u-pun
`
`
`1. leont
`Conn-«Ii
`09
`11.2
`1
`
`11.21
`Q11
`11.51
`1A
`13.11
`11.6
`20
`11.71
`11.72
`
`2. Validation
`1
`12.10
`Q10
`Q1 1
`
`2.16
`12.11
`13.13
`
`12.1
`13.13
`3
`7.14
`
`4
`11.44
`3. Chan Control
`1
`4. Re ection and Re-nse 111'Materials
`1
`2
`
`
`
`
`
`4.14
`
`7.44
`
`10.11
`
`11.4
`
`Dated: 10 June 2015
`
`Q7 Q&As
`
`
`
`20
`
`1 8.5l
`
`18.14
`18.2
`
`QSA
`QSD
`Q63
`11
`Q53
`
`19.3
`
`20
`
`
`
`13.17
`
`17.60
`
`
`
`
`
`14.30
`
`P- 21
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`
`
`
`3
`
`4
`
`1 .3
`2
`9. APII for Use in Clinical Trials
`1
`0. Glenn
`1
`
`2.50
`
`
`
`8. S ecifil: Guidance for APIs Manufactured 11 Cell Culture/Fermentation
`1
`
`14.1
`14.2
`14.3
`14.40
`11.11
`14.41
`11.11
`
`14.43
`5. Com aim; Mid Recalls
`15.10
`1
`In
`
`15.12
`15.13
`2
`1.50
`
`15.14
`
`6. Calm-act Mum Lament (including Llhmtnries)
`1
`2.2
`16.12
`
`2.22
`
`2
`16
`Q10
`3
`16.12
`16.14
`
`7. Agents, Bunkers, Traders, Distributors, Rep“
`, and Relnbeller:
`
`1
`17.1
`
`2
`17.1
`16.12
`3
`17.2
`9.42
`20
`9.43
`17.40
`17.61
`17.20
`17.6
`
` 2
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`

` 7?
`
`W ' Center for Drug Evaluation
`
`and Research (CDER)
`
`Reviewer Guidance'
`
`Validation of
`
`Chromatographic Methods
`
`November 1994
`CMC 3
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`

`I.
`
`ll.
`
`III.
`
`IV.
`
`TABLE OF CONTENTS
`
`INTRODUCTION ...............................................
`
`TYPES OF CHROMATOGRAPHY .................................
`
`A.
`
`High Performance Liquid Chromatography (HPLC) ...............
`
`1.
`
`2.
`3.
`
`4.
`
`5.
`
`6.
`
`Chiral Chromatography ...............................
`
`Ion-exchange Chromatography .........................
`lon-pairlAffinity Chromatography ........................
`
`Normal Phase Chromatography .........................
`
`3 A
`
`Reversed Phase Chromatography .......................
`
`3
`
`Size Exclusion Chromatography ........................ .4
`
`1
`
`2
`
`2
`
`2
`
`3
`3
`
`B.
`
`C.
`
`Gas Chromatography (GC) ..................................
`
`Thin-Layer Chromatography (TLC) ............................
`
`REFERENCE STANDARDS ......_ ................................
`
`PARAMETERS FOR VALIDATION OF HPL CHROMATOGRAPHIC
`METHODS FOR DRUG SUBSTANCE AND DRUG PRODUCT ..........
`
`Accuracy .............................................. _.
`
`.
`
`Detection Limit and Quantitation Limit .........................
`
`4
`
`5
`
`5
`
`7
`
`8
`
`8
`
`A.
`
`B.
`
`C.
`
`D.
`
`Linearity ......................................_ .......... 11
`
`Precision ................................... V............. 13
`
`1.
`
`Repeatability ........................................ 13 '
`
`a.
`
`b.
`
`Injection Repeatability .
`
`.
`
`.
`
`.' ..................... 13
`
`Analysis Repeatability ........................... 15
`
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`

