Rafail Usatinsky, Ph.D. Cedarburg Pharmaceuticals, A Division of AMRI

December 2014

USP Methods Verification

Talking Points

• Role of analytical development in pharmaceutical

• Overview of USP <1226>

• Examples of USP monograph

• Common deficiencies

• USP methods adjustment

• Questions

Drug Product Development

• DISCOVERY (RESEARCH)

• PROCESS DEVELOPMENT (Synthesis/Formulation)

• PRE-CLINICAL (Tox Studies)

• SCALE-UP

• CLINICAL

• PROCESS VALIDATION

• PROCESS TRANSFER

• PRODUCTION (API/PRODUCT)

Analytical Support – API

Rxn Rxn Purification

SM -------------> IM --------------> Crude API ----------------> API

• Analysis of raw materials

• In-process control (reaction completion)

• Analysis of intermediates

• Analysis of drug substance (assay, impurities…)

• Impurities identification

• Reference standard qualification

• Stability studies

Product of Analytical Chemists

Analytical Results

Analytical Method Lifecycle • Method Development (Chemistry and Art)

– Literature search

– Determine goal (qualitative/quantitative analysis, analytes, levels)

– Contact process chemists for specific requirements

– DOE

• Method Optimization (Chemistry and Game)

• Method Validation (Chemistry and Expertise)

– Identify purpose of the method

– Determine parameters to validate

– Develop validation protocol

– Generate data and report

• Method Transfer and/or Method Verification (Chemistry and Work)

USP Monograph (API) Typical Tests

• Identification – IR (<197K>) – HPLC RT

• Assay – Titration – HPLC

• Inorganic Impurities – ROI (<281>) – Heavy metals (<231>,

<232)

• Organic Impurities

– HPLC

– GC

• Specific Tests

– Water (<921>)

– Specific Rotation (<781S>)

• Residual Solvents

• Microbial (<61>, <62>)

USP <1226> Summary

• The intent of this chapter is to provide general information to laboratories on the verification of compendial procedures that are being performed for the first time to yield acceptable results utilizing the laboratories’ personnel, equipment, and reagents.

• Not intended for retroactive application to already successfully established laboratory procedures.

• Verification consists of assessing selected Analytical Performance Characteristics, such as those which are described in chapter <1225>, to generate appropriate, relevant data rather than repeating the validation process.

USP<1226> Summary (Cont’d)

• Users of compendial analytical procedures are not required to validate these procedures when first used in their laboratories.

• Documented evidence of suitability should be established under actual conditions of use

• Verification of microbial procedures is not covered in this chapter; it is covered in USP general test chapters: <51>, <61>, <62>, <71>, and <1227>

USP <1226> Requirements

• Verification requirements should be based on an assessment of the complexity of both the procedure and the material to which the procedure is applied.

• Only those characteristics that are considered to be appropriate for the verification of the particular method need to be evaluated.

USP <1226> Requirements (Cont’d)

• The degree and extent of the verification process will depend on:

– The level of training and experience of the user

– Associated equipment or instrumentation

– The specific procedural steps

– The material to be tested

USP <1226> Requirements (Cont’d) • Assessment of compendial procedure suitability for:

– Chromatographic conditions

– Detector signal response

– Sample matrix

– Synthetic route

• Specificity

• Limit of quantitation and detection

• Precision

Additional Notes

• “An assessment of specificity is a key parameter in verifying that a compendial procedure is suitable for use in assaying drug substances and drug products.”

• “An assessment of the limit of detection or quantitation and precision for impurities procedures may be useful to demonstrate the suitability of the compendial procedure under the actual conditions of use.”

• Foreign Compendial Methods – verification of EP and BP methods. Equivalence to the USP monograph method (if available) should be demonstrated.

