Analytical Test Methods in Downstream Processing

analytical example Methods in downriver ProcessingAndrea Waldvogel trial impression of analytical Test Methods in Downstream ProcessingIntroductionQuality, safety and efficacy atomic itemize 18 the main principles of flavor assurance of biopharmaceutical drug products. Quality must be designed into the product or process since it cannot be tested into it. Therefore, a quality system must comprise of brass, swap throw, training, quality control and vendor assurance amongst others.1-3An effective governance does not only allow a high degree of confidence that the finished drug product consistently and reliably meets all quality requirements only also leads to economic bene pairs by reducing the cost associated with process monitoring, sampling and exam.4 Biopharmaceutical companies must arrange facility, utility and equipment test copy/qualification, process governance, computer and computer systems validation, cleaning validation and analytical rule validation.5 Thi s project root give focus on analytical method validation, also referred to as analytical procedure validation. uninflected methods are developed to measure characteristics such as molecular identity, purity, potency, and safety of raw hooeys, in-process samples and nett drug products and to monitor the manufacturing process. The number of tests should be adequate to say manufacturing consistency and the impact of changes on the quality of the drug product. All methods must be demonstrated to be fit for their intended purpose before they are employed.5,6 uninflected method validation means establishing documented evidence that provides high degree of assurance that a specific method, and the ancillary instruments included in the method, will consistently yield results that accurately reflect the quality characteristics of the product tested.7This report begins by providing an overview over some of the regulations and guidelines related to analytical method validation. The seco nd shareage introduces the modern lifecycle approach to method validation and section three gives an insight into analytical method validation in biopharmaceutical downstream processing. The final section concludes the report with a summary of the main points discussed.There are many different regulations, guidelines and pharmacopeial monographs concerned with analytical method validation. As it would go beyond the scope of this document to write about all of them, the report focuses on some to give an overview.1.1 RegulationsValidation is based on, but not prescribed by regulatory requirement. It is beaver viewed as an essential and integral part of Good Manufacturing Practice (GMP) for the assurance of quality. Compliance with validation requirements is necessary for obtaining approval for clinical trials and to market new products.4In the U.S. for example, 21 CFR Part 211.165(e) states8The verity, sensitivity, specificity, and reproducibility of test methods employed by the f irm shall be established and documented. Such validation and documentation may be gracious in accordance with 211.194(a)(2).21 CFR Part 211.194(a) (2)8A statement of each method used in the testing of the sample. The statement shall indicate the location of data that establish that the methods used in the testing of the sample meet proper standards of accuracy and reliableness as applied to the product tested. (). The suitability of all testing methods used shall be verified under actual conditions of use.The requirement of validation is also implied in 211.100(a)8There shall be written procedures for production and process control designed to assure that the drug products have the identity, strength, quality, and purity they purport or are represented to possess.1.2 GuidelinesThe first guidance documents on analytical method validation were published in the 1990s. In the course of time, a lot of revision occupation has taken place allowing the incorporation of new approaches to science.The harmonised ICH Q2(R1) Validation of Analytical forces school text and Methodology guideline, issued in 2005, is considered the primary reference for recommendations and definitions on validation characteristics for analytical procedures and has tended to take on the role of a regulatory expectation. In the join States, it has been used as a guidance along with the related compendial documents USP Analytical Procedure Validation, Analytical Procedure Verification, and Analytical Procedure Transfer. However, those documents do not provide support for the users to accurately understand and control sources of variability.6,9In 2013, a Stimuli to the Revision Process paper on Lifecycle precaution of Analytical Procedures published by the USP Validation and Verification Expert Panel proposed a Quality by Design (QbD) approach to method increment, validation, and murder verification of an analytical method via a lifecycle concept. They suggested that