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Competing Interests: Competing interest statement: C.L.S. is employed through Torreyana Corp, San Diego, CA; Blackhawk Genomics, Concord, CA; and Advagenix, Rockville, MD. Volunteer activities of C.L.S. include those for College of American Pathologists, Northfield, IL, and Clinical Laboratory Standards Institute, Wayne, PA. T.J.O. is a Member, Scientific Advisory Committee, MioDx and Integrated Nano-Technologies. A.W.I.L.’s laboratory receives sponsorships from AstraZeneca (HK) Ltd. and MSD (HK) Ltd. for providing selected companion diagnostic tests free to public patients. N.C. is an employee at Quest Diagnostics, Inc. F.A.M. is an employee and stock option holder at Castle Biosciences, Inc. A.B.C. is paid teaching faculty for the American Medical Informatics Association Clinical Informatics Board Review Course and receives small honoraria as well as travel reimbursement to speak at multiple scientific and professional medical society meetings. T.L.P. has been consulted (compensated) for Bio-Rad, Inc. and is Director of Pathology Strategies for the Sturge Weber Foundation (compensated). H.E.W. has employment through Viapath, a majority National Health Service-owned independent pathology service provider; has been a paid faculty member at Kingston University, accepted paid accommodation and subsistence as an invited speaker to Cytocell User Group meeting for the United Kingdom and Ireland, and accepted paid event registration as an invited speaker to Digital Pathology/Global Engage meeting. H.E.W.’s laboratory received scholarship funds from The International Council for Standardization in Haematology for providing JAK2 testing. The other authors (K.F., E.A.B., P.M., N.R.P., K.P.P., B.S., M.S., M.L.G., S.M.M., H.F., and S.O.) do not have any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this article.

Compendial testing methods are not required to be fully validated, but their suitability for testing should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in the United States. ANVISA (Agência Nacional de Vigilância Sanitária) also requires that compendial analytical methods shall have their suitability demonstrated for the intended use by a partial validation study. Suitability verifications or partial validation can be divided into two major categories: visual and instrumental methods. For visual methods, the color and opalescence of interferences should be checked. If the color or clarity/opalescence of the sample is outside of the range of the Pharmacopeia standards/reference solutions, the validity of the test results should be evaluated. Specificity is usually waived because the methods are not specific to products, and accuracy/precision can be addressed by comparing results from analyst to analyst. For instrument methods, specificity can also be waived for certain assays. Accuracy is addressed by implementation of instrument calibration and/or method control. Precision is required either in suitability verification or when testing the samples. Here, we present approaches for suitability verification and the scientific rationale supporting compendial methods: visible particulates, subvisible particles, pH, osmolality, color and clarity/opalescence. Current challenges and recommendations are also discussed specifically for the analysis of protein products., (© PDA, Inc. 2020.)

The Empirical Statistical Model (ESM) for decoy library searching fused the expected amino acid sequence of 18 non-human protein standards to a human decoy library. The ESM assumed a priori the standards were pure such that only the 18 nominal proteins were true positive, all other proteins were false positive, there was no overlap in the peptides of non-human proteins versus human proteins, and that the score distribution of individual peptides would resolve true positive from false positive results or noise. The results of random and independent sampling by LC-ESI-MS/MS indicated that the fundamental assumptions of the ESM were not in good agreement with the actual purity of the commercial test standards and so the method showed a 99.7% false negative rate. The ESM for decoy library searching apparently showed poor agreement with SDS-PAGE using silver staining, goodness of fit of MS/MS spectra by X!TANDEM, FDR correction by Benjamini and Hochberg, or comparison to the observation frequency of null random MS/MS spectra, that all confirmed the standards contain hundreds of proteins with a low FDR of primary structural identification. The protein observation frequency increased with abundance and the log 10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification., (Copyright © 2020. Published by Elsevier Inc.)

The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution., (© 2020 Cold Spring Harbor Laboratory Press.)