When Verification Not Required

• Common compendial test procedures:

– Loss on drying

– Residue on Ignition

– Wet chemical procedures (i.e. titrations)

– Simple instrument methods (i.e. pH, conductivity)

– Identification tests

Benztropine Mesylate (Example)

Tropine Options • TLC British Pharmacopoeia

– Outdated, not sensitive

– Limit test

• HPLC

– Lack of chromophore

• GC (only freebase)

– Reacts with MSA

• IC

– Water soluble

Assay Options (Compendial)

• Compendial method

– Extraction followed by titration

– Time consuming preparation

– Reproducibility issues

– Extensive verification

Assay Options (In-House) • HPLC method

– Adopt related substances conditions

– Full validation

Alkyl Mesylates

• Genotoxic Impurities

• Threshold of Toxicological Concern (TTC)

• TTC = 1.5 µg/day, associated with a 10-5 lifetime risk of cancer

Metals Evaluation

• Are metal reagents or metal catalysts used in the manufacturing process?

• Are there other potential sources of metal contamination in the manufacturing process (process equipment, water system, etc.)?

• Did you screen 3 industrial batches for residual metals?

• Are analytical methods verified/validated?

Common Deficiencies

• Solution stability not evaluated.

– Solution stability should be assessed during method verification. Alternatively, state that solutions will be prepared fresh immediately before use. In cases where the API is unstable to light exposure, the method should specify to protect sample and standard solutions from light.

• Sample chromatograms not provided.

– Sample chromatograms should be provided and should be properly scaled.

Common Deficiencies

• The method verified is significantly different from the compendial method.

• Changes in sample preparation

• Original methods were modified without further study of documentation to justify the changes made.

– No adequate documentation on method verification.

– Lack of review by quality unit

Common Deficiencies

• No verification provided to support a new API Supplier.

– Previously verified methods may need to be shown to be specific for any new impurities that may be present, as impurities may vary depending on the route of synthesis.

Adjustment vs. Change Tweaking Validated Method

• USP: “The specification of definitive parameter in monograph doesn’t preclude the use of other suitable conditions. Adjustments permitted only when… adjustments improve quality of the chromatography.”

• EP: “The extent to which the various parameters of a chromatographic test may be adjusted to satisfy the system suitability criteria without fundamentally modifying the methods.”

Method Adjustment

• Adjustments to the specified chromatographic conditions may be necessary in order to meet system suitability.

• In some circumstances, it may be desirable to use an HPLC column with different dimensions (length, i.d., particle size)

• Change in stationary phase is considered modification and will require full validation.

• Adjustments to the mobile phase composition in gradient elution may cause changes in selectivity and are not recommended

– Change in column packing, duration of initial isocratic hold and dwell volume are allowed

New USP Changes

pH of Mobile Phase

• The pH of the aqueous buffer used in the preparation of the mobile phase can be adjusted to within ±0.2 units of the value or range specified. Applies to both gradient and isocratic separations.

– pH is important for ionic compounds

– pH should be outside pKa ± 1.5 range (robust method)

– Small pH variations can be important for related compounds

• EP: ± 0.2 unit; ± 1.0 for neutral

Effect of pH (Fentanyl USP Method)

HPLC Conditions:

XTerra RP8 150 x 4.6 mm, 3.5 μm

A: HClO4/TEA in Water, pH 2.62

B: Acetonitrile

Flow rate: 1 mL/min

Detection: 206 nm

Sample/Std concentr: 0.1 mg/mL

Injection Volume: 30 µL

Gradient: multi-step gradient from 10% to 60% B over 19 min

pH variations ± 0.1 units lead to coelution of related substances and parent peak

min11 12 13 14 15 16 17

mAU

0

10

20

30

40

50

60

DAD1 A, Sig=206,4 Ref=360,100 (2003-10\11020301.D)

12.9

94

14.5

64

DAD1 A, Sig=206,4 Ref=360,100 (2003-10\11020302.D)

12.7

27

13.0

23

14.3

65

14.5

75

Buffer Concentration

• The concentration of the salts used in the preparation of the aqueous buffer employed in the mobile phase can be adjusted to within ±10% if the permitted pH variation is met. Applies to both gradient and isocratic separations.

– Buffer strength is important for ionic compounds

• EP: ±10%

Mobile Phase Ratio

• The following adjustment limits apply to minor components of the mobile phase (specified at 50% or less).

• The amounts of these components 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).

• Adjustment can be made to one minor component in a ternary mixture.

