Friday, 17 March 2017


The US Food and Drug Administration (FDA) has made a guide.
The following process validation definition.
"Process validation is establishing documentary evidence to provide a
a high degree of assurance that the specific process (eg manufacturing of the pharmaceutical dosage form) will consistently produce a product meeting
its intended specification and quality characteristics." According to FDA, product quality assurance comes from careful And some important factors to the system's attention, including: selection
The quality of parts and materials, adequate product and process design as well
(Statistical) control process through process and end product testing.
So it is through careful design (qualification) and validation Process and its control system, you can establish a high degree of confidence
A given batch or inheritance for all individual manufacturing units it is acceptable to meet the specifications of the batch. According to FDA's current Good Manufacturing Practices (CGMP) 21CFR 211.110 a:
Control procedures for monitoring outputs and verification should be established Performance may be responsible for the manufacturing process Causing changes in the properties of the material and the drug during processing product. Such control procedures shall include but are not limited to the following, when appropriate .
1. Change in tablet or capsule weight
2. disintegration time
3. Mix thoroughly to ensure uniformity and uniformity
4. Dissolution time and rate
5. The clarity, completeness, or pH of the solution
The first four items listed above are directly related to manufacturing And solid dosage forms. Items 1 and 3 are usually associated There are changes in the manufacturing process, while items 2 and 4 are usually By the choice of ingredients in the product formulation. versus Respect for content consistency and unit performance control (item 3), adequacy Mixing to ensure uniformity and uniformity are considered to be of high concern. The routine quality control procedures for finished product testing include
Three basic steps:
1. The establishment of normative and performance characteristics
2. Select the appropriate method, equipment and instrument
To ensure that the product is tested in accordance with specifications
3. Final product testing, using proven analysis and testing
Make sure that the finished product meets the specifications.
With the emergence of the concept of pharmaceutical process validation, the following
Added four additional steps:
4. Qualification of processing facilities and equipment
5. Qualification and verification through appropriate manufacturing processes
6. Key steps in auditing, monitoring, sampling or challenging
The process conforms to the process and the final product specifications
7. Re-verify when the product has significant changes
Or its manufacturing process.

Wednesday, 15 March 2017

Blood products removed / inactivated virus technology and methods and validation guidelines

"Blood products removed / inactivated virus technology and methods and verification guidelines" by the State Food and Drug Administration on May 9, 2002 National Drug Note [2002] No. 160 issued.

Blood products removed / inactivated virus technology methods and verification guidelines

Currently known blood fluid products contagious virus mainly of HBV , of HCV , of HIV-1 , of HIV-2, of HTLV and parvovirus B19. Not found through blood products infected CJD. However, a few studies have found that the experimental infection and, therefore, should pay close attention to CJD, vCJD in particular the developments.

In order to improve blood product safety , production processes to have a certain removal / inactivation of viruses portion capacity , the production process should be specific removal / inactivation of viruses methods. The technical guidelines Is the blood products (refers to the human plasma as raw material products) and the specific production process of removal / inactivation of the virus method validation guidelines, including viruses and virus removal instructions selection / inactivation method of authentication scheme design, and technical verification result judgment listed in Appendix declaration procedures .

2 First, select the removal / inactivation of virus by

Because different types of blood products potentially contaminated distinct possibility of the virus, to select for this virus removal / inactivation methods focus should also be different:

2.1 (a) coagulation factor products category

There should be a specific production process to remove / inactivate lipid-enveloped and non-lipid-enveloped virus, can be combined with one or more of removal / inactivation of viruses.

2.2 (b) class of immunoglobulin products

For immunoglobulins type products (including intravenous injection of human immunoglobulin , human immunoglobulin and the specific human immunoglobulin) production process should inactivate lipid-enveloped virus specific method. However, to further improve the safety of such products to consider, promote production process by adding specific for the removal of non-lipid-enveloped virus / inactivation methods.

2.3 (c) albumin

Low temperature ethanol production process and specific removal / inactivation of viruses methods, such as Pasteur disinfection method.

3 Second, the common removal / inactivation of viruses evaluation method

3.1 (a) pasteurization (pasteurization)

3.1.1 1. Human albumin products

Decades of clinical application of the results showed that albumin of pasteurization of HIV and liver disease virus is safe. Viral inactivation conditions are perfect, do not require virus inactivation verification. But it must be used for pasteurization facilities to verify that pasteurization parameters to meet the requirements (including product temperature profile of uniformity and inactivation time).