Low volume aseptic filling of parenterals, particularly monoclonal antibodies is becoming increasingly important with the development of more and more intravitreal drugs and high concentrated formulations. Especially monoclonal antibodies are very delicate products to fill and the use of the right fill finish equipment plays an important role during process development. Protein aggregation can occur under conditions described in literature and can be influenced by the fill finish processing. The mechanism of product stress inside the filling systems is yet not fully understood. This study evaluated three different dosing systems to assess protein degradation caused by the shear rate during low volume filling of monoclonal antibodies. The newly developed quantitative liposomal shear stress model revealed the highest shear rate in the radial peristaltic pump, followed by the rotary piston pump and the linear peristaltic pump. In contrast to that, we found the highest sub-visible particle counts (>2 µm) in the rotary piston pump. We used computational fluid dynamics for a better and deeper understanding of filling processes inside the different dosing systems. Our results document that the rotary piston pump creates a recirculation zone inside the cylinder, where the protein formulation could be trapped and be exposed to the shear stress multiple times resulting in a cumulative shearing. This finding could serve as an explanation for the highest sub-particle counts in low volume filling using a rotary piston pump., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Edible insects such as black soldier fly larvae (BSFL) are alternative protein sources for animal feeds due to their high-protein content and potential low environmental footprint. However, protein quality and AA content may vary across insect species and age. Our objective was to determine the effects of age on nutrient and AA digestibility of BSFL intended for use in pet foods using the precision-fed cecectomized rooster assay. All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation. Twenty-four cecectomized roosters (four roosters per substrate) were randomly assigned to test substrates [BSFL0 = day 0 (day of hatch); BSFL11 = day 11; BSFL14 = day 14; BSFL18 = day 18; BSFL23 = day 23; BSFL29 = day 29]. After 24 h of feed withdrawal, roosters were tube-fed 20 g of test substrates. Following crop intubation, excreta were collected for 48 h. Endogenous corrections for AA were made using five additional cecectomized roosters. All data were analyzed using a completely randomized design and the GLM procedure of SAS 9.4. DM and OM digestibilities were not different among substrates, but acid-hydrolyzed fat digestibility tended to be greater (P < 0.10) for BSFL23 and BSFL29 than BSFL14 and BSFL18. Although all substrates had a high digestibility, BSFL0 and BSFL11 had the lowest (P < 0.05) digestibilities for most indispensable and dispensable AA. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to AAFCO nutrient profiles and NRC recommended allowances for dogs and cats. In general, BSFL18 had the highest, and BSFL11 had the lowest DIAAS-like values for most indispensable AA. Threonine, methionine, and tryptophan were often the first-limiting AA. Our results suggest that BSFL are a high-quality protein and AA source, but that age can affect the AA digestibility and protein quality of this alternative protein source., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Visible particles may potentially pose safety and efficacy concerns if inadvertently administered to patients; therefore, it is crucial to monitor and characterize these particles. These particles may be composed of proteinaceous or non-proteinaceous material. Although particles made of non-proteinaceous material are unacceptable in drug products, proteinaceous particles may be acceptable on a case-by-case basis if they are characterized and shown to not pose any quality, efficacy, or safety concerns. The focus of this manuscript is on the proteinaceous particles that may potentially form in some biopharmaceuticals. Monitoring and tracking proteinaceous particles in these biopharmaceuticals can be challenging, but a universal protein-like particle standard might be able to help. The aim of this work is to evaluate abraded ethylene tetrafluoroethylene (ETFE) as a visible protein-like particle standard and demonstrate a semiquantitative method to show how this surrogate can be used to effectively monitor proteinaceous particles during formulation and analytical development. Studies indicated that the ETFE particles in solution better mimic the appearance and behavior of protein particles than the commonly used polystyrene microsphere standards and therefore could be a viable standard for visible proteinaceous particles. Such standards and the semiquantitative method illustrated could be used effectively during development to nondestructively identify potential stability problems. LAY ABSTRACT: Routine visual inspection of protein biopharmaceuticals is crucial to ensure the quality and consistency of drug products. Visible particles may potentially pose safety and efficacy concerns if administered to patients; therefore, it is important to monitor and to minimize them as much as possible. Visible proteinaceous particles, composed of aggregated protein in biopharmaceuticals, may be acceptable on a case-by-case basis if they are characterized and shown not to pose any quality, efficacy, or safety concerns. Monitoring and tracking these visible proteinaceous particles are challenging and could be aided by the use of a universal protein-like particle standard. In this work, a new visible protein-like particle surrogate made of ethylene tetrafluoroethylene (ETFE) will be introduced, and its use will be explored by developing a semiquantitative method to monitor proteinaceous particles in protein products. These studies show that ETFE particles possess desirable traits to become a viable protein-like particle standard that could be used during formulation development and to nondestructively identify potential stability problems., (© PDA, Inc. 2019.)

The aim of our study was to correlate the serum concentration of human epididymis protein 4 (HE4) in lung cancer patients with the bone metastases detected by whole-body bone scintigraphy. The serum concentrations of HE4 were determined by electrochemiluminescence immunoassay method in 60 patients with lung cancer and in 10 persons without malignant disease (control group). All participants were examined by whole-body bone scintigraphy with hybrid gamma camera of type BrightView XCT. We found bone metastases in 25.0% of patients by whole-body bone scintigraphy and probable bone metastases in 18.3% of patients. We did not observe bone metastases in 56.7% of patients and in nobody from control group. We observed that 73.33% patients with bone metastases had more than 3 bone metastasis deposits. Patients had significantly increased concentration of HE4 (p < 0.0001). All three subgroups of patients (bone metastases, probable bone metastases, no evidence of bone metastases) had significantly increased concentration of HE4 compared to controls. The highest concentration of HE4 had 9 patients with small-cell lung cancer of whose 4 patients had bone metastases, 4 patients had probable bone metastases and one patient was with no evidence of bone metastases. We found that HE4 has a discriminatory ability to differentiate groups of patients and healthy controls, as well as within scaffold scintigraphy in patients with lung cancer (p = 0.0002). The serum concentration of human epididymis protein 4 was significantly increased in patients with lung cancer in comparison with persons of control group. A quarter of lung cancer patients had identified bone metastases by whole-body bone scintigraphy and approximately 20% of patients had probable bone metastases. The increasing serum concentrations of human epididymis protein 4 can have importance in the diagnosis of bone metastases in patients with lung cancer, in particular in small-cell lung cancer.