the tralatitious approaches describe in the U.S. Pharmacopeial monographs , , and should be revised and assembled into a single new general information chapter Lifecycle Management of Analytical Procedures and a new general chapter specifying the prefatory requirements. This would, for the first time, formally link method emergence and method validation within pharmacopeia.6,10In 2016, a general chapter prospectus on The Analytical Procedure Lifecycle was posted on the U.S. Pharmacopeial Notices and a draft of a new USP General Chapter Statistical Tools for Procedure Validation was published in the U.S. Pharmacopeial Forum (U.S. Pharmacopeial Convention).11,12In princely 2017, a new general USP Chapter Validation of Compendial Methods will become official. This is an effort to better align the validation concept with the revised FDA guidance for industry Analytical Procedures and Methods Validation for Drugs and Biologics issued in 2015. However, instead of including a section on Lifecycl e Management of Analytical Procedures only a reference has been added. Depending on the development of the chapters and , USP may be revised again.13Growing awareness that the implementation of an analytical method with adequate quality stairs designed into the procedure during the development phase led to the development of a lifecycle approach for analytical procedure validation.102.1 Stages of the Modern Lifecycle ApproachThe modern lifecycle approach is based on the Quality by Design (QbD) approach outlined in ICH Q8(R2) guideline and defines activities and deliverables for every stage of method validation. The following diagram provides an overview.Figure 1 QbD Approach for Analytical Methods2.1.1 Stage 1 Procedure Design, Development, and apprehensionTo be able to design quality into a method to ensure that the method is reliable and meets the analytical target profile (ATP) defined at the starting signal of this stage, an understanding of how the procedure melt downs is key. Risk assessment should be undertaken to identify variables that could have an influence on the method. The knowledge of variables and their impact is not only classic for the development of a control strategy but also for the object of a design space. The design space will reduce the amount of revalidation work considerably when the method is used operationally. Key elements of this stage are shown in Figure 1 no. 1-3.10Proper method development including the evaluation of rigor is essential for an effective analytical procedure.10 Robustness is a measure of the methods capacity to remain unaffected by small variations in method parameters and provides an indication of its reliability during normal usage.9 At this stage, system suitability parameters are established which help to ensure that the analytical method remains valid whenever used.9Without developing a spicy method and an understanding of how a change of key parameters will impact its procedure, the actual metho d validation step will be difficult.102.1.2 Stage 2 Procedure Performance QualificationThe lifecycle approach uses the term procedure performance qualification instead of method validation.Procedure performance qualification is the verification of the performance of the analytical procedure (either a new one or a revised procedure) against the requirements of the ATP.10If the procedure development has been through correctly, this step should simply be a confirmation that it is fit for the intended purpose. In cases where further controls withdraw to be added to ensure reliable results the analytical control strategy, developed during stage 1, will need an update.10Method validation work should be performed by a user laboratory under the same conditions as it will be used to honour with existing GMP regulations.102.1.3 Stage 3 Implementation and Continued Procedure Performance VerificationThis stage involves checking how the procedure works during operational use and that it remain s in a state of control.10For this purpose, inputs on reliability and performance of the method gathered from operators and customer complaints will be evaluated. Performance indicators such as system suitability, quality control samples and out-of-specification (OOS) results are tracked and trended.10The method should be continually improved through corrective and preventive action to reduce the number of out-of-specification (OOS) results. Any change to improve the overall performance needs to be assessed using change control procedures. As shown in Figure 3, the nature of the change specifies what actions have to be taken.6,10,14Figure 2 Change Types and appropriate Actions2.2 Traditional (Current) Approach vs Lifecycle ApproachIn their Stimuli to Revision paper, the USP Expert Panel recommends the adoption of a lifecycle approach for the steering of analytical procedures. In their conclusion, they outline the advantages of a lifecycle approach by comparing it to the traditional (current) approach to analytical procedure validation (Figure 1).6Downstream processing in biopharmaceutical manufacturing involves many steps from recovery over purification to fill finish. Next to in-process monitoring of process parameters such as pH and temperature, analytical testing for the determination of quantity, identity, strength, potency, purity (product- and process related impurities), bioburden and endotoxin has to be performed on raw materials, intermediates, drug substances and finished drug products. Some of the analytical methods in downstream processing are HPLC, gel electrophoresis, PCR, ELISA, Bradford, hemagglutination (HA) and plaque assay.All critical steps in a process have to be validated and less critical steps have to be under control. The criticality of an analytical method is dogged by risk assessment. There are various delegacys to perform method validation. The manufacturer is responsible for choosing the suitable validation procedure and justify ing it.1,7,93.1 Types of Analytical ProceduresThe four most common types of analytical procedures are identification tests, qualitative and quantitative tests for impurities and assay. taste involves the quantitative measurement of the major component(s) in the drug substance and drug product.93.2 Team SelectionThe validation project manager is responsible for the selection of a Cross-Functional-Team (CFT) from various related departments and functional areas. He or she is also in charge of assigning responsibilities and assuring that all personnel involved are proficient properly.73.3 Analytical Method Validation ProtocolThe first step in method validation is the preparation of a communications protocol that defines the work to be done to demonstrate that the method is fit for its intended use.7,10The analytical method validation protocol should contain the following sectionsPurposeShort description of what is to be over(p)Scope of the project specifying the test methods and pro ductsOverviewGeneral description of the test methodSummary of the characterisation studiesIdentification of method type and validation approachTest method performances and validation protocolIntended use of each test method applicationAnalytical performance characteristics for each test method applicationResourcesEnd user laboratory where the method validation is to be performedEquipment and materials to be used in the method validationSpecial instructions on handling, stability, and reposition for each materialAppendicesReferences, signature, and a review worksheet for all personnelSpecific tasks for all personnel and documentation of their trainingListings of all equipment and software necessary to perform the method validationDocument and materials worksheets used in method validationTest method procedures (SOPs)Before the method validation can begin the protocol must be agreed upon by the CFT and approved.73.4 Performance singularitys TestsPerformance characteristics and thei r acceptance criteria are defined during the characterisation studies at the development stage of the analytical method. Depending on the method and its intended use, some performance characteristics tests may be omitted, the number of replicates may be increased or reduced, or acceptance criteria may be adapted. All decisions have to be based on scientifically sound judgment. It is important that well characterised reference materials, with documented purity, are used for testing performance characteristics.7,9The following table outlines the performance characteristics and their meaning, test procedures, how data should be reported and acceptance criteria according to ICH Q2(R1) and FDA Guidance for Industry on Analytical Procedures and Method Validation. accuracyCloseness of test results to the true valueFor drug substances, accuracy measurements are obtained by comparing test results to the analysis of a standard reference material or to a second, well-characterized method.For d rug products, accuracy is evaluated by analysing synthetic mixtures (containing all excipient materials in the correct proportions) spiked with known quantities of analyte.Guidelines recommend that data be placid from a borderline of nine determinations over at least three concentration levels covering the contract range.The data should be reported as the percent recovery of the known, added amount, or as the difference in the midst of the mean and true value with confidence intervals (such as 1 SD).Acceptability criteria are defined by end users but rarely fall outside 97-103% of the nominal value. Statistical analysis can be applied using a one sample t-test.PrecisionDegree of agreement among test results when the method is applied repeatedly to multiple samplings of a homogeneous samplePrecision is commonly described in terms of repeatability, intermediate precision, and reproducibilityRepeatability is investigated by analysing a minimum of nine determinations using the same equipment