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Generic Pharmaceuticals Application BookGeneric Pharmaceuticals Application Book

M et hod d ev elo PM ent

M et hod vAlidAt ion

StABil it y indicAt inG

M et hodS

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diSSolut ion t eSt inG Bio equivAl enc e unifo rMit y

of cont entc l eAninG

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WAT ERS AND GENERIC PHARMAC EUT ICALS

Waters partners with laboratory-reliant companies around the world to

provide the appropriate technologies and services that fully support your

operational needs.

Whether that means optimizing your current laboratory output, or

implementing a scalable and forward-looking solution that empowers you

to achieve the innovation, productivity and profitability goals that define

your company’s success, Waters works with you every step of the way.

The process of developing and supporting a generic pharmaceutical,

from ANDA submission to raw material acceptance and product release,

requires practical analytical solutions to manage quality and compliance

while meeting aggressive financial performance goals.

Waters offers complete and specialized laboratory solutions for generic

pharmaceutical companies that cross the entire generic development

and manufacturing process: from sample preparation, chromatography,

mass spectrometry, software and informatics to compliance, applications

expertise and support services.

MET HOD DEV ELOPMENT

To assess raw material purity, finished product quality and stability and

to ensure production plant cleanliness, labs require analytical methods

that are capable of separating and detecting an active pharmaceutical

ingredient (API) from its impurities, degradants and excipients.

Method development is time-consuming and complicated: your labs need

to evaluate multiple combinations of mobile phase, pH, temperature,

column chemistries and gradient profiles to arrive at a robust, reliable

separation for every activity.

Waters solutions streamline the method development process:

■ ACQUITY UPLC® System: Separation speed and efficiency allows for

the rapid development of potential methodologies

■ UltraPerformance LC® methods: With analysis times as short as

one minute, methods can be optimized in just one or two hours,

significantly reducing the time required to develop and validate

■ ACQUITY UPLC BEH columns: High stability allows for a wide range

of column temperatures and pHs to be explored

■ ACQUITY UPLC Column Manager: Easily evaluate column temp-

eratures from 10 °C below room temperature to 90 °C

■ ACQUITY UPLC Console Calculator: Rapidly transition existing

liquid chromatography analyses to more rapid UPLC methodologies

M ET HOD DEV ELO PM ENT

The simple task of column and pH scout-ing can be easily automated using the ACQUITY UPLC System with the four-posi-tion ACQUITY UPLC Column Manager.

In this example, this confi guration has been used use to assess the selectivity of four dif-ferent ACQUITY UPLC BEH chemistries (Phenyl C

18, RPC

18 and C

8) for the separation

of the impurities of Ranitidine HCl, a com-mon histamine H2-receptor antagonist.

With a 5 to 95% methanol gradient at pH 9, the ACQUITY UPLC BEH Phenyl Column gave the best resolution of all of the peaks of interest.

By exploring the temperature and pressure capabilities of the ACQUITY UPLC System, the separation of a chromatographic test mix is reduced from 11 minutes to just 5 minutes with no loss in resolution.

MET HOD VALIDAT ION

All impurity profiling, stability indicating and uniformity of content

assays must have a robust method for long-term assessment of product

quality. Method validation requires the determination of specificity,

linearity, dynamic range, limit of detection, limit of quantification, as

well as the accuracy, precision and robustness of the assay. This tedious

process often requires more than 100 separate analyses, all of which

need to be mathematically assessed.

Traditionally, this assessment of chromatographic data has been

performed using statistical software. The entire method validation

process can take anywhere from days to weeks to months when using

HPLC and this data analysis approach. It is the primary bottleneck in

analyst time – and, as with any manual process, it is prone to errors, data

traceability problems and data compliance concerns.

