發(fā)光免疫類檢測試劑注冊技術審查指導原則EN

1、Attachment 2Guideline for Technical Review of Registration of Luminescent Immunoassay Class Detection ReagentsI.PrefaceThis Guideline advances guiding technical requirements on the main raw materials, production processes and reaction system, and product quality control that are specifically for lum

2、inescent immunoassay class detection reagents. This Guideline is a general requirement for luminescent immunoassay detection reagents. The Applicant shall determine whether the content is applicable in accordance with the specific properties of the product. If not applicable, the Applicant shall und

3、ergo justification with specific explanation, and the corresponding scientific basis shall be provided. This Guideline shall act as a guidance document for applicants and personnel of examination authority. However, this Guideline shall not include the administrative matters involved during the regi

4、stration examination and approval and shall not be executed as a mandatory regulation. Should there be other methods meeting the appropriate regulatory requirements, these methods can be used as well. However, detailed research information and verification documentation shall be provided. This Guide

5、line shall be utilized in compliance with the relevant laws and regulations under the premise.This Guideline is developed under the existing regulations, standards, and the currently accepted knowledge basis. With the continuous improvement of the regulations and standards and the continuous develop

6、ment of scientific technology, the relevant content of this Guideline shall undergo timely adjustments accordingly.II.Scope of ApplicationThis guideline is mainly applicable for technical review of registration of class III in vitro diagnostic reagents utilizing luminescent immunoassay analysis tech

7、nique to undergo testing on substances, namely proteins. This Guideline can be referenced and executed for the manufacturing and quality control of class II detection reagents.III.General Requirement(I)General Principle1.Applicable quality standards shall be established for various raw materials and

8、 auxiliary materials used in R&D and manufacturing and shall conform to the requirements of the relevant regulations.2.Manufacturing enterprises of diagnostic reagents shall be equipped with the corresponding professional technical personnel, instruments and equipment, and appropriate manufacturing

9、environment with obtainment of “Medical Device Manufacturing License”. Moreover, the manufacturing enterprises shall establish the corresponding quality management system in accordance with the requirements stated in the “Detailed Rules for Implementation of Manufacturing of in vitro Diagnostic Reag

10、ents (Interim)” with formation of written documents and records to implement and maintain effective operation. Additionally, the manufacturing enterprises shall pass the examination of the “Examination and Evaluation Standards for Quality Management System of Manufacturing Enterprises of in vitro Di

11、agnostic Reagents (Interim)”. The enterprises shall clearly state the range of applicability of the detection reagents and shall be approved by the state drug regulatory department. 3.R&D of diagnostic reagents shall be carried out in accordance with scientific and practical principles. General scie

12、ntific principles shall be complied for selection and determination of various reaction conditions.4.The materials and technical processes used in the R&D and manufacturing process shall fully consider all matters pertaining to the safety aspect that maybe involved. 5.The overall target of manufactu

13、ring and quality control of the detection reagents: assuring the safe use of the product with stable quality, controllable technical process, and effective use.(II)Quality Control of Raw Materials1.Major biological raw materialsThe biological raw materials that are mostly closely related to the qual

14、ity of the product mainly include various natural antigens, recombinant antigens, monoclonal antibodies, polyclonal antibodies, polypeptides, and hormones. This class of raw materials may be used for coating micro-aperture reaction plate, labeling relevant enzymes (horse radish peroxidase and alkali

15、ne phosphatase), neutralizing reaction used antigen or antibody, and preparing calibrators (standard controls). Quality inspection is carried out on this class of biological raw materials according to the requirements of the technical process generally to assure that they have attained the specified

16、 quality standards. The technical process must be relatively stable if the main biological raw materials are self-manufactured by the enterprises. If they are outsourced, the suppliers must be fixed without any changes of suppliers at will. In the event that the main raw materials (including technic

17、al process) or their suppliers are changed, application for change must be submitted in accordance with the requirements of the relevant state regulations. Routine inspection items for the main biological raw materials generally include:(1)AppearanceNaked eye observation is carried out with most of

18、biological raw materials showing clear and transparent liquid without presence of foreign substances, cloudiness, and deposition or particles after shaking. Moreover, they may be white powder without any colored impurities. Special biological raw materials shall consist of corresponding observation

19、standard for appearance.(2)Purity and molecular weightAfter SDS-PAGE electrophoresis, electrophoresis scanner is used to undergo analysis. Other suitable methods, such as high performance liquid chromatography, may be used. Appropriate concentration of polyacrylamide gel is selected to undergo elect

20、rophoresis in accordance with the molecular weight of the tested biological raw material. Generally, the added sample quantity for every electrophoresis channel is 5g. After electrophoresis, the gel may be stained with Coomassie brilliant blue or silver staining. Electrophoresis scanner is used on s

21、tained gel to analyze the purity and molecular weight of the raw material. The purity shall attain the corresponding quality standard. The size of the molecular weight shall be located in the proper band position. (3)Protein concentrationTesting of protein concentration may be carried out with metho

