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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
iM Pu rit y P rof il inG
diSSolut ion t eSt inG Bio equivAl enc e unifo rMit y
of cont entc l eAninG
vAlidAt ion
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
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