The increasing application of Single-Use Systems (SUSs) in pharmaceutical manufacturing lines poses a potential risk of polymer-related impurities leaching into the process stream and persisting through the manufacturing process. To minimize any potential toxicity and impairment to the product's quality, safety thresholds are strictly regulated and enforced in particular for parenteral solutions. At present, impurities are estimated from extractable profiles, which are generated for each SUS with thermal or static extraction. In this study we employed target leachable-testing by taking samples directly from an industrial filling line probed during real-life processing of three parenteral drugs (n = 2) under actual process-conditions, to estimate the concentration of leachables throughout drug-manufacturing. At five different points, samples were drawn to study the individual impact of SUSs on the leachable accumulation within the drug-filling process. The drug products were examined for leachables using stir-bar-sorptive-extraction (SBSE) with polydimethylsiloxane (PDMS) and ethylene glycol (EG)-PDMS coated stir-bars. Subsequent extraction from the stir-bars and analysis of the substances was performed with TD-GC-MS and solvent-back-extraction (SBE)-UPLC/QTOF-MS/MS analytics. Our study revealed the following main results: 1) Leachables were found in extremely low concentrations, all below toxicological thresholds (highest leachable concentration in the final drug product 1 (DP1): 0.274 ppm < drug specific threshold: 6.0 ppm | DP2: 0.010 ppm < 0.2 ppm | DP3: 0.011 ppm < 0.5 ppm). All compounds identified in the leachables study were found to be non-genotoxic. 2) Most of the leachables (68%) that were found were already observed at the beginning of the filling process, delivered by the API Neither a common source of leaching could be identified within the filling-line nor a specific product influence on quality or quantity of leachables. 3) No leachable increase could be observed over the filling process. On the contrary leachable concentrations declined with 83%, which was partly due to dilution by buffer-feed and to a proven absorption of leachables by filters and silicon tubing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The circulating level of the peptide hormone insulin-like factor 3 (INSL3) is a promising diagnostic marker reflecting Leydig cell function in the male. Few commercial immunoassays of varying quality exist. Therefore, we decided to develop and validate a precise method for quantification of INSL3 by mass spectrometry., Methods: We developed an assay in which the INSL3 A-chain is released from the INSL3 A-B heterodimer by chemical reduction and alkylation. The alkylated INSL3 A-chain is quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), as substitute for serum INSL3. The method was compared to a validated and sensitive in-house serum INSL3 immunoassay using 97 serum samples from 12 healthy boys during pubertal transition. Adult levels were determined based on sera from 72 adult healthy males aged 18-40 years., Results: An LC-MS/MS assay with limit of detection and limit of quantification (LOQ) of 0.06 and 0.15 ng/mL, respectively, and intra-assay CVs <9% in the relevant ranges was obtained. The LC-MS/MS compared well with the in-house immunoassay (Deming regression slope: 1.28; Pearson correlation: R=0.86). INSL3 concentrations increased with pubertal maturation in healthy boys. INSL3 concentrations were above the LOQ in all samples from the adult men. The mean (±2 SD range)for serum INSL3 concentrations in the adult men was 2.2 (0.5-3.9) ng/mL., Conclusions: We have developed a robust and sensitive method suitable for quantitation of serum INSL3 in a clinical setting using LC-MS/MS instrumentation available in modern clinical laboratories. The method paves the way for future studies into the clinical role of serum INSL3 measurements.

This article introduces the white paper from the 2017 Viral Clearance Symposium. The 5th Viral Clearance Symposium in Penzberg, Germany, addressed regulatory perspectives presented by officials from Health Canada, the US Food and Drug Administration, and Paul-Ehrlich-Institut as well as upstream and facility risk mitigation, downstream unit operations, and viral clearance strategies to support novel molecule formats, accelerated scenarios, and continuous processing. LAY ABSTRACT: This article introduces the summarized findings and next steps from the 2017 Viral Clearance Symposium in Penzberg, Germany., (© PDA, Inc. 2018.)

The process capability and potential for various forms of chromatography to remove viruses have been discussed extensively in the literature, including the observed variability in performance of some unit operations such as Protein A and cation exchange (CEX). Some unit operations such as anion exchange (AEX) have shown robustness over a wide range of operating conditions. The robustness and effectiveness of the AEX step combined with a detailed understanding of the mechanisms that result in virus and impurity partitioning versus protein partitioning (e.g. conductivity, loading, pH range) support the feasibility of a generic or modular claim for AEX. A more fundamental understanding of the mechanisms for other chromatographic media (Protein A, CEX, and mixed-mode) could lead to more effective and more robust log reduction value (LRV) claims for these steps as well. Specific examples of CEX and mixed-mode chromatography were explored in the session and were also discussed in detail at the 2013 and 2015 Viral Clearance Symposia. Although some gaps remain in the mechanistic understanding of these unit operations, significant progress has been made and was reported at the 2017 Viral Clearance Symposium. It is important to note that recent publications on the mechanisms of viral clearance for mixed-mode chromatography and a framework for measurement of relative hydrophobicity have provided insights and new tools to better define the operating space and critical process parameters. The session also explored the use of next-generation mixed-mode adsorbers and the potential mechanisms contributing to the observed viral clearance. Gaps were also identified (e.g. integrity test when size-based mechanisms are used) and should be addressed to ensure robust viral clearance for these integrated and productive emerging unit operations. LAY ABSTRACT: Preparative chromatography is the most widely used unit operation for purification of therapeutic proteins. This session focused on the potential for various forms of chromatography to remove viruses. To advance to the next level of process, understanding the virus removal mechanism of different types of chromatography was addressed., (© PDA, Inc. 2018.)

