Introduction to Modern Liquid Chromatography, Third Edition part 61 pot

However, another option for AMT is to involve the receiving laboratory from the beginning in the actual validation of the test method to be transferred.. By completing a co-validation st

that involve additional manufacturing sites and/or contract laboratories In general, comparative testing is most often used for late-stage methods and for the transfer of more complex methods

12.7.1.2 Co-validation between Laboratories

Comparative testing (Section 12.7.1.1) traditionally requires a validated method

as a prerequisite to AMT However, another option for AMT is to involve the receiving laboratory from the beginning in the actual validation of the test method

to be transferred By completing a co-validation study, the receiving laboratory is considered qualified to perform the test method for release testing To perform this transfer option, the receiving laboratory must be involved in identifying the intermediate precision validation characteristics to be evaluated and the experimental design By inclusion of data from all laboratories involved in the study, it is possible

to have the validation report serve as proof of AMT, without requiring a separate validation study by the receiving laboratory

12.7.1.3 Method Validation and/or Revalidation

Another technique that can be used for AMT involves the receiving laboratory’s repeating some or all of the originating laboratory’s validation experiments As with co-validation (Section 12.7.1.2), by completing part of a validation study, the receiving laboratory is considered qualified to perform routine release testing With this process, the laboratory staff and quality unit determine how much testing is required to satisfy AMT

12.7.1.4 Transfer Waiver

A transfer waver is used when a formal AMT is not needed (e.g., compendial methods) and for some other situations that may warrant omission of a formal AMT A transfer waiver is considered when:

• the receiving laboratory currently tests the product with another method

• a test method is in use for a dosage form comparable to the new product

• the test method (or one very similar) is already in use for another application

• the new method involves changes that do not significantly alter the use of an existing test method

• the personnel accompany the transfer of the test method from one laboratory

to another

When a transfer waiver is indicated, the receiving laboratory can use the test method without generation of any comparative data However, the reasons for the waiver must be documented

12.7.2 Essentials of AMT

Many interrelated components are necessary to achieve a successful AMT As in any validation process, documentation is essential both for the process and the results All steps, from the AMT protocol and ending to the transfer report, must be documented for compliance purposes

12.7.2.1 Pre-approved Test Plan Protocol

A protocol must be in place that describes the general transfer process and the acceptance criteria, before implementing an AMT This document usually takes the form of a standard operating procedure (SOP) that describes the details of the AMT protocol or test plan specific to the product and method This document should clearly define the scope and objective of the AMT, all of the respective laboratories’ responsibilities, a list of all the methods that will be transferred (if the AMT comprises more than one method), a rationale for any test methods not included (i.e., the transfer waiver), as well as acceptance criteria It should also include the selection process for materials and samples to be used in the AMT The protocol should include certificates of analysis for any samples and reference materials used Representative, homogeneous samples should be used that are identical for both laboratories Selection of proper samples is very important; usually samples that are not ‘‘official’’ production lots (e.g., pre-GMP materials) or a ‘‘control lot’’ are chosen so that an out-of-specification (OOS) investigation is not required if an unexpected result is obtained Remember, the purpose of the method transfer is to assess method performance, not to identify changes in samples or matrix

Instrumentation and associated settings should also be described A best-case scenario would have each laboratory use common instrumentation (e.g., transfer the instrumentation from the sending laboratory to the receiving laboratory); if this

is not the case, and it rarely is, then the sending laboratory should consider the use of instrumentation common to the receiving laboratory (e.g., same brand and model) to identify any potential issues prior to a formal AMT Intermediate precision validation studies also commonly take instrument differences into account

12.7.2.2 Description of Method/Test Procedures

The documentation should include not just the mechanics of performing the test method but also validation data and any idiosyncrasies in the method Any precau-tions that must be taken to ensure successful results should be included in the method description The test method should be written in a manner that ensures only one

pos-sible interpretation (e.g., use v /v notation if volume measurements are made instead

of weighing) Clear equations and calculations, if appropriate, should be specified; example calculations often help eliminate misinterpretation of the instructions 12.7.2.3 Description and Rationale of Test Requirements

This should include specific information, such as the number of replicates and lots to

be tested, as well as the rationale for how each characteristic was chosen This section should describe any system suitability parameters established for the test method

