Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis.

Concentration determination is a fundamental hallmark of protein reagent characterization, providing a means to ensure reproducibility and unify measurements from various assays. However, lot-to-lot differences in protein activity often still occur, leading to uncertainty in the accuracy of downstream measurements. Here, we postulate that those differences are caused by a misrepresentation of the protein concentration as measured by traditional total protein techniques, which can include multiple types of inactive protein species. To overcome this, we developed a standardized method to quantify a protein's active concentration via calibration-free concentration analysis (CFCA). As a pilot study, we compare the biophysical and immunoassay responses from three batches of recombinant soluble lymphocyte-activation gene 3 (sLAG3), as defined by either their total or active concentrations. Defining the sLAG3 reagents by their assay-specific concentration improved consistency in reported kinetic binding parameters and decreased immunoassay lot-to-lot coefficients of variation (CVs) by over 600% compared to the total protein concentration. These findings suggest that the total concentration of a protein reagent may not be the ideal metric to correlate in-assay signals between lots, and by instead quantifying the concentrations of a reagent's assay-specific epitopes, CFCA may prove a useful tool in overcoming lot-to-lot variability.

Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease.

Following recent success in genome-wide association studies, a critical focus of human genetics is to understand how genetic variation at implicated loci influences cellular and disease processes. Crohn's disease (CD) is associated with SNPs around IRGM, but coding-sequence variation has been excluded as a source of this association. We identified a common, 20-kb deletion polymorphism, immediately upstream of IRGM and in perfect linkage disequilibrium (r2 = 1.0) with the most strongly CD-associated SNP, that causes IRGM to segregate in the population with two distinct upstream sequences. The deletion (CD risk) and reference (CD protective) haplotypes of IRGM showed distinct expression patterns. Manipulation of IRGM expression levels modulated cellular autophagy of internalized bacteria, a process implicated in CD. These results suggest that the CD association at IRGM arises from an alteration in IRGM regulation that affects the efficacy of autophagy and identify a common deletion polymorphism as a likely causal variant.

Determining the Degree of Sulfo-tag Conjugation to AAV5 Vectors by LC-HRMS and Evaluating the Effects on Antibody Binding Affinity and Bridging Assay Sensitivity.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.

2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 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 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

Validation of preclinical pharmacokinetic and immunogenicity assays for an anti-PCSK9 antibody.

Pharmacokinetic data derived from assays that accurately and precisely quantitate a therapeutic drug in circulation are critical to appropriately designing suitable dosing schedules. This manuscript describes the validation and implementation of methods to quantitate a therapeutic anti-human PCSK9 monoclonal antibody in rat and monkey sera as well as immunogenicity methods to screen the possible presence of rat and monkey antibodies directed against the antibody. As soluble, endogenous PCSK9 can interfere with a PCSK9-mediated capture step in ELISA, an indirect target-capture assay was used that potentially could capture free and target-engaged therapeutic mAb. Immunogenicity assays were based on a standard bridge ELISA using the therapeutic antibody for capture and detection. Both pharmacokinetic and immunogenicity assays were implemented in preclinical studies of the therapeutic antibody. The methods presented here may enable further pharmacokinetic studies.

Strategies for method comparison when changes in the immunogenicity method are needed within a clinical program.

Aim: To present the reader with different approaches used to compare immunogenicity methods when changes are needed during a clinical program. Results: Five case studies are presented, in the first two case studies, the approach utilized a small sample size for the comparison. In the third case, all samples from a study were analyzed by both methods. In the fourth case, the intended use of noncomparable assays in an integrated summary drove design of experiments to establish the expected limits of pooling data. In the fifth case, a selectivity approach was used as an alternate to use of incurred samples. Conclusion: When data pooling across methods is needed, it is important to define the limits of comparability.

Development of CAPER peptides for the treatment of triple negative breast cancer.