`E.
`
`F.
`
`G.
`
`H.
`
`i.
`
`J.
`
`2,
`
`3.’
`
`Intermediate Precision ................................ 15
`
`Reproducibility ...................................... 16
`
`Range .................................................. 16
`
`Recovery ................................................ 16
`
`Robustness .............................................. 16
`
`Sample Solution Stability ................................... 17
`
`Specificity/selectivin....................................... 17
`
`System Suitability Specifications and Tests ..................... 21
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`Capacity factor ..... ' ................................. 22
`
`Precision/Injection repeatability ......................... 22
`
`Relative retention .................................... 22
`
`Resolution .......................... ................ 22
`
`Talling factor ........................................ 23
`
`Theoretical plate number .............................. 26
`
`K.
`
`General Points to Consider ....... ‘ ........................... 28
`
`V.
`
`VI.
`
`COMMENTS AND CONCLUSIONS y................................ 29
`
`ACKNOWLEDGEMENTS ........................................ 29
`
`VII.
`
`REFERENCES ................................................ 29 ,
`
`Ii
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`

`REVIEWER GUIDANCE1
`
`VALIDATION OF CHROMATOGRAPHIC METHODS
`
`I.
`
`INTRODUCTION
`
`The purpose of this technical review guide is to present the issues to consider when
`evaluating chromatographic test methods from a regulatory perspective. The
`document discusses the points to note and weaknesses of chromatography so that
`CDER reviewers can ensure that the methods performance claims are properly
`evaluated, and that sufficient information is available for the field chemist to assess the
`method. Analytical terms, as defined by the International Conference of
`Harmonization (ICH), 1993, have been incorporated in this guide.
`
`Chromatographic methods are commonly used for the quantitative and qualitative
`analysis of raw materials, drug substances, drug products and compounds in biological
`fluids. The components monitored include chiral or achiral drug, process impurities,
`residual solvents, excipients such as preservatives, degradation products, extractables
`and leachables from container and closure or manufacturing process, pesticide in drug
`product from plant origin, and metabolites.
`
`The objective of a test method is to generate reliable and accurate data regardless of
`whether it is for acceptance, release, stability or pharmacokinetics study. Data are
`generated for the qualitative and quantitative testing during development and post-
`approval of the drug products. The testing includes the acceptance of raw materials,
`release of the drug substances and products, in-process testing for quality assurance,
`and establishment of the expiration dating period.
`
`Validation of a method is the process by which a method is tested by the developer or
`user for reliability, accuracy and preciseness of its intended purpose. Data thus
`
`1This guidance has been prepared by the Analytical Methods Technical
`Committee of the Chemistry Manufacturing Controls Coordinating Committee (CMC
`CC) of the Center for Drug Evaluation and Research at the Food and Drug
`Administration. Although this guidance does not create or confer any rights for or on
`any person and does not operate to bind FDA or the industry, it does represent the
`agency’s current thinking on the validation of chromatographic methods. For additional
`copies of this guidance, contact the Division of Communications Management, HFD—
`210, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857 (Phone: 301-594-1012).
`Send one self-addressed adhesive label to assist the offices in processing your
`request. An electronic version of this guidance is also available via Internet the World
`Wide Web (WWW) ( connect to the FDA Home Page at WWW.FDA.GOV/CDER and
`go to the “Regulatory Guidance" section).
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`generated become part of the methods validation package submitted to CDER.
`
`Methods validation should not be a one-time situation to fulfil Agency filing
`requirements, but the methods should be validated and also designed by the developer
`or user to ensure ruggedness or robustness. Methods should be reproducible when
`used by other analysts, on other equivalent equipment, on other days or locations, and
`throughout the life of the drug product. Data that are generated for acceptance,
`release, stability, or pharmacokinetics will only be trustworthy if the methods used to
`generate the data are reliable. The process of validation and method design also
`should be early in the development cycle before important data are generated.
`Validation should be on-going in the form of re-validation with method changes.
`
`|l.
`
`TYPES OF CHROMATOGRAPHY
`
`Chromatography is a technique by which the components in a sample, carried by the
`liquid or gaseous phase, are resolved by sorption-desorption steps on the stationary
`phase.
`
`A.
`
`High Performance Liquid Chromatography (HPLC)
`
`HPL chromatographic separation is based on interaction and differential
`partition of the sample between the mobile liquid phase and the stationary
`phase. The commonly used chromatographic methods can be roughly
`divided into the following groups, not necessarily in order of importance:
`
`@SnPFpNT‘
`
`Chiral
`
`|on—-exchange
`|on--pair/affinity
`Normal phase
`Reversed phase
`Size exclusion
`
`1.
`
`Chiral Chromatography
`
`Separation of the enantiomers can be achieved on chiral stationary
`phases by formation of diastere‘omers via derivatizing agents or
`mobile phase additives on achiral stationary phases. When used
`as an impurity test method, the sensitivity is enhanced if the
`enantiomeric impurity elutes before the enantiomeric drug.
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`Ion—exchange Chromatography
`
`Separation is based on the charge-bearing functional groups,
`anion exchange for sample negative ion (X'), or cation exchange
