Access to ultra-long IgG CDRH3 bovine antibody sequences using short read sequencing technology.

The advances in high-throughput DNA sequencing and recombinant antibody technologies has presented new methods for characterizing antibody repertoires and significantly increased our understanding on the functional role of antibodies in immunity and their use in diagnostics, vaccine antigen design and as biological therapeutics. A subset of Bos taurus antibodies possesses unique ultra-long third complementary-determining region of the heavy chain (CDRH3) and are of special interest because they are thought to have unique functional abilities of broadly neutralizing properties - a functional role that has not been fully explored in vaccine development. Next generation sequencing technologies that are widely used to profile immunoglobulin (Ig) repertoires are based on short-read methods such as the Illumina technology. Although this technology has worked well in sequencing Ig V-D-J regions of most jawed vertebrates, it has faced serious technical challenges with sequencing regions in bovine Ig bearing ultra-long CDRH3 sequences, which are longer than 120 bp. To overcome this limitation, we have developed a sequencing strategy based on nested PCR products that allows sequence assembly of full-length bovine Ig heavy-chain (IgH) V-D-J regions. We have used this strategy to sequence IgH V-D-J regions of two Bos indicus breeds, Ankole and Boran. We confirm the presence of ultra-long CDRH3 sequences in IgG transcripts in both African cattle breeds, and provide preliminary evidence for differences and preferences in germline VH, DH and JH allele gene usage as well as differences in the length of the VH region in the two bovine breeds. Our method provides tools that should allow more robust analyses of ultra-long CDRH3 sequences aiding antibody and epitope discovery in different cattle breeds and their role in mediating immunity.

Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve.

Capillary sieving electrophoresis utilizing SDS (CE(SDS)) is one of the most applied methods for the analysis of antibody (mAb) size heterogeneity in the biopharmaceutical industry. Inadequate peak identification of observed protein fragments is still a major issue. In a recent publication, we introduced an electrophoretic 2D system, enabling online mass spectrometric detection of generic CE(SDS) separated peaks and identification of several mAb fragments. However, an improvement regarding system stability and handling of the approach was desired. Here, we introduce a novel 8-port valve in conjunction with an optimized decomplexation strategy. The valve contains four sample loops with increased distances between the separation dimensions. Thus, successively coinjection of solvent and cationic surfactant without any additional detector in the second dimension is enabled, simplifying the decomplexation strategy. Removal efficiency was optimized by testing different volumes of solvents as presample and cationic surfactant as postsample zone. 2D measurements of the light and heavy chain of the reduced NIST mAb with the 8-port valve and the optimized decomplexation strategy demonstrates the increased robustness of the system. The presented novel set-up is a step toward routine application of CE(SDS)-CZE-MS for impurity characterization of proteins in the biopharmaceutical field.

A proteolytic method for evaluating O-GlcNAcylation on proteins of similar molecular weight to antibody heavy chain after immunoprecipitation.

Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunoprecipitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies. IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25 kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins of interest formerly obscured by antibody heavy chain.

Heavy and light chain (AHL)-type cardiac amyloidosis: first histopathologic-proven case illustrating involvement of the heart.

Cardiac amyloidosis is most commonly comprised of either a monoclonal immunoglobulin or transthyretin; however, in practice, detailing of the former beyond light chain restriction is not typically performed. We present briefly the case of an 80-year-old man with concern for cardiac amyloidosis and a subsequent endomyocardial biopsy revealing significant deposition of amorphous Congo red-positive material. By immunofluorescence microscopy, the amyloidogenic material showed positive expression for IgG heavy chain and kappa light chain, with negative staining for IgM and IgA heavy chains and lambda light chain supporting a diagnosis of heavy and light chain (AHL)-type amyloidosis. Immunofluorescence staining for the IgG heavy chain subclasses supported and further classified the patient's AHL-type cardiac amyloidosis as being IgG4/kappa restricted. The presented case is the first to illustrate AHL-type cardiac amyloidosis via sampling of heart tissue.

Rapid Intact mass based multi-attribute method in support of mAb upstream process development.

N-linked glycosylation is a critical quality attribute for monoclonal antibodies (mAbs) as it affects product stability, immunogenicity, receptor binding and antibody effector function, clearance and half-life. It has been widely accepted that the glycosylation process is greatly impacted by several factors during bioreactor operations. Therefore, the timely acquisition of N-linked glycosylation information during cell culture process development is critical for the success of the endeavor. In this paper we describe a simple, rapid, and robust Multi-Attribute Method (MAM) based on intact mass analysis. This method, developed for an open access instrument, has been optimized for the analysis of light and heavy chains generated from dithiothreitol (DTT) reduction of intact mAbs sampled directly out of bioreactors. The N-linked glycosylation profile, identity confirmation of light chain and heavy chain, light chain glycation and non-glycosylated heavy chain (NGHC) can all be monitored by this method. Our results confirm that the N-linked glycosylation profile determined using Intact mass based MAM is comparable with a release glycan assay and LC-MS/MS peptide based MAM assay for the most abundant glycoforms. Furthermore, the results for the NGHC attribute determined with our method are comparable to results from capillary gel electrophoresis (CGE) and LC-MS/MS peptide based MAM.