Waters solutions eliminate bottlenecks in method validation:

■ UPLC® throughput: Analysis times are significantly reduced when

compared to HPLC, and adhere to the well-established principles of

HPLC for simplified method transfer processes

■ Method Validation Manager for Empower™ 2 Software:

■ Automates the entire chromatographic method validation

process in a privilege-controlled and compliance environment

■ Performs all validation results and statistical calculations

within Empower 2, eliminating the time-consuming manual

data transfer to other statistical software and the associated

problems of transcription errors and security concerns

■ Workflow-based approach provides status information that

guides the user through each step in the process

■ Automates error-checking to ensure that data complies with

your corporate validation requirements

■ Multi-component analyses and batch processing of method

validation results, with reports generated within standardized

Empower 2 templates

M ET HOD VALIDAT ION

Empower 2 Method Validation Manager automates each step of the chromatographic method validation process.

Residual analysis

Concentration accuracy

Robustness variance

Robustness testing

STABIL IT Y INDICAT ING MET HODS

Stability indicating methods are validated methods that can accurately

and precisely quantify the decrease of the active content due to degrada-

tion. They are a regulatory requirement as part of the pharmaceutical

manufacturing process. The regulatory methodology dictates that these

methods have to be specific and free from interference from impurities or

their degradation products.

The requirement for complete analyte resolution often results in low

HPLC throughput or the need for multiple instruments to compensate.

Additionally, as pharmaceutical compounds become more potent, they

are dosed at lower levels and require more sensitive detectors to monitor

and quantify resulting low-level impurities.

Waters solutions deliver the sensitivity, resolution and speed necessary for stability indicating methods:

■ ACQUITY UPLC System: With all of the conveniences of simple

methods transfer from HPLC, UPLC leverages the increased chro-

matographic efficiencies and speed advantages of sub-2 μm

column particles

■ ACQUITY UPLC TUV and PDA Detectors: Advanced optics and elec-

tronics provide best-in-class detection performance, even at the fast

scan speeds necessary for narrow UPLC peaks

■ ACQUITY UPLC PDA Detector with Empower 2 Software: Spectral

resolution combines with peak matching capabilities for the rapid

analysis and comparison of peaks to easily assign peak identity

STABIL IT Y INDICAT ING

M ET HODS

In this example, UPLC has been used for the rapid analysis of Simvastatin, a common cholesterol-lowering drug. The analysis is completed in just 4 minutes, with complete separation of the impurities from the active product.

0.064

0.032

0.096

AU

0.000

0.00 0.80 1.60 2.40 3.20 4.00Minutes

Impu

rity

1 - 0

.666

Impu

rity

2 - 0

.903

Impu

rity

4 - 1

.131

Impu

rity

5 - 1

.667

Impu

rity

6 - 1

.739

Impu

rity

8 - 2

.275

Impu

rity

9 - 2

.805

Sim

vast

atin

- 1.

241

Lova

stat

in -

1.05

4

Simvastatin Impurities Assay (238nm)

Autoscale2.40

0.00 2.001.00 3.00 4.00

Minutes

1.20

0.00

0.60

1.80AU

The entire process of peak detection, analyte confi rmation and quantifi cation is completely

automated in Empower 2 Software, as illustrated in this Simvastatin stability indicating method.

IMPURIT Y P ROFIL ING

The profiling, detection and quantification of drug substances and their

impurities in raw materials and final product testing is a critical part of

manufacturing generic drugs. Impurity profiling requires a high-resolution

chromatography system capable of reliably and reproducibly separating

and detecting all of the known impurities of the active compound.

Also critical to impurity profiling is the ability to accurately measure

low-level impurities at the same time as the higher concentration active

pharmaceutical component. This activity, however, can be complicated

by the presence of excipients in the sample, often resulting in long HPLC

analysis times to achieve sufficient resolution. Overall profitability can

be impacted by the speed at which these analyses have to be performed.

Waters solutions streamline impurity profiling studies:

■ ACQUITY UPLC System: Specifically addresses high-throughput

analysis requirements while maintaining high peak resolution

■ ACQUITY UPLC PDA Detector: Two analytical flow cells are avail-

able for maximum flexibility according to application requirements,

one for maximum chromatographic resolution and a second for high

sensitivity

■ Empower 2 Software: Incorporates the latest peak detection algo-

rithms and custom calculations to optimize data processing and

reporting

IM PU RIT Y P ROFIL ING

The ACQUITY UPLC System streamlines the batch analysis of raw materials quality assessment. Here the analysis of four different batches of Budesonide from different suppliers reveals that there are signifi cant differences in the number and magnitude of the impurity peaks in each batch sample.