3.1.2 2. Other blood products (liquid preparations)

Due to the composition of products, stabilizers (such as: amino acids , sugar, citric acid salt, etc.) and different concentrations of virus inactivating effect of both will have a certain impact. Therefore, when using pasteurization inactivated virus virus inactivation method must be verified.

3.2 (b) dry heat (lyophilized product)

80 ℃ heated for 72 hours to inactivate HBV, HCV, HIV and HAV and other viruses. It should consider articles moisture content, product composition (eg: proteins , sugars, salts and acids) on the inactivation of viruses. It should determine the difference between the parameters allowed by article bottles. Virus inactivation by dry heat tank at least once every six months to verify. Verifying the drying chamber should be located a plurality of measurement points (including products, highest and lowest temperature inside the point).

3.3 (c) an organic solvent / detergent (S / D) treatment method

Organic solvents , such as: phosphoric acid tributyl fat (TNBP) and non- ionic of the decontamination agent, such as: Triton X-100 or Tween-80 binding may inactivate lipid-enveloped viruses, non-lipid-enveloped viruses but ineffective. Common inactivation conditions is 0.3% TNBP and 1% Tween-80, treated at 24 ℃ at least 6 hours; 0.3% TNBP and 1% Triton X-100, treated at 24 ℃ for at least 4 hours. S / D treatment should be preceded by 1μm filter to remove the protein solution may be present in the particles (particles can harbor viruses affecting viral inactivation). After addition of S / D should be to ensure a uniform mixture . In the whole process of viral inactivation temperature should be controlled in a predetermined range. If after the addition of S / D filter shall be detected after filtration concentration S / D whether the occurrence of the change, if any changes should be adjusted appropriately. Tween-80 should be used plant-derived, and should be weighed weighing method.

3.4 (d) membrane filtration

Membrane filtration technology is only effective membrane pore diameter than an hour in order to effectively remove viruses virus. This method can not be used alone and should be used in combination with other methods.

Validation studies should take into account the protein concentration in the solution of important parameters, filtration rate, pressure and the amount of filtering and so on. We should test the integrity of the membrane after filtration and before filtration.

3.5 (e) a low pH incubation discharge method

Studies have shown that immunoglobulin production process low pH (eg pH4) process (sometimes plus pepsin ) can inactivate several lipid-enveloped viruses. Inactivation conditions (eg: the pH value , temperature and incubation time release, pepsin content, protein concentration, solute content and other factors) that may affect inactivation of virus, these studies should validate the test parameters allowed to vary the amplitude.

4 Third, the specific removal / inactivation of virus method validation

4.1 (a) indicates the selection of virus

First, you should choose through blood spread of related viruses (such as: HIV), the virus can not be associated, as far as possible to choose their physical and chemical properties similar indication virus; second, selected physical and chemical properties shall be representative of the virus (the virus size , nucleic acid type, and whether the envelope), which should include at least a kind of physical and / or chemical treatment significantly resistant viruses. During the removal / inactivation of viruses verification should be based on product characteristics and used in virus removal / inactivation process, refer to the following table lists the virus to choose the appropriate indication virus. The selection should include at least an indication of the virus HIV-1, HBV and HCV mold intended viral and non-lipid-enveloped viruses. Vesicular stomatitis virus (VSV) tolerable pH range relatively wide, low pH incubation can be used to verify this indication virus inactivation of virus release effect.

Blood-borne viruses and diseases associated verification optional indication virus (for example)

 virus Gene group Lipid-enveloped   (nrn) Examples indicating virus
 HIV RNA Have 80-100 HIV
 HBV DNA Have 45 Duck hepatitis B virus , pseudo- rabies virus
 HCV RNA Have 40-60   Bovine diarrhea virus, Sindbis virus
 HAV RNA no 27 HAV, polio , cerebral myocarditis (EMC) virus
 B19 DNA no 20 Canine parvovirus, porcine parvovirus

4.2 (b) Design

1. Removal / inactivation of virus validation studies should comply with GLP requirements.

2. Effects of the removal / inactivation of viruses effect parameters (including mechanical parameters and physicochemical parameters) allows the magnitude of change.

3. Study of viral inactivation kinetics , including the rate and inactivated viral inactivation curve.

4. Instructions should be as high viral titers (virus titers should ≥106 / ml).


6. If possible, a sample verification process every step should be taken as soon as possible directly to the virus titration , no further treatment (such as ultra-centrifugation, dialysis or save , remove inhibiting agents or toxic substances, etc.). If the sample must be further processed, or at different times of samples taken at the same time to be determined, it should consider the impact of these treatments on virus detection results.

