Stephen Flatman

The application of appropriate analytical methods is an essential requirement for the purification of therapeutic antibodies. A range of analytical methods need to be employed to effectively determine the purity, identity, integrity and activity of these important class of pharmaceuticals. These include notably electrophoresis, high performance liquid chromatography and immunoassays. Regulatory and industry demands in recent years have brought the need for improvements and many have been successfully implemented. This article reviews the current analytical methods applied to support the purification of monoclonal antibodies.

Recombinant human erythropoietin has been used to treat anemia associated with chronic renal disease. This paper provides a comprehensive comparative analysis of Dynepo and three other commercial erythropoiesis stimulating agents, Eprex, NeoRecormon and Aranesp. We found significant differences in the type, levels and amount of O-acetylation of sialic acids. Sialic acids and O-acetylation present provide protection from clearance from circulation. Aranesp had up to six O-acetyl groups attached to the sialic acids. Eprex and NeoRecormon had only minor amounts of O-acetylation while Dynepo had none. Dynepo had no Neu5Gc, which is potentially immunogenic for humans. Dynepo contained the least amount of disialylated and Aranesp the highest amount of tetrasialylated glycans. NeoRecormon and Eprex contained more trisialylated, but less tetrasialylated glycans than Dynepo and Aranesp. Dynepo had the highest amount of tetraantennary glycans and the lowest amounts of triantennary glycans with a β1-6-GlcNAc linkage. All the samples contained poly-N-acetyl-lactosamine repeats with Dynepo having the least. The major N-acetyl-lactosamine extensions in Dynepo and Aranesp were on biantennary glycans, whereas in NeoRecomon and Eprex they were on triantennary glycans. The sLex epitope was only detected in Dynepo.

Human uterine cervix possesses a high 12-lipoxygenase activity; this enzyme has been isolated in a purified form from the squamous epithelial region of human cervix and its major properties have been investigated. Enzyme activity was present in all subcellular fractions obtained by centrifugation; the highest specific activity was associated with the microsome fraction (160,000 X g pellet). Purification of the enzyme was achieved by acetone precipitation, ion exchange chromatography on CM-cellulose and affinity chromatography on linoleyl-aminoethyl-Sepharose. The product from the incubation of sodium [1-14C]arachidonate with crude enzyme extracts co-chromatographed with authentic 12-hydroxyeicosatetraenoic acid, but the purified enzyme gave a product that behaved like the 12-hydroperoxy derivative. The enzyme had optimum activity at pH 6.5, a Km of 15 microM for arachidonic acid and was stimulated by ATP and Ca2+. Enzyme activity was inhibited by esculetin, nordihydroguaiaretic acid, eicosatetraynoic acid, detergents at concentrations greater than 0.1% (w/v) and preincubation of substrate with GSH and GSH peroxidase. The occurrence of a high 12-lipoxygenase activity is discussed in relation to the specific physiological functions of this tissue.

Re-processing of “old” residue tailings has proved to be a profitable business for several operations, usually when incorporated into an existing facility to supplement declining ore arisings. These resources often prove to be a source of “cheap gold” since the high mining and feed preparation costs have already been incurred and only “variable” treatment costs are incurred in addition to the relatively low reclamation costs. Advances in processing technology, together with a degree of gold release through ageing of the ore over many years, often provide gold recovery in the range 50–60% (headgrade around 0.4–0.5 g/t), which is sufficient to cover the low processing costs and make a positive contribution to the profitability of an operation. Additional benefits such as releasing land for urban development or reducing environmental liabilities may also flow from this type of exercise. The paper covers key practical aspects of developing and implementing a residue re-treatment project beginning with evaluation of the resource, through the implementation phase and into final closure.

