Basic regulatory science behind drug substance and drug product specifications of monoclonal antibodies and other protein therapeutics

In this review,we focus on providing basics and examples for each component of the protein therapeutic specifications to interested pharmacists and biopharmaceutical scientists with a goal to strengthen understanding in regulatory science and compliance.Pharmaceutical specifications comprise a list of important quality attributes for testing,references to use for test procedures,and appropriate acceptance criteria for the tests,and they are set up to ensure that when a drug product is administered to a patient,its intended therapeutic benefits and safety can be rendered appropriately.Conformance of drug substance or drug product to the specifications is achieved by testing an article according to the listed tests and analytical methods and obtaining test results that meet the acceptance criteria.Quality attributes are chosen to be tested based on their quality risk,and consideration should be given to the merit of the analytical methods which are associated with the acceptance criteria of the specifications.Acceptance criteria are set forth primarily based on efficacy and safety profiles,with an increasing attention noted for patient-centric specifications.Discussed in this work are related guidelines that support the biopharmaceutical specification setting,how to set the acceptance criteria,and examples of the quality attributes and the analytical methods from 60 articles and 23 pharmacopeial monographs.Outlooks are also explored on process analytical technologies and other orthogonal tools which are on-trend in biopharmaceutical characterization and quality control.

Hepatitis B virus reactivation in patients treated with monoclonal antibodies

Hepatitis B virus(HBV)reactivation poses a significant clinical challenge,espe-cially in patients undergoing immunosuppressive therapies,including mono-clonal antibody treatments.This manuscript briefly explores the complex rela-tionship between monoclonal antibody therapy and HBV reactivation,drawing upon current literature and clinical case studies.It delves into the mechanisms underlying this phenomenon,highlighting the importance of risk assessment,monitoring,and prophylactic measures for patients at risk.The manuscript aims to enhance the understanding of HBV reactivation in the context of monoclonal antibody therapy,ultimately facilitating informed clinical decision-making and improved patient care.This paper will also briefly review the definition of HBV activation,assess the risks of reactivation,especially in patients treated with monoclonal antibodies,and consider management for patients with regard to screening,prophylaxis,and treatment.A better understanding of patients at risk can help clinicians provide optimum management to ensure successful patient outcomes and prevent morbidity.

New era in cancer immunotherapy: Twenty years to the discovery of monoclonal antibodies harnessing the immune system to eradicate tumors

The better understanding of the mechanism in which the immune system responds to the developing cancer provided the outcome in a new era in cancer immunotherapy. The tumor suppressive effect on the immune system is caused by negative T cell receptor signaling that abrogate immunity against the cancer cells. Novel monoclonal antibodies that target co-inhibitory receptors on T cells block the tumor induced inhibition of the immune system and enable the immune system to eradicate the tumors. The development of such antibodies started twenty years ago by the preparation of a monoclonal antibody termed BAT. A single administration of the antibody to tumor bearing mice resulted in striking anti tumor activity that was mediated by the lymphocytes. These studies provided a basis for the new era of cancer immunotherapy. The present review summarizes twenty years to the discovery of monoclonal antibodies harnessing the immune system to eradicate tumors.

Preparation and identification of monoclonal antibodies against the adenovirus vector

Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.

A NEW BIFUNCTIONAL CHELATING AGENT α,ε-N,N'-BIS(L-CYSTEINYL)-L-LYSINE FOR RADIOLABELLING OF MONOCLONAL ANTIBODIES WITH TECHNETIUM-99M

α,ε-N,N'-bis(L-cysteinyl)-L-lysine was synthesized and char- acterized for the first time.It was then employed as a bifunctional chelating agent to chelate technetium-99m and subsequently conjugated to fragment F(ab')_2 of anti-gastric tumor monoclonal antibody 3G9.The radiolabelled antibody was satisfactorily stable and immunoreactive.