7. Detection methods may include plaque formation, cell lesions (e.g. syncytia or foci formation), endpoint titration or other methods. These methods should have the appropriate sensitivity and reproducibility , each sampling point should take duplicate samples and a control to ensure the accuracy of the results.

8. If the production process of the products included in the above two-step or two-step virus removal / inactivation methods should verify the effect of virus inactivation, respectively.


4.3.1 1. Viruses

(1) removal / inactivation virus titre;

(2) the rate of viral inactivation, inactivation curve. Make a list and graphical reporting verification results.

4.3.2 2. Virus removal / inactivation of the allowable variation range of parameters

4.3.3 3. Protein aspects

(1) product quality should be consistent, " Biological Products Regulations" or the relevant regulations;

(2) using the appropriate method for the determination of protein structure and function changes in activity. Such as: product ratio Fc functional activity, IVIG is analyzed to learn whether the protein structure has changed; gel chromatography can detect proteins of molecular size and shape changes; changes in the shape of a protein can lead to the diffusion coefficient , sedimentation constant change and viscosity; SDS -PAGE, especially isoelectric focusing PAGE and binding (bidirectional electrophoresis ) is also a good method for detecting changes in protein structure.

(3) If a new removal / inactivation methods (including the replacement of viral inactivation method or use of the country has been recognized inside and outside have not been using a virus removal / inactivation methods), the need for protein half-life and the new immunogenicity studies. Requirements are as follows:

Half-life: with a suitable animal (such as rats or rabbits) and the same products without the virus removal / inactivation were half-life comparison; immunogenicity new: new immunogenic to check the changes in protein structure on a higher level. These changes are not necessarily damage protein function, but will cause the receptor immune response . The new laboratory testing immunogenicity is very difficult. Available through without viral inactivation and protein were immunized animals (such as rabbits), produced antibody cross-linking with and without virus inactivated by proteins. If virally inactivated antibody protein is not completely binding protein inactivated virus, indicating that without new antigen . Can not guarantee that the human immune system to recognize the epitope same experimental animals, it is recommended in the clinical stage Ⅳ (obtained after the production numbers) whether to carry out research of new antigens.

4.4 determination (iv) the effect of

Analyzing virus removal / inactivation of validity shall be comprehensive consideration, not only virus removal / inactivation is determined. Before determining effective, the following factors must be considered, carefully evaluate each verification results.

1. Verification test the suitability of the chosen virus, the virus verify whether the design is reasonable.

2. Reduce the amount of virus (log10) ≥4 logs, indicating that the step of removing / inactivating the virus effectively. When as a result of the detection method of reducing the amount of virus caused <4 as="" be="" blind="" can="" detection="" effective="" for="" identified="" inactivation="" is="" logs="" method.="" no="" p="" should="" three="" virus="">
3. Virus inactivation kinetics of viral inactivation can better display effect. Virus inactivation is usually not a simple reaction. Often the initial reaction rate, followed by slow. If viral inactivation rate decreased with time, indicating that the method may be ineffective, or the residual viral inactivation methods indicates resistant, indicating that the method of viral inactivation step is invalid.

4. The actual virus titer-based virus, indicating a virus sample 1: 9 ratio of the virus titer 0:00 mixing samples, was determined by comparing the removal / inactivation of the virus with the actual amount of residual virus as the virus removal the amount of virus inactivation / inactivation method (step) practical.

5. Virus detection sensitivity limit of degree.

for example:

(1) was added 6 logs virus, and the remaining 4 logs virus can be removed / log number of inactivated virus removal in the calculation of the total amount of the whole production process / inactivated virus being, but this step (method) removal / inactivation of viruses capacity is invalid.

(2) added 6 logs virus, but because of the cell preparation itself toxic effects makes the detection sensitivity limit of 4 logs, proved to be just removed 2 logs virus. In such cases we need to change the trial design revalidate.

(3) added 6 logs virus, but still remaining measured 2 logs virus, and the amount of clearing the virus can be repeated out, is not affected by craftsmanship and should be considered effective removal / inactivation of the virus method.

(4) Add 6 logs virus, then the virus is not detected. However, due to the sensitivity of the detection limit of 2 logs, I think only about 4 logs remove or inactivate the virus. May in fact be equal or greater than 4 logs, and therefore this method should be determined that the amount of virus removal ≥4 logs.

