Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.     

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10⁶ cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.     

Magnetic Precipitation: A New Platform for Protein Purification

One of the trends in downstream processing comprises the use of “anything‐but‐chromatography” methods to overcome the current downfalls of standard packed‐bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation is developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit are tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions are obtained when using PEG3350 as precipitant at 4 °C for 1 h, reaching 80% purity and 50% recovery of polyclonal antibodies from plasma, and 99% purity with 97% recovery yield of anti‐TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies.     

Adsorption of human IgG on to poly(N-isopropylacrylamide)-based polymer particles

Thermosensitive poly(N-isopropylacrylamide)-based polymer particles were synthesised, and screened for the adsorption of human immunoglobulin G (hIgG). At pH 9 the adsorption on microgel particles was strongly affected by temperature, approximately 40 mg hIgG/g support (90% of initial hIgG) being adsorbed at 40°C but only 10% of initial hIgG at 25°C. At pH 5 the maximum adsorbed amount (20 mg hIgG/g support) was similar for both temperatures. The adsorption of hIgG on to charged poly(methyl methacrylate)/poly(N-isopropylacrylamide) core-shell latexes was negligible (5–10 mg hIgG/g support) at the same temperature and pH conditions. The lower adsorption of hIgG onto the core-shell particles is explained by steric interactions due to the small size of the shell.     

人生长分化因子15单克隆抗体制备、鉴定及药用价值初探

目的:制备稳定分泌抗人生长分化因子15(GDF15)单克隆抗体(m Ab)的杂交瘤细胞系,并对其分泌的m Ab进行鉴定。方法:根据人GDF15氨基酸序列特征,设计合成了8条能够免疫产生GDF15特异性抗体的抗原多肽,与VLP载体偶联后,免疫雌性BALB/c小鼠,利用杂交瘤技术制备鼠源抗人GDF15的m Ab,用间接ELISA检测m Ab腹水效价。结果:获得针对7个抗原多肽的12株稳定分泌抗人GDF15的杂交瘤细胞系,腹水m Ab效价可达1×104~1×109。结论:获得了针对不同抗原多肽的抗人GDF15的特异性m Ab,为进一步研发以GDF15为靶点的单克隆抗体抗肿瘤药物奠定了基础。     

Large Scale Fabrication of Gold Nano-Structured Substrates Via High Temperature Annealing and Their Direct Use for the LSPR Detection of Atrazine

The present work is reporting on the fabrication of localized surface plasmonic resonant (LSPR) gold nano-structures on glass substrate by using different high annealing temperatures (500 °C, 550 °C, 600 °C) of initially created semi-continue gold films (2 nm and 5 nm) by the electron beam evaporation technique. Interestingly, well-defined gold nano-structures were also obtained from continuous 8 nm evaporated gold film - known as the value above gold percolated thickness - once exposed to high temperatures. The surface morphology and plasmonic spectroscopy of “annealed” nano-structures were controlled by key experimental parameters such as evaporated film thickness and annealing temperature. By using scanning electron microscopy (SEM) characterization of annealed surface it was noticed that the size and inter-particle distance between nano-structures were highly dependent on the evaporated thin film thickness, while the nanoparticle shape evolution was mainly affected by the employed annealing temperature. Due to the well-controlled morphology of gold nano-particles, prominent and stable LSPR spectra were observed with good plasmon resonance tunability from 546 nm to 780 nm that recommend the developed protocol as a robust alternative to fabricate large scale LSPR surface. An example of a LSPR-immunosensor is reported. Thus, the monoclonal anti-atrazine antibodies immobilizion on the “annealed” gold nano-structures, as well as the specific antigen (atrazine) recognition were monitored as variations of the resonance wavelength shifts and optical density changes in the extinction measurements.     

