Publications by authors named "Rand Jenkins"

35 Publications

First amyloid β1-42 certified reference material for re-calibrating commercial immunoassays.

Alzheimers Dement 2020 11 4;16(11):1493-1503. Epub 2020 Aug 4.

Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, Mölndal, Sweden.

Introduction: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aβ)1-42 (Aβ ). They are intended to be used to calibrate diagnostic assays for Aβ .

Methods: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays.

Results: The certified Aβ mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 μg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 μg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%.

Discussion: The Aβ CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aβ .
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http://dx.doi.org/10.1002/alz.12145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984389PMC
November 2020

Preanalytical approaches to improve recovery of amyloid-β peptides from CSF as measured by immunological or mass spectrometry-based assays.

Alzheimers Res Ther 2018 11 28;10(1):118. Epub 2018 Nov 28.

Division of Development Sciences, Department of OMNI Biomarker Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080, USA.

Background: Amyloid-β 1-42 (Aβ) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aβ are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aβ recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aβ peptides in stored samples.

Methods: CSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aβ concentrations were initially measured by immunoassay; subsequent determinations of CSF Aβ, Aβ, Aβ, Aβ, and Aβ concentrations were made with an absolute quantitative mass spectrometry assay. In a second study, CSF from healthy control subjects and patients with dementia was denatured with guanidine hydrochloride (GuHCl) at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay.

Results: Two distinct immunoassays demonstrated that CSF Aβ concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aβ recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aβ peptide mass spectrometry assay confirmed that Aβ peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aβ. A comparison of CSF collection and processing methods suggested that Aβ peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aβ/Aβ ratio was a useful method to reduce variability.

Conclusions: Analyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF Aβ measurements. Sample denaturation during aliquoting increases recovery of Aβ peptides and improves measurement accuracy. The Aβ/Aβ ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.
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http://dx.doi.org/10.1186/s13195-018-0445-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264029PMC
November 2018

2017 White Paper on recent issues in bioanalysis: aren't BMV guidance/guidelines 'Scientific'? (Part 1 - LCMS: small molecules, peptides and small molecule biomarkers).

Bioanalysis 2017 Nov 17;9(22):1807-1825. Epub 2017 Nov 17.

UK MHRA, London, UK.

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for Small Molecules, Peptides and Small Molecule Biomarkers using LCMS. Part 2 (Biotherapeutics, Biomarkers and Immunogenicity Assays using Hybrid LBA/LCMS and Regulatory Agencies' Inputs) and Part 3 (LBA: Immunogenicity, Biomarkers and PK Assays) are published in volume 9 of Bioanalysis, issues 23 and 24 (2017), respectively.
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http://dx.doi.org/10.4155/bio-2017-4975DOI Listing
November 2017

Do we have a mature LC-MS/MS methodology for therapeutic monoclonal antibody bioanalysis?

Bioanalysis 2017 Sep 13;9(17):1289-1292. Epub 2017 Sep 13.

Chromatographic Sciences Department, PPD Laboratories, Richmond, Virginia, USA.

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http://dx.doi.org/10.4155/bio-2017-4983DOI Listing
September 2017

Quantitation of levodopa and carbidopa in rat plasma by LC-MS/MS: The key role of ion-pairing reversed-phase chromatography.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Jun 3;1054:1-9. Epub 2017 Apr 3.

PPD Inc., Middleton, WI, United States. Electronic address:

A simple and selective bioanalytical method was developed for simultaneous determination of levodopa and carbidopa in rat plasma by LC-MS/MS. Levodopa and carbidopa are small polar molecules, posing challenges in the development of selective and efficient chromatography conditions. Perfluoropentanoic acid (PFPA), a volatile ion-pairing agent, was utilized to enhance chromatographic characteristics of both compounds in the reversed-phase mechanism. The ion-pairing chromatography played an essential role in mitigating matrix effects and achieving adequate separation between interfering background peaks and those of the analytes of interest, especially for levodopa. A 96-well based, automated liquid-liquid extraction, via the use Hamilton NIMBUS liquid handlers, was developed. Butyl alcohol, when mixed with ethyl acetate, greatly increased the recovery of both levodopa and carbidopa. The addition of PFPA further enhanced recovery for both analytes. Sodium metabisulfite, an antioxidant, was used to stabilize levodopa and carbidopa in rat plasma. The method was validated in the ranges of 50-10,000ng/mL and 25-5000ng/mL for levodopa and carbidopa, respectively, using levodopa-d3 and carbidopa-d3 as internal standards. The validated method was successfully applied to analyze rat plasma samples from in-life studies.
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http://dx.doi.org/10.1016/j.jchromb.2017.04.001DOI Listing
June 2017

Accuracy: a potential quandary in regulated bioanalysis of 'endogenous' analytes.