Conference Article| October 01 1983 Characterization of acetoacetyl-CoA reductase (3-oxoreductase) from Streptomyces coelicolor: Its possible role in polyhydroxybutyrate biosynthesis NEVILLE M. PACKTER; NEVILLE M. PACKTER 1Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar STEPHEN FLATMAN STEPHEN FLATMAN 2Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1983) 11 (5): 598–599. https://doi.org/10.1042/bst0110598 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation NEVILLE M. PACKTER, STEPHEN FLATMAN; Characterization of acetoacetyl-CoA reductase (3-oxoreductase) from Streptomyces coelicolor: Its possible role in polyhydroxybutyrate biosynthesis. Biochem Soc Trans 1 October 1983; 11 (5): 598–599. doi: https://doi.org/10.1042/bst0110598 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1983 Biochemical Society1983 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Article| October 01 1983 Partial purification of fatty acid synthetase from Streptomyces coelicolor STEPHEN FLATMAN; STEPHEN FLATMAN 1Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar NEVILLE M. PACKTER NEVILLE M. PACKTER 2Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1983) 11 (5): 597. https://doi.org/10.1042/bst0110597 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation STEPHEN FLATMAN, NEVILLE M. PACKTER; Partial purification of fatty acid synthetase from Streptomyces coelicolor. Biochem Soc Trans 1 October 1983; 11 (5): 597. doi: https://doi.org/10.1042/bst0110597 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1983 Biochemical Society1983 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Article| February 01 1985 Formation of storage lipids and actinorhodin, a phenolic antibiotic, in Streptomyces coelicolor NEVILLE M. PACKTER; NEVILLE M. PACKTER 1Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar STEPHEN FLATMAN; STEPHEN FLATMAN 1Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar ANDREW J. LUCOCK ANDREW J. LUCOCK 1Department of Biochemistry, University of Leeds, Leeds LS2 9JT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1985 Biochemical Society1985 Biochem Soc Trans (1985) 13 (1): 251–252. https://doi.org/10.1042/bst0130251 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation NEVILLE M. PACKTER, STEPHEN FLATMAN, ANDREW J. LUCOCK; Formation of storage lipids and actinorhodin, a phenolic antibiotic, in Streptomyces coelicolor. Biochem Soc Trans 1 February 1985; 13 (1): 251–252. doi: https://doi.org/10.1042/bst0130251 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1985 Biochemical Society1985 Article PDF first page preview Close Modal You do not currently have access to this content.

An overview is presented of the retreatment of gold tailings and other residues, with the large Ergo operation as a primary case study, comprising gold recovery from a mixture of sulfide- and non-sulfide-bearing residues that arose from mining activities on the Witwatersrand, South Africa, from the early 1900s until the mid 1960s. The phases of evaluation, testwork, flowsheet development, operations and rehabilitation are covered, including re-mining techniques, flowsheets, and metallurgical treatment of sand and slime.

Homogenates prepared from sections of human uterine cervix or endometrium were incubated with [1-14C]arachidonate and the products examined by radio-TLC. A major product chromatographing with 12-hydroxyeicosatetraenoic acid (12-HETE), and a number of more polar metabolites which were unaffected by 50 μM indomethacin but decreased to the same extent as 12-HETE by 50 μM nordihydroguaiaretic acid, were demonstrated by this technique. The addition of glutathione to the incubation mixture increased the production of 12-HETE, with a proportional decrease in the polar products. There was a large variation in 12-lipoxygenase activity measured in different cervix samples. The levels of the enzyme in the cervix were similar in two groups of uterine samples classified by the histological appearance of the endometrium as proliferative and secretory. However activity was significantly lower in samples taken from post-menopausal patients compared with pre-menopausal patients.

This chapter contains sections titled: Introduction Target Specifications Setting Specifications Physicochemical Properties Biological Activity Immunochemical Properties Purity and Impurities Stability Analysis The Role of Characterization During Process Development Effect of Process Changes on Product Characteristics: Case Studies Conclusions References

This chapter contains sections titled: Introduction The Standard Toolbox Western Blotting References

The chapter deals with gold and sulfur recovery. The reclamation methods and general processing techniques would, however, be applicable to the recovery of other minerals, although the specific flowsheets would obviously differ. Gold-mining residues fall into one of the three categories comprising slime (particle size of about 75% passing 74 μm), sand (about 10–20% passing 74 μm), and waste rock. These residue materials constitute a cheap source of gold, especially when exploited as a tonnage make up to fill excess treatment capacity within an existing processing facility. In this scenario, sand or slime can be processed at marginal treatment cost, and can make a substantial contribution to the bottom line profit of an operation. The prime justification for residue retreatment will always be to create profit for the company concerned. Other benefits to be considered include: release of land for urban development and removal of environmentally unfriendly dumps (and their associated long-term liability) for relocation to a more modern facility.