THE USE OF ANTI-HUMAN GLIOMA MONOCLONAL ANTIBODIES FOR TARGETING CHEMOTHERAPY OF BRAIN GLIOMAS(PREPARATION AND CYTOTOXIC PROPERTIES OF ANTIBODY-ADRIAMYCIN IMMUNOCONJUGATES)

Immunoconjugates are antibody-drug hybrid molecules which combine the exquisite selectivity or monoclonal antibodies with the potent toxicity of anticancer agents. A monoclonal antibody SZ39 against human brain gliomas was used as a drug carrier. Adriamycin (ADR) was bound covalently to SZ39 to form a SZ39-ADR conjugate. The cytotoxic activity of the SZ39-ADR conjugate was tested in vitro and demonstrated potent and specific killing of cells derived from a human malignant glioma. 50% inhibitory concentration (IC50) for SZ39-ADR to 'target' cells was 8.14×10-9 M. An index of specificity between 'target' and 'non-target' cells was calculated to be 88-fold. These data suggest that the SZ39-ADR may use as a potent and cell type-specific agent and is a likely candidate for the targeting chemotherapy of malignant gliotnas.

Preparation of Monoclonal Antibodies to Trimethoprim and ELISA Kit for Rapid Detection

[Objective] This study was conducted to find out an approach for determining trimethoprim residues in water. [Method] Trimethoprim antigen was prepared through a series of reactions from trimethoprim hapten which was generated through the reaction between trimethoprim and maleic anhydride. And trimethoprim monoclonal antibodies were prepared by animal immune, and used to prepare ELISA kit to detect trimethoprim residues in water. Finally, the limit of detection （LED） of the ELISA kit was determined. [Result] The standard curve covered a concentration range of 0-80 μg/L. The LeD of trimethoprim in water using the ELISA kit was 2.34 μg/kg; the IC50 （half maximal inhibitory concentration） was 4.8 μg/L; the recovery rate of added trimethoprim standard ranged from 60.5% to 79.7%; within-and among-batches RSD was less than 10%. The trimethoprim monoclonal antibody was specific, as the cross-reactivity rate of trimethoprim antibody and diaveridine was less than 1%. The stability tests revealed that the ELISA kit was stable after being stored at 4 ℃ for 12 months. [Conclusion] The results will provide references for controlling the abuse of trimethoprim.

Inhibiting effects of ^(131)I-labeled anti-CEA monoclonal antibodies injected intrasplenically on liver metastasis from human colonic adenocarcinoma

I-labeled anti-CEA monoclonal antibody injected intrasplenically has better effects on inhibiting liver metastasis from human colonic adenocarcinoma than that through tail vein in nu/nu mice model.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

AIM To prepare hybridoma cell lines which secrete anti HCV recombinant NS3 and NS5 proteins′ monoclonal antibodies, and to evaluate their usage in the study of the distribution of HCV NS3 and NS5 antigen in liver tissues. METHODS The hybridoma cell lines were raised using the spleen cells of BALB/C mouse immunized with recombinant NS3 and NS5 proteins according to the conventional protocols. The antibody secreting cells were screened using solid phase ELISA and cloned by limited dilution method. In order to determine the specificity of these hybridoma cell lines, the culture supernatant of these cells was western blot assayed with expression and nonexpresion E. coli and ELISA with other antigens, including HCV core and NS3 and HBsAg. Immunohistochemistry of 51 cases paraffin embedded liver tissues was performed to determine the distribution of HCV NS5 antigen as well as NS3 antigen in liver tissues. RESULTS Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were raised. They are named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Among them only 2B6 against NS3 protein can react with the polypipetides of C7 that is another recombinant polypipetides of NS3 gene. Others have no reaction with HCV core and HBsAg of HBV, and there is no cross reaction between NS3Ag and anti NS5Ag McAb and between NS5Ag and anti NS3 McAb. The immunohistochemistry results indicate that no HCV antigen was detected in the specimens of HBV infection in 20 cases. In 31 HCV infected specimens the positive rate of NS3Ag and NS5Ag are 51 6% (16/31) and 54 9% (17/31), respectively. There were six pure HCV infected specimens in these 31 specimens and half of them were HCV NS3Ag and NS5Ag positive. In the co infection of HBV and HCV group the positive rate of NS3Ag and NS5Ag were 52% (13/25) and 56% (14/25), respectively, almost the same with that of pure HCV infected group. The positive rates of HCV antigens were 70 6% (12/17) and 76 5% (13/17) in CAC patients. CONCLUSION The monoclonal antibodies we prepared are specific to the recombinant HCV NS3 and NS5 proteins and can be used in the clinical immunohistochemistry diagnosis.