Mobile Phase (Binary Example)

• Binary (50:50): 30% of 50 is 15% absolute, but exceeds the maximum permitted change of ±10% absolute in either component. Therefore, the mobile phase ratio may be adjusted only within 40:60–60:40.

• Binary (2:98): 30% of 2 is 0.6% absolute. Therefore the maximum allowed adjustment is within the range of 1.4: 98.6–2.6: 97.4.

Mobile Phase (Ternary Example)

• Ternary (60:35:5):

• For the second component, 30% of 35 is 10.5% absolute, which exceeds the maximum change of ±10% absolute in any component. Therefore the second component may be adjusted only within the 25% to 45% absolute.

• For the third component, 30% of 5 is 1.5% absolute. In all cases, a sufficient quantity of the first component is used to give a total of 100%. Therefore, mixture ranges of 50:45:5–70:25:5 or 58.5: 35: 6.5–61.5: 35: 3.5 would meet the requirement.

Column Length and Particle Size

• For isocratic separations, the particle size and/or the length of the column may be modified provided that the ratio of the column length (L) to the particle size (dp) remains constant or into the range between −25% to +50% of the prescribed L/dp ratio.

• Alternatively (particle-size adjustment to superficially porous particles), other combinations of L and dp can be used provided that the number of theoretical plates (N) is within −25% to +50%, relative to the prescribed column.

Column Length and Particle Size • Caution should be taken when the adjustment results in a

higher number of theoretical plates which generates smaller peak volumes, which may require adjustments to minimize extra-column band broadening by factors as instrument plumbing, detector cell volume and sampling rate, and injection volume.

• When particle size is not mentioned in the monograph, the ratio must be calculated using the largest particle size consigned in the USP definition of the column. For gradient separations, changes in length, column inner diameter and particle size are not allowed

Flow Rate

• When the particle size is changed, the flow rate may require adjustment, because smaller-particle columns will require higher linear velocities for the same performance.

– F2 = F1 × [(dc22 × dp1)/(dc1

2 × dp2)]

(F - flow rate (original and modified), dc – column diameter, dp – particle size)

• When a change is made from ≥ 3-mm to < 3-mm particles in isocratic separations, an additional increase in linear velocity (by adjusting flow rate) may be justified, provided that the column efficiency does not drop by more than 20%.

• Flow rate can be adjusted ± 50% (isocratic only)

• Changes in F, dc, and dp are not allowed for gradient separations

Flow Rate (Example)

Injection Volume

• Injection Volume (HPLC):

– The injection volume can be adjusted as far as it is consistent with accepted precision, linearity, and detection limits. Note that excessive injection volume can lead to unacceptable band broadening, causing a reduction in N and resolution. Applies to both gradient and isocratic separations.

Column Temperature

• Column Temperature (HPLC):

– The column temperature can be adjusted by as much as ±10°. Column thermostating is recommended to improve control and reproducibility of retention time. Applies to both gradient and isocratic separations.

Gas Chromatography

Common Questions

• How frequently should compendia methods be re-

verified?

– There is no requirements to re-verify the method if no significant changes have been made

– The method performance is confirmed by meeting system suitability criteria

Common Questions

• What to do if the compendial procedure can’t be verified?

– Provide additional training

– Use other equipment

– Repeat analysis

– Develop alternative in-house procedure

– Validate new procedure

Common Questions

• What are requirements for using in-house methods instead of USP for analysis of API? – It is possible use in-house methods to control your

final substance, provided that you demonstrate that the methods are equivalent to those of the USP and/or EP (the methods must be fully described and validated).

Common Questions

• Does a TLC method need to be verified?

– It’s recommended to verify specificity and sensitivity of the method prior to GMP analysis

• Do we need to verify a KF method?

– It could be necessary; for example for compounds that may react with KF titrant or not well soluble in organic solvents

Common Questions

• Can USP method be used for EP Monograph?

– Yes, as long as the method is equivalent

• What samples to select for method verification?

– Verification should be performed by using qualified reference standards and process representative samples

Common Questions

• How to document verification experiments and results?

– It depends on degree of verification

– Protocol may be required

– A report should be written summarizing the experiments and results

THANK YOU!