Hybridoma Culture Research Articles

A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N′,N′-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Simple and fast one-step FRET assay of therapeutic mAb bevacizumab using anti-idiotype DNA aptamer for process analytical technology

We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50–2000 μg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.

In-silico optimal operating policies of a batch or a fed-batch bioreactor for mAb production using a hybridoma cell culture

Production of monoclonal antibodies (mAb) is a well-known method to synthesize a large number of identical antibodies, of huge importance in medicine. In thus context, huge efforts have been spent to maximize the mAb production in industrial bioreactors by using hybridoma cell cultures. However, the optimal operation of these bioreactors is an engineering problem difficult to solve due to the highly nonlinear bioprocess dynamics, and a bioreactor involving a large number of decision (control) variables, subjected to multiple nonlinear process constraints, which often translates into a non-convex optimization problem. Based on an adequate kinetic model adopted from literature, this paper is aiming at in-silico, off-line deriving and comparing the optimal operating policies of a batch bioreactor (BR), and a fed-batch bioreactor (FBR) operated in several feeding alternatives (including substrates and the viable biomass) with using a hybridoma culture immobilized on a porous support (alginate) for mAb production. FBR with a variable time stepwise optimal feeding policy proved to reach better performances in terms of mAb production maximization with a minimal raw-material consumption.

Affinity purification of mAb from serum-containing hybridoma culture supernatant through a novel nanobody that discriminates mouse IgG from bovine IgG by recognizing the mouse kappa constant region (mCK)

When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.

Serum components influence antibody reactivity to glycan and DNA antigens

We previously generated three types of anti-glycan monoclonal IgM antibodies that react with certain structures on the glycans of glycosphingolipids and glycoproteins. As the nucleotide sequences for the variable regions of these IgM antibodies showed homology with those of anti-DNA antibodies deposited in public databases, we analyzed the reactivity of the anti-glycan IgM antibodies to DNA by ELISA. We found that anti-α2,6-sialyl LacNAc IgM in the supernatant of a hybridoma culture cross-reacted with DNA, and after purification of the IgM by zirconia column chromatography, the highly purified IgM showed increased cross-reactivity to DNA. As most of the contaminating bovine serum proteins in the culture supernatant were removed by the purification process, it is likely that a part of the removed components influences antibody reactivity to DNA. Purified anti-DNA antibodies prepared from lupus model NZB/W F1 and MRL/lpr mouse sera and normal human serum were then analyzed, and similar results showing increased reactivity to DNA were obtained. Furthermore, ELISA using these purified antibodies and various carbohydrate antigens showed that the antigen-binding specificity of these antibodies was altered by the purification process from serum-containing antibody preparations. Our results indicate that mammalian serum contains components that strongly influence antibody reactivity to carbohydrate antigens, including DNA.

A zirconia-based column chromatography system optimized for the purification of IgM from hybridoma culture supernatants

By using EDTPA-modified zirconia particles that selectively adsorb immunoglobulins in a column, we developed a chromatography separation system for efficient concentrating and purifying of IgM from hybridoma culture supernatants. Hybridoma culture supernatants containing IgMs were diluted 3-fold with 10 mM phosphate buffer (pH 7.0) and passed through the column. During this process, zirconia particles selectively adsorbed these IgMs, and most of the contaminating proteins flowed out into the flow-through. The adsorbed IgMs were easily eluted with a small volume of 400 mM phosphate buffer (pH 8.0), and high-concentration IgM solutions were prepared. Subsequent simple processing using a Capto™ Core 400 cartridge column provided highly purified IgM. The operation is easy, and the activity of IgM is maintained because the purification process is performed using only neutral ranges of phosphate buffers. Here, we showed that anti-globoside and anti-CDw75 IgM purified by this method can be used to stain cervical cancer and Burkitt lymphoma cells that specifically express these respective tumor-associated carbohydrate antigens.

Substantially Improved Electrofusion Efficiency of Hybridoma Cells: Based on the Combination of Nanosecond and Microsecond Pulses.