Background: The European Federation of Clinical Chemistry and Laboratory Medicine European Biological Variation Study (EuBIVAS) has been established to deliver rigorously determined data describing biological variation (BV) of clinically important measurands. Here, EuBIVAS-based BV estimates of serum electrolytes, lipids, urea, uric acid, total protein, total bilirubin, direct bilirubin, and glucose, as well as their associated analytical performance specifications (APSs), are presented., Method: Samples were drawn from 91 healthy individuals (38 male, 53 female; age range, 21-69 years) for 10 consecutive weeks at 6 European laboratories. Samples were stored at -80 °C before duplicate analysis of all samples on an ADVIA 2400 (Siemens Healthineers). Outlier and homogeneity analyses were performed, followed by CV-ANOVA on trend-corrected data, when relevant, to determine BV estimates with CIs., Results: The within-subject BV (CV I ) estimates of all measurands, except for urea and LDL cholesterol, were lower than estimates available in an online BV database, with differences being most pronounced for HDL cholesterol, glucose, and direct bilirubin. Significant differences in CV I for men and women/women <50 years of age were evident for uric acid, triglycerides, and urea. The CV A obtained for sodium and magnesium exceeded the EuBIVAS-based APS for imprecision., Conclusions: The EuBIVAS, which is fully compliant with the recently published Biological Variation Data Critical Appraisal Checklist, has produced well-characterized, high-quality BV estimates utilizing a stringent experimental protocol. These new reference data deliver revised and more exacting APS and reference change values for commonly used clinically important measurands, thus having direct relevance to diagnostics manufacturers, service providers, clinical users, and ultimately patients., (© 2018 American Association for Clinical Chemistry.)

The requirement for visual inspection of pharmaceuticals has been a compendial expectation for over a century, with some advancement of visible particle control strategies in recent years. Current philosophies include a 100% inspection and an Acceptance Sampling Plan inspection. The particles found during these inspections are normally categorized simply by particle size (visible vs. subvisible), particle source (intrinsic vs. extrinsic) and particle type (inherent vs. extraneous). We believe that a more risk- and science-based approach is attainable, which is grounded in forensic data, toxicological/medical opinions and prior knowledge. We have provided an outline for how to determine patient safety impact of visible particles found in parenteral products and potential actions that could be taken within the quality system regarding lot disposition. We believe this approach focuses efforts on patient safety risks, enhances the use of prior knowledge and improves consistency in how particle observations are handled., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Although the emergence of gel-free approaches has greatly enhanced proteomic studies, two-dimensional gel electrophoresis (2-DE) remains one of the most widely used proteomic techniques for its high resolving power, relatively low cost, robustness, and high resolution. Preparation of high-quality protein samples remains the key in high-quality 2-DE for proteomic analysis. Samples with high endogenous levels of interfering molecules, such as salts, nucleic acids, lipids, and polysaccharides, would yield a low-quality 2-DE gel and hinder the analysis. Recently, a TRIzol-based protein extraction method has gained prominence and has attracted attention due to its promising performance in high-quality 2-DE. The authors evaluate the use of this approach for four valuable dried food products, namely two dried seafood products (abalone slices and whelk slices) and two traditional Chinese tonic foods (ganoderma and caterpillar fungus). The results indicate that 2-DE gels obtained through the TRIzol-based method are of high-quality and are comparable to those obtained through the trichloroacetic acid⁻acetone method in terms of spot number, spot intensity, and resolution. The TRIzol-based method is generally applicable to dried food samples and is simple and fast, which greatly streamlines the protein extraction procedure. Additionally, it enables the concurrent extraction and analysis of RNA, DNA, and protein from the same sample.

Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts toward developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants. Current understanding of factors affecting charge variant formation during manufacturing remains inadequate, and sometimes, even substantial commitment of resources may still not fully achieve the desired or consistent profiles. As such, this review attempts to provide a comprehensive resource for the biologics community by summarizing the impact of charge variants and CQA management, analytical methods for charge variant detection, as well as strategies in downstream and upstream PD for controlling charge variant profiles., (© 2018 Wiley Periodicals, Inc.)

Protein-particle conjugates and mixtures have been investigated extensively for their diverse applications in biotechnology. However, general methods to measure protein concentration of protein-particle solutions are lacking. Typically, proteins in turbid solutions require separation or staining with another chromophore to quantitate their concentration. Here we demonstrate a label-free, direct approach to measure protein concentrations in turbid solutions using a UV-vis integrating cavity absorbance spectrometer. Three systems are used to test the ability to measure accurate protein concentrations: proteins adsorbed to Alhydrogel, proteins in solution with gold nanoparticles, and proteins encapsulated within polymeric microspheres. Protein concentrations in each of the three protein-particle systems were successfully quantified using a calibration curve created from the absorbance at 280 nm.