The ACQUITY UPLC PDA detector gives exceptional spectral quality for compounds, regardless of concentration. Here, the active ingredient is present at 1.6 AU, while the known impurity shows an absorbance of 0.002 AU at 0.01% of the API. Both were quantifi ed in a single robust analysis with excellent spectral resolution.

0.38210.3

0.42216.3

5.76215.3

5.74244.3

4.16241.3

2.49246.3

0.42215.3

0.63245.3

0.38212.3

0.71211.3

0.42216.3

0.38211.3

0.44210.3

0.63245.3

1.79210.3

1.95240.3

2.49245.3 3.10

244.3

3.95210.3

4.16241.3

Spectrum EP 500 µg/ml11162006_budesonide_014

11162006_budesonide_013

11162006_budesonide_015

11162006_budesonide_016

3.0e-2

3.0e-2

3.0e-2

0.42216.3

0.38212.3

0.63244.3

0.95215.3

3.10214.3

2.25248.3

4.17241.3

3.96244.3

2.0e-2

2.0e-2

2.0e-2

2.0e-2

1.0e-2

1.0e-2

1.0e-2

1.0e-2

0.00

0.00

0.00

0.00

3.96238.3

4.17241.3

0.00

0.00

0.00

0.00

0.50

0.50

0.50

0.50

1.00

1.00

1.00

1.00

1.50

1.50

1.50

1.50

2.00

2.00

2.00

2.00

2.50

2.50

2.50

2.50

3.00

3.00

3.00

3.00

3.50

3.50

3.50

3.50

4.00

4.00

4.00

4.00

4.50

4.50

4.50

4.50

5.00

5.00

5.00

5.00

5.50

5.50

5.50

5.50

6.00

6.00

6.00

6.00

6.50

6.50

6.50

6.50

7.00

7.00

7.00

7.00

8.00

8.00

8.00

8.00

7.50

7.50

7.50

7.50

AU

Time

AUAU

AU

2: Diode Array240

Range: 6.311e-1

Batch A

Batch C1

Batch B

Batch C2

2: Diode Array240

Range: 6.512e-1

2: Diode Array240

Range: 7.128e-1

2: Diode Array240

Range: 7.487e-1

1.60

Prilocaine

AU

1.20

0.80

Minutes

0.00

0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80 3.20 3.60 4.00

0.40

0.42

0.0000

0.0005

0.0010

0.0015

0.0020 o-Toluidine0.01% ImpurityExpanded View

0.540.48 0.84 0.90 0.960.780.720.660.60Minutes

AU

0.616 Extracted o-Toluidine Spectrum0.0048

232.1

285.4

201.7

0.0036

0.0024

0.0012

0.00220.00

AU

240.00 260.00 280.00 300.00nm

2.319 Extracted Prilocaine Spectrum

0.80

202.9

0.60

0.40

0.20

0.00220.00

AU

240.00 260.00 280.00 300.00nm

Budesonide

DISSOLUT ION T EST ING

Dissolution testing is essential in the formulation, development and

production process for quality control and release of generic drugs. The

dissolution profile is used to demonstrate reliability and batch-to-batch

uniformity of the active ingredient. Often this is a bottleneck in the overall

evaluation process since many laboratories still rely on manual sampling

at specified time points to construct this profile. Additionally, newer and

more potent formulations require increased analytical sensitivity. Test-

ing higher potency drugs is particularly important in sustained-release

dosage formulations where dissolution can often be the rate-limiting

step in medicine delivery.

