A new humanized antibody is effective against pathogenic fungi in vitro.

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for ß-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C. auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need.

The development of neutralizing antibodies against SARS-CoV-2 and their common features.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide severe coronavirus disease 2019 (COVID-19) pandemic since December 2019. There is a great demand for effective therapies for the prevention and treatment of COVID-19. Developing therapeutic neutralizing antibodies (NAbs), which could block viral infection, is such a promising approach, as NAbs have been successfully applied to the treatment of other viral infections. The recent advances of antibody technology have greatly accelerated the discovery of SARS-CoV-2 NAbs, and many of which are now actively tested in clinical trials. Here, we review the approaches applied for SARS-CoV-2 NAb development, and discuss the emerging technologies underlining the antibody discovery. We further summarize the common features of these antibodies including the shared neutralizing epitopes and sequence features.

A Novel Pharmacodynamic Biomarker and Mechanistic Modeling Facilitate the Development of Tovetumab, a Monoclonal Antibody Directed Against Platelet-Derived Growth Factor Receptor Alpha, for Cancer Therapy.

Tovetumab (MEDI-575) is a fully human IgG2κ monoclonal antibody that specifically binds to human platelet-derived growth factor receptor alpha (PDGFRα) and blocks receptor signal transduction by PDGF ligands. The affinity of tovetumab determined using surface plasmon resonance technology and flow cytometry demonstrated comparable binding affinity for human and monkey PDGFRα. In single and repeat-dose monkey pharmacokinetic-pharmacodynamic (PK-PD) studies, tovetumab administration resulted in dose-dependent elevation of circulating levels of PDGF-AA, a member of the PDGF ligand family, due to displacement of PDGF-AA from PDGFRα by tovetumab and subsequent blockade of PDGFRα-mediated PDGF-AA degradation. As such, PDGF-AA accumulation is an indirect measurement of receptor occupancy and is a novel PD biomarker for tovetumab. The nonlinear PK of tovetumab and dose-dependent increase in circulating PDGF-AA profiles were well described by a novel mechanistic model, in which tovetumab and PDGF-AA compete for the binding to PDGFRα. To facilitate translational simulation, the internalization half-lives of PDGF-AA and tovetumab upon binding to PDGFRα were determined using confocal imaging to be 14 ± 4 min and 30 ± 8 min, respectively. By incorporating PDGFRα internalization kinetics, the model not only predicted the target receptor occupancy by tovetumab, but also the biologically active agonistic ligand-receptor complex. This work described a novel PD biomarker approach applicable for anti-receptor therapeutics and the first mechanistic model to delineate the in vivo tri-molecular system of a drug, its target receptor, and a competing endogenous ligand, which collectively have been used for optimal dose recommendation supporting clinical development of tovetumab.

Population pharmacokinetic analysis of phase 1 bemarituzumab data to support phase 2 gastroesophageal adenocarcinoma FIGHT trial.

PURPOSE: To report population pharmacokinetic (PK) analysis of the phase 1 study (FPA144-001, NCT02318329) and to select a clinical dose and schedule that will achieve an empirical target trough concentration (Ctrough) for an anti-fibroblast growth factor receptor 2b antibody, bemarituzumab. METHODS: Nonlinear mixed-effect modeling was used to analyse PK data. In vitro binding affinity and receptor occupancy of bemarituzumab were determined. Simulation was conducted to estimate dose and schedule to achieve an empirical target Ctrough in a phase 2 trial (FIGHT, NCT03694522) for patients receiving first-line treatment combined with modified 5-fluourouracil, oxaliplatin and leucovorin (mFOLFOX6) for gastric and gastroesophageal junction adenocarcinoma. RESULTS: Bemarituzumab PK is best described by a two-compartment model with parallel linear and nonlinear (Michaelis-Menten) elimination from the central compartment. Albumin, gender, and body weight were identified as the covariates on the linear clearance and/or volume of distribution in the central compartment, and no dose adjustment was warranted. An empirical target of bemarituzumab Ctrough of ≥ 60 µg/mL was projected to achieve > 95% receptor occupancy based on in vitro data. Fifteen mg/kg every 2 weeks, with a single dose of 7.5 mg/kg on Cycle 1 Day 8, was projected to achieve the target Ctrough on Day 15 in 98% of patients with 96% maintaining the target at steady state, which was confirmed in the FIGHT trial. CONCLUSION: A projected dose and schedule to achieve the target Ctrough was validated in phase 1 of the FIGHT trial which supported selection of the phase 2 dose and schedule for bemarituzumab.

