Validation of biological actiVity testing procedure of recombinant human interleukin-7

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation characteristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations.This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes.It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used.Validation characteristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research.The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.k e y w o r d s: recombinant human interleukin-7 (rhIL-7), biological activity, peripheral blood mononuclear cells (PBMCs), validation.
O ne of the most essential steps of develop ment of medicinal products (MPs) is pre clinical studies -a complex of investigation procedures and operations for the study of toxici ty and specific activity of potential bioactive sub stances.Appropriate conduct of these studies has to guarantee safety and high therapeutic efficacy of the developed MPs.The main instruments of preclinical studies are various pharmacological methods invol ving the use of several analytical measurements and tests on various biological objects.That is why these methods may be classified as bioanalytical.
Validation is a procedure which gives a high de gree of assurance that a particular process, method , or system consistently lead to results that meet pre determined acceptance criteria.Validation of ana lytical method represents experimental evidences that the method is suitable for its intended purpose [1].An important issue of validation of bioanalytical procedures is the absence of precise methodic guide lines on selection of the necessary parameters and performance methods.Besides, the use of pharma copeias and/or reference samples (as a basic element of validation of chemical analytical procedures) is mostly impossible during assessment of biological samples due to their absence.Analytical procedures' suitability evaluation is one of the most essential ele ments of quality assurance system of pharmaceutical and biotechnological products [2].
The goal of this study was validation of bio analytical procedure for control of biological activi ty of recombinant human interleukin7 (rhIL7) in accordan ce with requirements of national and inter national guidelines.

Isolation of human peripheral blood mononuclear cells (PBMCs) and their stimulation. Blood m e t h o d s m e t h o d s
was obtained from verified donors (under Article 16 of the Law of Ukraine "On donor service of blood and blood components") using heparin (10 U/ ml of blood was used) or EDTA as anticoagulants.The blood volume was 14 ml.Blood separation was performed in a centrifuge with bucket rotor (Liston 2204 Classic) for 30 min at 1500 rpm at room tem perature using density gradient Histopaque 1077 (Sigma, USA).PBMCs were collected with a ster ile pipette and washed twice with 10 ml of rinsing medium (RPMI1640, Biowest, USA, containing 2% fetal calf serum) by centrifugation for 10 min at 1000 rpm at room temperature.Then the cells were resuspended in 4 ml of the culture medium (RPMI with 10% fetal calf serum, 10 µg/ml of phytohemag glutinin (PHA) (Sigma, USA, L -8754) and 50 mM mercaptoethanol), and the number of viable cells was calculated upon staining with 0.4% trypan blue in Goryaev counting chamber.The cells were diluted to the concentration of (14)×10 6 cells/ml with the culture medium and cultivated in Т45 flasks (10 ml/ flask) for 5 days at 37 ºС in a 5% СО 2 atmosphere.After PHA stimulation, the cell suspension was cen trifuged at 1000 rpm for 10 min and the cells were washed once as described earlier.Then the cells were again resuspended in 35 ml of culture medium , and the number of viable cells was calculated upon staining with 0.4% trypan blue in Goryaev counting chamber.The cells were diluted to the concentration of 2×10 6 cells/ml and added into 96well plates, in troducing 50 µl into each well.
Biological activity control procedure.Referen ce sample solutions were prepared as follows: rhIL7 (PeproТech, USA) was used as the reference sam ple; a solution No 1 was prepared by dilution of the reference sample with phosphate buffer рН 7.4 and addition of 0.1% BSA to the concentration of 1 ng/µl.The following dilutions (ng/ml): 0.125, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 were prepared from the solution No 1 with PBMCs culture medium.
Used in test sample rhIL7 was obtained by re combinant DNA technology in e. coli cells and puri fied using stepwise chromatographic purification at LLC "Universal Agency PROPHARMA" [3].Test sample solutions were prepared as follows: first a so lution No 1 was prepared by dilutions in phosphate buffer pH 7.4 from the obtained sample with addition of 0.1% BSA to the rhIL7 concentration 1 ng/µl.The following dilutions (ng/ml): 0.25, 0.5, 2.0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 were prepared from the solution No 1 in PBMCs culture medium.
The obtained dilutions of a reference sam ple and test sample were transfered into wells of PBMCcontaining plate in the volume of 50 µl in triplicate and incubated for 4 days at 37 ºС in an in cubator with 5% СО 2 .After that, 15 µl of methyl thiazole tetrazolium bromide (MTT) solution (5 mg/ ml) was added into each well and incubated for 4 h at 37 ºС in the incubator with 5% СО 2 .After that, 200 µl of DMSO was added into each well to dis solve formazan crystals.Optical density (OD) was measured at 570 nm (spectrophotometer 6320D VIS, Jenway).The relation plot of optical density against IL7 concentration logarithm was generated (the plot had the appearan ce of sigmoid curve).The effective dose (ED 50 ) was calculated from linear part of the plot and represen ted 50% proliferative response of the maximum proliferative response obtained in the linear part of the plot.For ED 50 calculation, the sig moid curve data obtained were approximated using statistical software packages [4].
Mathematical (statistical) methods.The op timized method of biological activity control was verified in accordance with procedures described in guidelines of the International Conference on Har monization of Technical Requirements for Regis tration of Pharmaceuticals for Human Use (ICH), Validation of Analytical Procedures Q2 (R1) [5], as well as Microsoft Excel software.The following vali dation parameters were reviewed: specificity, linea rity and range, accuracy, precision, and robust ness [1, 6,7].
All calculations were performed in "normali zed" coordinates.This allowed formulating unified criteria related with content tolerance limits only, but independent of the specific properties of individual values.Normalized coordinates were established as follows: (1) where C i -test substance concentration in ith so lution (or sample), C st -concentration of the same substance in the reference solution, a i -analytical signal of the substance tested for ith test solution, a st -analytical signal of the same substance for the reference solution.
Subsequently, all calculations and criteria were performed for normalized values Х і and Y і .
Linear relation was calculated using the least square method ) where a -absolute term for the calculated regression line (y intercept); b -slope for the calculated regres sion line.
Concentrations tested during linearity study were characterized by standard deviation.
RSD y (%), calculated using the formula: where С і -ith solution concentration; С -mean concentration of solutions; g -sample volume (the number of points on a line) [6].

