Blog

Standard Operating Procedure (SOP) and Guideline for performing Cleaning Validation (CV) of drug product manufacturing equipments.

Cleaning Validation (CV)

1.0   Objective :

• • To lay down the procedure for Cleaning Validation (CV) of manufacturing equipments.
  • This Guideline describes the validation of cleaning procedures (CV) for the removal of contaminants associated with the previous products, residues of cleaning agents as well as the control of potential microbial contaminants.

2.0   Scope :

• • This Guideline is applicable for Cleaning Validation (CV) of manufacturing equipments in production.

3.0   Responsibility :

• • Quality Control Department & Microbiology :

• • Preparation and review cleaning validation (CV) protocol and report.
  • Preparation and review of cleaning validation (CV) product matrix / equipment train.
  • Evaluation of cleaning validation (CV) for new product / new equipment.
  • Calculation of the maximum allowable carry over.
  • Calculation of shared equipment surface area between products
  • Collection of samples for cleaning validation (CV) .
  • Monitoring of cleaning validation (CV) activities.
  • Review of analytical reports
  • Collection of the sample for microbiological analysis by microbiologist.
  • Analysis of the cleaning validation (CV) samples.
  • Performing recovery study for cleaning validation (CV) .
  • Compilation and review the data To follow the procedure
  • Review the cleaning validation (CV) matrix, protocol and report.
  • To review the analytical data.

• • Production Department:

• • Review of cleaning validation (CV) protocol and report.
  • Provide equipment details, Equipment cleaning SOPs.
  • Provide information to build database.
  • Identifies difficult to clean locations on equipment.
  • Clean the equipment by trained operators.
  • Execute the cleaning validation (CV) schedule.
  • Follow the procedure

  • Head – Engineering:

  • Review the cleaning validation (CV) matrix
  • Review cleaning validation (CV) protocol and report.
  • Calculate & review the equipment contact surface area.

  • Quality Assurance Department :

  • To approve the cleaning validation (CV) matrix.
  • To approve cleaning validation (CV) protocol and report.

4.0   Procedure for Cleaning Validation (CV) :

• • Principle – Cleaning Validation (CV):

• • Pharmaceutical products can be contaminated by other pharmaceutical products, by cleaning agents, by microorganisms or by other material (e.g. air-borne particles, dust, lubricants, raw materials, intermediates, auxiliaries).
  • In many cases, the same equipment may be used for processing of different products.
  • To avoid contamination of the pharmaceutical product, adequate cleaning procedures are essential.
  • Cleaning Validation (CV) is documented evidence that an approved cleaning procedure will provide equipment which is suitable for processing of pharmaceutical products.
  • Objective of the Cleaning Validation (CV) is the confirmation of a reliable cleaning procedure so that the analytical monitoring may be omitted or reduced to a minimum in the routine phase.

• • General Procedure for Cleaning Validation (CV):

• • Normally only cleaning procedures for product contact surfaces of the equipment need to be validated.
  • Consideration should be given to non contact parts into which product may migrate.
  • For example Outer surface, seals, flanges, are mixing shaft, fans of ovens, heating elements etc.
  • Cleaning procedures for product changeover in the case of marketed products should be fully validated.
  • Generally in case of batch-to-batch production it is not necessary to clean after each batch.
  • However, cleaning intervals and methods should be determined.

  • Several questions should be addressed when evaluating the cleaning process. For example:

    • At what point does a piece of equipment or system become clean?
    • What does visually clean mean?
    • Does the equipment need to be scrubbed by hand?
    • What is accomplished by hand scrubbing rather than just a solvent wash?
    • How variable are manual cleaning processes from batch to batch and product to product?
    • What is the most appropriate solvent or detergent?
    • Are different cleaning processes required for different products in contact with a piece of equipment?
    • How many times need a cleaning process be applied to ensure adequate cleaning of each piece of equipment?

• • Cleaning procedures for products and processes which are very similar, do not need to be individually validated.
  • It is considered acceptable to select a representative range of similar products and processes concerned and to justify a validation programme which addresses the critical issues relating to the selected products and processes.
  • A single validation study under consideration of the “worst case” can then be carried out which takes account of the relevant criteria.
  • This practice is termed “Bracketing”.
  • At least three consecutive applications of the cleaning procedure should be performed and shown to be successful in order to prove that the method is validated.
  • Control of change to validated cleaning procedures is required.

