Inter-batch Variability Research Articles

Prothrombin time standardisation in canine samples with regard to inter-batch and inter-reagent variability

The objectives of this study were to evaluate the batch-to-batch and inter-reagent variability of prothrombin time (PT) measurements in canine plasma and to investigate the effect of different methods of standardisation. PT standard test (PT(ST)) and PT modified test (PT(MT); 1:20 sample dilution, fibrinogen supplementation) were measured using five different batches of each of two commercial reagents (the first a human placenta thromboplastin and the second a rabbit brain thromboplastin). In addition, inter-reagent variability was studied for PT(MT) in 86 canine samples (four standardised reagents) based on clotting times and after calculation of percentage activity, prothrombin ratio (PTR) or International Normalised Ratio (INR). PT(ST) and PT(MT) measurements showed only a low variation between different batches of the two reagents (CV≤5%). Calculation of percentage activity or PTR reduced differences between different reagents when compared to results reported as coagulation times. Calculation of INR using the International Sensitivity Index values provided by manufacturers for use in humans showed less favourable results. In conclusion, standardisation to reduce inter-batch variability seems unnecessary for the studied reagents. Possibilities for PT standardisation in order to achieve a reagent-independent result are limited.

Treatment of Batch in the Detection, Calibration, and Quantification of Immunoassays in Large-scale Epidemiologic Studies

Many laboratory assays measure biomarkers via a 2-stage process. Direct measurement yields relative measures that are subsequently transformed to the unit of interest by using a calibration experiment. The calibration experiment is performed within the main experiment and uses a validation set for which true values are known and relative values are measured by assays to estimate the relation between relative and absolute values. Immunoassays, polymerase chain reaction, and chromatographic approaches are among assays performed in this manner. For studies with multiple batches, data from more than a single calibration experiment are available. Conventionally, calibration of assays based on the standard curve is performed specific to each batch; the calibration experiment from each batch is used to calibrate each batch independently. This batch-specific approach incorporates batch variability but, due to the small number of calibration measurements in each batch, may not be best suited for this purpose. Mixed-effects models have been described to address interassay variability and to provide a measure of quality assurance. Conversely, when interbatch variability is negligible, a model that does not incorporate batch effect may be used to estimate an overall calibration curve. We explore approaches for use of calibration data in studies with many batches. Using a real data example with biomarker and outcome information, we show that risk estimates may vary depending on the calibration approach used. We demonstrate the potential for bias when using simulations. Under minimal interbatch variability, as seen in our data, conventional batch-specific calibration does not best use information available in the data and results in attenuated risk estimates.

Opening the Black Box of Biomarker Measurement Error

Opening the Black Box of Biomarker Measurement Error

Surrogate functionality of celluloses as tablet excipients

Background: The variety of excipients from different sources and prices to which we have access gives rise to the necessity to evaluate their functional characteristics. The aim of this work is to determine some physical and technological characteristics of celluloses from different sources, India and United States, to ascertain their functionality as tablet excipients. Methods: The used surrogate functionality properties are particle morphology and particle size distribution, compactibility, ejection pressure, and the disintegration properties of pure excipients and their compressed tablets. Results: The innovators Avicel and Croscarmellose show advantages over the generic celluloses Alfacel and Carmacel. Avicel PH 101 and 102 show an average of 26% greater compactibility than both types of Alfacel, whereas the compactibility of Croscarmellose is greater than that of Carmacel in about 50%. Avicel tablets compacted at a compaction pressure of 47 MPa exhibit shorter disintegration times (3.7 minutes) than Alfacel tablets (28 minutes), whereas Carmacel show better disintegrant properties than Croscarmellose. This occurs regardless of the similar particle morphology, size, and size distribution. As expected, all celluloses show low ejection pressures. Conclusion: The surrogate functionality properties of the generic celluloses are still considered as satisfactory to be used as tablet excipients, although they are inferior in some aspects to innovator celluloses. Alfacel and Carmacel have the potential to be used as filler, binder, and disintegrant, in the design of tablets. Moreover, one should bear in mind that the differences reported here may be altered because of a possible inter-batch variability and variations in the moisture content.