Comparative characteristics of assessment methods of human interleukin-7 biological activity.
A set of validation characteristics to be determined is dependent on the method, product type or test/ measurement object, as well as biological test sys tems the study will be conducted in.Typical valida tion characteristics include: accuracy, precision (re peatability, intermediate precision, reproducibility), specificity, limit of detection, linearity, and range.The set of characteristics and methodological ap proaches to their determination are dependent on each specific procedure, but the presence/absence of each characteristic has to be justified.
Test method of control of the rhIL7 biological activity is based on its ability to cause Tlymphocyte proliferation.Human peripheral blood mononuclear cells (PBMCs) obtained from donor blood or cell lines may be used for testing of rhIL7 biological activity.At present, there are several interleukin 7dependent cell lines in the world, which may be used for testing of rhIL7 biological activity: 1xN/2b (stromal cell line), 2Е8 (mouse Blymphocytes), D1 (knockout mice thymocytes), DW34 (preВcells), PB1 (preВcells), Pno (Тcells).Table 1 contains brief comparative characterization of cell lines which may be used for testing of biological activity of rhIL7 [3,8,9].
Cells used for control were prestimulated with PHA.If finite cell lines are used, cellular growth al ways has to be maintained in the presence of human interleukin7 reference sample with known activity, thus we have selected PBMCs for biological activity testing from the viewpoint of technicaleconomical Method using tritiumlabeled thymidine, МТТmethod parameters.Proliferation assessment may be per formed by two methods using either 3(4,5dimethyl thiazole2yl)2,5biphenyl tetrazolium bromide (МТТ test), or tritiumlabeled thymidine (thymidine test) [3,10].Use of labeled thymidine for detection has several disadvantages related with the need in handling radioactive material, as well as its disposal, thus, we have given preference to MTT test for bio logical activity control.Method specificity assessment.Specificity of the method is its ability to assess biological activi ty of a particular test substance in the presence of other components which may be found in a sample.As biological activity testing is one of identification methods used for control of active pharmaceutical ingredients and finished medicinal products based on rhIL7, specificity is the proof of the fact that the test substance is actually identified.