Waters solutions automate dissolution testing:

■ Waters Dissolution Solution: For fully automated online HPLC

and/or UV/PDA analysis, capable of interfacing with a large number of

dissolution baths, including Hanson, Distek, Vankel, Erweka and Sotax

■ Waters Dissolution Solution with UPLC: Collects samples for post-

run analysis utilizing UPLC, ideal for today’s low-level high potency

multi-component medicines

■ Empower 2 Software with Dissolution Option:

■ Uses a simple wizard-driven interface to customize dissolution

method set-up, data collection, processing and reporting of results

■ Controls all system components, providing a compliant-ready

solution for the laboratory and fast methods development for

all components without the need for standard runs

DISSOLUT ION T EST ING

The Empower 2 Software dissolution run set-up wizard is shown along with its fl exible reporting capabilities. The software automatically monitors bath conditions, for complete compliance.

The data shown illustrates the capabilities of the Waters Dissolution System for the analysis of the components of a common oral contraceptive containing Ethinyl Estradiol and Norethindrone, in one fully automated HPLC run using both fl uorescence and UV detection.

Norethindrone (NE)2.5 µg/mL

Ethinyl Estradiol (EE)0.058 µg/mL

Fluorescence

Minutes

550.00

0.000

0.060

0.000

Fluo

resc

ence

AU

Excitation 280 nmEmission 312 nm

UV241 nm

4.00

4.00

Minutes

Dissolution Plot: EE90.00

80.00

70.00

60.00

50.00

%

Q Factor A (60.0, 80.0)

10.00 70.0020.00 30.00 40.00 50.00 60.00

Dissolution Plot: NE

Vessel 2Vessel 1

Vessel 3

Vessel 6

Vessel 4Vessel 5

Claimed Amount A: 0.03

100.00

90.00

80.00

70.00

60.00

110.00

10.00 70.0020.00 30.00 40.00 50.00 60.00

Transfer Time (min)

Claimed Amount A: 1.50

Q Factor A (60.0, 80.0)

Transfer Time (min)

%

Vessel 2Vessel 1

Vessel 3

Vessel 6

Vessel 4Vessel 5

BIOEQUIVALENC E

Bioequivalence studies play a critical role in the development of a new

generic medicine because of the necessity to prove that the drug reaches

the site of action at a similar rate and extent as the original branded

product. However, HPLC/MS/MS as the traditional industry-standard ana-

lytical technique is handicapped by ion suppression and other co-eluting

matrix effects, medium throughput, limited sensitivity and insufficient

resolution for the analysis of multi-component assays.

Since generated data is used in regulatory submissions to authorities,

analyses have to be performed according stringent to GLP standards.

This requires sophisticated and integrated laboratory solutions that

include qualification documentation and security-based software for

data collection.

Waters solutions address the end-to-end efficiency and regulatory needs of bioequivalence studies:

■ UPLC/MS/MS: Excellent chromatographic resolution and sensitivity,

even at faster analysis rates, means that this fully compliant-ready

configuration is ideally suited for the challenges of a bioanalysis

■ Oasis® Solid Phase Extraction (SPE) Sorbent Chemistries: With

a variety of Oasis SPE chemistries and formats (cartridges, 96-well

plates and micro-elution plates), Waters delivers comprehensive

sample clean-up and concentration for every application need

■ ACQUITY UPLC Sample Organizer: Maximizes the efficiency of the

UPLC/MS/MS system by accommodating large numbers of samples in a

temperature-controlled environment, ensuring maximum throughput

■ Quattro Premier™ XE and ACQUITY™ TQ Detector: Robust atmo-

spheric pressure ionization with the ability to switch between positive

and negative modes in just 20 milliseconds, allowing the rapid,

simultaneous detection of basic and acidic compounds with no loss

in sensitivity

■ MassLynx™ Software with the QuanLynx™ Application Manager:

Easy data processing and review for complex data sets

B IO EQUIVAL ENC E

Oasis SPE methodology yields a signifi cant improvement in analyte response when compared to simple protein precipitation. The MRM data for Terfenadine shows a 34% MS signal intensity loss for the precipitated sample, vs. the Oasis SPE sample with less than 5% ion suppression when compared to a standard in solution.

This comparison between an HPLC/MS/MS method and a UPLC/MS/MS method demonstrates the advantages of UPLC/MS/MS for bioanalysis. The ACQUITY UPLC System with the Quattro Premier XE produced a method with a 1.5 minute run time – more than three times faster than HPLC/MS/MS, with a three-fold reduction in the LOQ.