`for sample positive ion (X+). Gradient elution by pH is common.
`
`Ion—pair/Affinity Chromatography
`
`Separation is based on a chemical interaction specific to the target
`species. The more popular reversed phase mode uses a buffer
`and an added counter-ion of opposite charge to the sample with
`separation being influenced by pH, ionic strength, temperature,
`concentration of and type of organic co-solvent(s). Affinity
`chromatography, common for macromolecules, employs a ligand
`'(biologically active molecule bonded covalently to the solid matrix)
`which interacts with its homologous antigen (analyte) as a
`reversible complex that can be eluted by changing buffer
`conditions.
`'
`
`Normal Phase Chromatography
`
`Normal phase chromatography is a chromatographic technique
`that uses organic solvents for the mobile phase and a polar
`stationary phase. Here, the less polar components elute faster
`than the more polar components.
`
`Reversed Phase Chromatography
`
`The test method most commonly submitted to CDER is the
`reversed phase HPLC method. UV detection is the most common
`detection technique.
`
`Reversed phase chromatography, a bonded phase
`chromatographic technique, uses water as the base solvent.
`Separation based on solvent strength and selectivity also may be
`affected by column temperature and pH.
`In general, the more
`polar components elute faster than the less polar components.
`
`UV detection can be used with all chromatographic techniques.
`The concern for this type of detector is the loss of sensitivity with
`lamp aging, and varying sensitivity at the low level depending on
`design and/or manufacturer. A point to note is that observations
`on the HPL chromatograms, by UV detection in combination with
`reversed-phase HPLC, may not be a true indication of the facts for
`
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`the following reasons:
`
`0
`
`0
`
`0
`
`Compounds much more polar than the compound of interest
`may be masked (elute together) in the solvent front/void
`volume.
`
`Compounds very less polar than the analyte may elute
`either late during the chromatographic run or are retained in
`the column.
`
`Compounds with lower UV extinction coefficients or different
`wavelength maxima may not be detectable at the low level
`relative to the visibility of the analyte since only one
`wavelength is normally monitored.
`
`6.
`
`Size Exclusion Chromatography
`
`Also known as gel permeation orfiltration, separation is based on
`the molecular size or hydrodynamic volume of the components.
`Molecules that are too large for the pores of the porous packing
`material on the column elute first, small molecules that enter the
`pores elute last, and the elution rates of the rest depend on their
`relative sizes.
`
`Gas Chromatography (GC)
`
`Gas chromatography is based on the volatilized sample transported by
`the carrier gas as the moving phase through the stationary phase of the
`column where separation takes place by the sorption/desorption process.
`
`Samples for gas chromatographic analysis are normally low molecular
`weight compounds that are volatile and stable at high temperature.
`In
`this respect, residual solvents in drug substances and drug products are
`suitable for gas chromatographic analysis. Chemical derivatives can also
`be formed to achieve volatility and thermal stability;
`
`Common detectors are flame ionization (FID) for carbon—containing
`compounds, electron capture (ECD) for halogenated compounds, flame
`photometric (FPD) for compounds containing sulphur or phosphorous
`and nitrogen-phosphorous (NPD) for compounds containing nitrogen or
`phosphorous. Chiral separation also can be achieved by gas
`chromatography. Separation by the packed column is rapidly being
`replaced by the capillary column that provides improved resolution and
`analysis speed. The location of the analyte on the gas chromatogram is
`
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`described by retention time (R) which is similar to HPLC.
`
`C.
`
`Thin-Layer Chromatography (TLC)
`
`Thin-layer chromatography is the simplest of the more common
`chromatographic techniques. Separation is based on migration of the
`sample spotted on a coated (stationary phase) plate with one edge
`dipped in a mixture of solvents (mobile phase). The whole system is
`contained in an enclosed tank.
`
`Detection techniques include fluorescence, UV and sprays (universal and
`specific) for compounds that are not naturally colored. The location of the
`analyte on the TLC plate is described by the Rf value which is the ratio of
`the migration distance of the compound Of interest to the mobile phase
`front.
`
`Of the three techniques, gas, liquid and thin—layer, TLC is the most universal test
`method as all components are present on the plate and with appropriate detection
`techniques, all components can be observed. However, it normally is not as accurate
`or sensitive as HPLC. TLC has a higher analytical variation than HPLC, although one
`sees the "whole picture“ when appropriate detection schemes are selected.
`
`III.
`
`REFERENCE STANDARDS
`
`A reference standard is a highly purified compound that is well characterized.
`Chromatographic methods rely heavily on a reference standard to provide accurate
`data. Therefore the quality and purity of the reference standard is very important. Two
`types of reference standards, chemical and nuclidic, exist. With the latter, the radio-
`label purity should also be considered as well as the chemical purity.
`
`As described in the Guideline for-Submitting Samples and Analytical Data for Methods
`Validation, the two categories _of chemical reference standards are as follows:
`
`0
`
`o
`
`USP/NF reference standard that does not need characterization, and
`
`non-compendial standard that should be of the highest purity that can be
`obtained by reasonable effort and should be thoroughly characterized to
`assure its identity, strength, quality and purity.
`
`The points to note are:
`
`0
`
`Most USP/NF reference standards do not state the purity of the
`compound.
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`