Triple negative breast cancer (TNBC) is a heterogeneous disease, which lacks expression of the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor 2 receptor (HER2). This subtype of breast cancer has the poorest prognosis with limited therapies currently available, and hence additional options are needed. CAPER is a coactivator of the activator protein-1 (AP-1) (interacting specifically with the c-Jun component) and the ER and is known to be involved in human breast cancer pathogenesis. Recent published data have demonstrated a role for CAPER in TNBC and, as such, disrupting the function of CAPER with c-Jun could be a novel approach to treat TNBC patients. The data presented here shows the development and in vitro testing of CAPER-derived peptides that inhibit the coactivator activity of CAPER with c-Jun. These CAPER peptides result in a decrease in cell number and an increase in apoptosis in two TNBC cell lines, MDA-MB-231 and BT-549, while having no effect on the non-tumorigenic cell line MCF 10A. Additionally, two modes of action were demonstrated which appear to be cell line dependent: 1) a modulation of phosphorylated c-Jun leading to a decrease in Bcl-2 in MDA-MB-231 cells and a decrease in p21 in BT-549 cells and 2) a decrease in DNA repair proteins, leading to impaired DNA repair function in MDA-MB-231 cells. The data presented here supports further development of CAPER-derived peptides for the treatment of TNBC.

Generic Anti-PEG Antibody Assay on ProterixBio's (Formerly BioScale) ViBE Platform Shows Poor Reproducibility.

PEGylation is a modification commonly used to increase the half-life of therapeutic proteins. The strategy for immunogenicity testing of these compounds should include methods to detect both anti-protein and anti-PEG antibodies. We previously reported a method for the detection of anti-PEG antibodies using ProterixBio's (formerly BioScale) acoustic membrane microparticle (AMMP) technology. Our initial method development work showed the assay was capable of detecting antibodies in human serum with a sensitivity of 1 µg/mL with good reproducibility (CV < 7%). Since the publication of this initial paper, additional experimentation was performed in an effort to validate the assay for support of clinical sample analysis. This additional data indicate that the method has high variability (CV% > 20) and is unsuitable to support clinical sample analysis.

Accelerating Regulated Bioanalysis for Biotherapeutics: Case Examples Using a Microfluidic Ligand Binding Assay Platform.

The Gyrolab™ xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods. To improve bioanalysis testing timelines and increase the speed at which biotherapeutics are delivered to patients, we evaluated the technology for its potential to deliver high-quality data at reduced testing timelines for regulated bioanalysis. Six LBA methods were validated to support bioanalysis for GLP toxicokinetic or clinical pharmacokinetic studies. Validation, sample analysis, and method transfer are described. In total, approximately 4000 samples have been tested for regulated bioanalysis to support 6 GLP toxicology studies and approximately 1000 samples to support 2 clinical studies. Gyrolab™ xP had high run pass rates (≥83%) and high incurred sample reanalysis (ISR) pass rates (>94%). The maximum total error observed across all QC levels for a given assay was <30% for all six LBAs. High instrument response precision (CV ≤5%) was observed across compact discs (CDs), and methods were validated to use a single standard curve across multiple CDs within a Gyrolab™ xP run. Reduced bioanalysis timelines were achieved compared to standard manual plate-based methods, and methods were successfully transferred across testing labs, paving the way for this platform for use in late-stage clinical development.

Development of a Generic Anti-PEG Antibody Assay Using BioScale's Acoustic Membrane MicroParticle Technology.

Immunogenicity testing for PEGylated biotherapeutics should include methods to detect both anti-protein and anti-PEG antibodies (anti-PEG). Although some methods have been published for the detection of anti-PEG antibodies, the information is incomplete and, in some cases, reagents used (such as Tween-20) are known to interfere with detection. This rapid communication describes the use of BioScale's Acoustic Membrane MicroParticle (AMMP®) technology using the ViBE® Workstation to measure anti-PEG antibodies in human serum samples. Briefly, a sample spiked with monoclonal human IgG anti-PEG antibody is diluted in buffer and incubated with paramagnetic beads coated with linear chain mPEG to capture anti-PEG antibodies. The complex is then captured on an acoustic membrane coated with Protein A. The change in mass on the membrane caused by the binding of the complex to the membrane results in a signal proportional to the mass of anti-PEG antibodies. The data indicate that an assay with a sensitivity of less than 1000 ng/mL for IgG is achievable. This level of sensitivity is better than current published reports on IgG anti-PEG antibody detection.