(5) the virus inactivation kinetics is a very important observation index. Such as pasteurization (60 ℃, 10 hours), if the virus quickly to minimize the residual amount detection limit values, indicating that this method works well to inactivate the virus. If a virus inactivation rate is slow, the lowest detection limit value until the end of inactivation, can not be considered an effective viral inactivation method. That is to say, the evaluation results can not be verified only consider reducing the amount of virus, taking into account also the virus inactivation kinetics.

5 Fourth, the production process to remove / inactivate the virus validation capabilities

Production process to remove / inactivate viruses proficiency testing conducted with reference to specific removal / inactivation of the virus method validation requirements. Require special consideration of the issue are the following:

(A) may only have to remove / inactivate viral effect of the production steps to verify.

(Ii) simulation of the production process of various parameters as possible consistent with the actual production process, such as pH, temperature, protein concentration and other constituents of the reaction time , chromatography column bed height and flow rate and column bed height ratio, the elution pattern and the effect of the production steps (such as yield, specific activity, composition). We should analyze the production process deviations of various parameters on the impact of the virus removal / inactivation effect.

Each step (iii) the production of different types of viruses selective removal / inactivation.

(Iv) a nucleic acid amplification technology (such as the PCR ) detection of viral nucleic acid sensitivity is relatively high, but the biggest limitations of the detection technology is indistinguishable been inactivated or not inactivated virus. Therefore, this technique can not be used to verify the amount of virus inactivation, the virus can only be used in the production process of removal of verification.

(E) generally in the production process to remove / inactivate viruses is possible to calculate the total amount of clearing the virus, but not identified as an effective virus removal step. Because the production process is usually some changes, difficult to control and validation, and virus distribution depends entirely on the specific physical and chemical properties of the virus, these viruses affect the physical and chemical properties and gel media interaction properties and precipitation. Since variance can thus virus surface characteristics (such as: glycosylation), indicating the distribution of the virus with the target in the form of the virus may be completely different. Proliferation-associated virus in the laboratory may be on a different distribution of the wild strain.

(F) the purpose of verification is to determine the capacity of the production process to remove / inactivate the virus, obtain an estimate of the total production of the whole removal / inactivation of viruses. The total amount of each step is generally reduced to reduce the amount of virus sum . However, due to the virus validation limitations, such as step by step to reduce the amount of virus ≤1 log it should not be calculated in the total.

6 V. removal / inactivation of virus re-verification process

The following conditions need to be re-verified virus removal / inactivation method:

(A) for the first time producer , to be removed in the production process specific / virus inactivation method revalidation;

(B) the introduction of new technology or the original production process carried out a major reform, the need to remove the virus production process proficiency testing; the removal of specific / inactivated virus removal method / inactivated viral effect verification;

(3) when process changes but not a major reform process, removing the need for specific / virus inactivation method revalidation;

(D) In ​​the middle of inactivated product composition or pH changes, to be removed for specific / virus inactivation method revalidation.

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7 Appendix: blood products, viral removal / inactivation verification reporting procedures

1, the State Drug Administration of  Pharmaceutical and Biological Products licensed clinics and other recognized testing laboratory verification virus removal / inactivation effect.

2, manufacturers of blood products should be required to present technical guidelines for its production process and the specific virus removal / Off

Live method was validated. Such as unconditional, other units can be commissioned to verify the removal / inactivation of the virus effect.

3, manufacturers of blood products in the complete removal / inactivation of viruses effect after verification, the Chinese Pharmaceutical and Biological Products of the proposed verification application, along with products production processes, quality standards , a virus inactivation methods and standard operating rules (SOP), at least pilot scale continuous production of three batches of virus inactivation before intermediate product (sample removal / inactivation of viruses before) and manufacturing records and other relevant information.

4,  Pharmaceutical and Biological Products of the production unit verification application submission and review relevant technical information; performs virus removal / inactivation methods related parameters (such as provided by its three batches before viral inactivation intermediate product: pH, protein concentration, moisture and stabilizer content, purity, etc.) detection.

5, intermediate goods production batches continuous virus  Pharmaceutical and Biological Products licensed clinics recognized testing laboratory to production units submitted before the inactivated virus inactivation method is effective verification. Identified in authorized testing laboratory verification after the completion of the verification result communicated  Pharmaceutical and Biological Products.

6, after the virus removal / inactivation validation or verification at the same time,  Pharmaceutical and Biological Products reporting units to provide continuous production of three batches of product ( batch when virus verified with a lot of different articles submitted, but must be adopted the same production process and virus removal / inactivation process for the production of products) to conduct a comprehensive review of quality (according to " biological products regulations" and the relevant legal standards for the seizure, issued by the inspection report).