Authors:
Rand G Jenkins

Bioanalysis 2016 Dec;8(23):2393-2397

PPD Laboratories, 2244 Dabney Road, Richmond, VA 23230, USA.

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http://dx.doi.org/10.4155/bio-2016-0247DOI Listing
December 2016

2016 White Paper on recent issues in bioanalysis: focus on biomarker assay validation (BAV) (Part 1 - small molecules, peptides and small molecule biomarkers by LCMS).

Bioanalysis 2016 Nov 7;8(22):2363-2378. Epub 2016 Oct 7.

Novartis, Emeryville, CA, USA.

The 2016 10 Workshop on Recent Issues in Bioanalysis (10 WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis including Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This white paper is published in 3 parts due to length. This part (Part 1) discusses the recommendations for small molecules, peptides and small molecule biomarkers by LCMS. Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will be published in the Bioanalysis journal, issue 23.
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http://dx.doi.org/10.4155/bio-2016-4992DOI Listing
November 2016

Validation of a biotherapeutic immunoaffinity-LC-MS/MS assay in monkey serum: 'plug-and-play' across seven molecules.

Bioanalysis 2016 Aug 11;8(15):1565-1577. Epub 2016 Jul 11.

Genentech, Inc., South San Francisco, California, USA.

Background: Biotherapeutics development requires validated assays in biological matrices for pharmacokinetic assessment. Historically, ligand-binding assays have been the predominant platform available. Recently, alternative hybrid methods, combining ligand-binding analyte enrichment with LC-MS detection have emerged. Methodology & results: The validation of an immunoaffinity (IA)-LC-MS/MS method to quantify a monoclonal antibody biotherapeutic in cynomolgus monkey serum is described. This method includes immunoaffinity capture of the antibody in serum, followed by enzymatic digestion and detection of a framework peptide. Using similar method conditions, six additional biotherapeutic assays were readily validated in different nonhuman mammalian species, including mouse, rat and monkey.

Conclusion: The immunoaffinity-LC-MS/MS assay validation results across seven antibody therapeutics, using comparable conditions, illustrate the 'plug-and-play' nature of the IA-LC-MS/MS mAb framework peptide assay format.
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http://dx.doi.org/10.4155/bio-2016-0117DOI Listing
August 2016

CSF Aβ - an excellent but complicated Alzheimer's biomarker - a route to standardisation.

Clin Chim Acta 2017 Apr 20;467:27-33. Epub 2016 May 20.

Inst. of Neuroscience and Physiology Dept. of Psychiatry and Neurochemistry The Sahlgrenska Academy at University of Gothenburg Mölndal, Sweden; Clinical Neurochemistry LaboratorySahlgrenska University Hospital, MölndalSE-431 80 Mölndal Sweden. Electronic address:

The 42 amino acid form of amyloid β (Aβ-) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimer's disease. Several immunoassays for CSF Aβ are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.g., clinical diagnostics) and selection of individuals for enrolment in clinical trials (patient stratification) remains challenging. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has initiated a working group for CSF proteins (WG-CSF) to facilitate standardisation of CSF Aβ measurement results. The efforts of the IFCC WG-CSF include the development of certified reference materials (CRMs) and reference measurement procedures (RMPs) for key biomarkers. Two candidate RMPs for quantification of Aβ in CSF based on liquid chromatography tandem mass spectrometry have been developed and tested in two ring trials. Furthermore, two commutability studies including native CSF pools, artificial CSF and spiked materials have been completed. On the basis of these studies, human CSF pools containing only endogenous Aβ at three concentrations were selected as the format for future CRMs that are now being processed.
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http://dx.doi.org/10.1016/j.cca.2016.05.014DOI Listing
April 2017

Characterization of matrix effects in developing rugged high-throughput LC-MS/MS methods for bioanalysis.