Mechanistic considerations for the use of monoclonal antibodies for cancer therapy

Since the approval of rituximab in 1997, monoclonal antibodies(mAbs) have become an increasingly important component of therapeutic regimens in oncology. The success of mAbs as a therapeutic class is a result of great strides that have been made in molecular biology and in biotechnology over the past several decades. Currently, there are 14 approved mAb products for oncology indications, and there are ten additional mAbs in late stages of clinical trials. Compared to traditional chemotherapeutic agents, mAbs have several advantages, including a long circulating half-life and high target specificity. Antibodies can serve as cytotoxic agents when administered alone, exerting a pharmacologic effect through several mechanisms involving the antigen binding(Fab) and/or Fc domains of the molecule, and mAbs may also be utilized as drug carriers, targeting a toxic payload to cancer cells. The extremely high affinity of mAbs for their targets, which is desirable with respect to pharmacodynamics(i.e., contributing to the high therapeutic selectivity of mAb), often leads to complex, non-linear, target-mediated pharmacokinetics. In this report, we summarize the pharmacokinetic and pharmacodynamics of mAbs that have been approved and of mAbs that are nearing approval for oncology indications, with particular focus on the molecular and cellular mechanisms responsible for their disposition and efficacy.

Preparation of Anti-cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

Cardiac troponin I(cTnI) was separated and purified from human left ventricular tissue by affinity chromatographic method and used to immunize Balb/c mice by intraperitoneal injection and four hybridoma cell lines, which secreted monoclonal antibody(mAb) against human cTnI, were obtained by cell fusion, identification and cloning twice. Three mAbs(9F5, 2F11, 8C12) were produced from the ascites of Balb/c mice injected intraperitoneally the hybridoma cells and characterized by means of a surface plasmon resonance(SPR) biosensor. An optimal and specific sensing membrane for troponin I was prepared with staphylococcal protein A(SPA) as the intermediate layer and mAb against human cTnI as the capture antibody. On the basis of the sensing membrane, two modes of operation of the SPR biosensor were developed, i.e ., a direct detection of antigen antibody affinity and a sandwich assay. In the sandwich assay detection mode, the mAbs competition was measured by monitoring whether the secondary antibody had been attached to the cTnI already captured by the first antibody on the sensor surface. The SPR biosensor was shown to be able to directly detect the antigen antibody affinity and the order of the affinity was found to be 9F5>2F11>8C12. In the sandwich detection mode, it was found that the different epitopes on the cTnI molecules were recognized by the three mAbs respectively, but the asymmetrical competition was shown between 2F11 and 8C12 and no competition was found between 9F5 and 2F11 or 8c12. Based on these results, a double monoclonal sandwich immunoassay for cTnI was developed by using the optimal antibody pair of 9F5 and 2F11 and the SPR biosensor with SPA substrate membrane, which showed an excellent sensitivity of 0.8 μg/L for both the buffer and the serum samples compared with the direct detection of cTnI for the buffer with the lowest detection limit of 4 μg/L and conventional ELISA with the sensitivity of 1.9 μg/L.

Preparation and Application of Monoclonal Antibodies Specific for Salicylic Acid

Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.

Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people＇s health and environmental protection, the hapten 4-[[（2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy） carbonyl]- amino]-butanoic acid （BFNB） of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier （BFNB-bovine serum albumin, BFNB-BSA） conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay （ELISA）, based on BFNB-ovoalbumin conjugates （BFNB-OVA）. Purified monoclonal antibody （McAb） was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line （5D3） secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1：2.048 × 10^3 and 1：1.024 × 10^6, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 10×9 L mol^-1, with an IC50 value of 1.18 ng mL^-1 and a detection limit of 0.01 ng mL^-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized （4.59 × 10^-4% for 2,3-dihydro-2,2- dimethyl-7-benzofuranol and less than 3.0 × 10^4% for others）. The prepared McAb had a very high affinity and specificity, and it could be used to develop ELISA for rapid determination of carbofuran.

