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.

Establishment of Monoclonal Antibody Competitive ELISA Using Monoclonal Antibody Against VP1 Protein of Asia 1 Type Foot-and-Mouth Disease Virus

Using the purified VP1 protein of Asia 1 type foot-and-mouth disease virus as the antigen, the purified monoclonal antibody was labeled by the sodium periodate method and the monoclonal antibody competitive ELISA was established in this study. Ten positive porcine foot-and-mouth disease serums and more than two hundreds negative serum were tested, and the results were the same as the background of samples. The sensitivity test and replicate test indicated that this method was stable and sensitive, which was suitable for monitoring Asia 1 type porcine foot-and-mouth disease virus antibody.

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.

Preparation of Monoclonal Antibody against P65 Protein of Mycoplasma hyopneumoniae

P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.

Preparation and Identification of Specific Monoclonal Antibody against Porcine Circovirus Type 2

BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.

A MONOCLONAL ANTIBODY RECOGNIZING NON DERIVATIVE 13 HYDROXY GIBBERELLINS AND THEIR GLUCOSIDES *

The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA 3 at carbon 3. This antibody showed high affinity for GA 3 glucoside as well as for 13 hydroxy gibberellins (GA 1, GA 3, GA 5, etc). The affinity of MAb AB10 for 13 hydroxy GAs was significantly reduced by methylation of the 7 oic acid but not by glycosylation of 3 hydroxyl group. Based on this antibody, both of competitive enzyme linked immunosorbent assays (ELISAs) for GA 3 glucoside and for GA 3 were developed. These two ELISAs displayed linear detection ranges from 0 2 pmol to 20 pmol. Using these assays, the fluctuation of GA 3 like and GA 3 glucoside like substances in the leaves of Rumex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6 benzyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.

A Comparative Study on Detection of the Expression of Alternative Oxidase in Tobacco Callus with the Monoclonal Antibody Against Alternative Oxidase and Antibody Against-Synthetic Polypeptide Antibody

从经过不同温度处理的烟草 (NicotianarusticaL .)愈伤组织中提取并纯化线粒体蛋白 ,分别与交替氧化酶的单克隆抗体和抗合成多肽抗体进行免疫杂交。结果表明 :交替氧化酶的含量随温度的下降而显著上升 ;单克隆抗体的特异性较高于抗合成多肽抗体 ,但后者与交替氧化酶同样有良好的亲和性。因此 ,用合成多肽方法制备的抗体可以用于交替氧化酶的研究中。

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.

Role of adhesion molecules and dendritic cells in rat hepatic/renal ischemia-reperfusion injury and anti-adhesive intervention with anti-P-selectin lectin-EGF domain monoclonal antibody

AIM: To investigate the role of P-selectin, intercellular adhesion molecule-1 (ICAM-1) and dendritic cells (DCs)in liver/kidney of rats with hepatic/renal ischemiareperfusion injury and the preventive effect of anti-Pselectin lectin-EGF domain monoclonal antibody (anti-PsLEGFmAb) on the injury.METHODS: Rat models of hepatic and renal ischemiareperfusion were established. The rats were then divided into two groups, one group treated with anti-PsL-EGFmAb(n = 20) and control treated with saline (n = 20). Both groups were subdivided into four groups according to reperfusion time (1, 3, 6 and 24 h). The sham-operated group (n = 5) served as a control group. DCs were observed by the microscopic image method, while P-selectin and ICAM-1 were analyzed by immunohistochemistry.RESULTS: P-selectin increased significantly in hepatic sinusoidal endothelial cells and renal tubular epithelial cells 1 h after ischemia-reperfusion, and the expression of ICAM-1 was up-regulated in hepatic sinusoid and renal vessels after 6 h. CD1a+CD80+DCs gradually increased in hepatic sinusoidal endothelium and renal tubules and interstitium 1 h after ischemia-reperfusion, and there was the most number of DCs in 24-h group. The localization of DCs was associated with rat hepatic/renal function.These changes became less significant in rats treated with anti-PsL-EGFmAb.CONCLUSION: DCs play an important role in immune pathogenesis of hepatic/renal ischemia-reperfusion injury.Anti-PsL-EGFmAb may regulate and inhibit local DC immigration and accumulation in liver/kidney.

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective：Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem.It is necessary to develop standard rabies virus diagnostic tools,especially for diagnosing the strains prevalent in China.Methods：Monoclonal antibodies（MAbs）specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay（ELISA）,isotyping,affinity assay,immunofluorescence assay（IFA）,and immunocytochemistry.The MAb,whose affinity was higher for antigen,was used to establish an antigen captureELISA（AC-ELISA）detection system and test the efficiency by using clinical samples.Results：The heavy chain subclasses of two MAbs were all determined to be IgG2a.The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis,whereas the 3C7 MAb showed the highest affinity for antigen.IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples.Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.Conclusion：It was potentially useful for the further development of highly sensitive,easily handled,and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic,especially in China.