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.

Addressing matrix effects in ligand-binding assays through the use of new reagents and technology.

BACKGROUND: Ligand-binding assays (LBAs) used in the quantification of biotherapeutics for pharmacokinetic determinations rely on interactions between reagents (antibodies or target molecule) and the biotherapeutic. Most LBAs do not employ an analyte extraction procedure and are susceptible to matrix interference. Here, we present a case study on the development of a LBA for the quantification of a PEGylated domain antibody where matrix interference was observed. The assay used to support the single ascending dose study was a plate-based electrochemiluminescent assay with a lower limit of quantification of 80 ng/mL. To meet sensitivity requirements of future studies, new reagents and the Gyrolab™ Workstation were evaluated. RESULTS: Assay sensitivity improved nearly threefold in the final method utilizing new antibody reagents, a buffer containing blockers to human anti-animal antibodies, and the Gyrolab Workstation. CONCLUSION: Experimental data indicate that all factors changed played a role in overcoming matrix effects.

Evaluation of acoustic membrane microparticle (AMMP) technology for a sensitive ligand binding assay to support pharmacokinetic determinations of a biotherapeutic.

Achieving the required sensitivity can be a challenge in the development of ligand binding assays for pharmacokinetic (PK) determinations of biotherapeutics. To address this need, BioScale's Acoustic Membrane Microparticle (AMMP) technology was evaluated for the quantification of a PEGylated domain antibody (dAb) biotherapeutic. Previous uses of this technology had shown utility in biomarker and process development applications and this is the first application, to our knowledge, for PK determinations. In this evaluation, AMMP was capable of delivering a sensitivity of 0.750 ng/mL, which surpasses the sensitivity requirements for the majority of assays to support PK determinations. This evaluation demonstrates that this emerging technology has the ability to produce the required sensitivity, reproducibility, and selectivity needed to meet the industry's standards for PK analysis.

Development and validation of an LC-MS/MS assay for the quantitation of a PEGylated anti-CD28 domain antibody in human serum: overcoming interference from antidrug antibodies and soluble target.

AIM: To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study. MATERIALS & METHODS: The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28. RESULTS & CONCLUSION: The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.

An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

Pharmacokinetic immunoassay methods in the presence of soluble target.

Soluble targets represent a special challenge when employing ligand binding assays to support pharmacokinetic analysis of monoclonal therapeutics. Target-engaged antibody is not available for binding in immunoassays employing anti-idiotype-specific antibodies or target for capture. We investigated several formats of total antibody assays that show reduced interference of soluble targets: direct target capture, indirect target capture and acid dissociation. While indirect target capture worked well for a regular affinity antibody against DKK1, a high affinity antibody against PCSK9 required an additional acid dissociation step. The choice of a suitable format was antibody and target dependent. Our results offer several choices to approach immunoassay development for soluble targets.

Donor-recipient mismatch for common gene deletion polymorphisms in graft-versus-host disease.

Transplantation and pregnancy, in which two diploid genomes reside in one body, can each lead to diseases in which immune cells from one individual target antigens encoded in the other's genome. One such disease, graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT, or bone marrow transplant), is common even after transplants between HLA-identical siblings, indicating that cryptic histocompatibility loci exist outside the HLA locus. The immune system of an individual whose genome is homozygous for a gene deletion could recognize epitopes encoded by that gene as alloantigens. Analyzing common gene deletions in three HSCT cohorts (1,345 HLA-identical sibling donor-recipient pairs), we found that risk of acute GVHD was greater (odds ratio (OR) = 2.5; 95% confidence interval (CI) 1.4-4.6) when donor and recipient were mismatched for homozygous deletion of UGT2B17, a gene expressed in GVHD-affected tissues and giving rise to multiple histocompatibility antigens. Human genome structural variation merits investigation as a potential mechanism in diseases of alloimmunity.