Assay validation requirements for free and liposomal ... · Assay validation requirements for free...
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Dr. Christoph Siethoff Swiss BioQuant AG Kägenstrasse 18, 4153 Reinach Switzerland Assay validation requirements for free and liposomal fraction in plasma - A challenge for bioanalysis AGAH worksop –Liposomal formulations (September 2012)
Transcript of Assay validation requirements for free and liposomal ... · Assay validation requirements for free...
Dr. Christoph Siethoff
Swiss BioQuant AG
Kägenstrasse 18, 4153 Reinach
Switzerland
Assay validation requirements for free and liposomal fraction
in plasma - A challenge for bioanalysis
AGAH worksop – Liposomal formulations (September 2012)
Indroduction
� Validation parameter FDA and EMA guideline for
bioanalytical method validation
� Incurred sample reanalysis and stability
� Stability considerations of liposomes
� Separation of encapsulated and unencapsulated
drug substance
� Matrix effects in case of LC-MS quantification
� Metabolites quantification
Bioanalytical guidelines
FDA guidance 2001
FDA guidance 2012 – coming soon
AAPS workshop planned in December 2012
EMA guideline 2012
Validation Parameter
� Linearity
� Selectivity/specificity
� Precision and accuracy
� Stability of the analyte in stock and working solutions
� Stability of the analyte in matrix (short-term and long-term)
� Freeze-and-thaw stability (normally 3 to 4 cycles)
� Post processing stability
� Recovery (not required by EMA, but important for free drug)
� Carry-over of the method
� Dilution integrity for ALQ samples
Bioanalytical method validation
Validation Parameter
� Matrix effects or matrix factor - investigations
• Different matrix lots (normally 6 lots)
• Co-medications
• Haemolysed and hyperlipidaemic plasma
• Matrix from renally or hepatically impaired populations
Bioanalytical method validation
MF =Peak response in the presence of matrix / Peak
response in the absence of matrix
Validation Parameter
� Incurred sample reanalysis
Bioanalytical method validation
Variability (%) = ∗−
RV
originalrepeat )(100
The mean of the original value and the repeat result is considered to be
the reference value (RV). The ISR results are considered to be accepted if
≥ 2/3 of the variability values are within 20 %.
The variability is calculated using the following formula:
Challenges for bioanalysis of liposome drug products
� Liposomes as drug delivery systems exhibit
different distribution and pharmacokinetics than
free drug molecules.
� As for all other drug applications validated
bioanalytical methods should be available when
evaluating the pharmacokinetics and bioavailability
of a liposome drug.
� Why challenging?
There is more than one reason
� Comparative human pharmacokinetic investigations should
demonstrate not only the similarity of exposure of the non-
encapsulated and liposome encapsulated drug but they
should also demonstrate similar distribution and
elimination characteristics. Validated methods to determine
encapsulated and free concentration of the active
substance in biological samples (e.g. whole blood, plasma)
should be employed in pharmacokinetic studies.
� The concentration of encapsulated and non-encapsulated
drug substance should be determined at each sampling time
point.
Requirements for liposome drug productsEMA reflection paper 2011
� A single-dose study is recommended to assess the in vivo
stability of the liposome. The concentration-time profile should
be evaluated at multiple time points over an adequate period
of time.
� The following pharmacokinetic studies should be conducted:
• A single-dose pharmacokinetic study
• A multiple-dose study evaluating the pharmacokinetics
• a dose-proportionality study over the expected therapeutic dose
range after administration of the liposome drug product
Requirements for liposome drug productsFDA draft guidance 2002
� A validated in vitro test method should be established that
uses an appropriate simulated physiological medium and/or
human plasma and acceptance criteria for the in vitro release
of the drug substance from the liposome.
� It is not only important for assessing the (1) quality of a
liposome drug product, (2) adequacy of the process controls,
(3) release characteristics of the product over time, and (4) the
effect of CMC changes.
� For the determination of unencapsulated drug the in vitro
stability of the liposome is important as well.
� In vitro short term stability and analysis of unencapsulated
drug at t=0 h and e.g. 4 h should be investigated.
In Vitro Stability Considerations
Separation of encapsulated and unencapsulated drug
What should we measure?
There are different options:
� Total
� Encapsulated
� Unencapsulated
� Free
� Free and protein bound
(unencapsulated)
Reminder: The bioanalytical method should be able to measure encapsulated and
unencapsulated drug substance.
Doxorubicin Hydrochlorid 2 mg/mL injection
encapsulated unencapsulated
AUC 0-t (ng*h/mL) 3'500'000 32'000
c max (ng/mL) 32'000 370
t=337 h
Data from CHMP assessment report
Separation of encapsulated and unencapsulated drug
Drug release drug-protein binding
� Liposomes release drugs and unbound, protein-bound and
liposomal drug pools exist simultaneously.