`o
`
`0
`
`0
`
`-
`
`The purity correction factor for non-USP, reference standards is
`recommended to be included in the calculation of the test method.
`
`In addition to structurally-related impurities from the synthesis process,
`other process impurities like heavy metals, residual solvents, moisture
`(bound and unbound), pesticides for products of plant origin, and
`degradation products can also contribute to the lack of purity in the
`reference standard.
`
`The drying of the reference standard before use, if stated in the method,
`will eliminate residual solvent(s), unbound moisture and sometimes
`bound moisture (depending on the drying conditions). The drying step is
`always included for hygroscopic compounds. On the other hand, drying
`can result in the loss of a hydrate or cause degradation in heat-sensitive
`compounds.
`
`Chromatographic test methods use either external or internal standards for
`quantitation.
`
`A.
`
`An external standard method is used when the standard is analyzed on a
`separate chromatogram from the sample. Quantitation is based on a
`comparison of the peak area/height (HPLC or GO) or spot intensity (TLC)
`of the sample to that of a reference standard of the analyte of interest.
`
`The external standard method is more appropriate for samples as follows:
`
`1.
`
`2.
`
`3.
`
`Sample with a single target concentration and narrow
`concentration range, e.g., acceptance and release tests.
`
`Simple sample preparation procedure.
`
`Increased baseline time for detection of potential extraneous
`peaks, e.g., impurities test.
`
`B.
`
`With an internal standard method, compound of known purity that does
`not cause interference in the analysis is added to the sample mixture.
`Quantitation is based on the response ratio of compound of interest to the
`internal standard vs the response ratio of a similar preparation of the
`reference standard (HPLC or GC). This technique is rarely used for TLC
`methods.
`
`The internal standard method is more appropriate for samples as follows:
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`1.
`
`2.
`
`3.
`
`Complex sample preparation procedures, e.g., multiple
`extractions.
`
`Low concentration sample (sensitivity being an issue), e.g.,
`pharmacokinetics studies.
`
`Wide range of concentrations expected in the sample for analysis,
`e.g., pharmacokinetics studies,
`
`Although CDER does not specify Whether the method must use an internal or
`external standard for quantitation, it is commonly observed that HPLC methods
`for release and stability and TLC methods use external standards; and methods
`for biological fluids and GC methods use internal standards.
`
`The working concentration is the target concentration of the compound of interest as
`described in the method. Keeping the concentrations of the sample and the standard
`close to each other for the external standard method improves the accuracy of the
`method.
`
`Recommendations:
`
`1.
`
`2.
`
`Include the purity correction factor, if known, of the reference standard in
`the calculation.
`
`State the working concentrations of the standard and sample in the
`method.
`
`IV.
`
`PARAMETERS FOR VALIDATION OF HPL CHROMATOGRAPHIC
`METHODS FOR DRUG SUBSTANCE AND DRUG PRODUCT
`
`Though many types of HPL chromatographic techniques are available, the most
`commonly submitted method, the reversed-phase HPLC with UV detection, is selected
`to illustrate the parameters for validation. The criteria for the validation of this
`technique can be extrapolated to other detection methods and chromatographic
`techniques. For acceptance, release or stability testing, accuracy should be optimized
`since the need to show deviation from the actual or true value is of the greatest
`concern.
`-
`
`A.
`
`Accuracy
`
`Accuracy is the measure of how close the experimental value is to the
`true value.
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`Accuracy studies for drug substance and drug product are recommended
`to be performed at the 80, .100 and 120% levels of label claim as stated in
