2017 White Paper on recent issues in bioanalysis: a global perspective on immunogenicity guidelines & biomarker assay performance (Part 3 - LBA: immunogenicity, biomarkers and PK assays).

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV): (Part 3 - LBA, biomarkers and immunogenicity).

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 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. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

Validation of a ligand-binding assay for active protein drug quantification following the 'free analyte QC concept'.

AIM: Active drug assays are becoming increasingly important in protein drug development. We describe the validation of a ligand-binding assay for active protein drug quantification and address practical challenges as well as regulatory implications. RESULTS: A bioanalytical method for active protein drug quantification was successfully validated. Validation data prove that this method can be routinely used applying the commonly accepted acceptance criteria for ligand-binding assays. CONCLUSION: Active drug assays are a powerful tool to elucidate the pharmacokinetic/pharmacodynamic relationship as they take into consideration the influence of various matrix components, such as soluble ligand and anti-drug antibodies. However, not all aspects of the validation concept described in the guidelines for pharmacokinetic assays can be applied to active drug assays and thus regulatory guidelines should be adapted in consequence.

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.

Quantification of a bifunctional drug in the presence of an immune response: a ligand-binding assay specific for 'active' drug.

AIM: During development of biologics, safety and efficacy assessments are often hampered by immune responses to the treatment. The raised antidrug antibodies (ADA) might interfere with the bioanalytical method and complicate result interpretation if non-fully characterized bioanalytical methods were applied. METHODS: Here, we report an approach to characterize a ligand-binding assay (LBA) for the quantification of active drug exposure of a bifunctional therapeutic protein in the presence of antidrug antibodies, by correlating LBA results with those of a cell-based PK assay. RESULTS: A clear correlation between both assays could be observed when monoclonal and polyclonal antibodies against the toxin moiety of the drug were used as ADA surrogates, and results were confirmed with human ADA-positive sera. CONCLUSION: The observed correlation between the LBA-based and cell-based PK assay indicated the suitability of the developed LBA for the determination of active drug exposure in the presence of an immune response.

An ex vivo potency assay to assess active drug levels of a GLP-1 agonistic peptide during preclinical safety studies.

BACKGROUND: During development of biologics, safety and efficacy assessments are often hampered by immune responses to the treatment. To assess active exposure of a drug peptide in a toxicology study, we developed an ex vivo potency assay which complemented the total drug quantification assay. METHODOLOGY: Compound activity was assessed in samples of treated monkeys by cell-based cAMP measurements. For each animal, activity was compared with its predose sample to which the compound has been added at the postdose concentration as determined by a total LC-MS/MS assay. CONCLUSION: We were able to show that despite a high total test compound level, activity was reduced tremendously in antidrug-antibody-positive monkeys. Therefore, the applied ex vivo potency assay supplements drug quantification methods to determine active exposures.

Developability assessment during the selection of novel therapeutic antibodies.

Therapeutic antibodies and antibody derivatives comprise the majority of today's biotherapeutics. Routine methods to generate novel antibodies, such as immunization and phage-display, often give rise to several candidates with desired functional properties. On the contrary, resource-intense steps such as the development of a cell line, a manufacturing process, or a formulation, are typically carried out for only one candidate. Therefore, "developability," that is, the likelihood for the successful development of a lead candidate into a stable, manufacturable, safe, and efficacious drug, may be used as an additional selection criterion. Employing a set of small-scale, fast, and predictive tests addressing biochemical and biophysical features, as well as in vivo fate can help to identify a clinical candidate molecule with promising properties at an early stage of drug development. This article gives an overview of existing methods for developability testing and shows how these assays can be interlaced in the lead selection process.

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies.

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions.

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies.

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.

Low level sequence variant analysis of recombinant proteins: an optimized approach.

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA (desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.

Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins: what bioanalysts should know about their reagents.

Ligand-binding assays are the standard technology used for bioanalysis of therapeutic proteins, for example, for drug quantification (pharmacokinetics assays) and immunogenicity testing (antidrug antibody assays). Besides the selection of the most suitable technology platform (e.g., ELISA, electrochemiluminescence assays and surface plasmon resonance assays) and assay procedure, a pivotal prerequisite for good assay performance on any technology platform is the design, production and characterization of high quality reagents. To enable bioanalytical project support over the complete product life cycle, an appropriate long-term reagent supply is needed. This perspective describes our opinion on the requirements for generation and QC of critical reagents used in ligand-binding assays for drug quantification and antidrug antibody detection to enable high-quality assays and long-term supply, including reagent batch switches. The critical parameters during reagent design, production and long-term supply, along with the appropriate analytical methods for QC testing and appropriate certification, are discussed.