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs） （3C1, 3D9, 2F2, 2A8） against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria, However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens of 3C1 and 3D9 were located at the secretion on the surface of strain AP629. The binding antigens of 2F2 and 2A8 were noted on the membrane of the cells. MAbs 3C1 and 3D9 recognized the lipopolysaccharide fraction of strain AP629, and 2F2 and 2A8 recognized in western-blotting protein antigens with molecular weights of 113 and 128 kDa respectively. MAbs 3C1 and 3D9 have the potential for use in pathogen diagnosis, epidemiology and studies on the mechanism of how S. marisflavi infects A. japonicus. Imrnunohistochemistry with 3C1 or 3D9 identified strain AP629 in the body wall of infected A. japonicus. In the adult sea cucumbers that were infected via body wall injection, positive signals were observed at the site of skin ulceration, and at the connective tissue of the non-ulcerated body wall. In addition, some large blue-stained cells aggregated at the connective tissue colonized by large numbers of bacteria. In juveniles infected via immersion infection, positive signals were observed at the cuticle of the body wall only. Our results suggest that 3C1 and 3D9 could be used in various immunological assays to study the invasion mechanism of strain AP629 in A. japonicus, the law of bacterial colonization, proliferation in different tissues of A. japonicus, and correlation between secretion on the surface of strain AP629 and its pathogenesis to A. japonicus.

In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish (Cambarus proclarkii)

The neutralizing activities of eight monoclonal antibodies （MAbs） against white spot syndrome virus （WSSV） （2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4） were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs （hybridoma culture supernatant）, respectively. The mixture of WSSV and MAbs were injected into crayfish （Procambarus clarkii）. After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.

Monoclonal antibodies:Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

Hepatitis C virus(HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies(m Ab) of mice are predominantly used for the immunodiagnosis of several viral,bacterial,and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mA b are used in the immunotherapy of several blood malignancies,such as lymphoma and leukemia,as well as for autoimmune diseases. In this review article,we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mA b in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV,which is a major health problem throughout the world,particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mA b. Finally,we will discuss available and new trends to produce antibodies,such as egg yolk-based antibodies(Ig Y),production in transgenic plants,and the synthetic antibody mimics approach.

Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies

With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.

Recent progress in the molecular imaging of therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented.Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA,and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information,while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development.Recent progress in detecting therapeutic antibodies,in particular,the development of methods suitable for illustrating the molecular dynamics,is described here.

Detection of lymphocystis disease virus infection to flounder gill cells in vitro by monoclonal antibodies

Flounder gill （FG） cells were used to isolate lymphocystis disease virus （LCDV） and two monoclonal antibodies （Mabs） （1A8 and 3G3） against LCDV were used to trace LCDV infection to FG cells. FG monolayer cells was inoculated with LCDV supernatant, obtained from lymphocystis cells of diseased flounder, Paralichthys olivaceus. LCDV infection was detected with Mabs employing immunocytochemical assay （ICA） and indirect immunofluorescence assay test （IIFAT） technique. Detected by IIFAT, they were specifie for LCDV. The results of experimental infection illustrated that FG cells was sensitive to LCDV, and showed virus-infection positive detected by ICA. Cytopathic effect （CPE） occurred 1-2 days post inoculation （PI）, and half tissue culture infection dosage （TCID50） of vires supematant was 2^2.57 per 40μl. Tracing by IIFAT showed that LCDV positive signal first appeared at the cell membrane immediately PI, and then in cytoplasm at 24h PI, it reached the strongest positive at 48-72 h PI, and began to decrease at 96h PI.

Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

To prepare the PrP specific monoclonal antibodies （mAbs） that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein （HaPrP）. Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.