Compendial Methods: Suitability Verification, Challenges and Recommendations for Proteins.

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.

Bradford Assay for Determining Protein Concentration.

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.

Re-evaluation of the 18 non-human protein standards used to create the empirical statistical model for decoy library searching.

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 log10 precursor intensity distributions were Gaussian and nearly ideal for relative quantification.

Nutrient and AA digestibility of black soldier fly larvae differing in age using the precision-fed cecectomized rooster assay1.

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.

Low volume aseptic filling: Impact of pump systems on shear stress.

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.

The value of human epididymis protein 4 (HE4) as a serum tumor marker for accurate bone metastases finding by whole-body bone scintigraphy in lung cancer patients.

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.

Development of Protein-Like Reference Material for Semiquantitatively Monitoring Visible Proteinaceous Particles in Biopharmaceuticals.

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.

New studies on leachables in commercial scale protein drug filling lines using stir bar sorptive extraction coupled with TD-GC-MS and UPLC/QTOF-MS/MS analytics.

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.

Proceedings of the 2017 Viral Clearance Symposium.

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.

Proceedings of the 2017 Viral Clearance Symposium, Session 2.2: DSP Unit Operations-Purification Unit Operations.

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.

Evaluation of the Use of TRIzol-Based Protein Extraction Approach for Gel-Based Proteomic Analysis of Dried Seafood Products and Chinese Tonic Foods.

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.

The EuBIVAS: Within- and Between-Subject Biological Variation Data for Electrolytes, Lipids, Urea, Uric Acid, Total Protein, Total Bilirubin, Direct Bilirubin, and Glucose.

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 (CVI) 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 CVI for men and women/women <50 years of age were evident for uric acid, triglycerides, and urea. The CVA 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.

A Risk- and Science-Based Approach to the Acceptance Sampling Plan Inspection of Protein Parenteral Products.

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.

Development and validation of a mass spectrometry-based assay for quantification of insulin-like factor 3 in human serum.

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.

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles.

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.

Label-Free, Direct Measurement of Protein Concentrations in Turbid Solutions with a UV-Visible Integrating Cavity Absorbance Spectrometer.

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.