Gold nanoparticles-coated magnetic microspheres as affinity matrix for detection of hemoglobin A1c in blood by microfluidic immunoassay

A novel microfluidic immunoassay system for specific detection of hemoglobin A1c (HbA1c) was developed based on a three-component shell/shell/core structured magnetic nanocomposite Au/chitosan/Fe(3)O(4), which was synthesized with easy handling feature of Fe(3)O(4) by magnet, high affinity for gold nanoparticles of chitosan and good immobilization ability for anti-human hemoglobin-A1c antibody (HbA1c mAb) of assembled colloidal gold nanoparticles. The resulting HbA1c mAb/Au/chitosan/Fe(3)O(4) magnetic nanoparticles were then introduced into microfluidic devices coupled with a gold nanoband microelectrode as electrochemical detector. After that, three-step rapid immunoreactions were carried out in the sequence of HbA1c, anti-human hemoglobin antibodies (Hb mAb) and the secondary alkaline phosphatase (AP)-conjugated antibody within 20 min. The current response of 1-naphtol obtained from the reaction between the secondary AP-conjugated antibody and 1-naphthyl phosphate (1-NP) increased proportionally to the HbA1c concentration. Under optimized electrophoresis and detection conditions, HbA1c responded linearly in the concentration of 0.05-1.5 μg mL(-1), with the detection limit of 0.025 μg mL(-1). This system was successfully employed for detection of HbA1c in blood with good accuracy and renewable ability. The proposed method proved its potential use in clinical immunoassay of HbA1c.     

Green synthesis of gold nanoparticles using Nyctanthes arbortristis flower extract

The present study explores the reducing and capping potentials of ethanolic flower extract of the plant Nyctanthes arbortristis for the synthesis of gold nanoparticles. The extract at different volume fractions were stirred with HAuCl₄ aqueous solution at 80 °C for 30 min. The UV–Vis spectroscopic analysis of the reaction products confirmed successful reduction of Au³⁺ ions to gold nanoparticles. Transmission electron microscope (TEM) revealed dominant spherical morphology of the gold nanoparticles with an average diameter of 19.8 ± 5.0 nm. X-ray diffraction (XRD) study confirmed crystalline nature of the synthesized particles. Fourier transform infra-red (FTIR) and nuclear magnetic resonance (NMR) analysis of the purified and lyophilized gold nanoparticles confirmed the surface adsorption of biomolecules during preparation and caused long-term (6 months) stability. Low reaction temperature (25 °C) favored anisotropy. The strong reducing power of the flower extract can also be tested in the green synthesis of other metallic nanoparticles.     

Preparation and characterization of magnetic microspheres for the purification of interferon alpha-2b

Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN alpha-2b monoclonal antibodies (mAb) were selected for the purification of interferon alpha-2b (IFN alpha-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN alpha-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN alpha-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 x 10(8) IU/mg. Identity and molecular mass of purified IFN alpha-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.     

Epitope mapping, expression and post-translational modifications of two isoforms of CD33 (CD33M and CD33m) on lymphoid and myeloid human cells

We have tested the usefulness of several commercial anti-CD33 monoclonal antibodies (mAb) to determine the expression and localization of the two CD33 isoforms on several hematopoietic cell lines. The expression of the isoform CD33m, a CD33 transmembrane splice variant lacking the ligand-binding V immunoglobulin (Ig)-like domain, was detected by RT-polymerase chain reaction, western blot, confocal microscopy and flow cytometry on the membrane of several human cell types. CD33m was only detected by the anti-CD33 mAb HIM3-4 on the cell surface, whereas WM53, P67.6, 4D3, HIM3-4, WM54, D3HL60.251 or MY9 detected the CD33M isoform, indicating that HIM3-4 is the only mAb recognizing CD33 C(2) Ig domain. Accordingly, HIM3-4 binding to CD33 did not interfere with the binding of other antibodies against the CD33 V-domain. P67.6 mAb interfered with recognition by the rest of antibodies specific for the V domain. HIM3-4 staining could be increased after the sialidase treatment of all CD33(+) cells. However, this increase was stronger in activated T cells, suggesting a CD33 masking state in this cell population. Confocal microscopy analysis of CD33m HEK 293T-transfected cells revealed that this protein is expressed on the cell membrane and also detected in the Golgi compartment. CD33 is constitutively located outside the lipid raft domains, whereas cross-linked CD33 is highly recruited to this signaling platform. The unique ability of HIM3-4 mAb to detect the masking state of CD33 on different cell lineages makes it a good tool to improve the knowledge of the biological role of this sialic acid-binding Ig-like lectin.     