Preparation and Evaluation of the Fully Humanized Monoclonal Antibody GD-mAb Against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is the major cause of pulmonary and bronchial inflammation in infants, young children, and immunocompromised adults, but therapeutic options to control RSV are limited. In the present study a single chain antibody against RSV (GD-scFv) was screened using phage display library panning technology and a full-length monoclonal antibody (GD-mAb) was developed from GD-scFv based on the sequence encoding Ig VH and Ig VL. The anti-RSV potential of GD-mAb was evaluated in vitro and in mice. Our results indicated that both GD-scFv (4.25 ± 2 nM) and GD-mAb (3.13 ± 0.89 nM) showed high binding capability and strong binding specificity to GD protein. GD-mAb effectively neutralized RSV and reduced the plaque number in a concentration-dependent manner through a plaque reduction neutralization assay. In mice, GD-mAb lowered the lung index and reduced the lung virus titer in the mouse lung (p < 0.05). Antibody treatment reduced the phosphorylated protein level in pathways of TLR4/NF-κB, MAPKs, and PI3K/Akt (p < 0.05) and correlated with an absence of pro-inflammatory factors TNF-α, IL-1ß, and IL-6 in the mouse lung and serum (p < 0.05). In summary, these data suggest that GD-mAb may be an effective therapeutic agent for the treatment of RSV infections. Importance  Currently, only a few therapeutic options are available to control respiratory RSV in humans. In this study, our group developed a full-length monoclonal antibody (GD-mAb) and reported a high binding specificity of the RSV surface glycoproteins G. Moreover, GD-mAb effectively neutralized RSV in vitro, and significantly lowered the lung index and reduced the lung virus titer in an infected mouse lung, which suggests that GD-mAb may serve as an effective antiviral agent for RSV infection.

An ELISA for detection of complement-bound circulating immune complexes in mice.

Measurements of complement-bound circulating immune complexes (cCICs) in pre-clinical studies may provide important information about the etiology of certain pathology findings suggestive of being immune complex mediated. This article describes the development and qualification of a universal methodology to measure cCIC in mice after dosing with species foreign proteins. The assay is a sandwich enzyme-linked immunosorbent assay - exclusively based on commercially available reagents - that could detect mouse IgG bound to complement C3 independent of the test-substance present in the plasma sample. Heat-aggregated serum was used as positive control. The assay was qualified by assessment of acceptance criteria, stability of positive control, precision, and specificity. Finally, the performance of the assay was tested using plasma from mice administered either of three different proteins, i.e bovine serum albumin (BSA), a fully human monoclonal antibody, and a humanized monoclonal antibody.

Antigen-Specific Human Monoclonal Antibodies from Transgenic Mice.

Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germline configuration. The engineered mouse genome can undergo productive rearrangement in the B-cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animThis chapter summarizes the most common chromatographic mAb andals expressing human Ig genes. This chapter describes the type of transgenic-knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs.

Novel Antibody Drug Conjugates Targeting Tumor-Associated Receptor Tyrosine Kinase ROR2 by Functional Screening of Fully Human Antibody Libraries Using Transpo-mAb Display on Progenitor B Cells.

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes ("Transpo-mAb Display") for ROR2 binding. Individual cellular "Transpo-mAb" clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.

Humanised recombinant antibody fragments bind human pancreatic islet cells.

