Application of a risk analysis method to different technologies for producing a monoclonal antibody employed in hepatitis B vaccine manufacturing

CB.Hep-1 monoclonal antibody (mAb) is used for a recombinant Hepatitis B vaccine manufacturing, which is included in a worldwide vaccination program against Hepatitis B disease. The use of this mAb as immunoligand has been addressed into one of the most efficient steps of active pharmaceutical ingredient purification process. Regarding this, Quality Risk Management (QRM) provides an excellent framework for the risk management use in pharmaceutical manufacturing and quality decision-making applications. Consequently, this study sought applying a prospective risk analysis methodology Failure Mode Effects Analysis (FMEA) as QRM tool for analyzing different CB.Hep-1 mAb manufacturing technologies. As main conclusions FMEA was successfully used to assess risks associated with potential problems in CB.Hep-1 mAb manufacturing processes. The severity and occurrence of risks analysis evidenced that the percentage of very high severe risks ranged 31.0-38.7% of all risks and the huge majority of risks have a very low occurrence level (61.9-83.3%) in all assessed technologies. Finally, additive Risk Priority Number, was descending ordered as follow: transgenic plants (2636), ascites (2577), transgenic animals (2046) and hollow fiber bioreactors (1654), which also corroborated that in vitro technology, should be the technology of choice for CB.Hep-1 mAb manufacturing in terms of risks and mAb molecule quality.     

Establishment and validation of an immunoenzymatic assay to determine mouse IgG levels based on a rat monoclonal antibody

In this paper we describe an immunoenzymatic assay based on a rat monoclonal antibody (Ram kappa) developed to determine mouse IgG concentration, which is widely used for samples obtained on purification processes, like supernatant waste and the content of IgG in the vaccine (rHBsAg). This assay involves the use of a rat antibody-horseradish peroxidase-conjugated for the revealing of the antigen-antibody reaction. The rat antibody was produced in cell culture using a dialysis tube (DT). The immunoassay was standardized following several concepts, such as specificity, precision, and linearity. The result obtained permitted us to replace the use of polyclonal antibodies to determine the kappa light chain mouse antibodies by a rat monoclonal antibody of high sensibility and reproducibility. The assay permitted a reliable measurement of murine kappa Ig up to 0.68 ng/ml and was capable of quantifying 6.25 ng/ml. Due to the high frequency of the kappa light chain in mouse antibodies this system acquires a great application.     

A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines

Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30–13.68%) and reproducible (%CV range = 1.49–10.81%) with a LOQ of 2.5 μg/mL.     

Standardization and validation of an alkaline phosphatase-linked immunoassay to quantify a plant-derived antibody directed against the hepatitis B virus surface antigen

Immunopurification is one of the most effective chromatography steps to purify the hepatitis B surface antigen, which have successfully been used as an active pharmaceutical ingredient of hepatitis B vaccines. Plant-derived antibodies could be an appropriated ligand for such purposes because plants are the most cost-effective production systems and have the additional advantage that plant viruses cannot infect humans. In this work, a polyclonal antibody alkaline phosphatase-linked immunoassay was standardized and validated to quantify a plant-derived antibody directed against the HBsAg. The validation of an immunoassay to quantify plantibodies is a relatively complex task due to the complexity of the plant extract, the low level of expression of this molecule, and the potential interferences of endogenous peroxidases contributed by plants. These results allow estimating the plant-derived antibody concentration up to 3.81 ng/mL with high specificity, precision, and repeatability. The working range of the standard curve was between 3.81 and 60 ng/mL, and the intra- and inter-variation coefficients were between 10% and 20% in a production process's sample dependent way. This enzyme-linked immunosorbent assay is considered valuable to improve the design of the purification process and also to obtain a better estimation of the antibody expression level and process's recovery.     

CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor

The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg ml^-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml^-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics of the mAb harvested from the bioreactor during the 43 days of cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

Stirrer tank: an appropriate technology to immobilize the CB.Hep-1 monoclonal antibody for immunoaffinity purification

The CB.Hep-1 monoclonal antibody was coupled to CNBr-activated Sepharose CL 4B at three different immobilization scales for purification of recombinant hepatitis B surface antigen. Standard laboratory apparatus to obtain immunosorbents of 1 l (scale I) and 3 l (scale II) as well as a stirrer tank to prepare 6 l immunosorbents (scale III) were used. The binding capacity at scale III was 2- and 1.5-fold higher with respect to the scales II and I, while a reduction in the ligand leakage of 5- and 2-folds was observed. Immunosorbents from scale II showed a significantly reduced adsorption, and an increased ligand leakage. Differences in the coupling efficiency were not observed. Antigen purity eluted from the immunosorbents was always above 85%.     

