Ned Mozier - Academia.edu (original) (raw)

Papers by Ned Mozier

Journal of Pharmaceutical and Biomedical Analysis, Jun 1, 2013

The measurement of low levels of bacterial lipopolysaccharides (endotoxins) in protein samples ca... more The measurement of low levels of bacterial lipopolysaccharides (endotoxins) in protein samples can be a challenge due to potential interference from the inherent properties of the protein, its formulation or other substances that may be present. Other factors include the expression system which may have endotoxin species distinct from the standard, as well as different purification bioprocesses. The endotoxin measurement assays also have a number of variables. Those studied include differences between laboratories, reagents and standards, and detection modalities. A variety of protein samples from a range of expression systems was included in the evaluation. Endotoxin levels are relatively stable when samples are stored frozen with test variations between 1 and 38% among different aliquots. Test variation between labs was not significantly different when the same procedure was followed (intermediate precision) by trained analysts. Most testing modalities gave results within a 50-100% variation, a difference generally regarded as within assay variability. However, about 25% of the samples showed significant differences between testing modalities and/or reagents. The sources of these differences were further examined by traditional as well as novel sample treatments. These findings demonstrate that for some samples, endotoxin may be over-or underestimated and a more thorough pre-treatment or testing modality may be required.

Parenteral drugs, including products produced by biotechnology, are required to have a pyrogen te... more Parenteral drugs, including products produced by biotechnology, are required to have a pyrogen test by the health authorities in most markets. As described in the prior chapter, the rabbit pyrogen test (RPT) was the earliest test codified by law or compendia, and was used exclusively in the past. For at least two decades, however, the bacterial endotoxin test (BET) has served as an alternative test on the premise and overwhelming evidence that most pyrogens are lipopolysaccharides (LPS). It remains the obligation of the drug manufacturer to demonstrate that the BET is suitable as the sole pyrogen test, typically by confirming that at least three commercial scale batches are non-pyrogenic in the RPT and validating the BET. More recently, the FDA division reviewing biologics has begun requesting additional studies to determine if the BET for a given product is prone to the phenomenon described as low endotoxin recovery (LER) (Chen and Vinther, Presentation at PDA annual meeting, 2013, Orlando). If LER is observed, often with high dose products with formulations containing a surfactant and a chelator, FDA often require manufacturers demonstrate the absence of pyrogenicity by a variety of means until the LER is resolved (Hughes et al., BioPharma Asia, 2015, 4(2):14–24; Chen et al., Lowe Endotoxin Recovery, PDA Technical Report No.82, 2019, pp. 1–128). This may require spiking products to ascertain sensitivity in the RPT. Consequently, the demand has increased for additional RPT studies for LER products, even if they are nonpyrogenic (unspiked).

Biotechnology and Bioengineering, Nov 22, 2017

† This article has been accepted for publication and undergone full peer review but has not been ... more † This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as

Journal of Clinical Microbiology, Nov 1, 2009

Toll-like receptor (TLR)-expressing cells, for the first time, detected and identified a microbia... more Toll-like receptor (TLR)-expressing cells, for the first time, detected and identified a microbial contaminant in a product made in Escherichia coli using an old manufacturing process. It was suspected of having a microbial contaminant(s) because, although it tested negative by standard pyrogen assays, it was associated with adverse events in early clinical trials. The assay readout is the induction of NF-B and/or cytokines in response to TLR activation. Four coded samples, labeled A to D, including a sample prepared by the older manufacturing process, were submitted. The cell lines were activated only by samples B and D. Sample D stimulated only Mono-Mac 6 and HEK-human TLR4 (hTLR4) cells and was later identified as lipopolysaccharide. Except for TLR3 cells, sample B stimulated cells bearing the different TLRs (TLRs 2, 4, 5, 7, 8, and 9) and nontransfected HEK293 cells. These data suggested that flagellin was the microbial contaminant, since TLR5, the receptor for flagellin, is known to be expressed constitutively on HEK293 cells. Moreover, purified flagellin from Salmonella enterica serovar Typhimurium behaved like sample B, stimulating HEK293 and HEK-hTLR5 cells but not HEK-hTLR3 cells, and this stimulation by flagellin and sample B was blocked by an anti-hTLR5 neutralizing antibody. Western blots showed bands positive for flagellin and sample B with the molecular sizes expected for the flagellins from S. Typhimurium and E. coli, respectively. Mass spectrometry data were consistent with the presence of flagellin in the manufacturer's sample B. Taken together, these data indicate that the microbial contaminant in sample B was flagellin and may have been associated with adverse events when the recombinant product was administered.