Bioanalysis 2016 May 15;8(10):1021-34. Epub 2016 Apr 15.

PPD® Laboratories, 2240 Dabney Rd, Richmond, VA 23230, USA.

Aim: There is an ever-increasing demand for high-throughput LC-MS/MS bioanalytical assays to support drug discovery and development.

Results: Matrix effects of sofosbuvir (protonated) and paclitaxel (sodiated) were thoroughly evaluated using high-throughput chromatography (defined as having a run time ≤1 min) under 14 elution conditions with extracts from protein precipitation, liquid-liquid extraction and solid-phase extraction. A slight separation, in terms of retention time, between underlying matrix components and sofosbuvir/paclitaxel can greatly alleviate matrix effects.

Conclusion: High-throughput chromatography, with proper optimization, can provide rapid and effective chromatographic separation under 1 min to alleviate matrix effects and enhance assay ruggedness for regulated bioanalysis.
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http://dx.doi.org/10.4155/bio-2016-0005DOI Listing
May 2016

Development and validation of HILIC-ESI/MS/MS methods for simultaneous quantitation of several antipsychotics in human plasma and blood.

Bioanalysis 2016 Apr 23;8(8):765-94. Epub 2016 Mar 23.

Chromatographic Sciences Department, PPD Laboratories, 2244 Dabney Road, Richmond, VA 23230-3323, USA.

Background: Knowledge of antipsychotic drug levels at point of care (POC) may significantly aid therapeutic decision-making. To support the development of future POC devices and to validate the use of fingerstick capillary blood sampling, two robust hydrophilic interaction LC-ESI/MS/MS methods were developed and validated. Two PK studies were completed evaluating the correlation between fingerstick blood and plasma concentrations with corresponding venous blood and plasma concentrations for several commonly prescribed atypical antipsychotics and selected metabolites. Sensitive and reliable LC-MS/MS bioanalytical assays were developed to support these studies.

Results: Three methods, requiring only 25-μl matrix volumes, were developed using supported liquid extraction with hydrophilic interaction LC-MS/MS detection and validated according to regulatory guidance.

Conclusion: Robust and efficient LC-MS/MS assays were established and were effective in providing antipsychotic drug matrix comparator results in the intended clinical studies.
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http://dx.doi.org/10.4155/bio.16.27DOI Listing
April 2016

Ultrasensitive sub-pg/ml determination of tiotropium bromide in human plasma by 2D-UHPLC-MS/MS: challenges and solutions.

Bioanalysis 2016 19;8(5):385-95. Epub 2016 Feb 19.

PPD® Laboratories, 2240 Dabney Rd, Richmond, VA 23230, USA.

Background: To adequately support pharmacokinetic evaluations of tiotropium bromide in planned clinical studies. It was desirable to measure it with a LLOQ of sub pg/ml.

Results: A sensitive bioanalytical method for the determination of tiotropium in human plasma sample was successfully developed and validated in the range of 0.2-100 pg/ml. The method was successfully applied to support two clinical studies of over 3000 samples. The overall incurred sample reanalysis passing rate was 93.7%.

Conclusion: The combination of a dual stage liquid-liquid extraction and a 2D ultra-HPLC greatly reduced matrix effects and increased assay sensitivity. When developing effective ultrasensitive assays, it is imperative to balance the aspects related to sensitivity with those that will ensure assay ruggedness.
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http://dx.doi.org/10.4155/bio.15.256DOI Listing
December 2016

Comparison of a stable isotope labeled (SIL) peptide and an extended SIL peptide as internal standards to track digestion variability of an unstable signature peptide during quantification of a cancer biomarker, human osteopontin, from plasma using capillary microflow LC-MS/MS.

J Chromatogr B Analyt Technol Biomed Life Sci 2015 Sep 26;1001:156-68. Epub 2015 Jul 26.

Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA 23298, USA. Electronic address:

Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide 'GDSVVYGLR' was used as a signature peptide for this method. The optimized method involved immunocapture of the analyte protein using hOPN specific antibodies followed by trypsin digestion to obtain the signature peptide. Analysis was carried out on a Waters IonKey/MS system using a flow rate of 2.5μL/min. Immunocapture buffer was used as a surrogate matrix for the validation studies. The method was validated over a range of 25-600ng/mL. Intra-assay and inter-assay accuracies were within ±13%. Intra-assay and inter-assay precision were within 17%. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. Inherent digestion variability i.e., under controlled conditions, was within ±20% with both IS peptides. In digestion variability studies, where trypsin activity was varied (20-180%), the use of the extended SIL peptide as an internal standard limited the variability to within ±30% of the normalized response. Alternatively, when the SIL peptide was used as the internal standard, the variability ranged from -67.4% to 50.6% of the normalized response. The applicability of the validated method was demonstrated by quantification of OPN from plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. The plasma OPN concentrations in healthy individuals ranged from 38 to 85ng/mL with a mean concentration of 55.4±15.3ng/mL. A 1.5-12 fold increase in OPN concentrations, ranging from 85 to 637ng/mL, was seen in breast cancer patient samples.
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http://dx.doi.org/10.1016/j.jchromb.2015.05.040DOI Listing
September 2015

Scientific or regulated validation: a tiered approach? Meeting report from a joint EBF/DVDMDG workshop.

Bioanalysis 2015 ;7(14):1703-10

Pharma Navigators LLC, Lawrenceville, NJ, USA.

Tiered approach is rapidly gaining interest in the regulated bioanalytical community. Alternative approaches to the workflows as proposed in the regulatory Guidance (US FDA, EMA) are being used in discovery and early drug development, but with a growing array of assay types and studies requiring bioanalytical support in early drug development, the bioanalytical community is discussing how to bring best value to support these studies. Recently, international industry groups like European Bioanalysis Forum and Global Bioanalysis Consortium have discussed and published on the opportunity and need to include tiered approach more systematically in the early drug development support. On the back of these discussions, the Delaware Valley Drug Metabolism Discussion Group together with the European Bioanalysis Forum organized a meeting in Langhorne (PA, USA) to discuss the hurdles and added value of tiered approach with stakeholders from the Bioanalysis, quality assurance and PK community. The discussions focused on proposing scientific validation for studies where there is currently a mixed use of regulatory and tiered approach workflows. The meeting was well attended and the presentations and panel discussions contributed to a better understanding of what the industry is proposing as future practice.
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http://dx.doi.org/10.4155/bio.15.115DOI Listing
May 2016

Round robin test on quantification of amyloid-β 1-42 in cerebrospinal fluid by mass spectrometry.

Alzheimers Dement 2016 Jan 21;12(1):55-9. Epub 2015 Jul 21.

Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Salgrenska Academy, University of Gothenburg, Mölndal, Sweden.

Introduction: Cerebrospinal fluid (CSF) amyloid-β 1-42 (Aβ42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative.

Methods: We compared results for CSF Aβ42 quantification in a round robin study performed in four laboratories using similar sample preparation methods and LC-MS instrumentation.

Results: The LC-MS results showed excellent correlation between laboratories (r(2) >0.98), high analytical precision, and good correlation with enzyme-linked immunosorbent assay (r(2) >0.85). The use of a common reference sample further decreased interlaboratory variation.

Discussion: Our results indicate that LC-MS is suitable for absolute quantification of Aβ42 in CSF and highlight the importance of developing a certified reference material.
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http://dx.doi.org/10.1016/j.jalz.2015.06.1890DOI Listing
January 2016

Bioanalytical method validation considerations for LC-MS/MS assays of therapeutic proteins.

Bioanalysis 2015 ;7(11):1389-95

Chromatographic Sciences, PPD Bioanalytical Laboratories, 2244 Dabney Road, Richmond, VA 23230, USA.

This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC-MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC-MS/MS-based assays where proteins are quantified using the LC-MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC-MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria. Several key aspects of this bioanalytical approach that are discussed in the White Paper are treated here in additional detail. These topics include selectivity/specificity, matrix effect, digestion efficiency, stability and critical reagent considerations.
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http://dx.doi.org/10.4155/bio.15.69DOI Listing
March 2016

Workshop Report: AAPS Workshop on Method Development, Validation, and Troubleshooting of Ligand-Binding Assays in the Regulated Environment.