Albumin-based nanoparticles as magnetic resonance contrast agents: I. Concept, first syntheses and characterisation

To develop a platform for molecular magnetic resonance imaging, we prepared gadolinium-bearing albumin-polylactic acid nanoparticles in the size range 20-40 nm diameter. Iterative cycles of design and testing upscaled the synthesis procedures to gram amounts for physicochemical characterisation and for pharmacokinetic testing. Morphological analyses showed that the nanoparticles were spheroidal with rough surfaces. Particle sizes were measured by direct transmission electron microscopical measurements from negatively contrasted preparations, and by use of photon correlation spectroscopy; the two methods each documented nanoparticle sizes less than 100 nm and generally 10-40 nm diameter, though with significant intrabatch and interbatch variability. The particles' charge sufficed to hold them in suspension. HSA retained its tertiary structure in the particles. The nanoparticles were stable against turbulent flow conditions and against heat, though not against detergents. MRI imaging of liquid columns was possible at nanoparticle concentrations below 10 mg/ml. The particles were non-cytotoxic, non-thrombogenic and non-immunogenic in a range of assay systems developed for toxicity testing of nanoparticles. They were micellar prior to lyophilisation, but loosely structured aggregated masses after lyophilisation and subsequent resuspension. These nanoparticles provide a platform for further development, based on non-toxic materials of low immunogenicity already in clinical use, not expensive, and synthesized using methods which can be upscaled for industrial production.

Measuring behavior in mice with chronic stress depression paradigm

Many studies with chronic stress, a common depression paradigm, lead to inconsistent behavioral results. We are introducing a new model of stress-induced anhedonia, which provides more reproducible induction and behavioral measuring of depressive-like phenotype in mice. First, a 4-week stress procedure induces anhedonia, defined by decreased sucrose preference, in the majority of but not all C57BL/6 mice. The remaining 30-50% non-anhedonic animals are used as an internal control for stress effects that are unrelated to anhedonia. Next, a modified sucrose test enables the detection of inter-individual differences in mice. Moreover, testing under dimmed lighting precludes behavioral artifacts caused by hyperlocomotion, a major confounding factor in stressed mice. Finally, moderation of the stress load increases the reproducibility of anhedonia induction, which otherwise is difficult to provide because of inter-batch variability in laboratory mice. We believe that our new mouse model overcomes some major difficulties in measuring behavior with chronic stress depression models.

Induced pluripotent stem cells: a new tool for toxicology screening?

In recent years, stem cells have generated much interest as a potential tool for pharmacological and toxicology screening (CIRM 2008; ABPI 2008), due to various shortcomings of currently utilized assay models based on established cell lines, primary explanted somatic cells and laboratory animals (Cao et al. 2008; Vinoth et al. 2008). In vitro toxicology screening most commonly utilize established cell lines of cancerous/tumorigenic origin, that are highly adapted to in vitro culture conditions after countless passages, and which contain chromosomal and genetic aberrations that render them immortal (Phelps et al. 1996). Such inherent deWciencies make them non-representative of how a normal cell behaves physiologically in vivo. Indeed, it is common knowledge that immortalized transformed cell lines are more robust, proliferates faster, and have much less fastidious nutritional requirements compared to primary somatic cells explanted from living tissues (Phelps et al. 1996). Hence it may be preferable to utilize primary explanted cultures of somatic cells for toxicology screening, but these often are heterogeneous cultures that display a high degree of inter-batch variability, making it challenging to obtain consistent and reproducible results in toxicology screening (Cao et al. 2008; Vinoth et al. 2008). Additionally, primary cell cultures of human origin often suVer from inconsistent availability, depending on the will of patients to donate. It is often the case that primary cultures are established from discarded human tissues of pathological origin, which would skew their response to toxic challenge. Moreover, one of the main logistical improvements realized in cellbased screening in recent years has been the introduction of batch-based cryopreserved cell preparations in high throughput screening, which have greatly improved reproducibility in assay performance (Zaman et al. 2007). However, due to a high degree of heterogeneity and interbatch variability in primary explanted somatic cells, these often display much inconsistency in their post freeze–thaw viability and metabolic activity unlike established cell lines (Zaman et al. 2007), which could further confound the reproducibility and accuracy of screening assays based on primary explanted somatic cells. The logistical challenges of high throughput cell based assays cannot be underestimated and the introduction of any new screening paradigms needs to be accompanied by robust and practical working practices, conditions that are diYcult to be met with primary explanted somatic cells. Live animal models could provide yet another alternative for toxicology screening, but have a number of inherent Xaws. First, an animal model may not compare well with human physiology. Second, the use of live animals in routine toxicology screening of biomedical and cosmetic products may be ethically contentious, and can possibly aVect consumer conWdence. Last, live animals are expensive to purchase and maintain compared to in vitro cultured cells. B. C. Heng (&) Abbott Vascular Inc., 3200 Lakeside Drive, Santa Clara, CA 95054, USA e-mail: boonchinheng@gmail.com

Considerations for powering a clinical proteomics study: Normal variability in the human plasma proteome.

