Monoclonal antibody (mAb) coformulation containing two therapeutic proteins provides benefits of improved therapeutic efficacy and better patient compliance. Monitoring of the individual mAb stability in the coformulation is critical to ensure its quality and safety. Among post-translational modifications (PTMs), oxidation is often considered as one of the critical quality attributes (CQAs) as it potentially affects the structure and potency. Although hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC) have been used to monitor overall protein oxidation, mass spectrometry of peptide digests resolved by LC methods can afford superior selectivity and sensitivity for specific PTMs. With the advent of the Quadrupole Dalton (QDa) mass spectrometer as an affordable add-on detector, implementation of targeted oxidation assays in development and quality control (QC) laboratories is now feasible. In this study, as the first effort to implement MS-based methods for antibody coformulation in QC laboratories, we developed and validated a high-throughput and robust focused peptide mapping method using QDa for simultaneous site-specific monitoring of oxidation of methionine and tryptophan residues in heavy-chain (HC) complementary determining regions (CDRs) of two co-formulated mAbs. The method was validated in terms of accuracy, precision, linearity, range, quantitation limit (QL), specificity, and solution stability per recommendations in ICH Q2. The method robustness was systematically assessed involving multiple sample preparation and instrument method parameters. The method met the validation criteria in GMP laboratories with excellent robustness and was implemented in both GMP and development environments.

Multiple reaction monitoring (MRM) is a liquid chromatography-mass spectrometry (LC-MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC-MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC-MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra- and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP-mAb interaction on lipase quantification.

The isoforms Iso-2, Iso-3, and Iso-4 of Escherichia coli-derived recombinant human interferon alpha-2b (rhIFN α-2b), generated by posttranslational modifications of the protein during fermentation, present a major problem in terms of purification and the yield of the drug substance. We report here the structural characterization of these isoforms by mass spectrometry (MS) methods. An extensive MS study was conducted on Iso-4, which is composed of up to 75% of the in-process IFN, and on the native rhIFN α-2b. The trypsin-digested peptide mixtures generated from the two samples were analyzed by liquid chromatography (LC)-MS, and targeted peptides were further studied by LC-tandem MS (triple quadrupole mass spectrometer), high-resolution MS(n) (LTQ Orbitrap), and matrix-assisted laser desorption/ionization MS (MALDI-MS). The structure of Iso-4 was elucidated as a novel pyruvic acid ketimine derivative of the N-terminal cysteine (Cys1) of IFN α-2b, where the disulfide bond between Cys1 and Cys98 was fully reduced and the other disulfide bond pair, Cys29-ss-Cys138, was partially reduced. Similarly, Iso-2 was identified as a correctly disulfide-folded rhIFN α-2b with acetylation on Cys1, and Iso-3 was identified as an S-glutathionylated form (Cys98) of partially reduced rhIFN α-2b that was pyruvated on Cys1. Based on the characterization work, a reproducible conversion procedure was successfully implemented to convert Iso-4 to rhIFN α-2b.

PurposeThis paper aims to explore the growing role of customized case studies in executive education programs.Design/methodology/approachA general review is provided, mainly from the authors' experience, on designing and employing custom cases.FindingsIt is shown that the benefits of designing and using custom cases accrue throughout the program development, delivery and follow‐up phases and can have a positive impact not only on course participants, but also on senior management and teaching faculty.Research limitations/implicationsThe role of pedagogical tools in executive education remains a largely under‐researched area. Suggestions are made for future work.Practical implicationsIt is shown how the construction of a case can vary by both content and format to best suit program objectives. Guidelines are also provided for teaching effectiveness.Originality/valueThis appears to be the first, in‐depth assessment of the design and use of case studies in executive education.

The hepatitis C virus (HCV) is currently estimated to infect over 270 million people worldwide, with up to 4 million people newly infected each year. It is the leading cause of chronic liver disease and the most common reason for liver transplantation in the USA and Europe. The pegylated interferons (peginterferon alfa-2b: PEG-INTRON® and peginterferon alfa-2a: PEGASYS®), alone or in combination with ribavirin, are the current standard of care for patients with HCV. There are significant chemistry and manufacturing control (CMC) challenges in the manufacture and characterization of the pegylated interferons since these therapeutic drugs are composed of heterogeneous mixtures of mono-pegylated proteins with differing sites of attachment of polyethylene glycol (PEG) to the core interferon molecule, termed “positional isomers”. These CMC issues were comprehensively addressed during the development of PEG-INTRON®. Extensive characterization of the pegylated interferon in the drug substance and drug product, and the establishment of appropriate manufacturing controls, served as the foundation for a successful CMC strategy to obtain regulatory approval. This study describes the analytical strategies for quantitating the positional isomer populations which comprise PEG-INTRON®. Individual positional isomers were also isolated and characterized with respect to site of pegylation and in vitro biological potency. It was determined that different pegylation chemistries result in different mixtures of positional isomers. PEG-INTRON® is predominantly pegylated at histidine 34, demonstrated to be the most biologically active positional isomer of the pegylated interferons. In contrast, the antiviral activities of positional isomers generated by attachment of PEG to lysine residues were significantly lower. Thus, the differences in pegylation chemistry between PEG-INTRON® and PEGASYS® result in different distributions of pegylated positional isomers and differential biological activity profiles in vitro.