Molecular characterization and expression analysis of secretory immunoglobulin M (IgM) heavy chain gene in rohu, Labeo rohita.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila. These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

The quantification of antibody elements and receptors subunit expression using qPCR: The design of VH, VL, CH, CL, FcR subunits primers for a more holistic view of the immune system.

The expression levels of immunoglobulin elements and their receptors are important markers for health and disease. Within the immunoglobulin locus, the constant regions and the variable region families are associated with certain pathologies, yet a holistic view of the interaction between the expressions of the multiple genes remain to be fully characterized. There is thus an important need to quantify antibody elements, their receptors and the receptor subunits in blood (PBMC cDNA) for both screening and detailed studies of such associations. Leveraging on qPCR, we designed primers for all Vκ1-6, VH1-7, Vλ1-11, nine CH isotypes, Cκ, Cκ, Cλ1 &3, FcεRI α,ß, and γ subunits, all three FcγR and their subunits, and FcαR. Validating this on a volunteer PBMC cDNA, we report a qPCR primer set repertoire that can quantify the relative expression of all the above genes to the GAPDH housekeeping gene, with implications and uses in both clinical monitoring and research.

Characterization of ADCs by Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation technique that resolves ions based on their electrophoretic mobility in the presence of an applied voltage. It has been broadly applied for characterizing biotherapeutics including ADCs. In this chapter, step-by-step procedures for characterizing ADCs using CE will be described with focus placed on reduced and non-reduced capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for purity determination and imaged capillary isoelectric focusing (iCIEF) for charge heterogeneity analysis.

Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.

One challenge associated with the discovery and development of monoclonal antibody (mAb) therapeutics is the determination of heavy chain and light chain pairing. Advances in MS instrumentation and MS/MS methods have greatly enhanced capabilities for the analysis of large intact proteins yielding much more detailed and accurate proteoform characterization. Consequently, direct interrogation of intact antibodies or F(ab')2 and Fab fragments has the potential to significantly streamline therapeutic mAb discovery processes. Here, we demonstrate for the first time the ability to efficiently cleave disulfide bonds linking heavy and light chains of mAbs using electron capture dissociation (ECD) and 157 nm ultraviolet photodissociation (UVPD). The combination of intact mAb, Fab, or F(ab')2 mass, intact LC and Fd masses, and CDR3 sequence coverage enabled determination of heavy chain and light chain pairing from a single experiment and experimental condition. These results demonstrate the potential of top-down and middle-down proteomics to significantly streamline therapeutic antibody discovery.

Microfluidic chip technology applied to fine-needle aspiration cytology samples for IGH clonality assessment.

BACKGROUND: Most cases of non-Hodgkin lymphoma (NHL) can be diagnosed using a combination of fine-needle cytology (FNC) and flow cytometry together with immunoglobulin light chain restriction and/or specific phenotypic profiles. However, 5%-15% of B-cell NHLs lack these specific diagnostic features. In such cases, the diagnosis of NHL may be supported by molecular clonality testing based on the immunoglobulin heavy chain (IGH) assay of clonality by polyacrylamide heteroduplex analysis or by automated capillary electrophoresis via GeneScan analysis. Chip-based microfluidic technology (MT), based on miniaturized parallel capillary electrophoresis structures, is a viable alternative to capillary electrophoresis analysis, being less costly and cumbersome. In this study, we evaluated the performance of MT platform in IGH clonality assessment in a series of lymph node FNC samples. METHODS: Thirty-five consecutive lymph node FNCs were evaluated. In all cases, the first and the second passes were used to prepare a conventional smear and to collect material for flow cytometry analysis; residual material was collected for molecular clonality assessment, and PCR products were analyzed both by MT and GeneScan platforms. RESULTS: Molecular clonality assessment by MT had a sensitivity of 84.2% and a specificity of 76.9%; GeneScan analysis had a sensitivity of 88.8% and a specificity of 92.8%. The overall agreement between the two platforms was 85.7% (30/35). CONCLUSIONS: MT analysis proved to be a viable technique for IGH clonality assessment on FNC samples. Should our data be confirmed in larger studies, the MT procedure may be suitable for routine diagnostic practice, even on cytological samples.

Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain.

BACKGROUND: The variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health. RESULTS: VHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH 6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4 ng mL-1 and a limit of detection (LOD) of 0.3 ng mL-1 for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g-1 in rice and maize and 72 ng g-1 in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively. CONCLUSION: The VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.

Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning.

Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

Establishment of Immunoglobulin Heavy (IGH) Chain Clonality Testing by Next-Generation Sequencing for Routine Characterization of B-Cell and Plasma Cell Neoplasms.