7, the use of new virus removal / inactivation methods, after reviewing the information and virus inactivation verification ending, by the Pharmaceutical and Biological Products is responsible for the organization of blood products and other aspects of virology experts and include Center for Drug Evaluation, including staff relevant departments will participate in the technical feasibility studies of viral inactivation method declaration to demonstrate.

8. After verification,  Pharmaceutical and Biological Products will be removed / inactivated virus verify the results, comprehensive evaluation of comments and reply batches of products inspection report reporting unit;

9, reporting units of blood products will be declared to be adopted virus removal / inactivation methods research data (including literature), the virus self-test report and the  Pharmaceutical and Biological Products licensed clinics recognized testing laboratory to production units submitted removed / inactivation method validation results, comprehensive evaluation or demonstration opinions, join the virus removal products / inactivation process after the stabilization of the examination results, pharmaceutical and biological products issued three batches of products inspection reports be submitted to the State Drug Administration It is approved.

Chemicals quality control methods validation technical guidelines

1."Chemicals quality control methods validation technical guidelines" by the State Food and Drug Administration on March 18, 2005 the State Food and Drug Administration Note [2005] No. 106 issued.
2 I. Overview

To ensure drug safety, effective and controllable quality is the basic principle of drug development and evaluation should follow, wherein for medicines quality control is to ensure that the drug is safe and effective basis and premise. In order to achieve control of the quality of purpose, requires multi-angle, multi-level to control the quality of drugs, that is to for the drug on multiple projects to test, to fully investigate drug quality. In general, each test item can use different analysis methods , in order to make the test results are accurate and reliable analytical method must be used in science , accuracy and feasibility validation , analysis shows that in order to fully meet the test project objectives and requirements, which is commonly referred to validate the method.

Method validation is intended to judge the analysis method used is scientific, reasonable, whether the drug can effectively control the intrinsic quality. Essentially, the method validation is based on the detection requirements of the project, a certain pre-set content verification, and through the rational design of experiments to verify the analytical methods used to detect whether the project meets the requirements.

Method validation based on analysis of the process has an important role , and become an integral part of the quality of research and quality control. Only after the validation of analytical methods can be used to control the quality of drugs, so the method validation is to develop quality standards based. Method validation is the process of drug research important content.

This guidance focuses on methods to verify the nature of the analytical method validation requirements and the intended purpose combine systems and elaboration of regularity, it focuses on how scientific and rational design demonstration programs.

General principles of the guidelines include methods of verification, validation involves three main aspects, method validation of specific content, method validation and evaluation content.

The principles and other relevant technical guidelines constitute together a more complete quality control guidelines. With China's increasing levels of drug development, method validation will continue in-depth understanding of the guidelines will be gradually improved and revised. Because biological products and medicine particularity, this principle is mainly applied to chemicals.

3  general principles, methods validation

In principle, each detection analysis method adopted by the project, need to be validated.

The method of verification should be based on the content requirements of test items, combined with analytical methods used to determine the characteristics.

The same methodology used for different test items have different authentication requirements. For example, the use of HPLC methods for identification and quantification of impurities test preparations should be carried out to verify the different requirements of the former key requirements to verify the specificity, and the latter to verify key requirements specificity, accuracy , limit of quantification .

4 the method validation involves three main areas

4.1 (a) need to verify the test items

Test items for drug quality control to ensure safe and effective set of test items. according to

Setting objectives and verify the contents of the different requirements of the testing program, the present guidelines will need to verify the test items are divided into identification, impurity inspection (limit test, a quantitative test), quantitative determination (determination, dissolution , release , etc.), other specific test items and other four categories.

Object identification is to be determined analyte target compound , rather than other materials, analytical methods for identification require strong specificity.

Check the impurity is mainly used for the control of impurities other than the main component, such as organic impurities, inorganic impurities. Check test limits of impurities can be divided into two cases and quantitative tests. Analytical method for testing the limits of verification focused on specificity and limits of detection. Analytical methods for quantitative test validation emphasize specificity, accuracy and limit of quantification.

Quantitative assays include determination, dissolution testing and other preparations, since such projects require a higher accuracy, so the analytical methods used requires a certain degree of specificity, accuracy and linearity . Other items include the specific detection of the particle size distribution , specific rotation , molecular weight distribution, etc., due to the requirements of these test items with identification, checking impurities, and other different quantitative determination, analytical methods for verification of these items should have different requirements.

4.2 (b) Analysis

The guidelines referred to in the above analysis for each of the test items and test methods set established, including an analysis of the general principle of the method, apparatus and instrument parameters, reagents, system suitability test, the test for the solution prepared reference solution preparation, measurement, calculation and reporting of test results.

