Use of a Microfluidic Platform To Evaluate and Predict Reagent Performance in Microtiter Plate-Based Immunoassays.

Selection and characterization of antibodies are critically important in establishing robust immunoassays to support the development efforts of vaccines. Plate-based ELISA can be time- and resource-intensive to select initial antibody clones or characterize downstream resupply lots while providing limited information regarding the binding characteristics of the antibodies beyond concentration-response curves. This work applied the microfluidic Gyrolab to holistically evaluate immunoassay reagents through analyses of concentration-response curves as well as antibody-antigen interactions visualized in column images and affinity estimates. We exploited the automation capability of the Gyrolab to reduce the resources (time, reagents, and scientists) required for screening and evaluating antibody reagents. Using a flexible semi-universal assay format, we compared antibody clones for selection and resupply lots of sera and monoclonal antibodies in a simple "plug-and-play" manner without antibody modifications. We found that the performance of antibodies in the Gyrolab correlated well with the trends observed in traditional ELISAs, while the Gyrolab provided additional advantages over plate-based assays such as column images of antibody binding and affinity measurements.

Human monoclonal antibodies isolated from a primary pneumococcal conjugate Vaccinee demonstrates the expansion of an antigen-driven Hypermutated memory B cell response.

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.

A semi-universal assay platform to quantitate vaccines with potential applications for biotherapeutics.

AIM: Biologics development often requires multiple immunoassays to evaluate both assay reagents and potential drug candidates resulting in extensive analytical development. METHODOLOGY: We developed a semi-universal, 5-layer platform assay on Gyrolab using secondary antispecies or anti-isotype-specific capture and detection antibodies. We applied the assay to several multivalent vaccines. RESULTS: Method performance exhibited a median accuracy of 110%, reproducibility of 9% CV and intermediate precision of 11% CV. System suitability criteria were met for 92.5% of the samples and only one out of 31 replicate samples exhibited a %CV greater than 20%. CONCLUSION: The semi-universal Gyrolab assay allowed assay development without reagent labeling. The format could also be translated into a plate-based assay.

Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies.

IgG monoclonal antibodies (mAbs) consist of two Fab fragments and one Fc fragment. The Fab fragments contain the variable regions and are responsible for drug specificity (via antigen binding); the Fc fragment contains constant regions and is responsible for effector functions (via interactions with Fcγ receptors) and extended serum half-life (via interaction with the neonatal Fc receptor, FcRn). There are two conserved methionine (Met) residues located in the FcRn binding site of the Fc fragment. It has been shown previously that oxidation of these two Met residues decreases the binding affinity to FcRn. We have further evaluated the impact of Met oxidation on serum half-lives of two humanized IgG1 mAbs in transgenic mice with human FcRn. Variable oxidation levels were obtained by several procedures: exposure to an oxidizing agent, accumulation during extended refrigerated storage, or chromatographic separation. Our results show that Met oxidation can result in a significant reduction of the serum circulation half-life and the magnitude of the change correlates well with the extent of Met oxidation and changes in FcRn binding affinities. The relatively low levels of Met oxidation accumulated during 3 years of refrigerated storage had minimal impact on FcRn binding and no detectable impact on the serum half-life.

Application of miniaturized immunoassays to discovery pharmacokinetic bioanalysis.

INTRODUCTION: Pharmacokinetic properties of biotherapeutics are an important aspect of preclinical drug development. The lead identification and optimization space is characterized by aggressive timelines, large sample numbers, a variety of species and matrices, and limited reagent and sample volumes all of which represent challenges for traditional microtiter plate assays. Since the Gyrolab immunoassay platform can accommodate small sample volumes and automated assay processing, we evaluated the workstation as an alternative to the plate-based assays. METHODS: Three representative example assays--a generic anti-human IgG, a target specific and an anti-drug capture assay--were investigated in detail for accuracy and precision performance and their application to bioanalytical support for preclinical pharmacokinetic studies. Different animal matrices were tested in the assays and during study support. RESULTS: Gyrolab procedures could be closely modeled after regular microtiter plate assays. The small reagent volumes necessary for Gyrolab allowed studying serial bleeds of transgenic mice with only 10µL of blood sample. During development and during study support, the Gyrolab performance was similar to what can be expected from plate-based systems with accuracy and precision within 100 ± 20% or less. DISCUSSION: Overall, the technology was well suited to support quantitation of biotherapeutics using small volume samples from different preclinical species. Limited operator involvement for assay processing allowed for reduced staffing and training. However, high instrument costs and a single source of reagent supplies represent risks when moving assays further into long-term applications such as clinical studies. Despite interest in the bioanalytical field, this is the first detailed investigation of bioanalytical applications of Gyrolab in pharmacokinetic studies.