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.

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.

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.

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.

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.

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.

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.