The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.

Aim: Active drug assays are becoming increasingly important in protein drug development. We describe the validation of a ligand-binding assay for active protein drug quantification and address practical challenges as well as regulatory implications., Results: A bioanalytical method for active protein drug quantification was successfully validated. Validation data prove that this method can be routinely used applying the commonly accepted acceptance criteria for ligand-binding assays., Conclusion: Active drug assays are a powerful tool to elucidate the pharmacokinetic/pharmacodynamic relationship as they take into consideration the influence of various matrix components, such as soluble ligand and anti-drug antibodies. However, not all aspects of the validation concept described in the guidelines for pharmacokinetic assays can be applied to active drug assays and thus regulatory guidelines should be adapted in consequence.

Bipolar disorder (BD) is a complex and costly psychiatric disorder, which affects one hundred million people worldwide. Due to its heterogeneity, correct BD diagnosis is still a challenge. In order to overcome this issue, different bioanalytical strategies have been proposed in the literature recently. Among these strategies, proteomic approaches have arisen as some of the most promising in the area. Thus, recent applications suggest protein profiles to further refine the proteome of BD as well as the discovery of novel protein biomarkers to facilitate diagnostics. In this review, the state-of-art of proteomic research in BD is summarized. Furthermore, important aspects of proteomics for understanding of BD, such as sample type and size, sampling, sample preparation, gel-based and gel-free proteomics, proteomic quantitative and protein validation are overviewed.

Recently, a guideline has been published by the European Medicines Agency (EMA) on setting safe limits, permitted daily exposures (PDE) [also called acceptable daily exposures (ADE)], for medicines manufactured in multi-product facilities. The ADE provides a safe exposure limit for inadvertent exposure of a drug due to cross-contamination in manufacturing. The ADE determination encompasses a standard risk assessment, requiring an understanding of the toxicological and pharmacological effects, the mechanism of action, drug compound class, and the dose-response as well as the pharmacokinetic properties of the compound. While the ADE concept has broad application in pharmaceutical safety there are also nuances and specific challenges associated with some toxicological endpoints or drug product categories. In this manuscript we discuss considerations for setting ADEs when the following specific adverse health endpoints may constitute the critical effect: genotoxicity, developmental and reproductive toxicity (DART), and immune system modulation (immunostimulation or immunosuppression), and for specific drug classes, including antibody drug conjugates (ADCs), emerging medicinal therapeutic compounds, and compounds with limited datasets. These are challenging toxicological scenarios that require a careful evaluation of all of the available information in order to establish a health-based safe level., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.

Although the impact of hydrogen peroxide (HP) on proteins in liquid solutions has been studied extensively, the impact during lyophilization has been largely overlooked. The purpose of this work was to investigate the effect of HP on lyophilized proteins and HP removal by lyophilization. A protein formulation at 5 mg/mL and its placebo were spiked with HP up to 5.0 ppm and then lyophilized. HP concentration, protein oxidation, and aggregation were monitored before and after lyophilization, as well as during storage at 25°C. The lyophilization process removed on average 94.1% of HP from protein formulation, but only 72.5% from the placebo. There were also significant increases in protein oxidization and aggregation. The oxidation increment correlated with the decrease of HP concentration in both the protein formulation and placebo at all temperatures. Protein oxidation at different freezing temperatures was also studied in follow-up studies. Data from these studies suggest that (1) HP has a significant impact on oxidation and aggregation of protein during lyophilization; (2) significant oxidation can occur even when the protein formulation is frozen; (3) the oxidized protein is more prone to aggregation during lyophilization process., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Operation Pangea is an annual international week of action combating pharmaceutical crime. In this study, called Operation Resistance, we asked the national agencies in Europe to search for falsified antibiotics and biopharmaceutical injectables (peptides and proteins) amongst the medicines seized in Pangea 7 (2014). Reports were received from Belgium, Cyprus, Czech Republic, Denmark, France, the Netherlands, Portugal, Sweden, Spain, the United Kingdom, Norway, and Switzerland. The countries reported seizing about 21,000 dose units (e.g. tablets, capsules) of falsified antibiotics in total. Most of the antibiotics were unlicensed medicines with common antibiotic drugs. In this study week, very few falsified biopharmaceutical injectables were reported. Laboratories reported human growth hormone, sermorelin, melanotan II, and no active ingredients. The average shipment size seemed too large for personal use indicating that a substantial part was intended for resale. This study provides a snapshot of the falsified antibiotics and biopharmaceuticals that enter European countries. How much is actually reaching users during Pangea week - in on other weeks - remains unknown. The shipment sizes indicate falsified antibiotics and biopharmaceuticals are imported for both personal use and resale. The use of antibiotics from unreliable sources is a health risk, contributes to antimicrobial resistance, and may obscure a source of infection from health agencies. The falsified biopharmaceuticals are a health risk because they lack all labelling and may contain unlicensed drugs for injection. It seems important to raise awareness among health-care professionals that falsified medicines in Europe are not restricted to erectile dysfunction drugs. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Unlabelled: The drug product development process for proteins went through its infancy in the early eighties of last century and is in its maturity today. This has been driven largely by the rapid growth of the biotechnology industry, which led to the development and issuance of many regulatory guidelines/directories, especially those through the International Conference of Harmonization (ICH). These guidelines have certainly guided different aspects of a drug product development process. On the other hand, they were issued separately on different topics and in different time periods. An integration of all relevant guidelines into the corresponding areas in drug product development would greatly facilitate the development process. The purpose of this short review is to integrate the relevant (mainly ICH) regulatory guidelines into protein drug product development and to discuss remaining issues, which may lead to further revision of existing guidelines or development of new ones. Drug product development scientists need to collect adequate and relevant development data for a successful product registration. The key is the ability to justify the final drug product in terms of choice of the drug product formulation, container closure system, and manufacturing process., Lay Abstract: The drug product development process for proteins has matured today, largely due to the rapid growth of the biotechnology industry. In this process, many regulatory guidelines/directories were developed and issued, especially through the International Conference of Harmonization (ICH). However, they were issued separately on different topics and in different time periods. An integration of all relevant guidelines into the corresponding areas in drug product development would greatly facilitate the development process. The purpose of this short review is to integrate the relevant (mainly ICH) regulatory guidelines into protein drug product development and to discuss remaining issues, which may lead to further revision of existing guidelines or development of new ones. Drug product development scientists need to collect adequate and relevant development data for a successful product registration. The key is the ability to justify the final drug product in terms of choice of the product formulation, container closure system, and manufacturing process., (© PDA, Inc. 2016.)