Test methods may be used for chemical analysis methods and instrumental analysis methods. These methods have their own characteristics, the same test method can be used for different test items, but do not verify the contents of the same.

4.3 (c) to verify the contents

Verify that includes a specific method, linearity, range, accuracy, precision , detection limit, quantification limit, durability and system applicability.

5 , the method validation details

5.1 (a) Specificity

Specificity refers to the other ingredients (such as impurities, degradation products, accessories , etc.) may be present, the analytical methods used to correctly identify the detection characteristics of the material being analyzed.

Typically, identification, inspection impurity content determination in its specificity should be investigated. The method used is not enough exclusive, it should be supplemented by the use of multiple methods.

1, the differential response

Identification test should confirm analyte in line with its characteristics. Specificity test required to prove that the substance or may coexist with a structure similar to the compounds of distinction, to be confirmed for the analyte-containing sample was a positive reaction, while the negative control without the test component was a negative reaction, or a group of structurally similar points in the related compounds should also reacted negatively.

2, impurity inspection

As a purity test, the method used should ensure that the amount of analyte can be detected impurities, such as related substances, heavy metals , organic solvents and the like. Therefore, impurity inspection requirements analysis methods have some special properties.

In the case of impurities available, may be added to the test in a certain amount of impurities, contaminants and coexistence proof material can be separated and detected, and with proper standards of accuracy and precision.

In the case of impurities or degradation products can not be obtained through exclusive with another but have to justify isolation or detection principle different, or with a strong resolution capability of the method results compare to determine. Or the test sample with a bright light, temperature, humidity, acids, alkaline hydrolysis and oxidation method of destruction (preparations should consider the impact of excipients), comparative damage before and after the detection of the number and amount of impurities. If necessary, it can be a diode array detector and mass spectrometry, chromatography peak purity check.

3, Determination

Determination of the content or purpose is to get the test analyte in the potency of accurate results. In the case of impurities available for the determination of the main components may be added in the test sample impurities or excipients, examine whether the result of the determination by the interference , and not adding impurities and accessories for the test product comparison measurement results.

In the case of impurities or degradation products can not be obtained, and may be another experience permits or the pharmacopoeia methods are compared, comparing the results of two methods. May also be destructive test (strong light, high temperature, high humidity, acids, alkaline hydrolysis and oxidation) to give product containing impurities or degradation of the sample , using two methods to determine the content, compare measurement results. Chromatography peak purity check, if necessary, to prove the determination of peak component does not contain other ingredients.

5.2 (b) linear

Means the linear measurement range within the design, test results and the test sample analyte concentration (amount) was a direct linear relationship between the degree.

Linear is the basis for the quantitative determination, the project involves the quantitative determination of impurities such as quantitative test and determination are required to verify linearity.

A linear relationship should be determined within the measuring range design. Available a stock solution is diluted by the precision or were accurately weighed sample, prepare a series of series of test substance concentrations were measured, prepared at a concentration of at least 5. In response to the measured signal is plotted as a function of analyte concentration observed whether linear, linear regression using the least squares method.

If necessary, the response signal can be mathematically converted , then the linear regression calculation, and explain the basis.

5.3 (c) range

It means the range can reach a certain accuracy, precision and linearity test methods suitable sample analyte concentration or amount of high and low limit of range. Range is a predetermined value, before the start of the study the test should determine the scope of the validation test and prescription law. It can be used to meet the requirements formulated in different drug concentrations, according to the photographic measurement method should be tested.

The test results and analysis of the range of commonly used methods of the same unit (such as percent concentration ) expression. Relates to the quantitative determination of the test items are needed to verify the scope of such determination, content uniformity , dissolution or release, impurity quantitative testing.

Range should be based on the dosage form to determine the requirements and (or) testing program.

1, Determination

Test concentration range should be 80% to 100%, or wider.

2, dosage form content uniformity

Range should be 70% to 130% test concentration. According to the characteristics of the dosage form, such as aerosols , sprays , if necessary, the range may be relaxed.

3, dissolution or release

For dissolution, should range limit ± 20%; as specified limits, shall be the lower limit of -20% to + 20% upper limit.

For the release, as specified limits range from 1 hour to 20% to 90% after 24 hours, verify that range should be from 0 to 110%.

4, impurity

When impurity measurement range should be measured according to preliminary results, the elaboration of the prescribed limits of ± 20%.

If the determination of the simultaneous determination of impurities checks with area normalization method, the linear range should be 20% to limit the content of impurities below the limit (or cap) + 20%.

