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.

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.

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.

Effects of monoclonal antibodies against human stathmin 1 combined paclitaxel on proliferation of human hepatocellular carcinoma cell lines

Objective： We investigated the effects of monoclonal antibodies against stathmin 1 combined paclitaxel on the proliferation of HCC cells. Methods： HepG2 cells were treated with monoclonal antibodies against stathmin 1, paclitaxel alone or their combination, with the untreated cells used as the control, 24, 48, 72, 96 h later, the cell growth condition was observed by invert microscope and inhabitation rate was studied by MTT assay; The apoptosis was analyzed by flow cytometry with Annexin V/PI. Results： The population decreased and shape, size changed after treating with different concentration of experimental groups. Monoclonal antibodies against stathmin 1 and paclitaxel used alone or in combination both inhibited the proliferation of HepG2 cells, the inhibition ratio of their combination was more higher （P 〈 0.05）, and a synergistic effect of the two agents was noted in their combined action （P 〈 0.05）. Combined treatment of the cells resulted in significantly higher apoptosis rate than that in the other groups （P 〈 0.05）. Conclusion： Monoclonal antibodies against stathmin 1 and paclitaxel used alone or in combination both can inhibit proliferation of HepG2 cells and induce apoptosis. A synergistic effect is obsewed between the monoclonal antibodies against stathmin 1 and paclitaxel in their inhibition of HepG2 cell proliferation.

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

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

MONOCLONAL ANTIBODIES AGAINST HUMAN GASTRIC CANCER

Spleen cells from Balb/c mice immunized with five human gastric cancer cell lines in sequence were fused with murine myeloma cell line SP2/0, and hybridomas 3F4, 3G9 and 3H11, secreting monoclonal antibodiees (MoAbs) against gastric cancer, were obtained through selective culture and screening. These MoAbs have both good selectivity and a high positive rate of reaction for gastric cancer, reaching 5/5 and 84.8% to 93.5% for gastric cancer cells and tissues respectively. The reaction of MoAbs with normal cells and tissues was neglible.The corresponding antigens of the MoAbs were sensitive to digestion by trypsin and pronase and resistant to treatment with sodium periodate, indicating their nature as proteins. The antigen 3G9 could be visualized with Western blotting as two bands with molecular weights of 100KD and 70KD, however no band was found for antigens 3F4 and 3H11. There was a high expression of antigens in the majority of gastric cancer cells and tissues independent of his-topathological type of gastric cancer. A low expression of antigens was seen with other tumors and fetal gastrointestinal tissues. These could be considered as gastric cancer-associated antigens or on-cofetal antigens with a quite extensive distribution.

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.

GENERATION AND CHARACTERIZATION OF TWO MONOCLONAL ANTIBODIES AGAINST CYTOKERATINS

Thirty-five hybridoma cell lines against cytoke-ratins, isolated from Hela cells and human cellus respectively, were generated by fusion of immunized spleen cells of BALB/C mice with P3×63-Ag8.653, a mouse myeloma cell line. Two of them (HI and C53) were characterized by indirect immu-nofluorescence, ABC immunostaining and immuno-blotting. The results of immunofluorescence and ABC immunostaining suggested that both monoclonal antibodies were specific for keratin-type intermediate filaments. However, the two monoclonal antibodies showed different specificities in normal tissues and neoplasms as observed on both frozen and deparaf-finized sections. In normal tissues, H1 stained transitional epithelium and all types of simple epithelium except endothelium and mesothelium but did not stain stratified squamous epithelium. In contrast, C35 recognized only stratified squamous epithelium, but failing to react with simple epithelium. Both monoclonal antibodies did not react with nonepithelia cells and tissues. In neoplasms, H1 stained varieties of adenocaroinomas and C35 recognized merely squamous cell carcinomas. Therefore, all the epithelial tissues can nearly be recognized by combination of the two monoclonal antibodies. All the results indicated that H1 and C35 can be used in cell biology and histology studies, and can be used in differential diagnosis of adenocarcinoma and squamous cell carcinoma in pathology.

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.

CLINICAL EVALUATION OF MONOCLONAL ANTIBODIES AGAINST ALDOLASE-A IN THE DIAGNOSIS OF HEPATO-CELLULAR CARCINOMA

Serum ALD-A of 57 patients with HCC and 43 of other diseases were measured by ALD-A-McAb linked with 425I-staphylococcus A protein. The results showed that the ALD-A in patient with HCC was significantly elevated as compared with controls and that in patients with cholangiocarcinoma, gastrointestinal cancer without hepatic metastasis, cirrhosis. CAH and benign GI diseases. There was no statistical difference between ALD-A in patients with HCC and that in cases of cirrhosis with liver failure and that in cases of metastatic liver carcinoma. It was noted that diagnostic sensitivity of ALD-A in AFP (+) HCC was 73.9% and that in AFP (-) HCC was 81.8% 1-6 patients with HCC were treated by hepatic arterial embolization combined with chemotherapy. ALD-A in patients after the treatment decreased significantly than that before treatment, furthermore, advantages of the method are discussed.

IMMUNOELECTRON MICROSCOPIC STUDY ON THE LOCALIZATION OF MONOCLONAL ANTIBODIES ON GASTRIC CANCER CELLS

The localization of three monoclonal antibodies (MAb) against gastric cancer was studied on two human gastric cancer cell lines by immunoelectron microscopic technique. It has shown that the corresponding antigens of MAb 3G9 and 3H11 were distributed on the microvilli (M) and non-microvillus (NM) plasma membrane of target cells, with various M to NM ratios depending on the MAbs and target cells used. However, the corresponding antigens of MAb PD4 was only localized on the surface of round or finger-like bulges of target cells and never on the microvilli and non-microvillous plasma membrane. Since the nature and function of these tumor antigens have not been identified yet, the implication of the different distributions of these antigens remians to be clarifated.