`the Guideline for Submitting Samples and Analytical Data for Methods
`Validation.
`
`For the drug product, this is performed frequently by the addition of
`known amounts of drug by weight or volume (dissolved in diluent) to the
`placebo formulation working in the linear range of detection of the
`analyte. This would be a true recovery for liquid formulations. For
`formulations such as tablet, suppository, transdermal patch, this could
`mean evaluating potential interaction ofthe active drug with the
`excipients in the diluent. From a practical standpoint, it is difficult to
`manufacture a single unit with known amount of active drug to evaluate
`recovery. This test evaluates the specificity of the method in the
`presence of the excipients under the chromatographic conditions used for
`the analysis of the drug product.
`It will pick up recovery problems that
`could be encountered during the sample preparation and the
`chromatographic procedures. However, it does not count the effect of the
`manufacturing process.
`
`At each recommended level studied, replicate samples are evaluated.
`The RSD of the replicates will provide the analysis variation or how
`precise the test method is. The mean of the replicates, expressed as %
`label claim, indicates how accurate the test method is.
`
`Recommendations:
`
`Recovery data at least in triplicate at each level (80,100 and 120% of
`label claim)Is recommended. The mean is an estimate of accuracy and
`the RSDIs an estimate of sample analysis precision.
`
`Detection Limit and Quantitation Limit
`
`These limits are normally applied to related substances in the drug
`substance or drug product. Specifications on these limits are submitted
`with the regulatory impurities method relating to release and stability of
`both drug substance and drug product.
`Detection limit is the lowest concentration of analyte in a sample that can
`be detected, but not necessarily quantitated, under the stated
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`experimental conditions. Quantitation limit is the lowest concentration of
`analyte in a sample that can be determined with acceptable precision and
`accuracy under the stated experimental conditions.
`
`WithUV detectors, it is difficult to assure the detection precision of low
`level compounds due to potential gradual loss of sensitivity of detector
`lamps with age, or noise level variation by detector manufacturer. At low
`levels, assurance is needed that the detection and quantitation limits are
`achievable with the test method each time. With no reference standard
`
`for a given impurity or means to assure detectability, extraneous peak(s)
`could "disappear/appear." A crude method to evaluate the feasibility of
`the extraneous peak detection is to use the percentage claimed for
`detection limit from the area counts of the analyte. For example,
`detection limit claim of 0.01% for the analyte integrated area count of
`50,000 will give an area count of 5 that is not detectable.
`
`Though USP expresses detection limit and quantitation limit in terms of 2 ,
`or 3, and 10 times noise level respectively, this concept is not very
`practical. Noise level on a detector during the method development
`phase may be different when samples are assayed on different detectors.
`etc. The use of standard(s) in the test method at the quantitation limit
`level (proposed by the applicant) is assurance that the impurity can be
`observed and quantitated.
`
`Detector sensitivity can vary with the model number and/or manufacturer
`as illustrated in Table ‘_l for the analysis of a compound by two
`commercial detectors. The data should not be taken as the expected
`ratio of sensitivity of the two detectors.
`It is not known if other parameters
`which can also play a part, e.g., age of lamp, column, were considered
`when setting these limits.
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`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`lPR2016-00006
`
`lPR2020-00770
`
`United Therapeutics EX2007
`Page 4763 of 7335
`
`IPR2020-00770
`United Therapeutics EX2007
`Page 4763 of 7335
`
`