5.4 (d) Accuracy

Accuracy refers to the use of the method of measurement results with the true value or accepted reference value of the closeness between. Sometimes called realism.

Certain necessary conditions for the quantitative determination of accuracy, thus relates to the quantitative determination of the test items are needed to verify the accuracy of such determination, impurity quantitative testing.

Accuracy should be within a predetermined range established for the preparation generally recovery test to verify. Test design needs to be considered within a predetermined range, the preparation of three different concentrations of samples, each measured three times that measured nine times, reports a known amount of recovery (%) or the average of the difference between the measurement result and the true value of their confidence limits.

1, Determination

API available or known purity reference drug were determined to meet the requirements, or the results of this law has been established and the accuracy of the results of another method of measurement for comparison.

Available formulations containing a known amount of each component was measured in the mixture was measured. If you can not get all the components of the formulation, may be added to the formulation of a known amount of analyte is measured and, if necessary, with another established method is relatively accurate degree.

2, impurity quantitative test

Quantitative test impurities can be added to a known amount of impurities in the drug substance or drug were determined. If you can not get the impurities, it can be applied to the determination result with another mature methods compare as pharmacopoeia methods or validated methods.

Data can not be measured as impurities relative response factors, online measurement of impurities, such as using a diode-array detector by UV spectroscopy , when similar spectrum spectrum with the main ingredient of impurities, you can use the response factor of the drug impurities approximate calculation content (self-control method). And clearly the total amount of a single impurity and impurity corresponding to the main ingredient in a weight ratio (%) or area ratio (%).

5.5 (e) Precision

Precision means the specified test conditions, for the same homogeneous sample, after repeated sampling of proximity degree (degree of dispersion) between the results of a series of tests.

General use precision deviation, standard deviation or relative standard deviation. Expressed as standard deviation or relative standard deviation, the sampling frequency should be measured at least six times.

Precision from three levels of study: repeatability , intermediate precision and reproducibility.

1 Repeatability

Repeatability means under the same operating conditions, within a short time interval, by the same analyst

Members measurement results obtained precision.

Repeatability can be measured within a predetermined range, with at least nine measurement results were evaluated, such as the three different concentrations of sample preparation, each measured three times, or 100% of the level of concentration, with the measurement results were evaluated at least six times.

2, intermediate precision

Intermediate precision means in the same laboratory, due to the changing conditions within the laboratory, such as time, analysts, equipment, precision measurement results.

Verify the design of variable factors generally date, analysts and equipment.

3, reproducibility

Analysts refer to different measurement results between different laboratories precision.

When the analysis is the legal standard adopted should be reproducible test.

5.6 (f) the limit of detection

The detection limit refers to the minimum amount of analyte in the sample can be detected, but not necessarily accurate quantification.

The significance of validation indicators have investigated whether the method sensitive detection capabilities. And therefore impurity limit test method need to prove with sufficient low limits of detection, to be controlled in order to ensure the detection of impurities.

1, intuitive method

Visual evaluation can be used for non-instrumental analysis methods can also be used in instrumental analysis methods.

Determination of detection limit of a sample through a series of known concentrations of the analyte are analyzed, and accurate and reliable detection of the minimum amount of analyte concentration or a minimum established.

2, the signal to noise ratio method

The method can be used to display analysis of baseline noise, i.e., a sample of known low concentration signal is measured with the noise signal, and calculates the minimum detectable concentration or amount. General signal to noise ratio of 3: 1 or injection amount corresponding to the concentration of the instrument to determine the detection limit.

There is a method based on the standard deviation of the slope of the curve and the calculated response of the other methods.

No matter what method have been applied, a certain number of samples at a concentration equal to or close to the detection limit, were analyzed to determine the limit of detection and reliable.

5.7 (g) limit of quantitation

Means the limit of quantification of the analyte in the sample can be quantitatively determining the minimum amount, the measurement results should have a certain degree of accuracy and precision.

It reflects the limit of quantification method of analysis with sensitive quantitative detection capabilities. Impurity quantitative test, the limit of quantification need to study methods to ensure that the content of impurities can rarely be accurately measured.

SNR common method to determine the limit of quantification. General signal to noise ratio of 10: 1 or injection amount corresponding to the concentration of the instrument will be OK.

1, direct method

Visual evaluation can be used for non-instrumental analysis methods can also be used in instrumental analysis methods.

The case of the limit of quantification is generally through a series of samples containing known concentrations of analyte are analyzed in both accuracy and precision to meet the requirements, to determine the minimum amount of analyte that can be quantified.