12.7.2.4 Acceptance Criteria

Before the transfer takes place, documentation must stipulate how the results will

be evaluated Since statistical evaluations are usually employed, clear instructions

on the number of batches and replicates, for example, are needed It is common for simple statistics to be used for acceptance criteria, such as the mean and standard deviation from repeated use of the test method in the sending and receiving

laboratory More sophisticated statistics, such as the F-test, or Student’s t-test, are

also commonly applied The proper use of statistics can provide an unbiased and objective comparison of results using a predetermined procedure listed in the protocol documentation Appropriate statistical references (e.g., [20–22]) should be consulted for more detail Since specifications vary with the test method, instrumentation, sample, and other variables, specific performance criteria are not listed in the PhRMA guidance [19] A partial summary of the ISPE’s list [17] of recommended experimental design and acceptance criteria is presented in Table 12.12

12.7.2.5 Documentation of Results

The AMT Report summarizes the results of the AMT The report certifies that the acceptance criteria were met, and that the receiving laboratory is fully trained and qualified to run the test method In addition to a summary of all of the experiments performed and the results obtained, the report should list all of the instrumentation used in the transfer It is important to include in the AMT report any observations made while performing the transfer Observations in the form of feedback can be used to further optimize a test method or to address special concerns that might not have been anticipated by the sending laboratory

Sometimes, of course, the receiving laboratory may not meet the acceptance criteria in the AMT protocol When this situation arises, the transfer failure should

be addressed by an existing policy that dictates specifically how the situation should

be handled An investigation should be initiated and documented in the summary report, and appropriate corrective action should be taken

12.7.3 Potential AMT Pitfalls

Many of the common pitfalls encountered during AMT can be prevented with a little up-front work It cannot be stressed enough that the robustness studies performed during late method development or early method validation play a critical roll in the success of AMT During the robustness studies many of the critical elements might have been identified and noted as a precautionary statement in the test method Intermediate precision validation studies can serve to identify potential AMT issues

By anticipating that instruments, experience, training, and procedural interpretations can differ from laboratory to laboratory, many common pitfalls can be avoided 12.7.3.1 Instrument Considerations

Differences in instrumentation, such as component design and performance, are responsible for many adverse effects encountered during AMT Injector cycle times, detector wavelength accuracy, on-line mobile-phase accuracy and mixing character-istics, and gradient dwell-volumes are just a few of the differences that can result in different results for the same sample run by the same test method on two different instruments [11, 23] Methods that require that the HPLC system operate near its limits, such as high-throughput, high-resolution, or trace analysis methods can be particularly problematic Anticipation of such problems during method development can help simplify the AMT process

12.7.3.2 HPLC Columns

Columns have represented a significant source of variability in test method results, but there is much less concern with the current generation of columns The test method should specify the brand and other details of the column to be used, as

Table 12.12

Experimental Design and Acceptance Criteria for Analytical Method Transfer

Type of Method Number of

Analysts

Lotsaor Units Acceptance Criteria Notes

triplicate

Two-sample t-test

with intersite differences of ≤2%

at 95% CI are required.b

Each analyst should use different

instrumentation and columns, if available, and independently prepare all solutions All applicable system suitability criteria must be met.

Content

uniformity

2 1 lot Include a direct

comparison of the mean ±3% and variability of the results (%RSD) as a

two-sample t-test

with intersite differences of ≤3%

at 95% CI.

If the method for content uniformity is equivalent (e.g., same standard and sample concentrations, HPLC conditions and system suitability criteria) to the assay method, then a separate AMT

is not required Impurities,

degradation

products

2 3 lots in

duplicatec

For high levels, a

two-sample t-test is

required, with intersite differences

of ≤10% at 95%

CI; for low levels, criteria are based on the absolute difference of the means ±25%.

All applicable system suitability criteria should be met The LOQ should be confirmed in the receiving laboratory, and chromatograms should be compared for the impurity profile All samples should be similar with respect to age, homogeneity, packaging, and storage If samples do not contain impurities above the reporting limit, then spiked samples are recommended Dissolution na 6 units for

immediate release, 12 units for extended

Meet dissolution specifications in both laboratories, and the two profiles should be

comparable, or based on the absolute difference

of the means, ±5%.

A statistical comparison

of the profiles or the data at the test end time point(s) similar

to that used for the assay may be performed.

Table 12.12

(Continued)

Type of Method Number of

Analysts

Lotsaor Units Acceptance Criteria Notes

Identity 1 unit Chromatography:

confirm retention time Spectral identification and chemical testing can also be used, assuming operators are sufficiently trained and the instrumentation can provide equivalent results.