  • Documentation of Cleaning Validation (CV):

  • A Cleaning Validation (CV) Protocol is required for laying down the procedure on how the cleaning process will be validated.
  • It should include the following but not limited to:
    • The objective of the validation process.
    • Scope of Validation activities
    • Responsibility for performing and approving the validation studies.
    • Validation Team.

  • Cleaning Validation (CV) approach-

    • Product Matrix
    • Selection of Products
    • Selection of Equipments
    • Risk assessment of cleaning validation (CV)
  • Process Overview
  • Pre cleaning validation (CV) requirements
  • Precaution and Instruction
  • Calculation of MACO
  • Dose Criteria ( or safety based criteria or medical limit criteria)
  • 10 ppm criteria
  • Toxicity based criteria on LD50 value
  • Toxicity based criteria on ADE/PDE value
  • Microbial Contamination
  • Cleaning Validation (CV) Program
  • Selection of Cleaning Procedure
  • Clean-In- Place (CIP)
  • Clean- out-place (COP)
    • Manual Cleaning
    • Water Quantity

  • Selection of Analytical Method for Cleaning Validation (CV) –

    • Analytical Method Validation
    • Specificity Potential interferences
    • Selection of extraction solvent
    • Recovery Study
    • Recovery from Spiked plates/ Coupons or different MOC,s
    • Correction Factor (Recovery)
    • Sampling Plan, type of Sampling and Selection of Sampling Method
    • Evaluation of Cleaning Procedures

  • Sampling – Cleaning Validation (CV) –

    • Sampling technique for Chemical Analysis
    • Technique (sampling) for Microbial Analysis
    • Status Label
    • Sampling documentation
    • Determination of Sampling Points
  • Equipment Hold time Study
  • Testing Procedure
  • Ongoing Monitoring
  • Training

  • Acceptance Criteria

    • Selection of Worst case Product Review of Data
    • Justification of selection of Worst case
      • Smallest recommended daily dose (TD)
      • Solubility
      • LD₅₀
      • Calculation of swab Limit

  • Revalidation

    • Deviation and Failure investigations
    • Cleaning Validation (CV) final report
  • The Validation Protocol & reports shall be prepared by QA, reviewed by Quality Assurance, Head production, Head QC & Head Engineering and approved by Head QA and Head Operation.
  • A final Validation Report should be prepared.
  • The conclusions of this report should state if the cleaning process has been validated successfully.
  • Limitations that apply to the use of the validated method should be defined (for example, the analytical limit at which cleanliness can be determined).
  • The cleaning process should be documented in SOP.
  • Records should be kept for cleaning performed in such a way that the following information is readily available :
    • The area or piece of equipment cleaned,
    • The person who carried out the cleaning,
    • When the cleaning was carried out,
    • The SOP defining the cleaning process,
    • The product which was previously processed on the equipment being cleaned.
  • The cleaning records should be signed by the operator who performed the cleaning and checked by the Production chemist and should be reviewed by Quality Assurance.

• Personnel:

  • Operators who perform cleaning routinely should be trained in the application of validated cleaning procedures.
  • Training records should be available for all training carried out.
  • It is difficult to validate a manual, i.e. an inherently variable/cleaning procedure.
  • Therefore, operators carrying out manual cleaning procedures should be supervised at regular intervals.

• Equipment:

  • The design of the equipment should be carefully examined.
  • Critical areas (those hardest to clean) should be identified, particularly in large systems that employ semi-automatic or fully automatic clean-in-place (CIP) systems.
  • Dedicated equipment should be used for products which are difficult to remove, for equipment which is difficult to clean (e.g. bags for fluid bed dryers), or for products with a high safety risk (e.g. biologicals or products of high potency which may be difficult to detect below an acceptable limit).

• Microbiological Aspects:

  • The existence of conditions favorable to reproduction of micro organisms (e.g. moisture, temperature, crevices and rough surfaces) and the time of storage should be considered.
  • The aim should be to prevent excessive microbial contamination.
  • The period and when appropriate, conditions of storage of equipment before cleaning and the time between cleaning and equipment reuse, should form part of the validation of cleaning procedures.
  • This is to provide confidence that routine cleaning and storage of equipment does not allow microbial proliferation.
  • In general, equipment should be stored dry, and under no circumstances should stagnant water be allowed to remain in equipment subsequent to cleaning operations.