� That can result in very low concentrations for the free drug for
drugs with high degree of protein binding.
� Slight variations in experimental conditions can result in high
variability of the free drug concentrations.
� Drug-protein binding should to be taken into consideration to
evaluate the sample preparation approach.
Separation of encapsulated and unencapsulated drug
Different techniques were to determine the free or unencapsulated
drug use:
� Solid phase extraction for Doxorubicin (Wei et al. 2010)
� Ultrafiltration (and dialysis) for Amphotericin B (Bekersky et al. 2002)
� On-line column switching techniques for Doxorubicin (Yamamoto et al. 2011)
Solid phase extraction
Separation of encapsulated and unencapsulated drug
� The encapsulated drug passes through the sorbent but the free level is
retained.
� Unencapsulated drug (free and protein bound) will be determined.
� No unencapsulated drug should be detected in liposomal QC samples.
� Mild conditions should be used to avoid artificial high drug concentrations.
� Shearing forces can be responsible for rupture of the liposomes.
� SPE cannot be used for less lipophilic drugs like Cytosine arabinose or
Acyclovir.
Separation of encapsulated and unencapsulated drug
Roger D.D.Demers et al. (2009)
Guang-Li Wei et al. (2010) reported 1 % release due to the SPE process
SPE was performed in five batches with six replicates of QC samples
QC sample concentration of the encapsulated drug: 4000 ng/mL
Ultrafiltration
Separation of encapsulated and unencapsulated drug
• Drug specific adsorption issues need to be evaluated when using this approach.
• Drug-protein binding needs to be considered, only the free drug (non-protein
bound drug) will be determined.
• In general, the samples are not as clean as with SPE methods (suppression).
10 kDa cut-off membrane
Membrane Processing
Time (min)
3 kDa 45
10 kDa 20
30 kDa 10
Ihor Bekersky et al., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2002, p. 834–840
Separation of encapsulated and unencapsulated drug
Ultrafiltration – Amphotericin B
� Total, measured
� Encapsulated, calculated
� Unencapsulated, unbound and
protein bound, calculated from
in-vitro data
� Free (unbound), measured
30 kDa cut-off membrane
Separation of encapsulated and unencapsulated drug
Rapid equilibrium dialysisIn general, techniques like dialysis are more time consuming
Pierce (Thermo Fisher) Rapid Equilibrium Dialysis
Phospholipid, cholesterol,
polyethylene glycol (PEG)
Determination of total drug
Bursting of the lipsomes can be
performed using:
• Organic solvents
• Detergents
• Change of pH (acids)
• Increased temperature for thermal
sensitive liposomes
Intact Liposome Matrix effects
Matrix effects
� Matrix effects can be a source of significant errors
� Glycerophosphocholines (GPChos) and 2-lyso-
glycerophosphocholines (2-lyso GPChos) are known to fragment to
form ions at m/z 184 and m/z 104, respectively.
� Phospholipids can be used as markers to evaluate matrix effects
resulting in both ion suppression and enhancement.
� Matrix effects are more pronounced in electrospray than in APCI.
APCI might be more robust in many cases even if signal intensity is
lower compared to electrospray.
Matrix effects
� MRM monitoring for phospholipids during all method development steps:
m/z 496, 524, 704, 758, 786, 806 and product ion m/z 184
� Improvement of chromatographic separation or column switching
techniques
� Sample dilution would be an appropriate possibility
� Phospholipid depletion plates for sample preparation
(e.g. HybridSPE®-PPT, OstroTM)
� Liquid-liquid extraction or supported liquid extraction
� Stable isotope labeled internal standards are essential to correct for
possible matrix effects
Managing of Matrix Effects
Matrix effects
� Signal suppression related to PEG formulation - Electrospray
RT: 0.00 - 4.00
0 1 2 3 4
T ime (min)
0
10
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Re
lative
Ab
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da
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Ab
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da
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RT : 2.29
AA: 3818953
RT: 1.98
MA: 6440050
NL: 1.22E6
T IC F: + c ESI
sid=10.00 SRM ms2
454.140
[362.095-362.105]
MS ICIS
EC-70124_119
NL: 2.38E6
T IC F: + c ESI
sid=10.00 SRM ms2
481.190
[129.995-130.005]
MS EC-70124_119
RT: 0.00 - 4.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
T ime (m in)
0
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Ab
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Ab
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RT : 2.32
MA: 62527110
RT: 2.02
MA: 8593622
NL: 1.99E7
TIC F: + c ESI
sid=10.00 SRM
ms2 454.140
[362.095-362.105]
MS EC-70124_122
NL: 2.99E6
TIC F: + c ESI
sid=10.00 SRM
ms2 481.190
[129.995-130.005]
MS EC-70124_122
Sample
10 x diluted with blank plasma
CAL
Area ratio: 0.513 Area ratio: 6.656
Ratio: 13.0
Matrix effects
� Signal suppression related to PEG formulation - APCI
RT : 0.00 - 4.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