Cleaning

validation

2 spiked samples, one above, one below specification

Spiked levels should not deviate from the specification by an amount>3× the

validated standard deviation of the method, or 10% of the specification, whichever is greater.

Essentially a limit test Low and high samples

to confirm both positive and negative outcomes are required.

Source: Data from [17].

aA ‘‘lot’’ or ‘‘batch’’ is test material that has been manufactured at the same time with the same equip-ment; samples within a lot are considered to be identical; samples between lots may have (minor) varia-tions; ‘‘unit’’ is a single dosage unit (e.g., a single tablet, vial, syringe, or patch).

bConfidence interval (CI).

cTriplicate if the impurities and assay are determined in the same test procedure.

well as approved alternative columns (if any) Using the blanket statement ‘‘or equivalent’’ can lead to problems, especially for inexperienced workers, and should

be avoided The United States Pharmacopeia (USP) is addressing this issue by

publishing databases that use chromatographic tests to classify column selectivity [24] (for additional discussion of equivalent columns, see Sections 5.4.2, 6.3.6.1) With the aid of an appropriate database, users can quickly identify columns that are equivalent to the one currently in use The use of techniques such as this should help reduce problems in locating an alternative column that is truly equivalent when the original (specified) column is either not available or no longer provides the required selectivity

Column temperature is another source of variability; all test methods should use a column oven with mobile-phase pre-heating (Sections 3.7.1, 6.3.2.1) Retention-time variability and changes in chromatographic selectivity with temperature can be reduced through the use of a temperature-controlled column oven Column-oven temperature calibration and uniformity can vary between brands and models of ovens, so this should be addressed in the AMT as well

12.7.3.3 Operator Training

Training can be addressed at any time, but it is wise to train new users of the method before formal AMT Despite all the upfront work, errors are still made; either honest mistakes, or errors in procedure that result from test method ambiguities Procedures should be written (and proofread) so that there is only one possible interpretation of how to perform the test method, with enough detail so that nothing is left to chance

‘‘Official’’ or ‘‘validated’’ methods can be found in a number of places, such as the USP, or in the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC) Methods in both New Drug Applications (NDA) and Abbreviated New Drug Applications (ANDA) are also considered to be standardized, official, validated methods To use an official method ‘‘as is’’ for the first time, a laboratory must perform a verification to demonstrate that both the instrument and method performance criteria are met [25–27] However, if the desired results cannot

be obtained, an adjustment, or modification (change), to a standard method might

be needed

Although test methods in the USP (‘‘compendial methods’’) are considered to

be validated, adjustments to USP methods have been allowed in order that system suitability requirements are met; such instructions may be included in individual monographs But method changes usually require some degree of revalidation So

an important question is, At what point does an adjustment become a change

or modification? Historically, if adjustments to the test method are made within

the boundaries of any robustness studies that were performed, no further actions are warranted, as long as system suitability criteria are satisfied However, any adjustment outside of the bounds of the robustness study constitutes a change to the test method and may require a re-validation

In 1998 Furman et al proposed a way to classify allowable adjustments [28] But it was not until 2005 that official guidance appeared on the topic [27, 29–30] Although USP guidance on this topic was recently included into USP Chapter

621 on chromatography [12], the FDA Office of Regulatory Affairs (ORA) has had guidance in place for a number of years [27] Table 12.13 summarizes the adjustments allowed for various HPLC variables taken from both the USP and ORA documents Adjustments outside of the ranges listed in Table 12.13 constitute modifications, or changes, that are subject to additional validation Sound scientific reasoning should be used when determining whether to make a method adjustment

or a method change to a specific test method For example, if robustness studies have shown that the method conditions allow less change for a variable than that listed in Table 12.13, or when robustness testing have shown that more change is allowed, the robustness results (as summarized in the validation report) should prevail Although the criteria in Table 12.13 might seem quite straightforward, many

of the criteria do not completely account for recent advances in HPLC technology, especially columns with much smaller particle sizes (e.g., <2 μm) operated at

higher flow rates (linear velocities) and pressures (e.g.,>6000 psi) Also missing in

the guidelines is a discussion of gradient adjustments/modifications For example,

Table 12.13

Maximum Specifications for Adjustments to HPLC Operating Conditions

Variable Maximum Specificationa Comments

Buffer salt concentration ±10% pH variation must be met Concentrations of minor

mobile-phase components

Only components specified at 50% or less are

considered: ±30% or

±2% absolute, whichever

is larger; maximum change

of ±10% absolute; no component can be reduced

to zero

See Section 12.8.3 for examples and discussion.

