2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global 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 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

External magnet improves antitumor effect of vinblastine and the suppression of metastasis.

The use of magnetic drug targeting (MDT) to selectively deliver chemotherapeutic drugs to tumor cells is a widely investigated approach; however, the notion of targeting tumor endothelial cells by this method is a fairly new concept. Positively-charged (cationic) liposomes have an extraordinarily high affinity for tumor vessels, but heterogeneous targeting is frequently observed. In order to improve on the overall efficiency of targeting tumor vessels, we investigated the use of an externally applied magnetic field together with magnetic cationic liposomes (MCLs) for cancer treatment. We examined the antitumor effect of the chemotherapeutic agent vinblastine loaded in MCLs, using a murine model of melanoma. Two hours following i.v. administration of MCLs, we observed significant tumor vascular uptake with use of an external magnet (15.9 +/- 6.3%) compared to no magnet (5 +/- 1.3%). The administration of vinblastine-loaded MCLs with the magnet produced a significant antitumor effect, reducing the presence of tumor nodules in preferential sites of metastasis compared to untreated and free drug control groups. CD31 immunostaining revealed a decrease in the general length of tumor blood vessels, altered vascular morphology and interruptions in the tumor vascular lining for the vinblastine-loaded MCL groups. Drug-loaded MCLs with magnetic fields may represent a promising combination approach for cancer treatment.

Development and characterization of magnetic cationic liposomes for targeting tumor microvasculature.

Cationic liposomes preferentially target tumor vasculature compared to vessels in normal tissues. The distribution of cationic liposomes along vascular networks is, however, patchy and heterogeneous. To target vessels more uniformly we combined the electrostatic properties of cationic liposomes with the strength of an external magnet. We report part I of development. We evaluated bilayer physical properties of our preparations. We investigated interaction of liposomes with target cells including the role of PEG (polyethylene-glycol), and determined whether magnetic cationic liposomes can respond to an external magnetic field. The inclusion of relatively high concentration of MAG-C (magnetite) at 2.5 mg/ml significantly increased the size of cationic liposomes from 105+/-26.64 to 267+/-27.43 nm and reduced the zeta potential from 64.55+/-16.68 to 39.82+/-5.26 mv. The phase transition temperature of cationic liposomes (49.97+/-1.34 degrees C) reduced with inclusion of MAG-C (46.05+/-0.21 degrees C). MAG-C cationic liposomes were internalized by melanoma (B16-F10 and HTB-72) and dermal endothelial (HMVEC-d) cells. PEG partially shielded cationic charge potential of MAG-C cationic liposomes, reduced their ability to interact with target cells in vitro, and uptake by major RES organs. Finally, application of external magnet enhanced tumor retention of magnetic cationic liposomes.

The drug loading, cytotoxicty and tumor vascular targeting characteristics of magnetite in magnetic drug targeting.

Chemotherapy is a popular treatment approach against cancer but significant uptake of drugs by normal tissues is still a major limitation. Magnetic drug targeting (MDT) has been used to improve localized drug delivery to interstitial tumor targets. MDT is now being developed to improve drug delivery to tumor vessels. We thus seek to understand the role of magnetite (MAG-C) in drug loading, influence on cytotoxicity and vascular targeting characteristics. The inclusion of MAG-C at lower concentrations (0.5 mg/ml) in cationic liposomes did not alter the efficiency of loading etoposide, but at higher concentrations (2.5 mg/ml) incorporation decreased from 80+/-3.4% to 44+/-4.26%. MAG-C reduced the incorporation of dacarbazine. The incorporation was significantly lower compared to liposomal etoposide, both in the presence and absence of MAG-C. The incorporation efficiency of vinblastine sulfate in cationic liposomes was similar for low and relatively high MAG-C content; values for incorporation were 21+/-0.11 and 23+/-2, respectively. Polyethylene-glycol improved the efficiency of loading chemotherapeutic agents regardless of drug type. Additionally, cytotoxicity and tumor vascular targeting characteristics of liposome therapeutics were not influenced by MAG-C. The components used to prepare magnetic liposomes for MDT should be optimized for maximum therapeutic benefit.

Common deficiencies with bioequivalence submissions in abbreviated new drug applications assessed by FDA.

PURPOSE: A generic product must meet the standards established by the Food and Drug Administration (FDA) to be approved for marketing in the USA. FDA approves a generic product for marketing if it is proved to be therapeutically equivalent to the reference product. Bioequivalence (BE) between a proposed generic product and its corresponding reference product is one of the major components of therapeutic equivalence. These approvals may be delayed if the BE portion of the submission is determined to be deficient. Many of these BE deficiencies recur commonly and can be avoided. METHOD: We conducted a survey of the BE submissions to abbreviated new drug applications (ANDAs) over years 2001 to 2008 to identify the most commonly occurring BE deficiencies. RESULTS: Recurring deficiencies are found in a majority of the ANDAs reviewed by FDA's Division of Bioequivalence. The most common deficiencies were the two deficiencies related to dissolution (method and specifications) found in 23.3% of the applications and analytical method validation and/or report found in 16.5% of the applications. The approval of generic drugs would be greatly accelerated if these deficiencies could be avoided.

Importance of the liposomal cationic lipid content and type in tumor vascular targeting: physicochemical characterization and in vitro studies using human primary and transformed endothelial cells.

Using cationic liposomes to deliver cytotoxic molecules to the tumor microvasculature is currently being developed for the treatment of cancer and other angiogenesis-related diseases. To improve on their beneficial properties, the authors have examined whether the particular cationic lipid type and lipid content employed are important factors influencing cellular interactions and formulation effects. The authors prepared different PEG (polyethylene glycol)-modified cationic liposomes (PCLs) with varying percent cationic lipid content and lipid type, and evaluated liposome size, surface charge (zeta) potential, and cellular properties in vitro. The cell lines used were human umbilical vein (HUVEC), lung microvascular (HMVEC-L and HPVE-26), coronary microvascular (HMVEC-C), dermal microvascular (HMVEC-D), and immortalized dermal microvascular (HMEC-1) endothelial cells. In vitro experiments consisted of cellular uptake and cytotoxicity studies, fluorescence-activated cell sorting (FACS) analysis, fluorescence, and transmission electron microscopic analysis. Liposome size and zeta potential analysis of five different PCLs revealed significant differences in their physicochemical properties. Some cationic lipids formed relatively toxic liposomes compared to others. The efficiency of loading chemotherapeutic drugs (doxorubicin hydrochloride, etoposide), affinity of PCLs for endothelial cells, and formulation effects varied according to cationic lipid content and the lipid type. Cellular uptake was observed in lung, dermal, and coronary endothelial cells. Heparan sulfate proteoglycans were found present on HMEC-1 cells, which may have enabled PCL uptake. In conclusion, physicochemical properties of cationic liposomes and their ability to interact with endothelial cells are important factors to consider during the early stages of formulation development for the treatment of cancer and other angiogenesis-dependent diseases.