The cut point of the immunogenicity screening assay is the level of response of the immunogenicity screening assay at or above which a sample is defined to be positive and below which it is defined to be negative. The Food and Drug Administration Guidance for Industry on Assay Development for Immunogenicity Testing of Therapeutic recommends the cut point to be an upper 95 percentile of the negative control patients. In this article, we assume that the assay data are a random sample from a normal distribution. The sample normal percentile is a point estimate with a variability that decreases with the increase of sample size. Therefore, the sample percentile does not assure at least 5% false-positive rate (FPR) with a high confidence level (e.g., 90%) when the sample size is not sufficiently enough. With this concern, we propose to use a lower confidence limit for a percentile as the cut point instead. We have conducted an extensive literature review on the estimation of the statistical cut point and compare several selected methods for the immunogenicity screening assay cut-point determination in terms of bias, the coverage probability, and FPR. The selected methods evaluated for the immunogenicity screening assay cut-point determination are sample normal percentile, the exact lower confidence limit of a normal percentile (Chakraborti and Li, 2007) and the approximate lower confidence limit of a normal percentile. It is shown that the actual coverage probability for the lower confidence limit of a normal percentile using approximate normal method is much larger than the required confidence level with a small number of assays conducted in practice. We recommend using the exact lower confidence limit of a normal percentile for cut-point determination.

Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts; however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript, we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.

Quantification of free drug concentrations is highly challenging due to the dynamic drug-ligand equilibrium, which may result in incorrect results. Current QC concepts do not adequately cover all of the important influencing factors: the assay itself (format and procedure); the calibration concept; the sample preparation; and the sample storage. Here, we propose a 'free analyte QC concept' that enables quantitative testing of these four factors and, thus, provides best possible proof of correct free drug quantification. The principle of the free analyte QC concept and an example of its application for a free drug assay is described. A comparison of this novel approach with current approaches and how the new concept fits (or does not fit) with current regulatory guidelines is discussed.

A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy.

This paper summarizes the development of the extraction and characterization of allergens responsible for the induction of immunoglobulin (lg) E-induced allergies from the beginning of the 20th century, including the nomenclature of allergens. The majority of papers characterizing allergens and allergen extracts state that the lack of standardization of allergen extracts is the reason for the paper, and so it has been for more than 100 years. A natural part of that process might be the isolation of an allergen molecule and this starts the speculation of 'what makes that allergen an allergen?' To achieve the perfect standardization is a desirable end that is still awaited. So far none of these problems have been finally solved. I started in allergy shortly after the discovery of IgE in 1967. Since that time the history as I remember it is based on the literature, my interpretation of it, and of course may be a little biased due to personal prejudice! The history of the last 10-15 years has still not matured and it might be a little early to draw conclusions. However, at the end of this chapter I do dare to make a few conclusions after having followed the development in this field for 40 years. As this is history it is not meant to be either comprehensive or technically and scientifically precise in all aspects, but rather draws on some thoughts as to what in my mind have been important developments until now. Specific techniques are only mentioned by name and not intended to be discussed in depth. This activity has, however, pushed me to reflect on my hopes and speculations at the time of my introduction to the field of allergen chemistry. To my surprise I realize that far more than I ever expected at that time has been fulfilled. It has been extremely exciting to be a part of that development.