T ime (min)
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RT : 1.93
M A: 5336656
RT: 1.65
MA: 671014
NL: 1.74E6
T IC F: + c APCI
sid=10.00 SRM
ms2 454.140
[362.095-362.105]
MS EC-70124_150
NL: 2.45E5
T IC F: + c APCI
sid=10.00 SRM
ms2 481.190
[129.995-130.005]
MS EC-70124_150
RT : 0.00 - 4.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
T im e (min)
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Re
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Ab
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RT : 1.92
M A: 64733922
RT: 1.65
MA: 729646
NL: 2.13E7
T IC F: + c APCI
sid=10.00 SRM
ms2 454.140
[362.095-362.105]
MS EC-70124_142
NL: 2.73E5
T IC F: + c APCI
sid=10.00 SRM
ms2 481.190
[129.995-130.005]
MS EC-70124_142
Sample
10 x diluted with blank plasma
CAL
Area ratio: 7.120 Area ratio: 78.022
Ratio: 10.9 and a bias of approximately 20%
Matrix effects
RT : 0.00 - 4.97
0 1 2 3 4
T ime (m in)
0
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Ab
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2.10
3.25
3.963.79
3.11
2.88
2.67
0.680.61 0.95
4.33
NL:
8.08E8
T IC MS
EC-
70124_126
EC-70124_126 #57-68 RT : 1.90-2.27 AV: 12 NL: 6.47E6
T : + c ESI sid=10.00 Q1MS [100.000-800.000]
100 200 300 400 500 600 700 800
m/z
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Re
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Ab
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680.48
592.38702.48
570.40
724.49
548.43293.03 489.27
740.40
414.86
746.51
784.40392.87133.12
315.01
370.84177.10
208.02
� Signal suppression related to PEGs from formulation – Electrospray
� For the undiluted sample a factor of 2 was observed between the two
ionization modes
TIC
Match Matrix: Liposomes, an Unusual Case
� The matrix of the samples should be matched in the preparation
of the QC (and calibration samples).
� QC samples must also contain sufficient liposome material to
account for the amounts of liposome likely to be found in study
samples.
� Phospholipid material can dramatically affect extraction
efficiencies.
� Dosage of liposomes in animal TK studies is performed on much
higher levels (volume of liposome to volume of blood) than in
human clinical trials and therefore QC samples should adequately
match the study sample matrices.
Matrix effects and QC samples
� Bench-top stability at ambient or lower temperature for at
least the duration needed for sample preparation.
� Freeze-and-thaw stability for at least the number of cycles
needed for sample analysis, ISR and repeats.
� Temperature sensitive liposomes need to be handled at lower
temperatures.
� Stability of the liposome in the autosampler needs to be
investigated for a method where plasma is injected directly for
the determination of the unencapsulated drug.
Stability considerations – ex vivo stability
Stability considerations – ex vivo stability
Roger D.D.Demers et al. (2009)
Freeze-and-thaw effectsQC sample concentration of the encapsulated drug: 4000 ng/mL
� Stabilization of the liposomes with Glycerol, Guang-Li Wei et al. (2010)
Incurred sample reanalysis
For ISR: The ISR results are considered to be accepted if
≥ 2/3 of the variability values are within 20 %.
Possible for the total, but impossible for
the unencapsulated?
� Quantification of metabolites may facilitate to assess and compare
a release rate, since metabolism of the active substance takes
place only after release from the liposomes (EMA Reflection paper
2011).
� If there are several metabolites then the choice of metabolite
should be justified on kinetic grounds.
� If a significant clinical activity exists then it might be required to
compare the kinetics of metabolites.
� Method validations are required for the metabolites as well.
� Stable isotope labeled compounds should be used for the
metabolites as well for reasons already shown for the parent drug.
Quantification of metabolites
� A second assay needs to be validated, one for the encapsulated
drug and the other for the unencapsulated drug.
� A more sensitive method is required for the unencapsulated drug
and even more challenging for the free non-protein bound drug.
� Additional QC samples with encapsulated drug need to be
included for matrix effect investigations.
� Freeze-and-thaw cycles can change the free drug concentration.
� Bench top stability (release) of the liposomal drug in matrix.
Additional validation work required
� Liposomes should be handled carefully to avoid artificial
release of the drug substance.
� Measured unencapsulated drug concentrations depend on
the applied method and are probably not reliable.
� Matrix effects need to be investigated and may depend on
the nature of the liposome composition.
� Additional validation parameter are required to investigate
the stability of the liposome (in vitro, in vivo and ex vivo).
Summary
� Are there other options to get more reliable data for the
unencapsulated drug concentration?
Many thanks for your attention
One last Question
Dr. Christoph Siethoff
Swiss BioQuant AG
Kägenstrasse 18, 4153 Reinach
Switzerland