• Guidelines for Cleaning Validation (CV) :

  • It deals with the validation of equipment cleaning procedures used in the pharmaceutical industry to prevent cross-contamination or adulteration of drug products.
  • Active ingredients:
  • The most obvious area for evaluation in cleaning validation (CV) is removal of active ingredients from the equipment.
  • This evaluation can be carried out by a number of methods, but all have in common the need for adequate analytical methodology and the establishment of practical yet meaningful acceptance criteria for residuals.

  • Analytical methods requirements:

  • The analytical methods should be validated before the Cleaning Validation (CV) Study is carried out.
  • Methods used to detect residuals or contaminants should be specific for the substance to be assayed and provide a sensitivity that reflects the level of cleanliness determined to be acceptable.
  • Methods should be challenged in combination with the sampling methods used, to show that the contaminants can be recovered from the equipment surface and to show the level of recovery as well as the consistency of recovery.
  • This is necessary before any conclusions can be made based on the sample results.
  • A negative result may also be the result of poor sampling techniques

• Calculation for residuals:

  • Product Residue Contamination
  • The rationale for selecting limits of carryover of product residue shall be logically based on the materials involved.
  • The limits should be practical, achievable and verifiable. On the basis of following criteria acceptance limits i.e. Maximum Allowable Carry Over (MACO) shall be established:
  • Dose Based Criteria (or Safety Based Criteria):
  • As per this criteria not more 0.001 dose of any product shall appear in the maximum daily dose of the next product
  • The following equation shall be used for the calculation of MACO from previous product (let be a product ‘A’) to next product (let be a product ‘B’)

• 10 PPM Criteria:

  • As per this criterion, not more than 10 ppm of any product will appear in another product.
  • The following equation is used to calculate the limit of product ‘A’ if the next product on the production schedule is product ‘B’.

• Toxicity based criteria (LD₅₀ Criteria):

  • Calculate the maximum allowable carry over (MACO) based on toxicity criteria of active ingredient in mg per swab, for the piece of equipment by following the equation given below, if swab sampling is to be done.
  • NOEL shall be calculated by following equation:
•                                                                             LD₅₀  (mg / kg) x 50 (kg a person) 
•                                            NOEL  =          ———————————————— 
•                                                                                                          2000

•                                                                                       S.F x [NOEL (A)] x [BS (B) in mg]
• MACO (Total equipments in mg) =            ——————————————————
•                                                                                                   [LRDD (B) in mg]

Where,

• Toxicity Based Criteria (ADE/PDE Criteria):

  • Calculate the maximum allowable carry over (MACO) based on ADE/PDE following the equation given below, if swab sampling is to be done.
•                                                                                            PDE x BS (mg) 
•                                                            MACO =         ——————————
•                                                                                              LRDD (mg) 

Where,

• • On the basis of dose criteria, 10 ppm criteria and Toxicity Criteria the MACO limit shall be calculated.
  • After establishing MACO (with minimum value) the swab limits and / or rinse limits shall be established with respect to total product contact surface area and total rinse volume respectively.

•                                                                                MACO X Surface area of Equipment (A)
•            Rinse Limit (drug in mg/ml) = ————————————————————–
•                                                                               TS X Total water used for final rinse (A)

Where,

A: Equipment from which rinse sample taken

TS: Total product contact surface area (cm²)

• Visually Clean Criteria:

  • No quantity of residue should be visible on the equipment after cleaning procedure is performed.
  • Active ingredient in most of the products is visible at the approximately 100 µg per 10 cm sq of surface area. Below this level the residue is not visible to human eye.
  • Stringent Acceptance criteria shall be selected as worst case either from dose criteria or 10 ppm criteria or Toxicity criteria.
  • LOQ shall be considered during selection of acceptance criteria.

• Calculation of amount of residue present in rinse & swab:

  • Calculation of amount of residue present in rinse & swab done as per analytical method validation of individual molecules.
  • % Recovery Factor shall be applied to calculated results to get the actual residue.
•                                                                                           100
•                                 Recovery Factor =   ————————————-
•                                                                           % Recovery (From AMV)

Actual residue = Calculated result X Recovery factor

• Procedures for cleaning Validation (CV) Based on Bracketing & worst case Rating:

  • The cleaning processes of multiple product equipment are subject to requirements for cleaning validation (CV) .
  • In order to minimize the amount of validation requirement a worst case approach for the validation can be used.