We describe here the humanisation of two mouse monoclonal antibodies that bind to surface markers on human pancreatic islet endocrine cells. Monoclonal antibodies produced by the HIC1-2B4 and HIC0-4F9 mouse hybridomas bind distinct surface molecules expressed on endocrine cells and have been validated for a number of experimental methods including immunohistochemistry and live cell sorting by flow cytometry. Variable region framework and first constant region domain sequences were replaced with that from compatible human antibody sequences, and the resulting recombinant antigen-binding fragments were cloned and expressed in mouse myeloma cells. ELISA, fluorescent immunohistochemistry, and flow cytometry were used to assess the specificity of the humanised antibody fragments. Purification and binding analyses indicated that human islet endocrine cell binding was retained in the humanised antibody fragments. These humanised, recombinant antibody fragments have a lower risk of eliciting adverse responses from a patient's immune system and, therefore, have highly improved clinical potential. Thus, they may be used to image, target or carry cargo specifically to islet cells in human patients.

Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm.

Isolation and characterization of monoclonal antibody (mAb) variants to understand the impact of their structure on function is a typical activity during early-stage candidate selection that contributes to derisking clinical development. In particular, efforts are devoted to characterizing oligomeric variants, owing to their potential immunogenic nature. We report here a mAb variant consisting of a canonical mAb monomer associated in a non-covalent fashion with an antigen-binding fragment (Fab) arm amputated from its Fc domain. The truncated heavy chain is encoded in the cell line genome and is the likely product of a genomic recombination during cell line generation. The addition of the Fab arm results in severe loss of potency, indicating its interaction with the Fab domain of the monomer. The presence of such a variant can easily be mitigated by an adequate purification step.

[Humanization of Murine Monoclonal anti-hTNF Antibody: The F10 Story].

Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab.

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671.

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies.

Development of humanized scFv antibody fragment(s) that targets and blocks specific HLA alleles linked to myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease caused by sensitization of the immune system to self-antigens. We have previously shown that targeting MG-susceptible alleles can significantly inhibit proliferation of disease-specific T cells. In this work, we humanized a murine monoclonal antibody (mAb) LG11, capable of blocking MG-associated DQ beta 1 (DQB1) allele and reformatted it into single-chain fragment variable (scFv). A fully functional humanized scFv was obtained by optimizing variable domain orientations and linker lengths, along with the optimization of expression conditions and codons to suit Escherichia coli expression machinery. Characterization of humanized scFv (FL8) revealed that the reformatted scFv, despite recognizing the same epitope as the parent murine LG11 mAb, exhibited superior binding affinity (0.97 nM) compared to the LG11 mAb, towards the immunizing antigen (DQB1*0601/70-90) and was able to block the proliferation of T cells cultured from PBLs of MG-patients typed DQB1*0601. The scFv was also capable of binding a variant MG-associated allele (DQB1*0502/70-90) with moderate affinity (18.7 nM), a feature that was absent in the LG11. To our knowledge, this is the first report of humanizing a MG-associated human leukocyte antigen (HLA) scFv for preclinical studies.

Semi-synthetic vNAR libraries screened against therapeutic antibodies primarily deliver anti-idiotypic binders.

Anti-idiotypic binders which specifically recognize the variable region of monoclonal antibodies have proven to be robust tools for pharmacokinetic studies of antibody therapeutics and for the development of cancer vaccines. In the present investigation, we focused on the identification of anti-idiotypic, shark-derived IgNAR antibody variable domains (vNARs) targeting the therapeutic antibodies matuzumab and cetuximab for the purpose of developing specific capturing ligands. Using yeast surface display and semi-synthetic, CDR3-randomized libraries, we identified several highly specific binders targeting both therapeutic antibodies in their corresponding variable region, without applying any counter selections during screening. Importantly, anti-idiotypic vNAR binders were not cross-reactive towards cetuximab or matuzumab, respectively, and comprised good target recognition in the presence of human and mouse serum. When coupled to magnetic beads, anti-idiotypic vNAR variants could be used as efficient capturing tools. Moreover, a two-step procedure involving vNAR-functionalized beads was employed for the enrichment of potentially bispecific cetuximab × matuzumab antibody constructs. In conclusion, semi-synthetic and CDR3-randomized vNAR libraries in combination with yeast display enable the fast and facile identification of anti-idiotypic vNAR domains targeting monoclonal antibodies primarily in an anti-idiotypic manner.

Site-saturation mutagenesis library construction and screening for specific broad-spectrum single-domain antibodies against multiple Cry1 toxins.

Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites. Subsequently, site-saturation cooperative mutagenesis of the four key sites was performed using overlap extension PCR, and multiple site-saturation mutagenesis sdAb library with the capacity of 1.2 × 105 colony-forming units (CFU) was successfully constructed. With alternating five Cry1 toxins as coating antigen, two generic sdAbs (2-C1, 2-C9) were screened out from the mutagenesis library, which could detect six kinds of Cry1 toxins at least. Through ELISA analysis, the binding activity of 2-C9 was significantly enhanced, and its OD values versus Cry1Aa, Cry1Ab, Cry1B, Cry1C, and Cry1E increased to 1.34, 1.53, 1.82, 2.39, and 2.7 times, respectively, compared with maternal antibody A8. The IC50 values of 2-C9 against Cry1Aa, Cry1Ab, Cry1B, and Cry1C were lower than that of A8, which showed that the affinity of 2-C9 against Cry1 toxins was enhanced. The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry toxins.

Construction and Characterization of an Anti-Hepatitis B Virus preS1 Humanized Antibody that Binds to the Essential Receptor Binding Site.

Hepatitis B virus (HBV) is a major cause of liver cirrhosis and hepatocellular carcinoma. With recent identification of HBV receptor, inhibition of virus entry has become a promising concept in the development of new antiviral drugs. To date, 10 HBV genotypes (A-J) have been defined. We previously generated two murine anti-preS1 monoclonal antibodies (mAbs), KR359 and KR127, that recognize amino acids (aa) 19-26 and 37-45, respectively, in the receptor binding site (aa 13-58, genotype C). Each mAb exhibited virus neutralizing activity in vitro, and a humanized version of KR127 effectively neutralized HBV infection in chimpanzees. In the present study, we constructed a humanized version (HzKR359-1) of KR359 whose antigen binding activity is 4.4-fold higher than that of KR359, as assessed by competitive ELISA, and produced recombinant preS1 antigens (aa 1-60) of different genotypes to investigate the binding capacities of HzKR359-1 and a humanized version (HzKR127-3.2) of KR127 to the 10 HBV genotypes. The results indicate that HzKR359-1 can bind to five genotypes (A, B, C, H, and J), and HzKR127-3.2 can also bind to five genotypes (A, C, D, G, and I). The combination of these two antibodies can bind to eight genotypes (A-D, G-J), and to genotype C additively. Considering that genotypes A-D are common, whereas genotypes E and F are occasionally represented in small patient population, the combination of these two antibodies might block the entry of most virus genotypes and thus broadly neutralize HBV infection.

Preparative separation of monoclonal antibody aggregates by cation-exchange laterally-fed membrane chromatography.

Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed membrane chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction membrane chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small membrane pore size (3-5µm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange chromatography.

Choosing the right protein A affinity chromatography media can remove aggregates efficiently.

Protein A chromatography (PAC) is commonly used as an efficient capture step in monoclonal antibody (mAb) separation processes. Usually dynamic binding capacity is used for choosing the right PAC. However, if aggregates can be efficiently removed during elution, it can make the following polishing steps easier. In this study a method for choosing the right PAC media in terms of mAb aggregate removal is proposed. Linear pH gradient elution experiments of two different mAbs on various PAC columns are carried out, where the elution behavior of aggregates as well as the monomer is measured. Aggregates of one mAb are more strongly retained compared with the mAb monomer. Another mAb showed different elution behavior, where the aggregates are eluted as both the weakly and strongly retained peaks. In order to remove the two types of aggregates by stepwise elution two protocols are tested. The first protocol A consisted of the sample loading, the wash with the equilibration buffer and the low pH elution. The wash stage of the second protocol B included the wash with 1.0 M arginine. No detectable peaks are observed during the wash stage of protocol A whereas significant peaks are monitored during the arginine wash of protocol B. One of the PAC columns showed a smaller peak during the arginine wash. In addition, both aggregate removal and monomer yield are higher with protocol B compared with the other PAC columns. This method is found to be useful for choosing the right PAC column.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.

Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry.

As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure. Graphical Abstract ᅟ.