Modification of monoclonal antibody carbohydrates by oxidation,conjugation, or deoxymannojirimycin does not interfere with antibody effector functions

Site-specific attachment of metal chelators or cytotoxic agents to the carbohydrate region of monoclonal antibodies results in clinically useful immunoconjugates [Doerr et al. (1991) Ann Surg 214: 118, Wynant et al. (1991) Prostate 18: 229]. Since the capacity of monoclonal antibodies (mAb) to mediate tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) may accentuate the therapeutic effectiveness of immunoconjugates, we determined whether site-specific modification of mAb carbohydrates interfered with these functions. The chemical modifications examined consisted of periodate oxidation and subsequent conjugation to either a peptide linker/chelator (GYK-DTPA) or a cytotoxic drug (doxorubicin adipic dihydrazide). mAb-associated carbohydrates were also modified metabolically by incubating hybridoma cells in the presence of a glucosidase inhibitor deoxymannojirimycin to produce high-mannose antibody. All four forms (unaltered, oxidized, conjugated and high-mannose) of murine mAb OVB-3 mediated tumor cell lysis via CDC. Similarly, equivalent ADCC was observed with native and conjugated forms of mAb OVB-3 and EGFR.1. ADCC was achieved with different murine effector cells such as naive (NS), poly (I^*C)- and lipopolysaccharide-stimulated (SS) spleen cells, orCorynebacterium-parvum-elicited peritoneal cells (PEC). All murine effector cell types mediated tumor cell lysis but differed in potency such that PEC>SS>NS. Excellent ADCC activity was also demonstrable by human peripheral blood mononuclear cells with OVB-3-GYK-DTPA and high-mannose OVB-3 mAb. ADCC activity was detectable in vivo: both native and conjugated OVB-3 inhibited growth of OVCAR-3 xenografts in nude mice primed withC. parvum. In conclusion, modification of mAb carbohydrates did not compromise their in vivo or in vitro biological functions. Therefore, combination therapy using immunomodulators to enhance the effector functions of site-specific immunoconjugates could be seriously contemplated.     

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.     

Characterization of nanostructure of stimuli-responsive polymeric composite membranes

To elucidate the mechanism of stimuli-responsive permeability and to optimize the design, the nanostructure of polymeric composite membranes, developed in our laboratory, was characterized. The membranes were prepared to contain various amounts of stimuli-responsive nanoparticles of poly(N-isopropylacrylamide-co-methacrylic acid), with or without polyelectrolyte coating. Scanning electron microscopy and X-ray photoelectron spectroscopy were used respectively to examine the morphology and surface chemical composition, whereas atomic force microscopy and laser scanning confocal microscopy were employed to characterize the in situ surface and internal structure of the membranes in aqueous media of various pHs. The porous structure was evidenced in the presence of the nanoparticles. The surface content of the nanoparticles increased with increasing particle concentration while the polyelectrolyte coating was nearly undetectable. AFM images revealed that the particles in the membranes shrank with a concomitant increase in pore size as the buffer pH decreased. LSCM results indicated that particles were distributed through the membrane as interconnected clusters.     

Phagomagnetic immunoassay for the rapid detection of Salmonella

This work explores the use of the phage P22 in a phagomagnetic immunoassay for the rapid detection of Salmonella. The covalent attachment of wild-type phages was performed on two different magnetic carriers: carboxyl-activated magnetic nanoparticles (300 nm) and tosyl-activated magnetic microparticles (2.8 μm). The bacteria were captured and preconcentrated by the phage-modified magnetic particles, followed by the detection using specific anti-Salmonella antibodies conjugated to horseradish peroxidase as an optical reporter. Outstanding selectivity and sensitivity was obtained with this approach, achieving detection limits of 19 CFU mL^?1 in 2.5 h without any pre-enrichment, in milk samples. Moreover, if the samples were pre-enriched for 6 h, the method was able to detect as low as 1.4 CFU in 25 mL of milk. Therefore, the proposed strategy based on the combined use of phagomagnetic separation with immunological labeling is promising as a rapid and simple method for food safety.     