Cell
We have suggested testing the specificity of our rhIL7 in test sample versus rhIL7 (Peprotech, USA, Cat.N. 20007) in referen ce sample on human PBMCs .In accordance with requirements of national and international recommendations, RSD may not exceed 2% [1, 8].
Fig. 1 shows the results of specificity testing of the method -proliferation of human peripheral blood cells at increasing rhIL7 concentration.The obtained ED 50 values for the test and the reference samples were 2 and 5 ng/ml, respectively.This gave us grounds to believe that the test sample has the appropriate biological activity.It was lower than that observed in the reference sample.Neverthe less, these results are related solely with poorly opti mized test sample purification conditions.Thus, the obtained results are indicative of the fact that this method is specific with regard to rhIL7.assessment of range (R) and linearity.The fol lowing dilutions of the obtained rhIL7 in PBMCs culture medium: (ng/ml) 0.125, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 were used for the testing of the said validation characteristics.Control of each dilution was performed in triplicate.
Table 1 and Fig. 2 show the results of optical density dependence on rhIL7 concentration.Thus, according to the obtained results, it can be concluded that the admissible minimum range for recombinant human interleukin7 testing with this method is 0.125 ng/ml.
The data obtained were used to calculate linear regression equations listed in Table 2.The test linea rity was assessed by analysis of the obtained equations.Slope coefficient of linear regression plot was within the limits of 1.0111.028,which is an acceptab le result for quantitative bioanalytical proce dure.Correlation coefficients (r) between experimen tal and theoretical OD values were compared against the critical value for different significance levels [6].The obtained results (Table 2) are indicative of ac ceptable level of conformity between experimental data and OD results calculated using linear regres sion equation for test samples of recombinant human interleukin7.

-■--♦-reference sample
Test sample accuracy and precision assessment.It is well known that precision may be reviewed at different levels, in particular: repeatability (intra assay varia tion) characterizes variations in test procedures in the same conditions within a short time span; inter mediate precision (inter assay variation) takes into account intralaboratory variations; reproducibility characterizes the proximity degree of results at in terlaboratory experiment [1113].
In our study, the testing of accuracy and repeata bility expressed through coefficient of varia

Fig. 2. establishment of linear relation between optical density and rhIL-7 concentration
tion CV intra were carried out simultaneously.As biological activity test method is quantitative, the measurements were performed by testing 9 dilu tions, concentrations of which were uniformly dis tributed within the tested range of this procedure ng/ ml: 0.125, 0.25, 0.5, 1, Reference sample eD 50 calculation was performed using statistical software package originPro7.5.eD 50 (Donor 1) 0.7672±0.0896ng/ml ED 50 (Donor 2) 0.7352±0.0967ng/ml 2.0%, which is a very good parameter for bioanalyti cal procedure accuracy assessment.For precision as sessment, this analysis was conducted in parallel on PBMCs obtained from two different donors.Results of the relevant experiments are shown in Tables 3 and 4 and in Fig. 3. Repeatability mean values (CV intra ) of biological activity testing were 6.3% for the reference sample and 6.9 % for the test sample.Intermediate precision of the test, where PB  The suggested method for biological activity testing of rhIL7 in human PBMCs was validated for confirmation of its biological activity quantita tive characteristic.Validation has confirmed that this method showed the reliable data for all tested parameters such as specificity, accuracy, precision and linearity.The method linearity was satisfactory.Repeatability of test result was within the range of 6.3 to 6.9%, and intermediate precision was from 3.5 to 8.8%, which is an acceptable result (≤ 10%).The bioanalytical assay for biological activity testing of rhIL7 in donor PBMC has been recognized as vali dated with satisfactory results.К л ю ч е в ы е с л о в а: рекомбинантный интерлейкин7 человека, биологическая актив ность, монононуклеарные клетки перифериче ской крови (МКПК), валидация.