As the initial antibody technology, the preparation of hybridoma cells has been widely used in discovering antibody drugs and is still in use. Various antibody drugs obtained through this technology have been approved for treating human diseases. However, the key to producing hybridoma cells is efficient cell fusion. High-voltage microsecond pulsed electric fields (μsHVPEFs) are currently one of the most common methods used for cell electrofusion. Nevertheless, the membrane potential induced by the external microsecond pulse is proportional to the diameter of the cell, making it difficult to fuse cells of different sizes. Although nanosecond pulsed electric fields (nsPEFs) can achieve the fusion of cells of different sizes, due to the limitation of pore size, deoxyribonucleic acid (DNA) cannot efficiently pass through the cell pores produced by nsPEFs. This directly causes the significant loss of the target gene and reduces the proportion of positive cells after fusion. To achieve an electric field environment independent of cell size and enable efficient cell fusion, we propose a combination of nanosecond pulsed electric fields and low-voltage microsecond pulsed electric fields (ns/μsLVPEFs) to balance the advantages and disadvantages of the two techniques. The results of fluorescence experiments and hybridoma culture experiments showed that after lymphocytes and myeloma cells were stimulated by a pulse (ns/μsLVPEF, μsHVPEF, and control), compared with μsHVPEF, applying ns/μsLVPEF at the same energy could increase the cell fusion efficiency by 1.5–3.0 times. Thus far, we have combined nanosecond and microsecond pulses and provided a practical solution that can significantly increase cell fusion efficiency. This efficient cell fusion method may contribute to the further development of hybridoma technology in electrofusion.

Testing Hybridoma Cells for Mycoplasma Contamination.

Because mycoplasmas are a diverse group of organisms and are difficult to culture, several different strategies for detecting mycoplasma contamination have been developed. To date, no one test is suitable for detecting all of the possible mycoplasmas that may contaminate hybridoma or myeloma cultures. Therefore, it is sensible to consider using several methods. The most commonly used techniques are described here. Mycoplasma screening by growth on microbial medium can be performed on agar plates or in broth culture. Cultures are grown under both aerobic and anaerobic conditions because some common strains of Mycoplasma prefer the lack of oxygen (Mycoplasma buccale, Mycoplasma faucium, M. orale, and M. salivarium). Mycoplasma colonies form a characteristic "fried egg" appearance on agar plates, and this is the diagnostic feature used to confirm mycoplasma contamination. The colonies are small and are most easily seen with an inverted microscope. A quicker method for testing for mycoplasma takes advantage of the DNA-intercalating dye Hoechst 33258. Fixed cells are stained with the dye, and contaminated cultures are detected by the bright, punctate cytoplasmic staining of the Mycoplasma DNA. Finally, commercial kits for the detection of mycoplasmas using colorimetric assays or reporter cells are also described.

Monoclonal antibodies specific for the hemagglutinin-neuraminidase protein define neutralizing epitopes specific for Newcastle disease virus genotype 2.VII from Egypt

BackgroundNewcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs).MethodsAn HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes.ResultsSix out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes.ConclusionsThese results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.

Purification of anti-glycoconjugate monoclonal antibodies using newly developed porous zirconia particles

Here, we describe porous zirconia particles (PZPs) optimized for the purification of immunoglobulins. PZPs, with a pore size of approximately 10 nm, were designed to specifically interact with antibodies via surface modification with a phosphate functional group. A simple PZP purification method based on precipitation enabled efficient purification of mouse anti-glycosphingolipid globoside/Gb4Cer monoclonal IgM (κ-light chains) from hybridoma culture supernatants. Over 99% of contaminating proteins were removed by the PZP purification process, and approximately 50% of the IgM was recovered in the purified fraction after eluting the PZP-adsorbed antibodies with 100 mM phosphate buffer. Other IgG3 and IgM monoclonal antibodies that react with Gb4Cer or α2,6-sialyl LacNAc-modified glycoproteins could also be purified using PZPs and elution buffer at concentrations of 100–500 mM. All of the purified antibodies retained their antigen reactivity and specificity, indicating that PZP purification does not affect antibody function. As PZP purification is also suitable for purification of IgM consisting of λ-light chains and IgG derived from other mammalian species, it is expected to be applied to the purification of a variety of antibodies, including anti-glycoconjugate IgMs.

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures

SUMMARYFast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.

Selection of a Minimal Suboptimal Set of EFMs for Dynamic Metabolic Modelling

Mathematical modelling supports the understanding of basic biological mechanisms and is the basis for bioprocess simulation, prediction, control and optimization. Dynamic macroscopic models can be derived from the concept of elementary flux modes (EFMs), which provide a comprehensive representation of all possible pathways through a metabolic network. As the number of EFMs drastically increases with the size of the metabolic network, a procedure to reduce the number of EFMs and select the most informative ones is required. For this purpose, this study proposes a methodology to select a minimal suboptimal set of elementary flux modes allowing the development of reduced macroscopic models. The algorithm is divided into two steps. First, the concept behind the cosine-similarity algorithm is extended for a large number of EFMs to cut the initial set by removing all the collinear modes. Next, the algorithm is used to extract only the most informative modes from the reduced set by means of a series of optimization problems. The algorithm performance is illustrated with datasets from hybridoma cultures in batch and perfusion modes, and two metabolic networks with different levels of description.

Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3–CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

Model-Based Optimization of a Fed-Batch Bioreactor for mAb Production Using a Hybridoma Cell Culture.

Production of monoclonal antibodies (mAbs) is a well-known method used to synthesize a large number of identical antibodies, which are molecules of huge importance in medicine. Due to such reasons, intense efforts have been invested to maximize the mAbs production in bioreactors with hybridoma cell cultures. However, the optimal control of such sensitive bioreactors is an engineering problem difficult to solve due to the large number of state-variables with highly nonlinear dynamics, which often translates into a non-convex optimization problem that involves a significant number of decision (control) variables. Based on an adequate kinetic model adopted from the literature, this paper focuses on developing an in-silico (model-based, offline) numerical analysis of a fed-batch bioreactor (FBR) with an immobilized hybridoma culture to determine its optimal feeding policy by considering a small number of control variables, thus ensuring maximization of mAbs production. The obtained time stepwise optimal feeding policies of FBR were proven to obtain better performances than those of simple batch operation (BR) for all the verified alternatives in terms of raw material consumption and mAbs productivity. Several elements of novelty (i–iv) are pointed out in the “conclusions” section (e.g., considering the continuously added biomass as a control variable during FBR).

One-Step Antibody Arrays.

The sandwich-based immunoassay is renowned for its specificity and sensitivity for protein detection, where the antigen is "sandwiched" by a pair of antigen epitope-specific antibodies. The capture antibody-antigen-detection antibody complex will be formed if all the components are present in a proper reaction system. A one-step rapid sandwich-based antibody array can be developed through fixing the capture antibody on a glass slide with a fluorescence-labelled detection antibody. In this chapter, we describe the process of a one-step mouse immunoglobulin isotyping array for use in hybridoma culture supernatants.

Bovine serum albumin affects N-glycoforms of murine IgG monoclonal antibody purified from hybridoma supernatants.

Immunoglobulin G (IgG) is a class of monoclonal antibodies (mAbs) commonly produced in mammalian cell lines. These cell lines are grown in finely adjusted culture media, which contain components that may impact glycoforms. As variation of N-glycoforms can impact the biological properties of IgGs, medium composition should be controlled. Here, we studied the effects on IgG N-glycoforms of different components in hybridoma culture media, specifically compared bovine serum albumin (BSA) with other small molecules, using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight multistage mass spectrometry (MALDI-QIT-TOF MSn)-based approach. We show that small molecular additives caused little change in glycan species, though a number of these reagents, especially glutamine, affected levels of glycosylation. In comparison, the addition of macromolecular protein BSA significantly changed IgG N-glycan patterns, not only in species but also in glycosylation levels. Together, our finding suggests that BSA increases the complexity of IgG N-glycoforms, thus raising the difficulty in maintaining glycoforms consistency during antibody production. Therefore, the effect of BSA on IgG N-glycans should be considered when designing optimal medium formulations for IgG production. KEY POINTS: • Small molecular medium additives only affect glycosylation levels of IgG N-glycans. • BSA significantly changes IgG N-glycoforms as a medium additive. • BSA's skewing of IgG N-glycoforms should be considered in IgG production.

FcγR-binding affinity of monoclonal murine IgG1s carrying different N-linked Fc oligosaccharides

Glycosylation is one of the most common post-translational modifications which diversifies the structure and function of glycoproteins like immunoglobulin G (IgG). The effector function of IgG depends on N-glycan patterns located in the crystalline fragment (Fc). Fc gamma receptor (FcγR)-binding affinity is one of the most important effector functions in IgG, and it varies with different IgG isotypes. Murine IgG1 (mIgG1) triggers various immune effector functions via FcγRs, however, how N-glycans of mIgG1 impact interactions between mIgG1s and murine FcγRs remains largely unknown. Here, we generated mIgG1s with different N-glycan patterns by adding different types of N-glycan processing enzyme inhibitors to the hybridoma culture media, before comparing their FcγR-binding affinity using enzyme-linked immunosorbent assay (ELISA) analysis. We showed that N-glycans critically affect mIgG1 affinity to FcγRs. The removal of N-glycans nearly completely abolished mIgG1-FcγR binding. In comparison, when N-glycans are present, decreasing fucosylation levels enhanced the FcγR-binding affinity regardless of the types of N-glycans. Furthermore, high-mannose type and hybrid type N-glycans reduced FcγR-binding affinity, compared to complex type N-glycans. In conclusion, our findings clearly demonstrate that FcγR-binding affinity of mIgG1 is under the control of glycosylation. Importantly, we found that both the levels of specific glycosylation as well as the types of N-glycans affect FcγR-binding affinity. Together, these insights should greatly expand our understanding of N-glycans function in general, and assist in manipulating host immune responses by controlling antibody N-glycan patterns, which is important for designing therapeutic antibodies with improved characteristics.