Lipogels: single-lipid-bilayer-enclosed hydrogel spheres

We have fabricated Lipogels consisting of a single POPC lipid bilayer supported by a micrometer-sized, thermoresponsive, hydrophobically modified (HM), hydrogel sphere. The hydrogel consists of a lightly cross-linked poly(N-isopropylacrylamide) (pNIPAM) core surrounded by a highly cross-linked acrylic acid (AA)-rich p(NIPAM-co-AA) shell. The lipid bilayer was assembled by binding liposomes to HM microgels, followed by several cycles of freeze-thaw. The pNIPAM volume phase transition (VPT) at ～32 °C was present both before and after hydrophobic modification and after lipid bilayer coating. Fluorescence studies confirmed the fusion of liposomes into a continuous single bilayer. At a temperature above the VPT, it was found that the volume decrease in the hydrogel was coupled to the appearance of highly curved obtrusions of the uncompromised lipid bilayer into the surroundings. It is anticipated that these properties of Lipogels will prove to be useful in drug delivery applications and in fundamental biophysical studies of membranes.     

In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody

The major stress‐inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumours, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumour mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1‐fluorescein isothiocyanate mAb translocates into early endosomes and lysosomes. Intraoperative and near‐infrared fluorescence imaging revealed an enrichment of Cy5.5‐conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype‐matched control, in i.p. and s.c. located CT26 tumours, as soon as 30 min. after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane‐bound Hsp70, the fluorescence‐labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumour, reaching a maximum after 24 hrs and remained detectable at least up to 96 hrs after a single i.v. injection. The tumour‐selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane‐positive tumours. The anti‐tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody‐dependent cytotoxicity.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Speckled SiO2@Au Core–Shell Particles as Surface Enhanced Raman Scattering Probes

Silica particles of ~800 nm size were functionalized using 3-amino propyl triethoxysilane molecules on which gold particles (~20 nm size) were deposited. The resulting particles appeared to form speckled SiO₂@Au core–shell particles. The surface roughness, along with hot spots, due to nanogaps between the gold nanoparticles was responsible for the enhancement of the Raman signal of crystal violet molecules by ~3.2?×?10⁷ and by ~1.42?×?10⁸ of single-wall carbon nanotubes. It has also been observed that the electromagnetic excitation near surface plasmon resonance (SPR) of core–shell particles is more effective than off resonance SPR excitation.     

Cellular response to magnetic nanoparticles "PEGylated" via surface-initiated atom transfer radical polymerization

A new method to PEGylate magnetic nanoparticles with a dense layer of poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) by surface-initiated atom transfer radical polymerization (ATRP) is reported. In this approach, an initiator for ATRP was first immobilized onto the magnetic nanoparticle surface, and then P(PEGMA) was grafted onto the surface of magnetic nanoparticle via copper-mediated ATRP. The modified nanoparticles were subjected to detailed characterization using FTIR, XPS, and TGA. The P(PEGMA)-immobilized nanoparticles dispersed well in aqueous media. The saturation magnetization values of the P(PEGMA)-immobilized nanoparticles were 19 emu/g and 11 emu/g after 2 and 4 h polymerization respectively, compared to 52 emu/g for the pristine magnetic nanoparticles. The response of macrophage cells to pristine and P(PEGMA)-immobilized nanoparticles was compared. The results showed that the macrophage cells are very effective in cleaning up the pristine magnetic nanoparticles. With the P(PEGMA)-immobilized nanoparticles, the amount of nanoparticles internalized into the cells is greatly reduced to <2 pg/cell over a 5 day period. With this amount of nanoparticles uptake, no significant cytotoxicity effects were observed.