2, the signal to noise ratio method

The method can be used to display analysis of baseline noise, i.e., a sample of known low concentration measured signal and noise signal, and calculates the minimum detectable concentration or amount. Generally signal to noise ratio of 10: 1. There is a method based on the standard deviation of the slope of the curve and the calculated response of the other methods.

No matter what method have been applied, a certain number of samples at a concentration equal to or close to the limit of quantification, were analyzed to determine the limit of quantitation reliably.

5.8 (h) Durability

Durability refers to the measurement conditions occur changes little when the tolerance level measurement results are not affected. Durability mainly on the method itself is a variable factor test against interference can force. At the beginning of research and analysis methods, it should consider its durability. If the test conditions are demanding, it is recommended that the process be stated.

Typical changes include: liquid chromatography composition of the mobile phase, flow rate and pH value, different manufacturers or different batches of the same type of column, column temperature. GC carrier gas and flow rate, different brands or batches of column stationary phase, carrier body, column temperature, inlet and detector temperature. After testing, it should indicate whether the small changes in line with the system suitability test requirements to ensure effective method.

5.9 (ix) System suitability test

Some of the instrumental analysis methods, during method validation, it is necessary to analyze equipment, electronic instruments and experimental operation, test samples, etc. together as a complete system of assessment . The applicability of the system is the whole system of indicators to assess. Set the system suitability test parameters are to be based on the type of authentication method.

Chromatographic methods for analysis of high reliance equipment, electronic equipment, so all chromatographic methods should be carried out to verify the index, and the applicability of the system as an integral part of the analysis. The specific authentication parameters and methods refer to the relevant provisions of the Chinese Pharmacopoeia.

6 . The method of revalidation

In some cases, such as API synthesis process changes, preparation prescription change, analysis of partial changes, etc., are necessary for the analysis of full or partial re-verification to ensure reliable analytical method, a process known as METHODOLOGY verification.

Revalidation principles: corresponding re-certification according to the degree of change.

When the drug synthesis process changes, may introduce new impurities, impurity inspection methods and specificity determination method would need to be verified in order to prove that the substance inspection method capable of detecting newly introduced impurities, and the newly introduced impurities on Determination of the content of the main ingredient should be no interference.

When the prescription formulation composition changes, materials change may affect the identification of specificity, accuracy and determination of dissolution, so the need for identification, determination method revalidation. When changes occur drug origin, it may affect the specificity and accuracy of examination and determination of impurities, so the need for impurity testing method and determination method revalidation.

When the quality standards of a partial analysis of the project changes, such as the content was determined by high performance liquid chromatography, and the detection wavelength is changed, the need to re-detection limit, specificity, accuracy, precision, linearity and other content verification, to justify the revised analysis and feasibility.

Similarly, there are national standards for pharmaceutical quality research, based on the declaration of the drug synthesis, pharmaceutical formulations of excipients and other general consistency can not be guaranteed and marketed drugs, the need for quality standards in some of the projects for revalidation process.

The method is further validation of analytical methods to improve the process, should be re-verified according to the actual situation changes, so as to ensure the method used to take control of the intrinsic quality of medicines.

7 . the evaluation method validation

For method validation, there are several areas of concern.

(A) relating to the evaluation method validation General considerations

Overall , the method validation and verification purposes should focus on the general principles for, the selection of content and method validation test system design should, rational, rigorous verification process should be standardized.

Not every method of analysis of test items are required to verify all of the content, but we must pay attention to verify the contents should be sufficient enough to justify the analytical method used. As impurity limit test is generally required to verify the specificity and limits of detection, and for precision, linearity, limit of quantification and other projects involving quantitative determination, it is generally not required for verification.

(B) the integrity and systematic method of verification

Method validation correlation between the content as a whole. Therefore, regardless of the angle from the development point of view or evaluation, verification methods have focused on holistic and systematic.

For example, for the identification of the project required specificity, the general method of analysis is unlikely to completely discriminate analyte, this time using two or more analytical methods can enhance the overall specificity of the identification project.

In the process of verification there are more correlation between the content, they can complement each other. Such as drug content was determined by volumetric analysis time method , since the method itself reason, specificity slightly worse, but if detected at an impurity using exclusive stronger chromatography , it is generally believed that the entire detection method also has a strong specificity .

In short, because the actual situation is more complicated in the method validation process does not advocate dogmatic to be validated. In addition, more and more new methods continue to be used for quality control, and how to verify these methods require specific conditions, but can not copy this guidance.

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