Abstract Recombinant erythropoietins are used extensively in the management of anemia associated with chronic kidney disease (CKD). All the currently available erythropoietins are synthesized in Chinese hamster ovary (CHO) cell lines resulting in differences in glycosylation compared with human serum erythropoietin. We report the molecular characterization and pharmacokinetic properties of epoetin delta (Dynepo®, Shire plc), the only erythropoietin produced in a human cell line. A variety of techniques have been used to characterize epoetin delta, including amino acid sequencing, peptide mapping with reverse-phase HPLC/mass spectrometry, oligosaccharide profiling and MALDI-TOF of released glycans. Sialic acid and N-glycolylneuraminic acid (Neu5Gc) residues were quantified, following labeling of the released glycans, by reverse-phase HPLC analysis with fluorescence detection. Epoetin delta was produced as a highly pure and stable protein with the full human primary amino acid sequence. Neu5Gc residues were not detectable in epoetin delta within the validated limits of the assay but became readily detectable in CHO derived epoetins. Following characterization of the molecule early studies were done to determine the pharmacokinetic and pharmacodynamic properties of epoetin delta. Two studies investigated responses to epoetin delta in healthy individuals. The first study involved randomization of 21 men to either intravenous (i.v.) epoetin delta (15, 40 or 100 IU/kg) or placebo. The second was an open-label, crossover study, involving 32 volunteers randomized to receive single doses of epoetin delta 75 IU/kg given i.v. or subcutaneous (s.c.) by injection. Two further studies were carried out with CKD patients requiring hemodialysis who had previously received epoetin alfa. The first involved 40 patients randomized to epoetin delta or epoetin alfa (50 or 100 IU/kg), three-times per week for 4 weeks. The second was a single-dose study in 28 patients comparing epoetin delta 150 and 300 IU/kg given i.v. or s.c. Pharmacokinetic parameters were calculated from serum erythropoietin concentrations (determined by ELISA) by validated non-compartmental techniques using WinNonlin Professional v3.0A. Intravenous epoetin delta in healthy individuals displayed non-linear and dose-dependent pharmacokinetics, with a dose-dependent effect on serum hemoglobin. The bioavailability of s.c. epoetin delta is around 30%, and concentrations peak later and decline more slowly than with i.v. injection. In hemodialysis patients pharmacokinetic parameters were similar to those in healthy individuals, although AUC and half-life were increased. Compared with the 50 IU/kg dose, treatment with epoetin delta 100 IU/kg was associated with a trend to increased hemoglobin and hematocrit levels. Epoetin delta was well tolerated in all participants with the frequency of adverse events occurring in dialysis patients as expected for that population. Adverse events were similar with epoetin alfa and epoetin delta. No neutralizing anti-erythropoietin antibodies were detected in any patient. Epoetin delta was characterized at the molecular level and levels of Neu5Gc residues were undetectable within the validated limits of the assay. The pharmacokinetic profile of epoetin delta and effects on hemoglobin and hematocrit levels were suitable for the treatment of anemia in CKD patients by either s.c or i.v. administration.