Preparation of Anti-HER2 Monoclonal Antibody-paclitaxel Immunoconjugate and Its Biological Evaluation

Anti-HER2 monoclonal antibody （Sc7301）-paclitaxel （TAX） immunoconjugate was pre- pared and its specific binding to tumor cells was investigated in this study. Sc7301 was conjugated to TAX by the active ester method and then the TAX-Sc7301 immunoconjugate was obtained. After purification and labeling by Cyano-fluorescein isothiocyanate （FITC）, the specific binding of TAX-Sc7301 to HER2-positive tumor cells （SKOV3） and HER2-negative tumor cells （HepG2） was evaluated respectively. TAX-Sc7301 （20 nmol/L） showed distinct specific binding to SKOV3 cells rather than HepG2 cells. And the uptake of the immunoconjugate by SKOV3 cells was increased with the TAX-So7301 concentration （3-48 nmol/L） and the incubation time （P〈0.05）. It was concluded that the TAX-Sc7301 immunoconjugate is ootentially applicable as a targeted agent against HER2-10ositive tumor cells.

Enhanced radioimmunotherapeutic efficacy of a monoclonal antibody cocktail against SMMC-7721 human hepatocellular carcinoma

The improved tumoricidal effect of the radioatibody mixture ("cocktail") has been reported recently for the treatment of colon tumor. In the present study, we demonstrated the enhanced radioimmunotherapeutic efficacy of a monoclonal atibody (MAb) cocktail against human hepatocellular carcinoma. Therapeutic efficacy was determined by measuring the change in tumor size over a period, determining the percentage of growth inhibition of each treatment at various times after radioantibody therapy. boioimmunotherapy of SMMC-7721 human hepatoma xenografts in athymic nude mice with combination of 131I labeled Hepama-1 and 131Llabeled 9403 mouse MAbs was more effective than using either Hepeam-1 or 9403 Mab alone The MAb cocktail could target a greater number of hepstoma cells and increase the magnitude of hepatoma cen uptde of radioamibodies. The in vjtro results explain the enhanced effect of the MAb cocktail in in vjvo model system.

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 Atrazine Residue in Food Samples by a Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay

Atrazine（AT,2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine）has been detected in ground water in several areas of the United States for many years,as well as in China,wherein the growth rate of its gross

Liquid chromatography-mass spectrometry method for the quantification of an anti-sclerostin monoclonal antibody in cynomolgus monkey serum

Liquid chromatography tandem mass spectrometry(LC-MS/MS)has gradually become a promising alternative to ligand binding assay for the bioanalysis of biotherapeutic molecules,due to its rapid method development and high accuracy.In this study,we established a new LC-MS/MS method for the determination of the anti-sclerostin monoclonal antibody(SHR-1222)in cynomolgus monkey serum,and compared it to a previous electrochemiluminescence method.The antibody was quantified by detecting the surrogate peptide obtained by trypsin digestion.The surrogate peptide was carefully selected by investigating its uniqueness,stability and MS response.The quantitative range of the proposed method was 2.00-500μg/mL,and this verified method was successfully applied to the toxicokinetic assessment of SHR-1222 in cynomolgus monkey serum.It was found that the concentrations of SHR-1222 in cynomolgus monkeys displayed an excellent agreement between the LC-MS/MS and electrochemiluminescence methods(ratios of drug exposure,0.8-1.0).Notably,two monkeys in the60 mg/kg dose group had abnormal profiles with a low detection value of SHR-1222 in their individual sample.Combining the high-level anti-drug antibodies(ADAs)in these samples and the consistent quantitative results of the two methods,we found that the decreased concentration of SHR-1222 was due to the accelerated clearance mediated by ADAs rather than the interference of ADAs to the detection platform.Taken together,we successfully developed an accurate,efficient and cost-effective LC-MS/MS method for the quantification of SHR-1222 in serum samples,which could serve as a powerful tool to improve the preclinical development of antibody drugs.

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.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

PREPARATION AND CHARACTERIZATION OF TWO MONOCLONAL ANTIBODIES AGAINST A_α CHAIN'S C TERMINUS OF FIBRINOGEN

Two monoclonal antibodies (McAbs) against Aα chain's C terminus offibrinogen (Fg) have been prepared and designated SZ-78 and SZ-79. Both theantigens in binding assay and immunoblot analysis showed that the two McAbs recognized the epitopes located in residues 549-560 of the Aαchain. The two McAbs couldaccelerate rate of fibrin polymer assembly both in the purified system and in the humanplasma. From the pictures of transmission electronmicroscope, the average diametersof the fibers increase significantly to an average diameters of 375 nm after incubationwith the McAbs, while it was only 75nm without addition of the McAbs. There were al-so more branchings of fibers with addition of McAbs. These observations demonstratethat the amino acid sequences ofα 549-560 in the COOH terminus of the Aα chain mayplay an important role in the assembly of a fibrin clot, presumably being involved in lat-eral aggregation of protofibrils. The preparation of the McAbs supplies a usuful probe for the investigation of the