`

`Table 1.
`
`Comparison of Detector Sensitivity Limits in Two
`Commercial Detectors.
`
`_ Detector 1
`
`Detector 2
`
`
`
`
`
`
`
`Quantitatio
`:1 Limit
`
`Detection
`Limit
`
`0 . 21%
`
`0.07%
`
`0.16%
`
`,
`
`0.05%
`
`One also should be cautious that baseline noise is not interpreted as
`extraneous peaks. Undulations may be observed at the void volume if
`the diluent for the sample is different from the solvents (proportion and
`type) used in the mobile phase.
`
`If a reference standard for the compound of interest is available, a
`standard close to the quantitation limit or the specification could be used.
`For monitoring peak(s) with no reference standard for the impurity, a
`diluted reference standard of the drug substance is recommended. The
`method should then check that the high and low concentrations are
`operating in the linear range of detection of the drug substance.
`Otherwise the information that is expressed as % area or height of the
`It
`drug substance peak from the same HPL chromatogram will be biased.
`should also be noted that the extraneous peak using area count does not
`consider the detection response which depends on the UV extinction
`coefficient or absorptivity of the compound.
`
`Recommendations:
`
`1.
`
`2.
`
`Analysis repeatability and injection repeatability data at the
`quantitation limit.
`
`Use of an additional reference standard at the quantitation limit
`level in the test method.
`
`P- 13
`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`lPR2016—00006
`
`lPR2020—00770
`
`United Therapeutics EX2007
`Page 4764 of 7335
`
`IPR2020-00770
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`Page 4764 of 7335
`
`

`

`C.
`
`. Linearity
`
`The linear range of detectability that obeys Beer's Law is dependent
`«smonrthe compound analyzed and detector used. The working sample
`concentration and samples tested for accuracy should be in the linear
`range.
`'
`~
`
`Figures-’1 and 2 illustrate the behavior of UV response vs.
`.A point to
`.-concentration of a (a) linear and _(b) non-linear relationship.
`;note is that when monitoring impurity peaks expressed as percent
`'area' of the parent drug substance, the impurity observed may not be
`a true reflection of the theoretical amount if the non-linear section of
`:the concentration curve is employed.
`In addition, the actual amount
`will be obtained only if the extinction coefficient or absorptivity values
`are the same for both impurity and parent compound.
`Impurity
`reference standards are often needed.
`
`'
`
`Concentrations vs. Peak Areas of Standards to Illustrate
`Figure 1.
`Linearity.
`
`tame
`
`um
`
`wand“
`
`.
`'
`regression coefficient = 0.999998
`intercept = 0.103
`slope = 0.000011
`
`
`
`
`
`mama.
`.
`Marking
`
`
`.... Concentration
`8
`ruww
`L
`_
`_
`< _
`.
`x
`”WV”
`fl
`"
`II
`_
`_
`a
`u wwwm
`>
`,
`<
`'
`,
`4000909
`
`
`
`
`
`”Wm
`
`
`-I—l
`l-—-——l-——-‘l~-'--—l-——i--——i——I--——-I'-—--4 eu—l'
`“9
`[IV
`20
`so
`a
`ID
`IO
`7D
`In
`N
`IN
`In
`
`IV
`
`.
`
`,
`
`0
`
`uq-par ul
`
`1 1
`
`P- 14
`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`|PR2016—00006
`
`lPR2020-00770
`
`United Therapeutics EX2007
`Page 4765 of 7335
`
`IPR2020-00770
`United Therapeutics EX2007
`Page 4765 of 7335
`
`

`

`Figure 2.
`
`Concentrations vs. Peak Areas of Standards Outside the Linear Range.
`
`KIO‘D
`
`'31,)
`
`'
`
`.
`
`n no u m a m u n u m 7“
`o-l——I—i—+—i——I—i—i—+—+—i——+—--Hi———i-—+—--i ---+-—-l
`
`Concentration ue/nL
`
`
`Recommendations:
`
`- The linearity range for examination depends on the purpose of the test
`method. For example, the recommended range for an assay method
`-for content would be NLT i 20% and the range for an
`assay/impurities combination method based onrarea % (for impurities)
`would be +20% of target concentration down to the limit of
`quantitation of the drug substance or impurity. Under most
`circumstances, regression coefficient (r) is 2 0.999.
`Intercept and
`slope should be indicated.
`
`12
`
`P- 15
`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`lPR2016-00006
`
`lPR2020-00770
`
`United Therapeutics EX2007
`Page 4766 of 7335
`
`IPR2020-00770
`United Therapeutics EX2007
`Page 4766 of 7335
`
`