In-silico Optimization of a Batch Bioreactor for mAbs Production in Relationship to the Net Evolution of the Hybridoma Cell Culture

Bioreactor optimization is a common engineering problem difficult to be solved due to the large number of influential variables of high variability. Production of monoclonal antibody is a well-known method to synthesize a large number of identical antibodies (that is of uniform characteristics, also called monoclonal antibodies, mAb). Due to such reasons intense efforts have been invested to maximize the production of mAb by using hybridoma cell culture. Based on an adequate kinetic model from literature (experimentally checked) this paper focus on pointing-out the major role of the net evolution of the viable biomass (growth, and decay) in the location of the optimal operating setpoint (SP) of a three-phase mechanically agitated batch bioreactor (TPMAB) with immobilized hybridoma culture. This in-silico analysis opens the possibility I) to optimize the bioreactor performances by placing the SP in the most favourable location, by adjusting the substrate and biomass initial load in the bioreactor according to the preliminary determined characteristics of a modified / improved biomass; ii) to optimize the batch-to-batch operation mode (not approached here) according to the time-varying characteristics of the biomass culture, or iii) to determine the optimal operation of the bioreactor in a fed-batch operating mode (not approached here).

Optimization of Microparticle Reagents to Collect and Detect Antibody.

Bead reagents are used in a large number of assays in bioscience and biotechnology to collect and purify antibodies by immobilization. Bead-based immunoassays offer high-throughput analysis of multiple antibodies in a single sample. Although a variety of antibody-binding moieties on the collection beads have been studied, the physical and material properties of collection beads have not been optimized to isolate specific antibodies over a broad range of concentrations from complex environments containing cells. We present a study of how to optimally use microparticles coated with protein G to collect low concentrations of IgG antibodies from complex solutions. We study the impact of bead material, bead size, incubation time, and protein G density to more efficiently collect antibodies and detect specific antibodies via fluorescent antigen labeling. The minimum detectable limit and the minimum incubation time for antibody collection are used as metrics to evaluate the collection parameters. We found that larger silica beads can capture more antibodies from a low concentration of sample, with a minimum incubation time of 60 min to equilibrium binding, resulting in a minimum detectable concentration of antibodies of 26 nM. We show that simple biophysical optimization of antibody collection reagents can be used to improve the collection of low concentrations of antibodies in complex environments. We demonstrate that the technology may be useful for monitoring antibody secretions from hybridoma cultures.

Electrofusion by a bipolar pulsed electric field: Increased cell fusion efficiency for monoclonal antibody production

The excessive cell death rate caused by electrofusion with unipolar pulses (UPs) has been a bottleneck to increasing cell fusion efficiency in monoclonal antibody technology. Several studies have confirmed that compared with UPs, bipolar pulses (BPs) with microsecond pulse widths can increase electropermeabilization while reducing cell death. Given these characteristics, BPs were used to increase cell fusion efficiency in this study. Cell staining and hybridoma culture experiments were performed using SP2/0 mouse myeloma cells and lymphocytes. Based on the equal energy principle, UPs and BPs were delivered to electrodes at a distance of 3.81 mm, with electric field intensities ranging from 2 kV/cm to 3 kV/cm and pulse duration of 40 μs for the UPs and 20–20 μs for the BPs. The results of cell staining experiments showed that cell fusion efficiency was 3-fold greater with BPs than with UPs. Similarly, the results of the hybridoma culture experiments showed that the hybridoma yields were 0.26‰ and 0.23‰ (2.5 kV/cm and 3 kV/cm, respectively) in the UP groups and increased to 0.46‰ and 0.35‰ in the BP groups. Taken together, the results show that the efficiency of heterologous cell fusion can be greatly increased if BPs are used instead of the commonly applied UPs. This study may provide a promising method for monoclonal antibody technology.