PDA Journal of Pharmaceutical Science and Technology, 2020

Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers ... more Endotoxin testing by recombinant factor C (rFC) is increasing with the addition of new suppliers of reagents. By use of a recombinantly produced factor C , based on the sequence of a coagulation enzyme present in horseshoe crab amebocyte lysates, the rFC tests are designed as substitutes for the traditional Limulus amebocyte lysate (LAL)/Tachypleus amebocyte lysate tests based on horseshoe crab blood. Comparative testing of samples with both the LAL and recombinant reagents has shown a high degree of correlation, suggesting that use of rFC is comparable to the more traditional LAL tests and may be technologically superior. Recombinant factor C does not recognize the factor G pathway, the alternate coagulation pathway that the lysate reagents detect. This feature allows rFC to detect endotoxin more selectively. As a recombinantly produced material, it avoids the use of the horseshoe crabs required for lysate production, thereby protecting this species, which is at risk in some parts of the world. Recombinant factor C is expected to further benefit from a more sustainable supply chain based upon a robust biotechnological production process. We summarize here the results of many studies that evaluated the use of recombinant technology for the detection of environmental endotoxin. Additionally, we include a review of the current compendia and regulatory status of the recombinant technologies for use in the quality control of pharmaceutical manufacturing. Our analysis confirms that the recombinant technologies are comparable in protecting patient safety.

L'invention concerne des isomeres de position de proteine PEG de recombinaison. Plus precisem... more L'invention concerne des isomeres de position de proteine PEG de recombinaison. Plus precisement, elle concerne des isomeres de position de PEG d'un anticorps specifique de determinants antigenes du facteur de necrose tumorale humain alpha (TNFα). Plus precisement encore, elle concerne des isomeres de position PEG de CDP870. Elle concerne egalement des compositions comprenant ces isomeres ainsi que leurs utilisations therapeutiques dans le traitement d'une maladie induite par TNFα comprenant des troubles immunitaires aigus chroniques et immuno-regulateurs, ainsi que la polyarthrite rhumatoide ou l'osteoarthrose.

Endotoxin Detection and Control in Pharma, Limulus, and Mammalian Systems, 2019

Biotechnology and bioengineering, Feb 24, 2017

Host cell proteins (HCPs) are a heterogeneous mixture of impurities that should be minimized in b... more Host cell proteins (HCPs) are a heterogeneous mixture of impurities that should be minimized in bulk preparations of biotechnologically produced medicines. Immunoassays are commonly used to detect and measure HCPs in therapeutic products, and a successful assay is directly dependent on the quality of the polyclonal antibodies (pAbs) used. These pAbs are enriched from antisera of animals immunized with a broad mixture of HCPs, but there is limited information regarding the best strategy for purification of these critical reagents. The use of protein A or protein G affinity chromatography results in purified pAbs that are not entirely HCP-specific, while the use of HCP affinity chromatography results in a more specific pAb population but may be harder to recover fully. In theory, both approaches have advantages and disadvantages for generating optimal reagents. In this study, we compared reagents from these two purification procedures using the same starting material, as well as those...