  • Collect the following information before starting cleaning validation.

  • Materials needed to be cleaned from the equipment i.e. active ingredient.
  • Equipment chain used during manufacturing of a particular product.
  • A list of product that are manufactured using above chain of equipment in the facility.
  • Data of surface area of each piece of equipment that is in contact of the product, used in the manufacturing of the product(s) at the formulation site with respect to the batch size.
  • For each product collect the following details
  • Batch size in Kg.
  • Number of dosage units per batch.
  • Active ingredient
  • Smallest batch manufactured
  • Maximum daily dosage
  • Solubility in water.
  • Product contact surface area of each piece of equipment in the facility.
  • Cleaning Validation (CV) matrix shall be prepared as per Annexure No. VII.

  • Worst Case Rating:

  • Decide the worst case product(s) based on the following :
    • Select the worst case product based on lowest strength (high potency) lowest solubility of its active ingredient in water from the product matrix and followed by difficulty in cleaning.
    • Worst Case Rating shall be as follows:

Potency (TD) >Solubility >Toxicity (LD50)/ (PDE/ADE)> Difficult to clean

• Methods of evaluation:

  • There are several reasonable ways to evaluate the effectiveness of cleaning procedures and the choice of one over the others should be based on the unique characteristics of the equipment and product(s) involved.

  • Swab test:

  • After cleaning the equipment, product contact surfaces can be swabbed to evaluate surface cleanliness. Swabs used should be compatible with the active, in that they should not interfere with the assay, should not cause degradation of the compound, and should allow extraction of the compound for analysis.
  • The solvent used for swabbing should provide good solubility for the compound and should likewise not encourage degradation.

  • Sampling considerations:

  • The area to be sampled should be in its final condition, as it would be when ready to use.
  • In some cases, it is reasonable to swab the entire product contact surface.
  • However, when this is not reasonable, a known surface area should be tested, and the approximate overall surface area which is represented by the swab(s) should be known.
  • The area to be sampled should be selected using judgment about which areas are most difficult to clean.
  • To ensure accuracy of the overall procedure, it can be applied to a surface which has been deliberately contaminated with a known, low level of the active.
  • Obviously, the surface used for this challenge must be made of the same material as the equipment to be tested.

  • Advantage:

  • Advantage of direct sampling of that areas, hardest to clean and which are reasonable accessible can be evaluated, leading to establishing a level of contamination or residue per given surface area.
  • Additionally, residues that are dried out or are insoluble can be sample by physical removal.

  • Rinse samples:

  • Sampling and testing of rinse samples for residual active ingredient is commonly used method to evaluate cleanliness the solvent used should be selected based on the solubility of the active ingredient or at least provide adequate solubility.
  • If possible, steps should be taken to ensure the uniformity of the residual material in the rinse prior to sampling.
  • It is also critical that the volume of rinse solvent used be controlled.
  • For equipment designed to hold liquids, either the volume of rinse solvent used should be sufficient to ensure contact with all product contact surfaces, or the method of introducing the rinse solvent should ensure adequate contact with all surfaces.

  • Placebo samples:

  • The placebo sampling method provides the best simulation of actual production of a subsequent batch of product.
  • To use this technique, a suitable placebo formulation must first be chosen.
  • Factors to consider in making this selection include placebo manufacturability, solubility of the compound being studied (liquid) and accurate simulation of actual production conditions for liquid products, water is often the best placebo formulation.

  • Cleaning Agent:

  • A second important focus of cleaning validation (CV) is the removal of cleaning agents.
  • These are known equipment contaminants which are added, ironically, to assist in the cleaning operation itself.
  • In most cases, more than one cleaning agent is approved for use.
  • The removal of each may have to investigate to ensure that no problems will be encountered with their use.
  • For this reason, it is prudent to limit the number of approved cleaning agents to minimum required for effective cleaning in various situations.