Immunoglobulin heavy chain (IGH) clonality testing by next-generation sequencing (NGS) offers unique advantages over current low-throughput methods in the assessment of B-cell lineage neoplasms. Clinical use remains limited because assays are not standardized and validation/implementation guidelines are not yet developed. Herein, we describe our clinical validation and implementation of NGS IGH clonality testing and summarize our experience based on extensive routine use. NGS-based clonality testing targeting IGH FR1, FR2, FR3, and the conserved leader sequence upstream of FR1 was validated using commercially available kits. Data were analyzed by commercial and in-house-developed bioinformatics pipelines. Performance characteristics were evaluated directly comparing with capillary electrophoresis (CE) assays (BIOMED-2 primers). Assays were monitored after implementation (>1.5 years), concurrently testing by CE methods. A total of 1189 clinical samples were studied (94 validation, 1095 postimplementation). NGS showed superior performance compared with CE assays. For initial assessment, clonality detection rate was >97% for all malignancy types. Concordance with CE was 96%; discordances were related to higher sensitivity/resolution of NGS and improved detection in cases with high somatic hypermutation. Routine NGS clonality assessment is feasible and superior to existing assays, enabling accurate and specific index clone assessment and future tracking of all rearrangements in a patient sample. Successful implementation requires new standardization, validation, and implementation processes, which should be performed as a multicenter and multidisciplinary collaboration.

Detection of intracellular IgD using flow cytometry could be a novel and supplementary method to diagnose IgD multiple myeloma.

BACKGROUND: We examined whether detecting the heavy chain of cytoplasmic immunoglobulin D (IgD) by flow cytometry could be used as a supplemental method to diagnose IgD multiple myeloma (MM). METHODS: Bone marrow (BM) samples of thirty-five patients with MM were collected. Five of them were IgD MM, the rest of thirty were other subtypes of MM. Antibodies to four types of heavy chains of immunoglobulin (e.g., IgA, IgG, IgM, and IgD) were analyzed by flow cytometry in each patient's BM sample. RESULTS: The five IgD MM patients were all positive for cytoplasmic IgD. The percentage of IgD positive MM cells among nucleated cells varied from 0.4 to 12.9%. Cytoplasmic IgG was positive in eight patients with IgG MM (n = 9); cytoplasmic IgA was positive in all patients with IgA MM (n = 10); cytoplasmic IgM was positive in one patient with IgM MM (n = 1). No heavy chain was detected in light chain MM (n = 9) and non-secretory subtype (n = 1). CONCLUSIONS: Detection of cytoplasmic IgD by flow cytometry is a convenient, sensitive and supplemental method to diagnose IgD MM.

Analytical validation of the Hevylite assays for M-protein quantification.

BACKGROUND: The heavy/light chain (HLC) immunoassay quantifies the different heavy chain/light chain combinations of each immunoglobulin (Ig) class. This makes the HLC assay suited to quantify monoclonal immunoglobulins (M-protein) and for monitoring of patients with monoclonal gammopathies. This method is particularly advantageous for those samples in which electrophoretic quantification of the M-protein is not possible. METHODS: In this study we tested the analytical performance of the HLC assay in 166 routine clinical samples and in 27 samples derived from the Dutch external quality assessment (EQA) for M-protein diagnostics (74 participating laboratories). Analytical accuracy was assessed by verification that the sum of the HLC-pairs equaled total Ig concentration. Sensitivity of the HLC assay was determined in a direct method comparison with immunofixation electrophoresis (IFE). RESULTS: Comparison of HLC data with routine Ig diagnostics in 27 EQA samples showed very good correlation for both the quantification of polyclonal and monoclonal IgG, IgA and IgM (Pearson correlations [r] were 0.94, 0.99 and 0.99, respectively; slopes were 0.94, 1.07 and 0.98, respectively). The overall concordance between IFE and the HLC ratio was high (93%) with a Cohen κ coefficient of 0.84. Discrepancies between both assays were mainly caused by the higher sensitivity of IFE to detect monoclonality. CONCLUSIONS: We conclude that the HLC assay is an accurate method to quantify M-proteins that can improve monitoring of M-proteins in the beta fraction that cannot be quantified using electrophoretic techniques.

Quantitative analysis of glycation and its impact on antigen binding.

Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.

Sequence composition predicts immunoglobulin superfamily members that could share the intrinsically disordered properties of antibody CH1 domains.

Antibodies are central to the growing sector of protein therapeutics, and increasingly they are being manipulated as fragments and combinations. An improved understanding of the properties of antibody domains in isolation would aid in their engineering. We have conducted an analysis of sequence and domain interactions for IgG antibodies and Fab fragments in the structural database. Of sequence-related properties studied, relative lysine to arginine content was found to be higher in CH1 and CL than in variable domains. As earlier work shows that lysine is favoured over arginine in more soluble proteins, this suggests that individual domains may not be optimised for greater solubility, giving scope for fragment engineering. Across other sequence-based features, CH1 is anomalous. A sequence-based scheme predicts CH1 to be folded, although it is known that CH1 folding is linked to IgG assembly and secretion. Calculations indicate that charge interactions in CH1 domains contribute less to folded state stability than in other Fab domains. Expanding to the immunoglobulin superfamily reveals that a subset of non-antibody domains shares sequence composition properties with CH1, leading us to suggest that some of these may also couple folding, assembly and secretion.