AAPS J 2015 Jul 30;17(4):1019-24. Epub 2015 Apr 30.

MKelley Consulting LLC, West Chester, PA, USA.

A novel format was introduced at the recent AAPS NBC Workshop on Method Development, Validation and Troubleshooting in San Diego on 18th May 2014. The workshop format was initiated by Binodh De Silva; Marie Rock and Sherri Dudal joined the initiative to develop and chair the workshop. Questions were solicited by a variety of avenues, including a Linked-In Discussion Group. Once collated and clarified, the topics covered assay development, validation, and analysis of PK, Immunogenicity, and Biomarkers with an additional topic on alternative bioanalytical technologies. A panel of experts (workshop report co-authors) was assigned to each topic to bring forward thought-provoking aspects of each topic. The format of the workshop was developed to target the needs of bioanalytical scientists with intermediate to advanced experience in the field ranging to enable robust discussion and to delve deeper into the current bioanalytical hot topics. While the new format allowed for an interactive session with the topical discussion driven by the audience members, it did not foster equal discussion time for all of the proposed topics, especially Biomarkers and alternative LBA technologies.
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http://dx.doi.org/10.1208/s12248-015-9767-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476978PMC
July 2015

An extended stable isotope-labeled signature peptide internal standard for tracking immunocapture of human plasma osteopontin for LC-MS/MS quantification.

Biomed Chromatogr 2015 Nov 27;29(11):1780-2. Epub 2015 Apr 27.

Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, 23298, USA.

A stable isotope-labeled signature peptide, whose sequence corresponds to the human osteopontin (hOPN) specific antibody epitope, was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity-coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well from 150 to 4500 ng and analysis was carried out with internal standards added before and after the immunocapture step. The immunocapture variability ranged from -80.9 to 77.0% when the IS was added after immunocapture and from -37.5 to 20.3% when the internal standard was added before immunocapture. The lower variability demonstrates the ability of stable labeled isotope internal standard peptide to compensate for variation during immunocapture.
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http://dx.doi.org/10.1002/bmc.3471DOI Listing
November 2015

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

Bioanalysis 2014 ;6(22):2957-63

Covance Laboratories, Chantilly, VA, USA.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.
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http://dx.doi.org/10.4155/bio.14.287DOI Listing
July 2015

Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics.

AAPS J 2015 Jan 13;17(1):1-16. Epub 2014 Nov 13.

Chromatographic Sciences, PPD Bioanalytical Laboratories, 2244 Dabney Road, Richmond, Virginia, 23230, USA.

This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.
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http://dx.doi.org/10.1208/s12248-014-9685-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287296PMC
January 2015

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

Bioanalysis 2013 Dec 10;5(23):2903-18. Epub 2013 Oct 10.

Biogen Idec Inc.,Cambridge, MA, USA.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science. Such as the previous editions, this 2013 White Paper addresses important bioanalytical issues and provides practical answers to the topics presented, discussed and agreed upon by the global bioanalytical community attending the 7th Workshop on Recent Issues in Bioanalysis.
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http://dx.doi.org/10.4155/bio.13.238DOI Listing
December 2013

Development and optimization of on-line 2-dimensional chromatographic approaches for eliminating matrix effects and improving bioanalysis of peptides in human plasma using UHPLC-MS/MS.

Drug Test Anal 2014 May 5;6(5):415-25. Epub 2013 Aug 5.

Zagazig University, Faculty of Pharmacy, Department of Analytical Chemistry, Egypt.