Therapeutic pegylated interferon-αs (IFN-α) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-α molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-α molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-α2b that were monopegylated using 5–30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys1, His34, Lys31, Lys83, Lys121, Lys131, and Lys134. The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-α2b control. The highest residual activity was observed with the His34 positional isomers, and the lowest was observed with the Cys1 positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys134 > Lys83 ∼ Lys131 ∼ Lys121 > Lys31. The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-α to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His34 positional isomer suggests that this site may be favorable for pegylating IFN-α2b molecules.

The efficacy of several therapeutic cytokines in vivo is often restricted by their clearance rate, which can be retarded by the covalent attachment of polyethylene glycol (PEG)—a large, highly soluble, nontoxic adduct (, , , , ). However, pegylation is a double-edged sword. In addition to retarding clearance, it also frequently impairs the biological activity of the modified protein. Consequently, therapeutic biologicals are typically only minimally pegylated. For small cytokines, monopegylation provides a good balance. The most commonly used chemistry involves carboxyalkylation of nucleophilic nitrogens on the target protein. The product is a stable mixture of monopegylated proteins varying in the point of polymer attachment. The relative proportion of each positional isomer is based on protein properties, such as local-charge fields and solvent exposure. However, by modifying the pH of the pegylation reaction, the preferred target side chain can be shifted from ε amines (preferred at pH 10) to α amines and imidazoles (preferred at pH 6.5;) which may have positive effects on biological activity ().

The presence and absence of inter‐heavy‐chain disulphide linkages contribute to the existence of the tetrameric (H2L2) and half (HL) human IgG molecules, respectively [Schuurman, Perdok, Gorter and Aalberse (2001) Mol. Immunol. 38, 1–8]. Reduced effector response in the human IgG4 subclass presents an alternative therapeutic platform in a monoclonal‐antibody (mAb) development program. During the initial cell‐selection stage, titres of the recombinant human antibody present in crude cell‐culture supernatants are determined by ELISA, a technique requiring nanogram quantities of mAb. In the case of an IgG4 antibody, this material is represented mainly by the combination of the tetrameric (H2L2) and dimeric (HL) forms of the antibody. The determination of concentrations or ratios of tetramer and dimer usually requires at least one chromatographic purification step, and thus frequently this is evaluated later in the mAb development process when the number of potential clones has been reduced. In the present paper we describe a Western‐blot‐based method that detects and quantifies IgG4 half‐molecules, HL, from crude cell‐culture supernatants without purification so that H2L2/HL ratios can be included as a part of early clonal evaluation along with the screening of mAb titres. This method was demonstrated (1) to have a linear HL detection range of 0.5–10 ng, (2) to require microlitre volumes of culture and (3) to react specifically with human IgG4 produced from hybridoma and Chinese‐hamster ovary cell cultures. Moreover, this protocol is applicable to evaluate and monitor potential H2L2/HL variations as a result of changes during the process‐development stage of a mAb development program.

The clinical application of recombinant adenoviruses as vectors for gene therapy brings about the need to develop new analytical methodologies for monitoring the quality of the viral production as well as establishing structure–function relationships. A mass spectrometry-based assay has been developed for the characterization of structural proteins of the recombinant adenovirus type 5 vector encoding human p53 tumor suppressor gene. The fingerprinting of the viral proteome was accomplished by integration of MALDI-MS and/or MALDI-PSD-MS with SDS–PAGE and RP-HPLC, followed by database search using MS-Fit and MS-Tag algorithms. Viral proteins (molecular weights ∼10,000–100,000Da) corresponding to more than 95% of total protein mass were resolved and identified, which include hexon (II), penton base (III), peripentonal hexon-associated protein (IIIa), minor core protein (V), major core protein (VII), and other hexon-associated proteins (VI and VIII). An important finding of our studies was the identification of some precursor proteins (i.e., pVIII) and propeptides of precursor proteins (pVIII, pX, and pVI) present in the adenovirus sample. The information obtained allows direct and accurate assessment of the quality of recombinant adenoviruses.

Purpose. Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-α2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH.

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has approximately 30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-glycosylated, and contains two disulfide bonds as determined by comparative electrospray mass spectrometric (MS) analysis of the protein before and after reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine-containing peptic fragments, provided rapid determination of the existing disulfide bonds between cysteine residues 28-56 and 44-70. This disulfide arrangement is similar to that observed for the analogous four internal cysteine residues in hIL-4. The conservation of disulfide bond arrangements between hIL-13 and hIL-4, coupled with their alpha-helical structure and sequence homologies, confirms that IL-13 and IL-4 are structural homologues. It is also consistent with their reported similarities in biological function and receptor binding kinetics.

A method for increasing the yield of an interferon alpha composition, comprising converting a pyruvate adjunct isoform of interferon alpha into interferon alpha, by exposing said pyruvate adjunct isoform of interferon alpha to a solution having a pH of about 5, 5.

The CheA protein of the Salmonella typhimurium chemotaxis system is phosphorylated by ATP. Phospho-CheA transfers its phosphoryl group to a second chemotaxis protein, CheY. Unlike phospho-CheA, phospho-CheY is relatively unstable, rapidly decaying to phosphate and CheY. We propose that phosphorylation of CheY may play a role in its function as a tumble regulator to control motor behavior in response to attractant and repellent stimuli.