Journal of Immunological Methods, 2017

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) of... more Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) of several therapeutic antibody drugs and evaluation in ADCC bioassays is important in antibody drug development and maintenance. Three types of effector cells now routinely used in bioassay evaluation of ADCC are natural killer cells from human donors (FcγRIIIA + primary NK), FcγRIIIA engineered NK-92 cells and FcγRIIIA/NFAT-RE/ luc2 engineered Jurkat T cells. Engineered effector cells were developed to address need for improved precision and accuracy of classic NK cell ADCC bioassays. The main purpose of our study was to rationalize which of these ADCC effector cells best simulate the expected response in human subjects and to identify which effector cells and assays best fit ADCC bioassay needs during antibody drug development. We characterized differences between the effector cells and compared ADCC biological activities using the well-known humanized IgG1 antibody drug, trastuzumab. The three effector cell types studied expressed either V-158 or F-158 allotype of FcγRIIIA, hence six cell preparations were compared. Our results demonstrate highest surface expression of FcγRIIIA in primary NK and engineered NK-92 (V-158) cells with nearly all expressed on the cell surface. In contrast, expression in engineered Jurkat T cells was low with only a small percentage expressed on the cell surface. Studies evaluating binding of trastuzumab to effector cells demonstrated the highest affinity of FcγRIIIA in primary NK and NK-92 (V-158) cells. ADCC cytotoxicity studies showed greatest trastuzumab potency in primary NK and engineered NK-92 (V-158) cells and negligible cell lysis obtained using engineered Jurkat T cells. In contrast, the engineered Jurkat T (V-158) cells responded as effectively as primary NK (V/V) cells to nuclear factor of activated T cells (NFAT2) activation upon binding of trastuzumab to FcγRIIIA, demonstrating similar ADCC pathway activation in these cells despite the low surface expression of FcγRIIIA and its low affinity for trastuzumab. Dose-response range of trastuzumab in activation of NFAT2 (measured as pNFAT2 dephosphorylation) was very similar to response in classic ADCC assay for primary and NK-92 cells and to response in ADCC reporter assay for Jurkat T effector cells, bridging the assays. Trastuzumab potency in ADCC reporter assay using the engineered Jurkat T cells was close to that seen using either primary NK or engineered NK-92 cells in classic ADCC assay. In summary, all three effector cell systems differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, yet only primary NK and engineered NK-92 cells are capable of inducing ADCC-mediated cell lysis. Engineered Jurkat T effector cells have value to assure antibody manufacturing consistency and in other applications where accuracy and precision are important. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells better reflect the physiologically relevant ADCC mechanism of action. As an engineered cell line, NK-92 cells may behave more reproducibly than primary NK, but this must be balanced with the objective for biological relevance in decisions on which NK cells to use in assay.

Journal of Pharmaceutical and Biomedical Analysis, 2013

Journal of Clinical Microbiology, 2009

Selenite is detoxified by reduction to HâSe and methylation from S-adenosylmethionine (AdoMet) to... more Selenite is detoxified by reduction to HâSe and methylation from S-adenosylmethionine (AdoMet) to yield methylselenol, dimethylselenide (DMSe; exhaled), and trimethylselenonium ion (TMSe; excreted in urine). DMSe methyltransferase (DMSeMT) was assayed by its ability to generate (Â³H)TMSe from DMSe and (methyl-Â³H)AdoMet, with TMSe separated by cation-exchange HPLC. DMSeMT is found primarily in the cytosol. In terms of specific activity (umol/min/mg protein), the enzyme is most active in the lung (298) and liver (105), with much lower activity in kidney (8), heart (7), muscle (8), RBC (4), and is negligible in brain and spleen. Initial purification from liver cytosol was by DEAE and gel filtration column chromatography. Gel filtration and SDS polyacrylamide gel electrophoroesis indicate that the enzyme is monomeric with a molecular weight of 30,000 daltons. Activity assays on liver cytosol indicate a pH optimum of 6. DMSeMT is inhibited 50% by 25 uM Sinefungin, an AdoMet analog, and by its product, S-adenosylhomocysteine, at 40 uM. Purification to homogeneity will allow analysis of the ability of this enzyme to also catalyze the methylation of HâSe and/or to methylselenol.