Design,synthesis and application of benzyl‐sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

The human anti‐human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity‐ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4‐aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS‐Trz‐4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS‐Trz‐4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two‐step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S‐Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS‐Trz‐4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and enzyme‐linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

抗心型脂肪酸结合蛋白单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗心型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),并建立侧向免疫层析方法检测血浆中H-FABP。方法:用H-FABP蛋白免疫纯系Balb/c小鼠,采用杂交瘤技术建立能稳定分泌抗人H-FABP的单克隆杂交瘤细胞株。常规制备腹水,纯化后得到特异性抗H-FABP单克隆抗体,进行效价、特异性、亲和力的鉴定分析,并在ELISA平台进行抗体配对,用所筛选到的抗体对初步建立了检测H-FABP的侧向免疫层析方法。结果:成功获得12株稳定分泌抗体的阳性细胞株,并筛选出能相互配对,并应用于侧向免疫层析平台的抗体3D1和5F4,检测临床样品与对照试剂比较总符合率为100%。结论:筛选能稳定分泌抗体的细胞株,配对抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏的检测出临床样品中H-FABP,为临床应用快速检测H-FABP指标提供了方法和关键材料。     

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG₂ mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process. A correlation between the level of free thiol in the HCCF (as a result of antibody reduction) and aggregation during the low pH step was established, wherein higher levels of free thiol in the starting sample resulted in increased levels of aggregates during low pH treatment. The elevated levels of free thiol were not reduced over the course of purification, resulting in carry‐over of high free thiol content into the formulated drug substance. When the drug substance with high free thiols was monitored for product degradation at room temperature and 2–8°C, faster rates of aggregation were observed compared to the drug substance generated from HCCF that was purified immediately after harvest. Further, when antibody reduction mitigations (e.g., chilling, aeration, and addition of cystine) were applied, HCCF could be held for an extended period of time while providing the same product quality/stability as material that had been purified immediately after harvest. Biotechnol. Bioeng. 2017;114: 1264–1274. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.     

Screening adenoviruses in stool samples: evaluation of a genus-specific monoclonal antibody based enzyme immunoassay

To screen fecal samples for adenovirus antigens a genus-specific monoclonal antibody based enzyme immunoassay was developed. In a comparative analysis with commercial latex agglutination test, high sensitivity was demonstrated. The assay did not detect other viruses usually found in faeces suggesting its specificity. One hundred and eighty stool samples collected in Baranya County were tested and 13 (7.2%) of them showed reactivity. The application of our immunoassay combined with other, more sophisticated methods may help us to determine the serotype specificity of these adenovirus isolates and assess the importance of adenoviruses in viral gastroenteritis.     

检测HSA—GLP-1融合蛋白双抗体夹心ELISA方法的建立

目的：为检测血清中HSA-GLP-1融合蛋白整体分子的浓度,建立一种特异、灵敏的定量检测食蟹猴体内HSA-GLP-1融合蛋白浓度的双抗体夹心ELISA的方法。方法：采用双抗体夹心ELISA方法,以GLP-1单克隆抗体为包被抗体、HSA-GLP-1融合蛋白为夹心抗原、anti-HSA-Biotin为检测抗体,用Streptavidin-HRP进行免疫放大,TMB显色。结果：建立了检测HSA-GLP-1融合蛋白的ELISA方法,其线性范围为15.6~1000 ng/mL,最低检测限为15.6 ng/mL,与GLP-1、HSA、IL2-HSA均无交叉反应,板内和板间精密度均小于15%,准确度为±15%,冻融稳定性和稀释稳定性良好。结论：建立的HSA-GLP-1蛋白检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于HSA-GLP-1融合蛋白在临床前药代动力学试验的定量检测。     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

Efficient CD4 binding and immunosuppressive properties of the 13B8.2 monoclonal antibody are displayed by its CDR-H1-derived peptide CB1.

A systematic exploration of the V(H)2/V(kappa)12-13 variable domains of the anti-CD4 monoclonal antibody (mAb) 13B8.2 was performed by the Spot method to screen for paratope-derived peptides (PDPs) demonstrating CD4 binding ability. Nine peptides, named CB1 to CB9, were identified, synthesized in a cyclic and soluble form and tested for binding to recombinant soluble CD4. Among them, CB1, CB2 and CB8 showed high anti-CD4 activity. Competition studies for CD4 binding indicated that PDPs CB1, CB8, and the parental mAb 13B8.2 recognized the same complementarity determining region (CDR)3-like loop region. PDP CB1 was shown to mimic the biological properties of 13B8.2 mAb in two independent cellular assays, demonstrating inhibitory activities in the micromolar range on antigen presentation and human immunodeficiency virus promoter activation. Our results indicate that the bioactive CDR-H1 PDP CB1 has retained a significant part of the parental 13B8.2 mAb properties and might be a lead for the design of anti-CD4 peptidomimetics of clinical interest.     