and sample, covering the specified range of the procedure, or a minimum of six determinations at 100% of the test concentration and reported as percent relative standard deviation (RSD).Intermediate precision refers to the agreement among the results from a single laboratory, despite potential variations in sample preparation, analysts, or equipment.Reproducibility refers to the agreement among the results from different laboratories. Results are reported as % RSD, and the percent difference in the mean values between the analysts must be within specifications. Less than 2% RSD is often recommended, but less than 5% RSD can be delicious for minor components.SpecifityAbility to measure accurately and specifically the analyte of interest in the presence of other componentsIn drug assays, specificity takes into account the degree of interference from other active ingredients, excipients, impurities, degradation products, or matrices.In chromatography, it ensures that a chrom atographic peak corresponds to a single component. Specificity can be demonstrated by the resolution between peaks of interest.Limit of detection(LOD)Lowest concentration of an analyte in a sample that can be detectedIn a chromatography laboratory, the most common way to determine both the LOD and the LOQ is using signal-to-noise ratios (S/N), commonly 31 for LOD and 101 for LOQ.An appropriate number of samples must be analyzed to fully validate the method performance at the limit.Limit of quantitation(LOQ)Lowest concentration of an analyte in a sample that can be quantified with acceptable precision and accuracy under the stated operational conditions of the methodLinearityAbility of a method to provide results that are directly proportional to analyte concentration within a given rangeGuidelines specify that a minimum of five concentration levels be used to determine the range and linearity, along with certain minimum specified ranges depending on the type of method.The range is normally expressed in the same units as the test results obtained by the method (for example, nanograms per millilitre).Data to be reported generally include the equation for the calibration curve line, the coefficient of determination (r 2), residuals, and the curve itself.RangeInterval between the upper and lower concentrations of an analyte that have been demonstrated to be determined with acceptable precision, accuracy, and linearity using the methodRobustnessMeasure of a methods capacity to obtain comparable and acceptable results when perturbed by small but reach variations in procedural parametersIt provides an indication of the methods suitability and reliability during normal use.During a robustness airfield, method parameters (such as eluent composition, gradient, and detector settings) are intentionally varied to study the effects on analytical results. Common chromatography parameters used to measure and document robustness include critical peak pair resolution (R s), plate number (N) or peak width in gradient elution, retention time (t R), tailing factor (T F), peak area (and height) and concentration.Robustness studies are expected to be done during method development.Table 7 gives an overview of the performance characteristic tests that have to be performed on different types of analytical procedures.9Figure 6 Performance Characteristic tests performed on different Types of Analytical ProceduresThe performance characteristics are evaluated by comparing the results to the specifications defined at the development stage.An analytical method is considered to be validated when it meets the specifications defined at the development stage. Once an analytical method has been made a formal part of the manufacturing process, it is extremely difficult to remove it. In the event of changes in the drug substance, the composition of the finished product and in the analytical procedure, revalidation may be necessary.5,7,93.5 Validation DocumentationEvery v alidation step needs to be documented to be able to provide written evidence to the regulatory authorities that a specific method is fit for its purpose. Documentation associated with method validation are validation protocols, standard operating procedures (SOPs), specifications and validation reports.Downstream processing in biopharmaceutical manufacturing involves many analytical methods which help to ensure quality, safety and efficacy of the final drug product. Development, validation and control of a robust analytical method is a lengthy and difficult task. However, without written evidence that an analytical method is fit for its intended use the company will not obtain a marketing authorisation.Over time, many guidelines and pharmacopeial monographs have been issued and a lot of revision activity has happened especially following the Stimuli to Revision paper published in 2013. Even though, no comprehensive guideline or monograph incorporating the modern lifecycle approach h as