Biotechnology and Bioengineering, Nov 6, 2011

Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molec... more Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molecules originating from the host organism that may be present in samples from recombinant biological processes. Although similar in basic structural base pair units, rDNA may exist in different sizes and physical forms. Interest in measuring rDNA in recombinant products is based primarily on demonstration of effective purification during manufacturing, but also on some hypothetical concerns that, in rare cases, depending on the host expression system, some DNA sequences may be potentially infectious or oncogenic (e.g., HIV virus and the Ras oncogene, respectively). Recent studies suggest that a sequence known as long interspersed nucleotide element‐1 (LINE‐1), widely distributed in the mammalian genome, is active as a retrotransposon that can be transcribed to RNA, reverse‐transcribed into DNA and inserts into a new site in genome. This integration process could potentially disrupt critical gene functions or induce tumorigenesis in mammals. Genomic DNA from microbial sources, on the other hand, could add to risk of immunogenicity to the target recombinant protein being expressed, due to the high CpG content and unmethylated DNA sequence. For these and other reasons, it is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm low levels in the final drug substance using an appropriately specific and quantitative analytical method. The heterogeneity of potential rDNA sequences that might be makes the testing of all potential analytes challenging. The most common methodology for rDNA quantitation used currently is real‐time polymerase chain reaction (RT‐PCR), a robust and proven technology. Like most rDNA quantitation methods, the specificity of RT‐PCR is limited by the sequences to which the primers are directed. To address this, primase‐based whole genome amplification is introduced herein. This paper will review the recent advancement in rDNA quantitation and recent findings regarding potential risks of immunogenicity, infectivity, and oncogenicity of rDNA. Biotechnol. Bioeng. 2012; 109:307–317. © 2011 Wiley Periodicals, Inc.

Springer eBooks, Nov 28, 2014

Historically, vaccines have predominantly been manufactured through complex biological processes ... more Historically, vaccines have predominantly been manufactured through complex biological processes (growth in embryonic chicken eggs, bacterial fermentation, and mammalian cell culture) that can be challenging to control and reproduce. Although the introduction of Quality by Design principles is changing the “the process is the product” mindset, the development of appropriate release assays remains a critical element in ensuring the safety and efficacy of a vaccine throughout its shelf life. The development of relevant and robust potency assays requires careful consideration of the nature of the protective immune response to the targeted antigen as well as a detailed understanding of the structural features of the antigen that elicit the protective response.

BioTechniques, Sep 1, 2019

Biological activity is a critical quality attribute for biopharmaceuticals, which is accurately m... more Biological activity is a critical quality attribute for biopharmaceuticals, which is accurately measured using an appropriate relative potency bioassay. Developing a bioassay is a complex, rigorous undertaking that needs to address several challenges including modelling all of the mechanisms of action associated with the biotherapeutic. Bioassay development is also an exciting and fast evolving field, not only from a scientific, medical and technological point of view, but also in terms of statistical approaches and regulatory expectations. This has led to an industry-wide discussion on the most appropriate ways to develop, validate and control the bioassays throughout the drug lifecycle.

level: interMediate H ost cell proteins (HCPs) are process-related impurities derived from a host... more level: interMediate H ost cell proteins (HCPs) are process-related impurities derived from a host cell expression system that may be present in trace amounts in a final drug substance. During biologics development, it is important to demonstrate that a bioprocess is efficient in removing HCPs and that it provides consistent control of HCP levels. Several techniques are typically used for detection, quantitation, and risk evaluation of HCPs in biologics. The most common are enzyme-linked immunosorbent assays (ELISAs), Western blotting, sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and chromatographic separation methods (1). Genomics, proteomics, and bioinformatics also have been used recently in analysis and evaluation of host cell proteins (2). From our recent understanding, thousands of potential HCP species could be present in a bioprocess (3–5). In a typical two-dimensional gel electrophoresis of HCPs from mammalian cells or Escherichia coli, hundreds of re...