Proteomics is increasingly being applied to the human plasma proteome to identify biomarkers of disease for use in non-invasive assays. 2-D DIGE, simultaneously analysing thousands of protein spots quantitatively and maintaining protein isoform information, is one technique adopted. Sufficient numbers of samples must be analysed to achieve statistical power; however, few reported studies have analysed inherent variability in the plasma proteome by 2-D DIGE to allow power calculations. This study analysed plasma from 60 healthy volunteers by 2-D DIGE. Two samples were taken, 7 days apart, allowing estimation of sensitivity of detection of differences in spot intensity between two groups using either a longitudinal (paired) or non-paired design. Parameters for differences were: two-fold normalised volume change, α of 0.05 and power of 0.8. Using groups of 20 samples, alterations in 1742 spots could be detected with longitudinal sampling, and in 1206 between non-paired groups. Interbatch gel variability was small relative to the detection parameters, indicating robustness and reproducibility of 2-D DIGE for analysing large sample sets. In summary, 20 samples can allow detection of a large number of proteomic alterations by 2-D DIGE in human plasma, the sensitivity of detecting differences was greatly improved by longitudinal sampling and the technology was robust across batches.

HPLC–APCI–MS for the determination of vitamin K 1 in human plasma: Method and clinical application

A sensitive HPLC–APCI–MS method for the determination of vitamin K 1 (VK-1) in human plasma was established. Target ions at [M+H] + m/ z 451.5 for VK-1 and [M+H] + m/ z 331.4 for the I.S. (teprenone). Calibration curve was linear over the range of 0.3–1000 ng/ml. The lower limit of quantification was 0.3 ng/ml. The intra- and inter-batch variability values were less than 8% and 15%, respectively. The C max was 210.1 ± 86.7 ng/ml while the elimination half-life ( t 1/2) was 8.8 ± 1.7 h and time to the C max was 5.5 ± 0.8 h after administration of soft capsule containing 10 mg VK-1.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of xanthinol in human plasma and its application in a bioequivalence study of xanthinol nicotinate tablets

A sensitive, rapid liquid chromatographic–electrospray ionization mass spectrometric method for the determination of xanthinol in human plasma was developed and validated. Xanthinol nicotinate in plasma (0.5 mL) was pretreated with 20% trichloroacetic acid for protein precipitation. The samples were separated using a Lichrospher silica (5 μm, 250 mm × 4.6 mm i.d.). A mobile phase of methanol–water containing 0.1% formic acid (50: 50, v /v) was used isocratically eluting at a flow rate of 1 mL/min. Xanthinol and its internal standard (IS), acyclovir, were measured by electrospray ion source in positive selected reaction monitoring mode. The method demonstrated that good linearity ranged from 10.27 to 1642.8 ng/mL with r = 0.9956. The limit of quantification for xanthinol in plasma was 10.27 ng/mL with good accuracy and precision. The mean plasma extraction recovery of xanthinol was in the range of 90.9–100.2%. The intra- and inter-batch variability values were less than 4.8% and 7.9% (relative standard deviation, R.S.D.), respectively. The established method has been successfully applied to a bioequivalence study of two xanthinol nicotinate tablets for 20 healthy volunteers.

Stereoselective analysis of tiopronin enantiomers in rat plasma using high‐performance liquid chromatography‐electrospray ionization mass spectrometry after chiral derivatization

A specific and relatively sensitive high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) was developed for the quantitative analysis of tiopronin enantiomers in rat plasma. The method is based on the derivatization of (+)-tiopronin and (-)-tiopronin with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate (GITC) in acetonitrile. The separation of resulting diastereomic derivatives was performed on C18 column (150 mm x 2.0 mm ID, packed with 5.0 mum C(18) silica RP particle), using a mobile phase of methanol/water (containing 5.3 mM formic acid) with gradient elution. LC-MS was performed in the selected ion monitoring and positive ion mode using target ions at m/z: 575 for the diastereomic derivatives of tiopronin and m/z: 603 for the derivative of N-isobutyryl-D-cysteine (internal standard). The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The calibration curves were linear over the concentration range of 0.025-5 microg/ml for both enantiomers of tiopronin. For both enantiomers of tiopronin, the interbatch and intrabatch variability values were less than 15%, and the accuracy was within +/-17% in terms of relative error. The method was successfully applied to a pharmacokinetic study of rac-tiopronin in rat.