HPLC/MS/MS UPLC/MS/MS

UNIFORMIT Y OF CONT ENT

The analysis of a pharmaceutical product for the uniformity of content and

assay is necessary for raw material acceptance, drug formulation process

and QC for product release. The faster raw materials can be accepted into

production – or the faster that product quality can be determined – the

faster a batch can be released for shipment and profits can be realized.

Assays are typically governed by a registered method and pharma-

copoeial monographs, which have a rigid set of acceptance criteria.

These prescribed methodologies typically suffer from long analysis

times. New technologies require validated method transferability, but

these methodologies hold the key to higher throughput batch release.

Instrument reliability and service are the keys to operational success.

Waters solutions seamlessly and dependably solve content uniformity analyses:

■ ACQUITY UPLC System: Analysis times are significantly reduced in

comparison with HPLC, while meeting exacting regulatory guidelines

■ Connections INSIGHT®: An Intelligent Services Delivery Platform

that provides reactive service to predictive maintenance to ensure

maximum system uptime

■ Empower 2 Software: Custom calculations provide maximum flex-

ibility in a validated environment

0.22

0.20

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

0.00

2.00 4.00 6.00 8.00 12.00

9.281

14.00

Minutes

AU

16.00 18.00 20.0010.00

Simvastatin

0.07

0.00

0.00 0.50 1.00Minutes

1.50 2.00 2.50

0.21

AU

1.412

0.14

The Simvastatin USP Assay using HPLC shows a retention time of 9.2 minutes, with an effi ciency of 12,112 and a tailing factor of 0.94. The method was easily moved to UPLC using the ACQUITY UPLC console calculator. Using defi ned conditions of “Equal Effi ciency,” the resulting analysis produced an analyte retention time of just 1.4 minutes.

A typical report automatically generated by Connections INSIGHT shows that the ACQUITY UPLC System is working properly and that no user action is required.

UNIFO RMIT Y OF CONT ENT

CLEANING VALIDAT ION

During the pharmaceutical manufacturing process, equipment is

routinely cleaned after a production batch to avoid cross-contamination

in subsequent batches of different products. The effectiveness of the

cleaning process is confirmed by analytical measurements via a cleaning

validation method that provides evidence that the procedure removed

residues to pre-determined safety levels. This activity demands a

fast analysis that minimizes production down-time, that must also be

sensitive and specific for the accurate identification and detection of

low-level components in a sample. HPLC-based methods are often too

slow and cannot meet sensitivity and specificity requirements.

Waters solutions ensure faster cleaning validation analyses:

■ ACQUITY UPLC System: Produces high resolution separations with

short analysis times, critical to enabling a production facility to

minimize down-time

■ ACQUITY UPLC PDA and SQ Detector: For high sensitivity and

specific analyte confirmation

The cleaning validation assay for the active pharmaceutical Simvastatin in a rapid 2-minute analysis time is shown. The extra resolution generated by the Waters ACQUITY UPLC System allows for the resolution of the analytes of interest from interferences.

C L EANING VALIDAT ION

%

1.501.32

No interferences

Swab extracted with ACN

Swab extracted with 75:25 ACN: H2050 ng/mL (1) Lovastatin and (2) Simvastatin

0.50 2.001.00Time

1: SIR of 2 ChannelsES + TIC 8.40e6

1.50

Simvastatin

The ACQUITY SQD featuring the SQ Detector facilitates the detection of drug substances at very low levels, providing confi dence in the effectiveness of the cleaning process.

Waters, ACQUITY UPLC, Connections INSIGHT, Oasis, UltraPerformance LC and UPLC are registered trademarks of Waters Corporation. ACQUITY, Empower, MassLynx, QuanLynx, Quattro Premier and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2007 Waters Corporation. Printed in the U.S.A.February 2007 720001982EN JDH-CP

Waters Corporation34 Maple StreetMilford, MA 01757 U.S.A.T: 508 478 2000F: 508 872 1990www.waters.com

www.waters.com/generics