One of the requirements during the process of manufacturing development of a therapeutic protein is continuous monitoring of several key parameters namely, titre, structural integrity and purity at selected stages of the product life cycle. Traditionally conventional methods such as Protein A HPLC, SDS PAGE electrophoresis and ELISA assays are being used to obtain this information for antibody products. These methods are time consuming, labour intensive and individually offer only single parameter analysis required for the product quality assessment. At Lonza Biologics the 2100 Bioanalyzer has been used to determine product purity, integrity profi le, size and titre using minimum sample volume and time resource input. The 2100 Bioanalyzer is part of the new generation of miniaturised μ-TAS (Micro total analytical system) allowing the multiple capture of analytical parameters for product quality and cell antibody assembly effi ciency. This technology is essentially miniaturised capillary electrophoretic system with microfl uidics application confi ned to a chip. 2100 Bioanalyzer was used to monitor the quantity and quality of monoclonal antibodies (MABs) expressed using Lonza Biologics GS-expression system. Suitable medium selection was facilitated using 2100 Bioanalyzer screening. In-process culture supernatant and purifi cation samples from lab-scale and pilot scale processes were directly tested for product quality and quantity. The data generated using the 2100 Bioanalyzer were rapid and proved useful in the optimisation of the fermentation and purifi cation processes. This poster outlines the data obtained for IgG tested at various stages of the manufacturing development process. 2100 Bioanalyzer showed acceptable correlation with conventional techniques. We demonstrate that 2100 Bioanalyzer will have a positive impact on the quality and effi ciency of the production processes for therapeutic proteins. Introduction The number of therapeutic antibodies and recombinant proteins developed has risen dramatically over the past decade. This explosion in therapeutic antibody research was followed with immediate success as Rituxan® and Herceptin®, the fi rst therapeutic antibodies accepted by the FDA for non-Hodgkin’s lymphoma and breast cancer respectively and lately immunotherapeutics showed indications in the inhibition of prion replication and slowing down the disease (White et al, 2003). Over 90% of the potential therapeutic antibodies are at the clinical trial stages. This trend is likely to increase with the timing of proteomic research. The type of diseases targeted include arthritis (Kevorrkov & Futlik, 2000), Asthma (Nelson, 2003), HIV (Tsamis, 2003) and stroke (Wiessner, 2003). Clinical trials and in market therapeutic antibodies are produced in vast quantities depending on the market requirements and trial demands. Analysis of these products is vital throughout the product life cycle from development to fi nal release. The widely employed techniques for such purposes are SDS-PAGE electrophoresis, Protein A HPLC and ELISA assays which require multiple operators and can result in protracted time line. The use of these techniques is necessitated to have an understanding of the product quality issues and titre assessment. The current advances in proteomic research and increased therapeutic antibody development requires more effi cient technologies and platforms with multi analytical/parameter capabilities to deliver the data with minimum resource and at a rapid speed to facilitate the anticipated throughput, this is where the 2100 Bioanalyzer (Figure 1) proves its usefulness. This technique has been evaluated at Lonza Biologics for the analysis of monoclonal antibody (IgG4). This is referred to as Lab Chip technology and is based on microfl uidics where sample preparation, fl uid handling and biochemical analysis are carried out within the confi nes of a microchip. The chips comprise of microchannels fabricated in glass that create an interconnected network of gel matrix reservoirs and pathways. This technology has fully automated analytical and data processing capability. At Lonza Biologics we have employed the 2100 Bioanalyzer at selected stages of product manufacturing development and compared it with conventional techniques. Some promising data were obtained. Lonza Biologics, part of Swiss-based Lonza Group, is a contract manufacturer of biotherapeutics derived from mammalian cell culture. Results and Discussion • Antibody analysis using 2100 Bioanalyzer at early stages of the manufacturing development process (Figure 2a & 2b) showed that titre and antibody integrity information are essential in the selection process of cell line clones. • Rapid trending of the product titre and overall profi le of the fermentation medium containing the product in real time at line was only realised using the 2100 Bioanalyzer and specifi c Lonza Biologics sample preparation method. The titre, total % and quality of the product and other product related fragments were trended at line (Figure 3 & 4). • Product purifi cation processes require on-going monitoring of the status of the antibody after each purifi cation steps for titre, % purity and quality of the product. 2100 Bioanalyzer provided these data at line. The data have direct impact on the purifi cation process development strategy (Figure 5). • The 2100 Bioanalyzer was used for the medium selection process. Various medium specifi cations, including protein free media were used to grow CHO cells and the intact antibody, light chain (LC), and heavy chain (HC) products were analysed (Figure 6). • The 2100 Bioanalyzer was compared with Protein A HPLC and A280 techniques. The correlation obtained was R2= 0.98 and R2=0.99 respectively. Medium Selection Process Figure 6. Monoclonal antibody grown in various media specifi cations. The level of product generated is shown in the following electropherograms. The level of product in medium fi ve was higher than the other media. Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Antibody Assembly Development Process Figure 2a. Electropherograms for the profi le of culture supernatant containing intact monoclonal antibody and other fragments obtained from preliminary overgrown transfected cell lines. Figure 2b. Intact monoclonal antibody titre. FL0, FL1, FL2, FL3, FL4, FL5, FL6 Method and Sample Preparation • 4 μl of sample was added to 2 μl of sample buffer (± s-MCE). Sample was heated in boiling water for 1 minute. After cooling the sample was spun at 13K RPM for 15 seconds. This was followed by the addition of 84 μl of distilled water. • 6.0 μl of sample and ladder was applied into the protein chip wells where proteins are separated by the acrylamide sieving process. The protein chip was loaded onto the 2100 Bioanalyzer and each protein is then induced by fl uorescent excitation at 633 nm. • All the reagents are provided in the 2100 Bioanalyzer-protein 200 plus kit (http://www.chem.agilent.com/Scripts/PCol.asp?lPage=50) Figure 1. Correspondence Dr Nesredin A Mussa & Imtiaz Alam Research & Development, Lonza Biologics, Bath Road, Slough, SL1 4DX, Berkshire, UK Concluding Remarks • The data presented here shows for the fi rst time the applicability of 2100 Bioanalyzer for the simultaneous determination of the titre, integrity, and % purity of monoclonal antibodies throughout the manufacturing and purifi cation development process. • 2100 Bioanalyzer can be used for the analysis of both monoclonal antibodies and other recombinant proteins produced in mammalian cell culture. • The 2100 Bioanalyzer allows at line analysis at a rapid pace relative to the conventional techniques. This introduces improved effi ciency in the process of therapeutic antibody manufacturing development. • This ultimately reduces the time line required to manufacture products for clinical trials and market supply. Purifi cation Development Process Figure 5. Monoclonal antibodies undergo several stages of purifi cation. At each stage of purifi cation process development, the 2100 Bioanalyzer was used to assess product quality and purity. A Unpurifi ed load B Post-purifi cation step 1 C Post-purifi cation step 2 D Post-purifi cation step 3 Figure 4. A The titre of the intact antibody in culture supernatant analysed at-line. B The % total of the intact monoclonal antibody. C The rise in antibody fragments across fermentation process. The Multiple Application of 2100 Bioanalyzer in the Manufacturing Development Process of Therapeutic Antibody Fermentation Development Process Figure 3. Electropherograms of intact monoclonal antibody and other fragments in culture supernatant analysed using 2100 Bioanalyzer at line. Samples were collected at different intervals across two weeks pilot scale fermentation process. Day 1 Day 4 Day 6 Day 8 Day 12 Day 15