22、ds, such as the Lowry method, 280nm light absorption method, and Biuret method.(4)TiterThe determination of titer is generally performed through multiple proportion dilution in accordance with the measured result of the protein content. Titer shall meet the specified requirements. (5)Functional expe

23、rimentFunctional experiment refers to the condition of biological raw materials during the actual manufacturing of the reagent kit, which is generally used to assess the sensitivity, specificity and stability of the reagent kit using the raw materials. Moreover, correlation with the previous batch o

24、f raw materials is compared. 2.Biological auxiliary materialsBiological auxiliary materials generally refer to the class of biological raw materials used as protein preservative in the manufacturing process, which mainly includes bovine serum, goat serum, and bovine serum albumin. The quality standa

25、rd of these biological raw materials shall meet the requirements of standard stated in the “Chinese Quality Control Standards for Main Raw Materials and Auxiliary Materials of Biological Products (2000)”. Moreover, these materials must be suitable for the manufacturing of the enterprise.The followin

26、g testing shall be recommended: (1)Bovine serum and sheep serumAppearance: light yellow, clear, and slight viscous solution without hemolysis or foreign substances.Sterility testing: the serum is directly cultured in 37 for 7 days. Observation is carried out under bright light. No occurrence of clou

27、diness or sediment shall appear. Total protein content: Biuret method is used for determination. The protein content shall not be less than 32mg/ml.Globulin content: 1 ml of the testing serum is taken. Precipitation is carried out with saturated ammonium sulphate method. The precipitate is dissolved

28、 in 0.85% sodium chloride solution to 1 ml. Lowry method is used for measurement. The protein content shall be 2mg/ml.(2)Bovine serum albumin:Appearance: shall be light yellow, yellow, or milk white lyophilized powder without moisture absorption, clotting, and other impurity particles visibly.Solubi

29、lity: bovine serum albumin is dispensed into 10% solution. The solution time shall not exceeded 15 minutes under 826. The pH value for 1% bovine serum albumin solution is 6.57.1.Total protein content: Biuret method is used for measurement. The quality standard is 95.BSA content in total protein: Cel

30、lulose nitrate membrane electrophoresis is used. The standard is higher than 95%.Net content of BSA: total protein content is multiplied by BSA content in total protein. The standard is 90.(3)CaseinAcidity shall meet the quality standard necessary for manufacturing. (4)Enzyme used for labelingThe na

31、me of enzyme used for labeling shall be stated in the quality standard of the product (e.g. horse radish peroxidase, alkaline phosphatase). Moreover, testing shall be carried out in accordance with the testing method and quality standard of different manufacturers. The RZ value (OD403nm/OD280nm) of

32、purity of the enzyme shall be greater than 3.0. For bovine serum or goat serum, bovine serum albumin, and casein, functional experiments shall be carried out, which is using it as a raw material to prepare a certain concentrations of diluent as sample to undergo testing. No non-specific reaction sha

33、ll appear. Similarly, the suppliers of biological auxiliary materials must be fixed without any changes of suppliers at will.3.Chemical raw materialsQuality standard of chemical raw materials, including appearance, general salt testing, solution pH value, heavy metal testing, dissolution state, loss

34、 on drying, and residue on ignition, shall meet the requirements of analytical purity level stated in the “Chinese Quality Control Standards for Main Raw Materials and Auxiliary Materials of Biological Products (2000)”. Moreover, these materials must be suitable for the manufacturing of the enterpri

35、se. (1)Inorganic class: mainly includes sodium chloride, disodium hydrogen phosphate, and sodium dichloro phosphate.(2)Organic class: mainly includes Tween 20 and tris (hydroxymethyl) amino methane.(3)Special chemical raw materials: Eu-DTTA and Luminal.Purity analysis and validation of Eu-DTTA: the

36、various functions of Eu-DTTA shall meet the requirement of molecular structure with purity higher than 96%.4.Other raw materials(1)Strip of micro-aperture plateAppearance: no scratch, damage, dirty or burr edge is present on the surface of strip with smooth surface through visual observation under b

37、right condition. The plate strip and plastic frame of micro-aperture reaction strip shall fit each other appropriately. Materials: 200 L enhancement solution is added per aperture of micro-aperture reaction plate strip, of which the fluorescence value is tested using luminescence immunoassay analyze

38、r. The mean background fluorescence value is 1500. Absorptivity and precision: micro-aperture plate strip is coated with certain concentration of protein, and fluorescence value is tested. Result of CV value shall meet the functional quality standard of the relevant product. Generally, CV 5 for with

39、in a batch, and CV10 for between batches.(2)OthersCorresponding quality control standards for viscose paper, aluminum foil bag, instruction for use, external packaging cartridge, bottle and dryer shall be established in accordance with the “Guideline for Drafting of Instruction for Use of in vitro D