Comparison of different ligand densities for the manufacture of CB Hep-1 immunosorbents

Different ligand densities of monoclonal antibody (Mab) CB.Hep-1 were studied during covalent coupling on Sepharose CL-4B for recombinant hepatitis B surface antigen (rHBsAg) immunoaffinity purification. Ligand densities of 2.2, 3.2, 4.2 and 5.2 mg Mab/ml immunosorbents, respectively, were assayed during five cycles of immunoaffinity chromatography (IAC). Adsorption capacities averaged either 3.2 mg/ml (0.57 mg rHBsAg/ml immunosorbent/5.42 mg of total purified protein) or 5.2 mg/ml (0.56 mg rHBsAg/ml immunosorbent/5.05 mg total purified protein). Immunosorbents showed ligand leakage levels below 3 ng Mab/microg rHBsAg. Antigen purity was higher than 95% in all cases. The results suggest that a ligand density (LD) of 3.2 mg Mab/ml immunosorbent should be used for immunoaffinity chromatography because no significant differences were found in the ligand densities studied (P-value=0.012), which saves 40% of CB.Hep-1 immunosorbent manufacturing cost in comparison with 5 mg Mab/ml immunosorbent, which is currently used in large-scale production.     

Analytical characterization of a monoclonal antibody therapeutic reveals a three-light chain species that is efficiently removed using hydrophobic interaction chromatography

Size exclusion high performance liquid chromatography analysis of a human monoclonal antibody (mAb) showed the presence of a new species that eluted with a retention time between the dimeric and monomeric species of the antibody. Extensive characterization of this species, referred to as “shoulder,” indicated that it was a mAb containing an extra light chain and had a molecular weight of approximately 175 kDa. The extra light chain was found to be non-covalently associated with the Fab portion of the protein. The relative amount of shoulder (typically 1−3% of the total mAb present) varied with the Chinese hamster ovary cell line producing the mAb and was not influenced by the growth conditions. Our three-step mAb purification platform using protein A, anion exchange, and cation exchange process steps was successful at removing dimer and higher and lower molecular weight species, but not the shoulder impurity. It was found that hydrophobic interaction chromatography could be used in place of cation exchange to exploit the subtle differences in hydrophobicity between monomer and shoulder. We developed an antibody polishing process using Butyl Sepharose HP resin that is capable of removing the majority of high and low molecular weight impurities yielding 99% pure mAb monomer, virtually devoid of the shoulder species, with a step recovery of about 80%.     

Double-modified albumins as a tool for antibody purification

A general approach for anti-hapten antibody purification utilizing double-modified albumins is presented. Purification is based on simultaneous modification of an albumin with a hapten (e.g. fluorescein) and desthiobiotin. Three distinct albumins (BSA, HSA and ovalbumin) were modified accordingly and evaluated for their ability to purify the anti-fluorescein mAb from a mixture of commercial preparation and an E. coli cell lysate. The recovered mAb was obtained at relatively high purity (88-95%), in a wide range of target concentrations (0.66-0.02 mg/ml) within a total purification time of approximately 20 min. Substantial increase in the contamination background did not affect purity.     

Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype. Injection of H delta a/1-BSA into BALB/c mice, which express Ig of the a allotype, or into (BALB/c x CB20)F1 mice (a x b allotype heterozygotes) induced IgG1 anti-BSA antibody responses that peaked 8 to 9 days after injection, and were more than 1000 times larger than those induced by injection of BSA alone, and 100 times larger than those induced by injecting unconjugated BSA plus H delta a/1. H delta a/1-BSA was no more immunogenic than unconjugated BSA when injected into CB20 mice, which express Ig of the b allotype, while AF3.33-BSA greatly enhanced anti-BSA antibody production in CB20, but not in BALB/c mice. Mice serially immunized with three different Ag conjugated to H delta a/1 made large antibody responses to all three Ag, provided that the mouse strain used did not recognize allotypic determinants on H delta a/1 as foreign and produce a neutralizing antibody response. Intravenous and s.c. routes of inoculation produced responses of similar magnitude and relatively low variability; responses to footpad or intramuscular inoculation were more variable, and i.p. inoculation induced smaller responses. Injection of BALB/c mice i.v. with 100 micrograms of H delta a/1-BSA induced an IgG1 anti-BSA response of 5.6 mg/ml, which was approximately 70% of the total IgG1 response. Anti-BSA responses to 30 micrograms of conjugate or less were much smaller, but could be considerably enhanced by adding unconjugated H delta a/1 to the inoculum. This system will be useful for the rapid stimulation of large antibody responses to biologically important Ag, and for investigating mechanisms of Ag processing and B and T cell activation.     

Immunization with antigen bound to bispecific antibody induces antibody that is restricted in epitope specificity and contains antiidiotype.

Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.