High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

Various in vitro models are commonly used to study adipose tissue function. However, methods for protein normalization in dependence of differentiation and fat cell size are poorly defined. Therefore, the aim of this study was to characterize frequently used housekeepers during adipose differentiation in fat cells of varying cell size and between different subjects to allow a reliable and robust data interpretation. Human preadipocytes, SGBS, 3T3-L1, and mature cells were used to study the housekeeper profile. Glyceraldehyde-3-phosphate dehydrogenase was the most stable internal control in human preadipocytes, whereas all others showed substantial variation. In SGBS and 3T3-L1 cells none of the housekeepers revealed stable protein levels during differentiation. In mature adipocytes GAPDH and α-tubulin were found to be suitable as internal loading controls. Importantly, there was no substantial variation between different donors. However, Coomassie-staining turned out to represent an appropriate alternative as a stable and highly sensitive internal standard for all cell models tested. In conclusion, standard housekeeping proteins have substantial limitations in studies of adipogenesis and in dependence of fat cell size. Coomassie-staining turned out to be a sensitive and stable alternative as internal control for western blot studies during adipogenesis. However, inter-subject variability was low for the investigated housekeeper., (© Georg Thieme Verlag KG Stuttgart · New York.)

As a result of substantial instrumental automation and the continuing improvement of software, crystallographic studies of biomolecules are conducted by non-experts in increasing numbers. While improved validation almost ensures that major mistakes in the protein part of structure models are exceedingly rare, in ligand-protein complex structures, which in general are most interesting to the scientist, ambiguous ligand electron density is often difficult to interpret and the modelled ligands are generally more difficult to properly validate. Here, (i) the primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) the most common categories of building errors or overinterpretation are classified; (iii) a few instructive and specific examples are discussed in detail, including an electron-density-based analysis of ligand structures that do not contain any ligands; (iv) means of avoiding such mistakes are suggested and the implications for database validity are discussed and (v) a user-friendly software tool that allows non-expert users to conveniently inspect ligand density is provided.

Background: The size of the protein sequence database has been exponentially increasing due to advances in genome sequencing. However, experimentally characterized proteins only constitute a small portion of the database, such that the majority of sequences have been annotated by computational approaches. Current automatic annotation pipelines inevitably introduce errors, making the annotations unreliable. Instead of such error-prone automatic annotations, functional interpretation should rely on annotations of 'reference proteins' that have been experimentally characterized or manually curated., Results: The Seq2Ref server uses BLAST to detect proteins homologous to a query sequence and identifies the reference proteins among them. Seq2Ref then reports publications with experimental characterizations of the identified reference proteins that might be relevant to the query. Furthermore, a plurality-based rating system is developed to evaluate the homologous relationships and rank the reference proteins by their relevance to the query., Conclusions: The reference proteins detected by our server will lend insight into proteins of unknown function and provide extensive information to develop in-depth understanding of uncharacterized proteins. Seq2Ref is available at: http://prodata.swmed.edu/seq2ref.

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 μg protein per ampoule (95% C.I: 212.407-218.486 μg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards., (Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.)

Protein is a large component of the standing biomass of algae. The total protein content of algae is difficult to measure because of the problems encountered in extracting all of the protein from the cells. Here we modified an existing protein assay to measure total protein in microalgae cells that involves little or no extraction of protein from the cells. Aliquots of fresh or pretreated cells were spotted onto filter paper strips. After drying, the strips were stained in a 0.1% (w/v) solution of the protein stain Coomassie Brilliant Blue R-250 for 16 to 24 h and then destained. The stained protein spots were cut out from the paper, and dye was eluted in 1% (w/v) sodium dodecyl sulfate (SDS). Absorbance at 600 nm was directly proportional to protein concentration. Cells that were recalcitrant to taking up the dye could be either heated at 80°C for 10 min in 1% SDS or briefly sonicated for 3 min to facilitate penetration of the dye into the cells. Total protein measured in Chlorella vulgaris using this method compared closely with that measured using the total N method. Total protein concentrations were measured successfully in 12 algal species using this dye binding method., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To support clinical trials, bioanalytical methods are often transferred from one laboratory to another. With the rising number of large-molecule therapeutic proteins submitted for US FDA approval, the demand for large-molecule bioanalytical support and, subsequently, method transfer increases. Ligand-binding assays are the methods most commonly used to quantify endogenous and therapeutic proteins for the assessment of biomarkers and pharmacokinetic parameters. The goal of this review is to provide an overview of ligand-binding assay method transfer, essential parameters for partial method validation and to lay out a strategy to increase the chance of success. The recommendations herein are based on a summary of current publications and the authors' specific experiences, to help increase workload efficiency, maintain positive collaborations with partners and meet program timelines.

Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled (13)C6-arginine and (13)C6-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentrations as low as 2% relative to unmodified peptides. The method is robust, reproducible and produced a linear response for every peptide that was monitored. The method was also successfully used to distinguish between two batches of a mAb that were produced in two different cell lines while two batches produced from the same cell line were found to be highly comparable. Finally, the use of the SITRS method in the comparison of two stressed mAb samples enabled the identification of sites susceptible to deamidation and oxidation, as well as their quantitation. The experimental results indicate that use of a SITRS in a peptide mapping experiment with MS detection enables sensitive and quantitative comparability studies of proteins at high resolution.

