for quantitating mAb biotherapeutics in the presence of a target when standard acid-dissociation procedures are ineffective.

Matrix interference from Fc–Fc interactions in immunoassays for detecting human IgG4 therapeutics

BACKGROUND An assay measuring an IgG4 biotherapeutic in human serum used a drug-specific monoclonal antibody (mAb) capture reagent and an antihuman IgG4 mAb as detection reagent. However, serum IgG4 binding to the capture mAb via Fc-interactions was detected by the anti-IgG4 mAb, causing high background. RESULTS Two approaches were developed to minimize background; incorporating a mild acid sample preparation step or using the Fab of the capture antibody. Either strategy improved signal:noise dramatically, increasing assay sensitivity >20-fold. Biophysical analyses of antibody domains indicated that noncovalent Fc oligomers could inhibit the background. CONCLUSION Matrix interference from human IgG4 binding to the capture mAb was reduced with a Fab fragment of the drug-specific capture antibody or by incorporating a mild acid sample treatment into the assay.

Bridging immunogenicity assays for IgG4 therapeutics: mitigating interference from Fc–Fc interactions

AIM A bridging immunogenicity assay for a human IgG4 mAb therapeutic was transferred to an automation system to increase throughput. However, background signal increased five- to six-fold during the 6- to 8-h run. RESULTS Noncovalent Fc contacts formed between labeled IgG4 drugs in reagent solutions stored during the automation run. This generated substantial background signal, reducing assay sensitivity by approximately sixfold. Fc interactions also significantly impacted the confirmation assay. Fc contacts formed between labeled and unlabeled drug, significantly increasing signal inhibition (∼7-70%) in the 6-h run. CONCLUSION Storing labeled antibody solutions separately and combining them immediately before adding to samples reduced interference from Fc interactions. Preincubation time for reagent solutions should be strictly controlled for anti-drug antibody assays with IgG4 drugs to avoid false-positive results.

IMPACT OF ANTI-DRUG ANTIBODIES TO ALIROCUMAB ON LDL-C LOWERING EFFICACY AND SAFETY

All biotherapeutics have the potential to elicit an immune response, which can be monitored by testing for anti-drug antibodies (ADA). Neutralizing antibodies (NAb) are ADA with potential to inhibit biotherapeutic function. This analysis evaluated the impact of ADA on efficacy and safety of the