Determination of raltitrexed in human plasma by high performance liquid chromatography–electrospray ionization-mass spectrometry

A sensitive high performance liquid chromatography–electrospray ionization-mass spectrometry (HPLC–ESI-MS) assay was developed to determine raltitrexed in human plasma. After addition of benazeprilat as the internal standard (IS), methanol was used to produce a protein-free extract. Chromatographic separation was achieved with a Zorbax SB-C18 column (Narrow-Bore 2.1mm×150mm, 5-μm) using a mobile phase of acetonitrile–water containing 0.1% formic acid and 2% methanol (21.9:78.1, v/v). Raltitrexed was determined with electrospray ionization-mass spectrometry. HPLC–ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at [M+H]+m/z 459.1 for raltitrexed and [M+H]+m/z 397.1 for IS. Calibration curves were linear over the range of 2.0–3000ng/ml. The lower limit of quantification was 2.0ng/ml. The intra- and inter-batch variability values were less than 6.7% and 10.3%, respectively. The mean plasma extraction recovery of raltitrexed was in the range of 85.2–91.1%. The method was successfully applied to determine the plasma concentrations of raltitrexed in eight Chinese colorectal cancer patients.

HPLC–APCI–MS for the determination of teprenone in human plasma: Method and clinical application

A sensitive high performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (HPLC–APCI–MS) method for the determination of teprenone (GGA) in human plasma using menatetrenone as the internal standard (I.S.) was established. After protein precipitation with ethanol, the plasma sample was extracted by cyclohexane and separated by high performance liquid chromatography on a reversed phase C 8 HPLC column with a mobile phase of water–methanol (4:96, v/v). GGA was determined with atmospheric pressure chemical ionisation–mass spectrometry (APCI–MS). HPLC–APCI–MS was performed in the selected ion monitoring (SIM) mode using target ions at [ M + H] + m/ z 331.3 for GGA and [ M + H] + m/ z 445.4 for the I.S. Calibration curve was linear over the range of 0.3–1000 ng/ml. The lower limit of quantification was 0.3 ng/ml. The intra- and inter-batch variability values were less than 7.8% and 8.7%, respectively. The method was successfully applied in the pharmacokinetic study in which plasma concentrations of GGA in 20 healthy Chinese volunteers were determined up to 24 h after administration of capsule containing 50 mg GGA. The maximum GGA plasma concentration ( C max) was 246.9 ± 85.4 ng/ml, the elimination half-life ( t 1/2) was 3.38 ± 1.20 h, and the time to the C max was 5.35 ± 1.39 h.

Comparative evaluation of freeze‐dried and liquid antigens in the direct agglutination test for serodiagnosis of visceral leishmaniasis (ITMA‐DAT/VL)

The direct agglutination test (DAT) for visceral leishmaniasis (VL) with liquid (LQ) antigen is known to be only moderately reproducible because of inter-observer and batch-to-batch variability as well as its sensitivity to temperature and shaking during transport. We evaluated a DAT with freeze-dried (FD) antigen and compared it with the LQ antigen version. Blood samples of clinical VL suspects and healthy endemic controls were collected in Sudan, Nepal and India. Both test versions were performed in duplicate in the respective countries and in the reference laboratory. Interbatch variability and stability tests were conducted and agreement was examined within and between centres on a dichotomic scale by Cohen's kappa as well as on a continuous scale through Bland-Altman plots. The FD antigen remains fully active even after storage at 45 degrees C for 24 months. Using a cut-off titre of 1:6400, the agreement between the FD and the LQ formats was excellent. The major advantages of FD antigen are its better stability at higher temperatures and its longer shelf life, which make it much more suitable than the LQ version for use in the field.

An Improved Method of Elimination of DNA from PCR Reagents

The presence of exogenous DNA in commercially available polymerase chain reaction (PCR) reagent preparations is a serious problem when amplifying conserved regions of bacteria. The preferred and currently in-use method of decontamination using 8-methoxypsoralen (8-MOP) and UVA requires re-standardization of decontamination with increasing concentrations of 8-MOP and UVA irradiation timings, if the DNA load of reagents is high due to lot-to-lot differences. The objective of this study was to develop a decontamination method, which would (i) work at the minimum reported concentration of 8-MOP and UVA irridation timings; and (ii) take care of inter-batch DNA-load variability of reagents. The improved method described here was formulated after studying the exact molecular mechanism of action of 8-MOP with DNA. The successful working of the method was experimentally proven and validated with 6-7 new batches of PCR reagents. The sensitivity of eubacterial PCR, after using the new method of decontamination, to be used clinically was checked with both the spiked specimens and the actual clinical specimens. The new method was found to work at the same starting parameters of 8-MOP and UVA in such situations. The increased efficiency of this method was found to be due to the synergistic effect of both the selective treatment of Taq DNA polymerase and the split-irradiation approach.