Inhibition of intracerebral glioblastoma growth by local treatment with the scatter factor/hepatocyte growth factor-antagonist NK4.

PURPOSE: Scatter factor/hepatocyte growth factor (SF/HGF) and its tyrosine kinase receptor MET are strongly up-regulated in malignant gliomas. The SF/HGF-MET system contributes to glioma invasion and angiogenesis via autocrine and paracrine mechanisms. We analyzed whether local treatment with NK4, an antagonistic fragment of SF/HGF, could inhibit glioma growth in vivo. EXPERIMENTAL DESIGN: A guide-screw system was used to implant tumor cells intracerebrally and to perform therapeutic injections. Mice received daily intratumoral injections of NK4 or buffer as of day 1 or 7 after tumor cell injection until day 20. Functional effects of NK4 on glioma and endothelial cells were analyzed in vitro. RESULTS: Tumor volume was reduced by 61.1% in mice treated with NK4 compared with controls when treatment was initiated on day 1 (P < 0.05) and by 61.4% when treatment was initiated on day 7 (P < 0.001). Intratumoral microvessel density was reduced by 64.9% when treatment started on day 1 and by 36.7% when it started on day 7. The proliferative activity of the tumor cells was reduced by >30% regardless of when NK4-treatment was initiated. The apoptotic fraction of tumor cells was increased 2-fold and 1.5-fold when animals were treated with NK4 as of day 1 or day 7, respectively. In vitro, NK4 inhibited SF/HGF-induced glioblastoma, and endothelial cell migration and proliferation in a dose-dependent fashion. CONCLUSION: NK4 inhibits glioblastoma growth in vivo, most likely via antimitogenic, antimotogenic, proapoptotic, and antiangiogenic mechanisms. Given the strong up-regulation of SF/HGF and MET in human malignant gliomas, NK4 holds promise as a direct interstitial therapeutic agent for these fatal tumors.

Effects of hedgehog proteins on tissue engineering of cartilage in vitro.

The effects of three derivatives of the N-terminal signaling domain of hedgehog proteins on cartilage engineered in vitro were investigated, with specific focus on the ability to increase tissue growth rate and concentrations of major extracellular matrix components, that is, glycosaminoglycans (GAG) and collagen, and on the effects on morphological appearance of the tissue. Bovine articular chondrocytes were cultured on biodegradable polyglycolic acid (PGA) scaffolds with or without the addition of dipalmitoylated sonic hedgehog (dp-shh), dipalmitoylated indian hedgehog (dp-ihh), or sonic hedgehog dimer (shh-dimer) to medium with either 1% or 10% fetal bovine serum (FBS). All three hedgehog proteins dose-dependently increased construct weights (by up to 1.95-fold, dp-shh at 1,000 ng/mL) and the fraction of GAG over 4 weeks (by up to 2.7-fold, dp-shh at 1,000 ng/mL), as compared to control constructs. Dp-shh and dp-ihh elicited similar responses; a 10-fold higher concentration of nonacylated shh-dimer was necessary to reach comparable results. Positive hedgehog effects were more pronounced in medium containing 1% FBS than in medium containing 10% FBS; however, at either FBS concentration, cartilaginous tissues grown in the presence of hedgehog proteins appeared morphologically more mature. Hedgehog derivatives thus appear as promising candidates to improve the development and composition of engineered cartilage.

In situ gene expression analysis during BMP2-induced ectopic bone formation in mice shows simultaneous endochondral and intramembranous ossification.

We examined the molecular progression of ectopic bone development upon application of recombinant human bone morphogenetic protein-2 (rhBMP2), using a commercial collagen type I carrier, in the hind quarter muscles of mice. We performed a gene expression study using mRNA in situ hybridisation to compare embryonic cartilage and bone formation with BMP2-induced ectopic bone formation. As bone growth can be induced postnatally or in adult animals, we examined the expression of molecules regulating embryonic bone development. We found that the mRNAs of the same molecules, such as Indian hedgehog (IHH), parathyroid hormone (PTH)/PTH-related peptide receptor (PPR) and BMPs, that regulate embryonic cartilage and bone development, are expressed during BMP-induced ectopic bone formation, suggesting parallels in the mechanisms controlling these processes. Our studies support by molecular means the previous findings in rats that BMP2-induced ectopic bone formation in mice undergoes bone development involving both modes, endochondral and intramembranous ossification, simultaneously at different sites of the implant.