HIM_(1) AND HIM4, TWO MONOCLONAL ANTIBODIES POTENTIALLY USEFUL FOR AUTOLOGOUS BONE MARROW TRANSPLANTATION IN CHRONIC MYELOGENOUS LEUKEMIA

We developed two complement-fixing MoAbsHIMand HIM(murine)that were specifically reac-tive with chronic myelogenous leukemia (CML) cells.They were capable of fixing human or rabbit com-plement and suitable for CML cells purging of re-mission marrow from CML patients.HIMreactedwith majority leukemic cells form 7 out of 10 CMLpatients by complement-mediated cytotoxicity(C’MC)assay(positive cells 80%—90%),HIMreacted withmajority CML cells from 4 out of 5 CML by C’MCassay(positive cells 80%—90%).Treatment withHIMor HIMand human C’was capable of lysing97% of K562,U937,HL-60 and CML cells in a 20fold excess of unrelated cells by indirect FITC+EBstain.Using limited dilution culture,incubation withHIMand C’produced 1.5 logs inhibition of growthin K562 cells,and 1.9 logs in U937 cells,and withHIMand C’produced 2.9 logs inhibition in HL-60cells and 3.0 logs in U937 cells.Both MoAbs cocktailwas shown 1.8 logs in K562 cells and 3.2 logs in U937cells.They were no suppression on the growth o

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.

THE USE OF ANTIHUMAN GLIOMA MONOCLONAL ANTIBODIES FOR TARGETING CHEMOTHERAPY OF BRAIN GLIOMAS(POTENT SUPPRESSION OF MALIGNANT GLIOMA GROWTH WITH IMMUNOCONJUGATES IN VIVO)

Athymic nude mice bearing subcutaneous and intracerebral human glioma xenografts were used to assess the therapeutic efficacy of monoclonal anti-body-adriamycin immunoconjugates against malignant gliomas in vivo. Immunoconjugates showed a significantly stronger antitumor effect with a T/C (treated/ control tumor volume) of 30% as compared with free drug (T/C of 84%). The targeting treatment with immunoconjugates significantly prolonged 54% of median survival time of nude mice. Side effects of immunoconjugates on the normal bone marrow and small intestines were much slighter than those of the free drug. The results of this study indicate that the use of monoclonal antibodies as carriers of anti-tumor agents may have many therapeutic advantages and potential for the treatment of brain gliomas.

DETECTION OF CANCER-ASSOCIATED ANTIGEN IN FECES USING MONOCLONAL ANTIBODIES IN THE DIAGNOSIS OF COLON CARCINOMA

Monoclonal antibodies against colon and pancreatic cancer, CL-2, CL-3, PS-9, PS-10, were used to detect the associated antigens in feces of patients with gastrointestinal carcinoma and non-cancer diseases. Binding inhibition test by SABC-ELISA method were performed for the measurement of the antigen level. Results showed that the associated antigen detected in feces of patients with colon cancer were significantly higher than that of non-cancer disease or normal subjects. The positive rates were 61.1% as detected with CL-2; 53.4% with CL-3; 55.0%, PS-9; and 53.3% PS-10 in cancer patients while that in normal subjects were 7%; 9%; 8%; and 8% respectively. When 'cocktail' of CL-2, PS-9 and PS-10 were used, the positive rates were 92.5% in colon cancer and 14% in normal subjects. In seven out of the sixty patients with colon cancer studied who were graded as Dukes A, the results were all positive. The results seem superior to the serologic detection and may provide a promising new approach in the early diagnosis of colon cancer.

Development and detection application of monoclonal antibodies against Zucchini yellow mosaic virus

Aphid-borne Zucchini yellow mosaic virus （ZYMV） is one of the most economically important viruses of cucurbitaceous plants. To survey and control this virus, it is necessary to develop an efficient detection technique. Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines （16A11, 5A7 and 3B8） secreting monoclonal antibodies （MAbs） against ZYMV Zhejiang isolate were obtained. The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^-7 by indirect enzyme-linked immunosorbent assay （ELISA）. All MAbs were isotyped as IgG1, kappa light chain. Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues. According to this molecular weight, we consider this reactive protein As likely to be the HC-Pro protein. Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA （ACP-ELISA）, dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA （DAS-ELISA）, and immunocapture-RT-PCR （IC-RT-PCR）, for the sensitive, specific, and easy detection of ZYMV. The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1：163840, 1：2560, 1：327680 and 1：1 310720 （w/v, g mL-1） diluted crude extracts from the ZYMV-infected plants. We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids. A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays. Our results showed that 163 of the 275 samples （59%） were infected with ZYMV. This finding indicates that ZYMV As now widely present in cucurbitaceous crops in China. RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays. We consider that these detection assays can significantly benefit the control of ZYMV in China.

Studies on Purification of Methamidophos Monoclonal Antibodies and Comparative Immunoactivity of Purified Antibodies

To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 靏/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 靏/mL and 1.03 靏/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 靏/mL, and the linear working range and LOD were 10.91-11412.29 靏/mL and 3.42 靏/mL, respectively. Conclusion Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.