`

`Precision
`
`Precision is the measure of how close the data values are to each other
`
`for a number of measurements under the same analytical conditions.
`ICH has defined precision to contain three components: repeatability,
`intermediate precision and reproducibility. Ruggedness as defined in
`USP XXII <1225>, 1990 incorporates the concepts described under the
`terms "intermediate precision”, "reproducibility" and "robustness" of this
`guide.
`
`1.
`
`Repeatability
`
`a.
`
`Injection Repeatability
`
`Sensitivity is the ability to detect small changes in the
`concentration of the analyte in the sample. Sensitivity can
`be partially controlled by monitoring the specification for
`injection reproducibility (system suitability testing).
`
`The sensitivity or precision as measured by multiple
`injections of a homogeneous sample (prepared solution)
`indicates the performance of the HPLC instrument under the
`chromatographic conditions and day tested. The
`information is provided as part of the validation data and as
`a system suitability test. The specification, as the
`coefficient of variation in % or relative standard deviation
`
`(RSD), set here will determine the variation limit of the
`analysis. The tighter the value, the more precise or
`sensitive to variation one can expect the results. This
`assumes that the chromatograph does not malfunction after
`the system suitability testing has been performed. Keep in
`mind,‘ however, that it does not consider variations due to
`the drug product manufacturing and laboratory sample
`preparation procedures. As an illustration for injection and
`_Rt variation, Table 2 provides representative data collected
`when a leak developed in the chromatographic system
`during sampling. The set of four duplicate samples were
`injected sequentially. Variations in peak area and drift of
`retention times are noted. Sets of typical data from a well-
`behaved system for comparison are shown in Table 3.
`
`13
`
`P- 15
`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`lPR2016-00006
`
`lPR2020-00770
`
`United Therapeutics EX2007
`Page 4767 of 7335
`
`IPR2020-00770
`United Therapeutics EX2007
`Page 4767 of 7335
`
`

`

`Table 2.
`
`Representative Injection Repeatability Data for an HPL
`Chromatographic System that Developed a Leak During
`Sampling.
`'
`
`
`_“
`
`2155699
`004
`
`
`2120466
`
`
`A1
`A2
`
`B1
`B2
`
`C1
`C2
`
`562
`566
`
`5.52
`613
`
`6.48
`
`A Peak Area
`
`35233
`
`52555
`
`38213
`
`20128
`
`2255555
`2288355
`
`2265279
`
`2602016
`
`Illlllllliailllll
`lllllllliigilllll
`
`Table 3.
`
`Representative Injection Repeatability Data for Select
`Formulations from a Normally Functional HPL
`Chromatographic System.
`
`Inhalation
`Solution
`
`Solution for
`Inhalation
`
`10
`
`10
`
`
`
`1993162 i
`5029
`
`0.25%
`
`1722253 i
`6288
`
`1744320 i
`3133
`
`
`
`
`
`H
`
`P- 17
`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`IPR2016-00006
`
`|PR2020-00770
`
`United Therapeutics EX2007
`Page 4768 of 7335
`
`IPR2020-00770
`United Therapeutics EX2007
`Page 4768 of 7335
`
`

`

`Recommendations:
`
`As part of methods validation, a minimum of 10 injections
`with an RSD of 51% is recommended. With the methods
`
`for release and stability studies, an RSD of s 1% RSD for
`precision of the system suitability tests for at least five
`injections (n 2 5) for the active drug either in drug substance
`or drug product is desirable. For low level impurities, higher
`variations may be acceptable.
`
`b.
`
`Analysis Repeatability
`
`Determination, expressed as the RSD, consists of multiple
`measurements of a sample by the same analyst under the
`same analytical conditions. For practical purpose, it is often
`combined with accuracy and carried out as a single study.
`See section IV.A under Accuracy.
`
`intermediate Precision
`
`Intermediate precision was previously known as part of
`ruggedness. The attribute evaluates the reliability of the method in
`a different environment other than that used during development of
`the method. The objective is to ensure that the method will provide
`the same results when similar samples are analyzed once the
`method development phase is over.
`
`Depending on time and resources, the method canvbe tested on
`multiple days, analysts, instruments, etc.
`
`Intermediate precision in the test method can be. partly assured by
`good system suitability specifications. Thus, it is important to set
`tight, but realistic, systemsuitability specifications.
`
`Recommendations:
`
`As a minimum, data generated as described under section |V.A
`Accuracy, for two separate occasions, is recommended to indicate
`the intermediate precision of the test method.
`
`P- 18
`
`UT Ex. 2035
`SteadyMed v. United Therapeutics
`lPR2016-00006
`
`lPR2020-00770
`
`United Therapeutics EX2007
`Page 4769 of 7335
`
`IPR2020-00770
`United Therapeutics EX2007
`Page 4769 of 7335
`
`

`

`3.
`
`Reproducibility
`
`As defined by lCH, reproduci