Applications of Sampling Methods

• Cleaning Validation (CV) Approach:

  • The strategy for the cleaning validation must cover all these activities and ensure that all equipments are adequately cleaned before it is used for manufacturing of product.
  • To cover this wide range of activities, a matrix approach has been adopted for each cleaning procedure.
  • Whenever any new product introduced in the facility cleaning validation (CV) review and assessment will be carried out to evaluate the need of further cleaning validation (CV) or to show that existing cleaning method stands valid in future.
  • For any major change in the cleaning procedure change control shall be filed and initiated to revise the cleaning validation (CV) protocol to include the major changes.
  • The acceptance criteria shall be calculated by using 10 ppm criteria, dose based criteria and Toxicity based criteria and the criterion having lowest value shall be selected for study.
  • Worst-case approach shall be adopted.
  • Cleaning Validation (CV) is carried out on Non-dedicated equipments (product contact surfaces) which may become the source of contamination.
  • However, emphasis is also given on non product contact parts and area which may become the source of potential contamination.

• Acceptability limits for Cleaning Validation (CV):

  • Determination of acceptability limit is a critical factor while preparing cleaning validation (CV) protocol.
  • The calculation of acceptable level for previous product as contaminant is important while determining the acceptability limit for cleaning validation (CV) .
  • The Product matrix must include the list of all products manufactured in the equipment/facility and in which the cleaning validation (CV) shall be performed.
  • Carry-over of product residues should meet defined criteria, the most stringent limit of the following three criteria shall be considered for carry-over of product residues:

  • Visual inspection criteria:

  • Visual inspection of equipment/ subject shall be done before taking swab sample from the sampling location.
  • Examination (visually) of the whole equipment /subject from product contact as well as product non contact parts shall be done.
  • It shall be checked by production personnel and then verified by QA personnel.
  • The observation shall be maintained in format for recording of visual inspection record.
  • No quantity of residue should be visible to naked eye on the whole equipment/subject after cleaning procedures are performed (i.e. less than 100 mcg /100 cm²).
  • Visual examination of cleaning shall be done from the distance of NMT 5 feet with observing angle of 15⁰ to 90⁰ in suitable light condition at least 200 Lux intensity.
  • The equipment/subject shall demonstrate the absence of any noticeable “off” odor during visual inspection.

  • 10ppm criteria:

  • Not more than 10ppm of active pharmaceutical ingredient of previous product is permitted in next product.

  • Dose based criteria:

  • Not more than 1/1000 of minimum daily therapeutic dose of the previous product in the maximum daily dose of the next product, calculated with respect to the total weight of the dosage form.
  • It defines that 0.001 (Safety factor) of the normal therapeutic active dose of previous product must not appear in largest recommended daily dose of the subsequent product.

  • Toxicity based criteria:

  • Environmental Protection Agency and toxicologists suggest that an acceptable level of a toxic material may be that which is no more than 1/1000 of a toxic dose for solid oral products (Topical products 10- 100, Oral products 100 – 1000 and Parenteral products 1000 – 10000) of an amount which is not known to show any harmful biological effect in the most sensitive animal system known, e.g., no effect.
  • The acceptability limits for microbiological sample shall be determined based on;

• • Remark: Total Microbial Count for manufacturing area shall be studied after completion of  cleaning, by Active /Passive monitoring (Only for reference purpose).

• Hold Time Studies:

  • The objective of hold time study is for establishing time limit between equipment cleaning and reuses it to ensure that the equipment remains clean till the next use.

• Dirty Equipment Holding Studies:

  • The interval between the end of production and the beginning of the cleaning process shall be established through equipment holding studies prior to cleaning.
  • It is important to consider the effect that weekends, holidays and delays might have on the cleaning schedule.
  • For example, a piece of equipment that is utilized first in the morning shift may contaminate with product, until the second shift starts. Having the product ‘dry’ on equipment, therefore, considered as worst case.
  • To establish the expiry of cleaning in view of microbiology, equipment shall be kept ideally after Type B cleaning for 72 hours (3 days) and microbiological swab shall be taken and analyzed at different intervals (0 hour, 24 hours, 48 hours and 72 hours).

• Cleaned Equipment Hold Time Studies (Microbiological Study only)

  • Concerns relative to microbial control are lessened in the production of non-sterile products but are still important.
  • Practices which minimize the potential for contamination by ‘objectionable organisms’ are common in the manufacture of non-sterile formulations.
  • To establish the expiry of cleaning in view of microbiology, equipment shall be kept ideally after Type A cleaning for 96 hours (4 days) and microbiological swab shall be taken and analyzed at different intervals (0 hour, 24 hours, 48 hours, 72 hours and 96 hours).
  • This will be considering as worst case and microbial load should remain well within limit.