UV-detector wavelength No deviations A validated procedure must

be used to verify that wavelength error is ≤ ± 3 nm.

Column inner diameter ±50% (ORA) ±25% (USP) For USP, see [12].

Injection volume Reduced as far as consistent

with accepted precision and detection limits

Increase to as much as 2× volume is specified as long

as there are no adverse chromatographic effects.b

Particle size Reduced by as much as 50%

Column temperature ±10% USP, ±20% ORA

Source: Data from [7, 12, 26–30].

aSee Section 12.8 for examples.

bAdverse chromatographic effects include factors such as baseline, peak shape, resolution, linearity, and retention times.

although solvent composition is addressed, compensation for gradient dwell-volume (Section 9.2.2.4) when changing between different column dimensions must be considered for equivalent results In addition identical column chemistry, while not explicitly stated, is implied

Adjustments to HPLC systems in order to comply with system suitability requirements should not be made in order to compensate for column failure or system malfunction To prevent specification ‘‘creep,’’ adjustments are only made from the original test method conditions and are therefore not subject to continuous adjustment Adjustments are permitted only when suitable reference standards are available for all compounds used in the system suitability test and only when those standards are used to show that the adjustments have improved the quality of the chromatography so as to meet system suitability requirements The suitability

of the test method under the new conditions must be verified by assessment of the relevant analytical performance characteristics Since multiple adjustments can have a cumulative effect in the performance of the system, any adjustments should

be considered carefully before implementation Finally, one word of caution: just because limits for a variable are listed in Table 12.13 does not mean that they can

be made with impunity; system suitability is the final test of the appropriateness of any change within the limits of Table 12.13

12.8.1 pH Adjustments

As shown in Table 12.13, the pH value of the buffer in the mobile phase can be adjusted by as much as ±0.2 pH units For example, a mobile-phase pH of 2.5

could be adjusted in the range of 2.3 ≤ pH ≤ 2.7 Adjustment of the pH should,

however, take into account the pKa’s of the compounds of interest; since for a pH near the pKa, even a 0.1 unit change in the pH can result in significant (>10%)

changes in retention time [31] Studies show that for many compounds, the±0.2 unit allowed change makes sense only if the test method is operated well away from the compound pKa (e.g., pH< 4 for basic compounds; or at pH < 3 or pH > 7 for

acidic compounds) [32]

12.8.2 Concentration of Buffer Salts

The concentration of the salts used in the preparation of the aqueous buffer used

in the mobile phase can be adjusted to within ±10%, provided the pH variation (Section 12.8.1) is met For example, a mobile-phase buffer containing a 20-mM phosphate buffer could be adjusted in the range of 18 to 22 mM See Section 7.2.1 for additional discussion of buffer effects

12.8.3 Ratio of Components in the Mobile Phase

The adjustment of the ratio of mobile phase components (% buffer or organic solvent) is allowed within certain limitations Minor components of the mobile phase (≤50%) can be adjusted by ±30% relative However, the change in any component cannot exceed ±10% absolute (i.e., in relation to the total mobile phase) For example, for a 50:50 water:MeOH mobile phase, a 30% change is 15% absolute, which exceeds the limit of±10% absolute for any one component Therefore an adjustment only in the range of 60:40 to 40:60 is allowed For a mobile phase of 95:5 water:MeOH, 30% of 5% is 1.5% absolute, but since up to±2% is allowed, a mobile phase of 93:7 to 97:3 could be used

Adjustment can be made to only one minor component in a ternary mixture For example, with a mobile phase of 60:35:5 buffer:MeOH:ACN, a 30% change

in MeOH (35%) would be 10.5% absolute, exceeding the±10% absolute allowed,

so the MeOH content could be adjusted in the range of 25–45% For the ACN, 30% of 5% is 1.5%, but ±2% is allowed, so the ACN content could be adjusted from 3 to 7% In the case of this ternary mixture, either the MeOH or the ACN concentration could be changed, but not both In each case a sufficient portion

of the highest concentration component should be used to give a total of 100% Additional examples of adjustments for binary and ternary mixtures are outlined in USP Chapter 621 and can be consulted for more detail [12]

Although Table 12.13 may allow an adjustment of the mobile phase, it should

be emphasized that this can alter chromatographic selectivity Therefore proceed with caution when making mobile-phase adjustments to existing methods