Inter-batch variability of extended culture media does not result in changes in embryo quality at the blastocyst stage

Background: An intangible factor in assisted reproductive technology (ART) outcomes may be lot-to-lot variation in culture media used. The study of outcomes obtained with different batches of media may clarify the impact of this variable on the ability of cleavage stage embryos to develop appropriately in extended culture.

Inter-batch variability of extended culture media does not result in changes in embryo quality at the blastocyst stage

Background: An intangible factor in assisted reproductive technology (ART) outcomes may be lot-to-lot variation in culture media used. The study of outcomes obtained with different batches of media may clarify the impact of this variable on the ability of cleavage stage embryos to develop appropriately in extended culture.

Development of bioassays using the bovine model to measure the efficiency of SCNT in humans

Development of bioassays using the bovine model to measure the efficiency of SCNT in humans

Reconstructed Skin Kits: Reproducibility of Cutaneous Irritancy Testing

The aim of this study was to determine the reproducibility of data obtained from in vitro irritation testing using three industrial reconstructed human epidermis models, EpiDerm^TM, Episkin^TM and SkinEthic^R, and one in-house model developed at Wella/Cosmital. A common protocol was established based on the measurement of cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and of extracellular release of proinflammatory mediators and cytosolic enzymes after a range of exposure times to sodium lauryl sulfate (SLS). This time course protocol was applied to 6 different batches of each skin model using triplicate tissue cultures per test condition. The parameters analyzed for intra- and inter-batch reproducibility were the cell viability determined as MTT reduction capacity and the ET-50 values in the 6 batches, as well as the release of the cytokine IL-1α and of the cellular enzymes LDH and GOT in 3 batches only. The MTT viability results showed that EpiDerm was the most resistant to the SLS treatment and at the same time the most reproducible model, SkinEthic was the most sensitive to SLS and the least reproducible, and Episkin and the Cosmital model were intermediate. Measurements of IL-1α release showed a relatively high intra- and inter-batch variability in all the skin models. It was not possible to detect the extracellular release of the enzymes LDH and GOT in the Episkin assay medium. With the 3 other models, the release of LDH and GOT varied in about the same range as that of IL-1α. For all the parameters in this study, the inter-batch variability was generally greater than the intra-batch variability. A possible reduction in the number of batches and replicates for future applications in routine irritancy testing is discussed on the basis of the results obtained using 6 batches in triplicate.

Comparison of German St. John's Wort Products According to Hyperforin and Total Hypericin Content

To compare the hyperforin and hypericin content of currently available St. John's wort products and to determine their batch-to-batch reproducibility. Representative products were obtained either directly from the manufacturer or purchased from pharmacies in and around Frankfurt, Germany. For five batches from each of the eight manufacturers, 10 individual dosage forms (tablets or capsules) were analyzed for both hyperforin and hypericin content. Laboratories of the Institute of Pharmaceutical Chemistry at Johann Wolfgang Goethe University, Frankfurt, Germany. PRODUCTS: Eight German St. John's wort products containing from 250 mg to 612 mg dry extract were studied. Three of these products are capsules, four are film-coated tablets, and one is a sugar-coated tablet. Two of the products (Jarsin 300 and Neuroplant 300) are also available in the United States. Hyperforin concentrations were analyzed by high-performance liquid chromatography. Total hypericin concentrations were determined by polarography, an electrochemical method. Concentrations were compared among different batches of the same product and among products from different manufacturers. The products contained widely differing amounts of hypericin and hyperforin, even after correcting for differences in the amount of extract per dose. Some products demonstrated consistent concentrations of hyperforin and hypericin from batch to batch, others exhibited pronounced interbatch variability. The St. John's wort preparations studied exhibited large differences in hypericin and hyperforin content and are not interchangeable for the treatment of mild-to-moderate depression. Pharmacists should take this variability into account when counseling patients on the use of St. John's wort products.