• Training:

  • Operator training is critical, especially for manual cleaning.
  • During cycle development, operators should be trained in the requirements of the evolving or existing SOPs.
  • Proper training consists of understanding the SOP, apprenticeship with qualified, trained operators and review to ensure that the training is successful.
  • The effective training or qualification of the operators may be confirmed by monitoring of the equipment after cleaning, including, where necessary, analytical testing for residuals.
  • It is important not only has that operator training occurred, but also that the training be well documented.
  • Operators should be retrained each time a cleaning procedure is changed and the new training must be documents, just as in the case of a change to a manufacturing procedure.

• Revalidation of Cleaning Procedure:

  • Any change impacting a parameter of a previously establishes validation study may require revalidation.
  • Appropriate assessment by manufacturing and QA to determined the necessity of revalidation is required, on a periodic basis, consistent with the Validation Master Plan.
  • Re-validation shall be performed in case of any change, (at least the following but not limited to)
    • Cleaning verification after every 1 year on one batch of worst case product after compilation of study.
    • Introduction of a new facility, equipment, process or product.
    • Change in cleaning procedure.
    • MACO limit change.
    • Change in cleaning agent used for cleaning (If applicable)
    • Major Modification in processing equipment.
    • As per regulatory requirements.

  • Deviations and Investigations:

  • Any deviation observed during cleaning validation (CV) shall be recorded and investigated as per respective SOP.
  • If the observed deviation does not have any major impact on the validation the final conclusion shall be provided.
  • If the observed deviation has major impact on the validation, deviation shall be reported to the concerned department for the corrective action and validation activity shall be redone.

  • Cleaning verification:

  • Cleaning Verification shall be done after 1 year for one batch.
  • A document for cleaning verification shall be prepared before execution of study on annexure-II. The document shall have pre-approval before execution and post approval shall be done after compilation of report.

  • New product/ Equipment Evaluation:

  • When a new product is introduced in the plant an evaluation is made to determine if cleaning validation (CV) is required.
  • If the new product carryover limit is above the previously determined carryover limit and the new product is more soluble and less potent than the target component of the previous product, then cleaning validation is generally not required.
  • This will be documented in the cleaning validation plan.
  • Introduction of any new product in existing product matrix shall be carried out through new product evaluation sheet (Annexure -IV) and shall be updated in Cleaning validation (CV) product matrix (Annexure- VII)
  • Review and update the cleaning validation (CV) product matrix before a new product is introduced in production facility accordingly.
  • If any new molecule is selected as worst case, re-validate the cleaning process.
  • Introduction of any new equipment in existing train shall be carried out through new equipment evaluation sheet (Annexure -V) and shall be updated in cleaning validation (CV) matrix (Annexure- VII) accordingly.
  • Review and update the cleaning validation (CV) equipment train if any change in existing equipment train, re-validate the cleaning process if required.

5.0   Reference – Cleaning Validation (CV) :

• • PIC/S – ‘Recommendations on Validation Master Plan, Installation and Operational 18. Qualifications, Non-Sterile Process Validation & Cleaning Validation’
  • APIC – ‘Guide on Aspects of Cleaning Validation in Active Pharmaceutical Ingredient Plants (December 2000)
  • Validation Master Plan
  • Guideline on setting health based exposure limits for use in risk identified in the manufacture of different medical products in shared facilities. By EMA/CHMP/CVMP/SWP/169430/2012 in Nov 2014

6.0   Glossary:

Annexure I   : Template for Cleaning Validation Protocol

Annexure II: Template for Cleaning Validation Report

Annexure III: Cleaning Validation Protocol Number Issuance Log

Annexure IV: New Product Evaluation Sheet

Download the pdf : New Product Evaluation Sheet

Annexure V: New Equipment Evaluation Sheet

• Compare the above data with data provided in cleaning validation protocol, and draw the conclusion whether above equipment falls under the existing matrix or required cleaning verification / validation or change in acceptance criteria is required.

Annexure VI: Planner for cleaning validation study

Annexure VII: Cleaning validation Matrix

Download the pdf :Cleaning Validation Matrix