40、iagnostic Reagents” and the “Provisions on the Administration of Instruction Leaflet, Labeling, and Packaging Labeling of Medical Device” promulgated by the SFDA. (III)Manufacturing of various components of reagent kitManufacturing of main component of the luminescent immunoassay analytic reagent ki

41、t is consisted of procedures of reaction plate of coating, preparation of marker, processing of various solutions, lyophilizing, sub-packaging, etc.; and the completion of which is through two quality control processes of testing of half-finished product and testing of finished product to assure the

42、 compliance of quality.1.Preparation of solid phase carrier (as large difference is existed between coating carriers of different products, here the description is performed using standard 96 micro-aperture reaction plate as an example)(1)Preparation of coating plateQualified coating plate is prepar

43、ed, and the lot number, amount and status identification is recorded.Quality control items: size, appearance and packaging.(2)Process of coating solutionCoating buffer is processed, coated antibody or antigen is added into working concentration, after being mixed, the required coating solution is co

44、mpleted, and the coating solution of working concentration should be used within specified period.Quality control items: formula of coating buffer, pH and component of coating substance.(3)Coating of coating plateCoating solution is added into coating plate according to technique requirements, and t

45、he number of coating plate being coated is recorded.Quality control items: volume, temperature, time and process monitor of coating.(4)Preparation of working solution for plate processing (plate processing may not be performed according to technique requirement of individual unit)Working solution fo

46、r plate processing may be prepared according to formula.Quality control item: formula and pH of working solution for plate processing.(5)Preparation of blocking solutionBlocking solution is prepared according to formula.Quality control item: formula and pH of blocking solution.(6)Plate processing an

47、d blockingAfter completion of packaging, the coating solution within aperture is drawn, blocking solution is added when plate processing has been completed (plate processing may not be performed according to technique requirement of individual unit).Quality control item: monitor of blocked volume, t

48、emperature, time and process.Quality control item: uniformity of coating is tested prior to blocking.(7)UnwateringReaction plate after being blocked, the liquid within the aperture should be unwatered.Quality control item: monitor of process.(8)DryingReaction plate should be dried according to requi

49、rement of technique.Quality control item: monitor of temperature, humidity, time and process, etc.(9)Sealed packagingThe dried reaction plate may be sealed packaged using aluminum foil bag, with dryer in it (the dryer may not be put into it according to technique requirement of individual unit).Qual

50、ity control item: performance of sealing, label and efficiency date, etc.(10)Testing of reaction plate (half finished product)Sampling testing is performed with the reaction plate after packaging and sealing, includes appearance, variation within plate, and variation between plates.2.Titration proce

51、ss(1)Preparation of enzyme conjugate (this procedure may not be performed according to actual circumstances of specific product)Label horse radish peroxidase (or other enzyme) using related antibody (or antigen), utilizing common used sodium periodate - ethylene glycol method, applicable preservativ

52、e should be added into enzyme labeled antibody (or antigen) and stored in lower temperature.Quality control item: labeling method, process control.(2)Validation of enzyme conjugateFunctional experiment Enzyme conjugate may be used for titration of product after being diluted with dilution. The resul

53、t should be complied with the quality standard of relative reagent kit.StabilityThe enzyme conjugate is diluted with dilution, perform 2-8 and thermal stability experiment. The titration result should be complied with the quality standard of relative reagent kit.(3)Dilution of enzyme conjugatePrepar

54、ing according to formula of enzyme conjugate dilution, stored in 2-8, and use within specified time.Quality control item: formula of enzyme conjugate dilution, pH.(4)Titration of enzyme conjugate working concentrationEnzyme conjugate is diluted using enzyme conjugate dilution to different concentrat

55、ions. Titration is performed using the prepared reaction plate. Detect the serial standard and relative control; determine the working concentration of enzyme conjugate which has reached the optimum state.(5)Preparation of working solution of enzyme conjugateMix to uniformity with the required amoun

56、t of enzyme conjugate and enzyme conjugate dilution according to titration concentration.Quality control items: testing prior to filling, using the matched reaction plate, and linear measurement should be performed with calibrator for appearance, sensitivity, control measured value and quantitative

57、product.(6)Filling of enzyme conjugate working solutionFilling of enzyme conjugate working solution according to technique requirementsQuality control items: confirmation of the reagent name, lot number, quantity, volume to be filled, and sealing performance after sealing prior to filling.(7)Testing

58、 of enzyme conjugate working solution (half finished product)Sampling detection is performed with the enzyme conjugate working solution after filling, which includes appearance, volume to be filled, sensitivity, calibrator dose reaction curve linear, measured value of control.3.Preparation of calibrator and negative/positive control(1)DilutionPreparing according to formula of dilution, storing in 28 or -20, and using within efficiency date.Quality control item: formula of dilution, pH.(2)Preparation of calibrator, negative/positive controlPreparation of calibra