been issued yet.Although proper development of robust and effective analytical methods is more time-consuming and expensive, it has many advantages. It leads to more efficient validation, variability is reduced and controlled and analytical method-related out-of-specification results and failure investigation are minimised. Additionally, changing method parameters within the design space facilitates continual improvement as it does not require regulatory re-approval.Validation is a team effort. Members of the CFT need to be properly trained. Their first and most demanding task is the preparation of a protocol which defines the scope of the validation project and provides all details necessary for a thriving validation. It also defines, depending on the type of the analytical procedure, which performance characteristics need to be tested. The use of well characterised reference materials with known purity is important. Analytical method validation is considered to be complete wh en all acceptance criteria are met and a validation report has been written. BibliographyReferences1 Choudhary, A. (2009). Validation in Pharmaceutical Manufacturing. Pharmaceutical Guideline. Accessed on 1 touch 2017. operable on net profit http//www.pharmaguideline.com/2010/12/validation.html2 International Conference on Harmonization (2009). Harmonised Tripartite Guideline ICH Q8(R2) Pharmaceutical Development. Accessed on 1 March 2017. Available on Internet https//www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q8_R1/Step4/Q8_R2_Guideline.pdf3 Stockbridge, P. (2008). Biopharmaceutical Fill and Finish Technical and Operating Challenges for the Latest Formulations and Devices. BioProcess International. Accessed on 7 March 2017. Available on Internet http//www.bioprocessintl.com/2008/biopharmaceutical-quality-assurance-184041/4 Nandhakumar, L. Dharmamoorthy, G. Rameshkumar, S. Chandrasekaran, S. (2011). An Overview of Pharmaceutical Validation Quality Assu rance View Point. IJRPC, 1(4). Accessed on 1 March 2017. Available on Internet http//www.caidat.org/m4atomp3/2561456335400862.pdf5 Lutz, H. (2005). Introduction to Validation of Biopharmaceuticals. BioPharm International. Accessed on 1 March 2017. Available on Internet http//www.biopharminternational.com/introduction-validation-biopharmaceuticals6 USP Validation and Verification Expert Panel (2013). Lifecycle Management of Analytical Procedures Method Development, Procedure Performance Qualification, and Procedure Performance Verification. Stimuli to the Revision Process Article. Accessed on 1 March 2017. Available on Internet https//www.usp.org/sites/ disregard/files/usp_pdf/EN/USPNF/revisions/lifecycle_pdf.pdf7 Shabir, G. A. (2004). Step-by-Step Analytical Methods Validation and Protocol in the Quality System Compliance Industry. IVT Network Analytical Method Validation, pp. 4-14. Accessed on 2 March 2017. Available on Internet http//www.ivtnetwork.com/sites/default/files/Analytic al%20Method%20Validation.pdf8 U.S. Food and Drug Administration. Code of Federal Regulations, Title 21, Parts 211.165(e), 211.194(a) and 211.100(a). Accessed on 2 March 2017. Available on Internet http//www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart=2119 International Conference on Harmonization (2005). Harmonised Tripartite Guideline ICH Q2(R1) Validation of Analytical Procedures, Text and Methodology. Accessed on 1 March 2017. Available on Internet http//www.ich.org/products/guidelines/quality/article/quality-guidelines.html10 McDowall, R. D. (2014). GLP and GMP Approaches to Method Validation Going the same Way?. Spectroscopy, 29(4). Accessed on 1 March 2017. Available on Internet http//www.spectroscopyonline.com/glp-and-gmp-approaches-method-validation-going-same-way11 U.S. Pharmacopeial Convention (2016). General Chapter Prospectus The Analytical Procedure Lifecycle. USP-NF, Notices. Accessed on 2 March 2017. Available on Internet http//www.usp.org/usp -nf/notices/1220-analytical-procedure-lifecycle12 U.S. Pharmacopeial Convention (2014). New USP requirements for Analytical Method Validation. USP-NF, Notices. Accessed on 2 March 2017. Available on Internet http//www.usp.org/usp-nf/pharmacopeial-forum13 ECA honorary society (2017). Revised USP Chapter Validation of Compendial Methods approved. Accessed on 2 March 2017. Available on Internet http//www.gmp-compliance.org/gmp-news/revised-usp-chapter-1225-validation-of-compendial-methods-approved14 Huber, L. (2015). Recent Updates and Trends in Analytical Method Validation. PPP of The Agilent critical Compliance Seminar. Accessed on 7 March 2017. Available on Internet http//www.agilent.com/cs/library/flyers/Public/Recent_regulatory_updates_and_trends_in_analytical_method_validation.pdfIllustrationsFigure 1 Huber, L. (2015). Recent Updates and Trends in Analytical Method Validation. PPP of The Agilent Critical Compliance Seminar. Accessed on 7 March 2017. Available on Internet http// www.agilent.com/cs/library/fl