Here we present the Coon OMSSA Proteomic Analysis Software Suite (COMPASS): a free and open-source software pipeline for high-throughput analysis of proteomics data, designed around the Open Mass Spectrometry Search Algorithm. We detail a synergistic set of tools for protein database generation, spectral reduction, peptide false discovery rate analysis, peptide quantitation via isobaric labeling, protein parsimony and protein false discovery rate analysis, and protein quantitation. We strive for maximum ease of use, utilizing graphical user interfaces and working with data files in the original instrument vendor format. Results are stored in plain text comma-separated value files, which are easy to view and manipulate with a text editor or spreadsheet program. We illustrate the operation and efficacy of COMPASS through the use of two LC-MS/MS data sets. The first is a data set of a highly annotated mixture of standard proteins and manually validated contaminants that exhibits the identification workflow. The second is a data set of yeast peptides, labeled with isobaric stable isotope tags and mixed in known ratios, to demonstrate the quantitative workflow. For these two data sets, COMPASS performs equivalently or better than the current de facto standard, the Trans-Proteomic Pipeline., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.

2D-DIGE experiments are a high-throughput technique for measuring protein abundances based on gel separation. Traditionally three samples are multiplexed per gel: two biological test samples and a third internal standard sample consisting of a pool of all test samples. We demonstrate that the use of an internal standard helps to account for technical variation caused by spatial intensity biases that exist in the gels and propose a novel data-preprocessing technique, a spatial intensity bias removal (SIBR), which can approximate these biases using only the data of biological replicates loaded on the gel. Using this technique, we show that by replacing the internal standard with additional biological replicates, a significant increase in statistical power can be achieved compared to traditional 2D-DIGE designs. This boost in statistical power can be used to reduce the false positive rate for identifying differential protein abundances without compromising sensitivity, or to improve sensitivity without compromising false positive rate. A software implementation of SIBR can be downloaded at http://ibiza.biw.kuleuven.be/SIBR .

Fluorescence microscopy is a method widely used in life sciences to image biological processes in living and fixed cells or in fixed tissues. Quantification and calibration of images in fluorescence microscopy is notoriously difficult. We have developed a new methodology to prepare tissue "phantoms" that contain known amounts of (i) fluorophore, (ii) DNA, (iii) proteins, and (iv) DNA oligonucleotide standards. The basis of the phantoms is the ability of gelatin to act as a matrix for the conjugation of fluorophores as either a free-flowing liquid or a gelatinous solid depending on temperature (> or = 40 and < or = 4 degrees C)., (2010 Elsevier Inc. All rights reserved.)

Therapeutic proteins (TPs) may affect the disposition of drugs that are metabolized by cytochrome P450 (CYP) enzymes, as is evident from a review of data in recently published literature and approved Biologic License Applications. Many TPs belonging to the cytokine class appear to differentially affect CYP activities. Cytokine modulators may affect CYP enzyme activities by altering cytokine effects on CYP enzymes. The alteration in CYP enzyme activities seems to result from changes in transcription factor activity for CYP enzyme expression or changes in CYP enzyme stability, which have been observed during altered immunological states such as infection and inflammation. Human growth hormone also appears to differentially affect CYP activities through unknown mechanisms. Because TP-drug interaction research is an evolving area, limited information is available during drug development on TP-drug interactions mediated by CYP inhibition or induction. The authors of this review suggest that effort be made to understand TP-drug interactions for the safe and effective use of TPs in combination with small-molecule drugs.

Biologics represent a large and growing segment of the therapeutic medicinal market. Sub-visible particles present in these products are a product quality attribute and a potential patient safety concern yet to be fully explored. Early and consistent particle quantitation and control throughout the product life cycle of these drugs from development to commercial lot release is critical in mitigating any concerns. This requires appropriate analytical methods which can be applied to biopharmaceuticals across a large variety of protein concentrations and modes of administration. The compendial light obscuration method for quantitating sub-visible particles in small volume parenterals is not ideally suited for therapeutic biologics. Approaches to modify the current compendial method so that it is applicable to biologics, including appropriate sample preparation, reduced assay sample volume, increased sizing information, and development of an appropriate sampling plan, are presented in this article. Successful applications of a modified light obscuration method to therapeutic protein products are demonstrated, and a strategy to utilise complimentary methods and techniques at different phases of product development is discussed.

Due to the essentiality of lysine for fish, its availability is commonly used as a predictor of the protein nutritional quality of fish feed. The objective of this work was to establish a high-throughput analytical method for protein quality control in fish feed through the measurement of available lysine by planar chromatography. Sample was first incubated with 1-fluoro-2,4-dinitrobenzene to obtain the dinitrophenyl-lysine derivative and then hydrolyzed with hydrochloric acid in order to release the derivative from proteins. Chromatography was performed on silica gel 60 F254 HPTLC plates using n-propanol-25% ammonia (7 + 3, v/v) as mobile phase. Quantitative analysis was performed by densitometry in the absorbance mode at 360 nm. Calibration showed a polynomial relationship with an R2 of 0.9991 in the range of 25 to 125.0 ng/band. Repeatability relative standard deviation (RSD) and intermediate precision (RSD) in matrix were 0.8 and 3.6%, respectively. Recoveries of spiked samples at two levels ranged from 72 to 85% with RSD from 3 to 8%. This method provides the salmon feed industry with a reliable, high-throughput, and low-cost means for routine quality control of available lysine.