Online 2-dimensional chromatographic approaches for eliminating matrix effects and optimizing bioanalysis of peptides using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were studied. Three therapeutic peptides (octreotide, desmopressin, and vasopressin) were selected as model analytes. Human plasma was precipitated with acetonitrile; peptides were analyzed on C(8), C(18), Phenyl and HILIC ACQUITY UPLC columns. For simpler online clean-up applications, a C(18) pre-column was coupled to the analytical column via a switching valve. For more complex heart-cutting applications, two analytical columns were used with optional online dilution to refocus the analyte peaks prior to the second dimension separation. This allows the use of MS incompatible mobile phases, such as TFA, in the first dimension separation. Online clean-up effectiveness was investigated by monitoring phospholipids. Flushing direction, mobile phase composition, flow rate and transfer window were evaluated. Phospholipids were readily retained on reversed-phase columns, and the peptides were reproducibly transferred, individually or as a group, to the second column using appropriate transfer windows. The best peak shapes were obtained when the second dimension column was more retentive (e.g. C(18) vs. C(8)). However, C(8) to HILIC gave broad unresolved peaks due to mobile phase mismatch. Trapped phospholipids were efficiently removed from either guard columns or first dimensional columns by forward- or back-flushing at high flows; however, back-flushing was more efficient with lower flow rates on larger columns.
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http://dx.doi.org/10.1002/dta.1521DOI Listing
May 2014

Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies.

Bioanalysis 2013 Jun;5(11):1363-76

Bristol-Myers Squibb, Analytical & Bioanalytical Development, Princeton, NJ 08543, USA.

Background: There is a need for general and reliable LC-MS assays capable of supporting the bioanalysis of a variety of human monoclonal antibody-based therapeutic drug candidates in animal PK/TK studies.

Results: We present herein improvements in our previously reported universal peptide approach to the bioanalysis of human monoclonal antibody protein drug candidates in animal studies. These improvements include incorporation of a second, light chain-based universal peptide into the assay, thus introducing the concept of a dual universal peptide assay; and incorporation of a universal stable isotope-labeled monoclonal antibody into the assay.

Conclusion: Improvements reported herein to the universal peptide assay will enable more reliable quantification of human monoclonal antibody protein drug candidates in animal studies.
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http://dx.doi.org/10.4155/bio.13.55DOI Listing
June 2013

Investigation of endogenous blood lipids components that contribute to matrix effects in dried blood spot samples by liquid chromatography-tandem mass spectrometry.

Drug Test Anal 2013 Aug 10;5(8):710-5. Epub 2012 Oct 10.

Zagazig University Faculty of Pharmacy, Department of Analytical Chemistry, Egypt.

Dried blood spot (DBS) sampling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a rapidly developing approach in the field of biopharmaceutical analysis. DBS sampling enables analysis of small sample volumes with high sensitivity and selectivity while providing a convenient easy to store and ship format. Lipid components that may be extracted during biological sample processing may result in matrix ionization effects and can significantly affect the precision and accuracy of the results. Glycerophosphocholines (GPChos), cholesterols and triacylglycerols (TAG) are the main lipid components that contribute to matrix effects in LC-MS/MS. Various organic solvents such as methanol, acetonitrile, methyl tertiary butyl ether, ethyl ether, dichloromethane and n-hexane were investigated for elution of these lipid components from DBS samples. Methanol extracts demonstrated the highest levels of GPChos whereas ethyl ether and n-hexane extracts contained less than 1.0 % of the GPChos levels in the methanol extracts. Ethyl ether extracts contained the highest levels of cholesterols and TAG in comparison to other investigated organic solvents. Acetonitrile is recommended as an elution solvent due to low lipid recoveries. Matrix effects resulted from different extracted lipid components should be studied and assessed carefully in DBS samples.
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http://dx.doi.org/10.1002/dta.1421DOI Listing
August 2013

Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

Bioanalysis 2012 Sep;4(17):2117-26

Advion Bioanalytical Laboratories, Quintiles, NY, USA.

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.
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http://dx.doi.org/10.4155/bio.12.192DOI Listing
September 2012

Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid β42.

Biomark Med 2012 Aug;6(4):409-17

Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, the Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital, Mölndal, Sweden.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid β42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid β42.
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http://dx.doi.org/10.2217/bmm.12.39DOI Listing
August 2012

4th Global CRO Council for Bioanalysis: coadministered drugs stability, EMA/US FDA guidelines, 483s and carryover.