12.8.4 Wavelength of the UV-Visible Detector

Deviations from the wavelengths specified in the test method are not permitted The procedure specified by the detector manufacturer, or another validated procedure, should be used to verify that error in the detector wavelength is≤ ± 3 nm

12.8.5 Temperature Adjustments

In the case of HPLC column temperature, a±10 change (or ±20%, depending on the guideline consulted) is allowed It should be noted that temperature differences can have significant selectivity and retention effects (Section 6.3.2.1)

12.8.6 Column Length, Diameter, and Particle-Size Adjustments

A few inconsistencies exist in the guidelines regarding flow rate, column internal-diameter and length, and particle-size adjustment criteria It is possible to reduce flow rate and internal diameter more than that listed in Table 12.13 (up to 50% allowed by ORA, 25% by USP) with no effect on retention (or selectivity) as long as the mobile-phase linear velocity is constant (isocratic separation assumed)

In its most recent update [12] the USP has allowed for greater adjustments if the linear velocity is maintained

Although column length, internal diameter, and particle-size adjustments are listed separately in Table 12.13, these variables really need to be considered together, and when correctly scaled in accordance with well-known theoretical principles (including constant linear velocity), equivalent separations will result even outside the recommended adjustment criteria [33] For example, if the length-to-particle size

ratio (L /d p) is kept constant, an identical separation well outside the recommended limits can be obtained for a 50-mm, 1.7-μm column as for a 300-mm, 10-μm column

(L /d p= 3 for both) as long as an increase in the flow rate inversely proportional to the particle size is maintained (and, of course, the stationary-phase chemistry must

be identical, extra-column peak broadening must be minimized, and the pressure must be maintained within acceptable limits) In cases such as this, where the regulatory guidelines are deficient, it is wise to write an addendum to the test method (or a separate SOP) that describes the allowable adjustments to a method, with appropriate evidence to support the adjustment

The terms ‘‘quality control’’ and ‘‘quality assurance’’ often are used interchangeably However, in a properly designed and managed quality system, the two terms have separate and distinct meanings, and functions Quality assurance can be thought of

as related to process quality, whereas quality control is related to the quality of the

product In a given organization, it does not matter what the functions are named,

but the responsibilities for these two activities should be clearly defined Both quality assurance and quality control make up the ‘‘quality unit,’’ and are essential to the production of analytical results that are of high quality and are compliant with the appropriate regulations

12.9.1 Quality Control

Quality control (QC) is the activity that determines the acceptability or unaccept-ability of a product, and is determined by the comparison of a product against the original specifications that were created before the product was manufactured In some organizations, the QC group is responsible for the use of the test method to perform analysis of a product Other tasks related to QC may include documented reviews, calibrations, or additional types of measurable testing (sampling, etc.) that reoccur more often than activities associated with quality assurance QC will usually require the involvement of those directly associated with the research, design, or pro-duction of a product For example, in a laboratory-notebook peer-review process,

a QC group would check or monitor the quality of the data, look for transcription errors, check calculations, and verify notebook sign-offs All of these activities help

to ensure an acceptable product

12.9.2 Quality Assurance

Quality assurance (QA) is determined by senior management policies, procedures, and work instructions—and by governmental regulations At the beginning of the validation process, QA may provide guidance for the development of or review of validation protocols and other validation documents During the analytical stage, QA’s job is to ensure that the proper method or procedure is in use and that the qual-ity of the work meets company and governmental guidelines and regulations QA can

be thought of as the activity that will determine how quality control tasks will be car-ried out—and then verify that they were performed properly As opposed to quality control checks (that focus on the product), quality assurance focuses on the process used to test a product QA is more likely to be performed by managers, by corporate level administrators, or third-party auditors through the review of the quality system, reports, archiving, training, and qualification of the staff that performs the work

Method validation constantly evolves and is just one part of the overall regulated-environment activities The validation process starts with instrument qualification (Section 17.2.1.1) before an HPLC instrument is placed online, and continues long after method development, optimization, and transfer—living on with the test method during routine use A well-defined and documented validation process provides regulatory agencies with evidence that the system and test method are both suitable for their intended use It also assures that the guidelines established meet method validation requirements and specifications

The bottom line is that all parties involved should be confident that an HPLC method will give results that are sufficiently accurate, precise, and reproducible for the analysis task at hand Formal method validation is just a set of tools to use to accomplish this task Whether or not a formal validation is required, performance

of good, justifiable science as part of an established quality system will help to ensure that the resultant test method, and the data that it generates will survive the