Bioanalysis 2012 Apr;4(7):763-8

The Global CRO Council for Bioanalysis (GCC) was formed in September 2010. Since then, the representatives of the member companies come together periodically to openly discuss bioanalysis and the regulatory challenges unique to the outsourcing industry. The 4th GCC Closed Forum brought together experts from bioanalytical CROs to share and discuss recent issues in regulated bioanalysis, such as the impact of coadministered drugs on stability, some differences between European Medicines Agency and US FDA bioanalytical guidance documents and lessons learned following recent Untitled Letters. Recent 483s and agency findings, as well as issues on method carryover, were also part of the topics discussed.
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http://dx.doi.org/10.4155/bio.12.48DOI Listing
April 2012

Determination of octreotide and assessment of matrix effects in human plasma using ultra high performance liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2011 Jul 1;879(22):2081-8. Epub 2011 Jun 1.

Zagazig University, Faculty of Pharmacy, Department of Analytical Chemistry, Egypt.

A selective UHPLC-MS/MS method for determination of the therapeutic peptide octreotide in human plasma was developed and validated. This assay used a UHPLC C(18) column with 1.7 μm particle size for efficient separation and an ion-exchange SPE for selective extraction. Octreotide and its labeled internal standard, [(13)C(6)Phe(3)] octreotide, were extracted from human plasma using a simple Oasis® WCX μElution SPE method and analyzed with a total chromatographic run time of 7.5 min. Matrix effects were studied during method development by direct monitoring of representative phospholipids. On-line removal of phospholipids using column switching and pre-column back-flushing was carried out to trap and remove any residual phospholipid matrix interferences. The UHPLC column provided baseline separation between the analyte and matrix peaks. The chromatographic conditions yielded optimal retention and excellent peak shape for both the analyte and internal standard. The assay was linear in the concentration range of 0.025-25.0 ng/ml, inter- and intra-assay precision and accuracy were within 6.1% and ±1.93%, respectively. Recovery was ∼73%. Post-extraction addition experiments showed that matrix effects were less than 4%. This method for octreotide in human plasma has been validated and utilized to support of clinical pharmacokinetic studies.
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http://dx.doi.org/10.1016/j.jchromb.2011.05.039DOI Listing
July 2011

A randomized, multiple-dose parallel study to compare the pharmacokinetic parameters of synthetic conjugated estrogens, A, administered as oral tablet or vaginal cream.

Menopause 2011 Apr;18(4):393-9

Teva Branded Pharmaceuticals USA, Woodcliff Lake, NJ, USA.

Objective: A randomized, parallel-design study was conducted to determine the pharmacokinetic profile of synthetic conjugated estrogens A (SCE-A) vaginal cream (0.625 mg SCE-A/g) when administered at intervals (1 g once daily for 7 d, then twice weekly) over a 27-day period as compared with the pharmacokinetic profile of 0.3 mg SCE-A tablets administered once daily orally for 27 days.

Methods: Blood samples were collected 48 hours before initial dosing for baseline levels and at multiple occasions during the study until 48 hours after final study dosing (day 29). Maximum plasma concentration, time to maximum plasma concentration (Tmax), and area under the curve from 0 to 24 hours were calculated at days 1, 7, and 27; in addition, area under the curve from 0 to 48 hours was calculated at days 7 and 27, and area under the curve weekly (AUCweekly) values were calculated for both groups. For purposes of comparison, ratios of AUCweekly values for vaginal cream and oral tablets were analyzed.

Results: Compared with an oral daily dose of 0.3 mg SCE-A, the steady-state systemic exposure from vaginal cream application was considerably less, with the cream-to-oral ratio being 0.45 for baseline-adjusted (BA) unconjugated estradiol, 0.30 for BA unconjugated estrone, and 0.04 for unconjugated equilin (AUCweekly). At steady-state, the systemic blood levels of BA unconjugated estrone, BA unconjugated estradiol and unconjugated equilin were significantly lower in women who received biweekly application of 1 gm vaginal cream compared to women who took an oral daily dose of 0.3 mg SCE-A tablet.

Conclusions: After intravaginal application of SCE-A vaginal cream, absorption of estrogens was lower compared with absorption after oral administration. At steady state, the systemic exposure of equilin, estradiol, and estrone was significantly lower after twice-weekly administration of 1 g SCE-A vaginal cream compared with that achieved with an oral daily dose of a 0.3 mg SCE-A tablet.
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http://dx.doi.org/10.1097/gme.0b013e3181f7a2d6DOI Listing
April 2011