Rose Gaines-Das

Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals

An important aspect of evaluating the safety of therapeutic biologicals is the assessment of the unwanted immunogenicity of such biologicals in recipients. Properly planned immunogenicity studies with appropriately devised strategies are critical if valid conclusions concerning the unwanted immunogenicity are to be derived. Such studies need to be conducted using carefully selected and validated procedures. Several techniques are available for detection and measurement of immunogenicity including immunoassays, radioimmunoprecipitation assays (RIPAs), surface plasmon resonance (SPR) and bioassays. A combination of methods for characterization of the induced antibodies is usually necessary for a detailed understanding of the type(s) of antibodies generated against a therapeutic product. This review considers the benefits and limitations of the various techniques available for antibody detection and outlines a strategy for the assessment of unwanted immunogenicity of therapeutic products.

Immunoassays for detecting cytokines: What are they really measuring?

In summary, many factors influence the estimates of cytokine levels provided by immunoassays. If immunoassays are to supply data which are valid and useful to researchers and clinicians, these factors must be fully investigated during assay design and construction.

Fate of injected interleukin 1 in rats: Sequestration and degradation in the kidney

The tissue distribution and route of clearance of human recombinant interleukin 1 alpha (IL 1 alpha) injected intravenously in rats was studied. The plasma half-life was approximately 2.5 min, and this was increased after nephrectomy, the kidney being the major organ through which the IL 1 alpha was excreted. Two iodinated fragments of IL 1 alpha, of approximately 5 and 9 kDa, were excreted by the kidneys whereas only intact, 17-kDa IL 1 alpha was detected in plasma, suggesting that the protein was being degraded after uptake by the kidney. The results of in vivo experiments in which surface endopeptidase-24.11 was inhibited with phosphoramidon and in vitro experiments in which rat kidney homogenates were incubated with radiolabeled IL 1 alpha suggest that the cytokine was endocytosed and then hydrolysed by lysosomal proteinases.

Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy

In this study, the development of neutralizing and non‐neutralizing GM‐CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli‐derived GM‐CSF and a colon carcinoma‐reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM‐CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM‐CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM‐CSF binding antibodies. However, only a proportion (40%) of the 19 antibody‐positive patients developed antibodies that neutralized the biological activity of GM‐CSF in an in vitro bioassay. The presence of GM‐CSF neutralizing antibodies was associated with a significant reduction in GM‐CSF‐induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non‐neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM‐CSF preparations derived using different expression systems (chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross‐react with endogenously produced GM‐CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.

Physicochemical and biological assays for quality control of biopharmaceuticals: Interferon alfa-2 case study

A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.

International Standards for salmon calcitonin, eel calcitonin, and the Asu1-7 analogue of eel calcitonin: calibration by international collaborative study.

Regulatory specifications in most countries require that the potency of salmon calcitonin (sCT) clinical products be expressed in International Units (IU) defined by the World Health Organization (WHO) International Standard. The first ampouled standard was prepared in 1972 and has been distributed world-wide since then. A batch of ampoules to serve as the replacement standard is now required. Other piscine calcitonins, eel calcitonin (eCT) and an amino-suberic acid analogue of eCT (Asu1-7 eCT) are now clinical products in some countries and international standards are required for these peptides which are similar to, but not identical with, sCT. This paper describes the preparation of three new ampouled standards and their biological calibration by international collaborative study comprising 17 participants from 10 countries. Following the recommendations in the final report of the collaborative study, the 2nd International Standard (IS) for sCT, the 1st IS for eCT and the 1st IS for Asu1-7 eCT were recently established by WHO, each with an assigned potency in IU, and are now available for issue.

The International Standard for Epidermal Growth Factor (EGF): Comparison of Candidate Preparations by In Vitro Bioassays and Immunoassays

Four preparations of recombinant human sequence epidermal growth factor (EGF), two of the full length 53-amino acid chain, and two of the 52-amino acid chain, lacking the carboxyl-terminal arginine, were evaluated, using a variety of in vitro bioassays and immunoassays, by 12 laboratories in seven countries, for their suitability to serve as the international standard for EGF. The study shows that some assay systems appear to discriminate between the full length and the 52-amino acid forms of EGF and that use of a common standard in place of the various in-house standards leads to a substantial decrease in between-laboratory variances in estimates of potency of EGF samples. On the basis of the results reported here, the World Health Organization (WHO) established one of the preparations of the full-length EGF molecule (coded 91/530) as the first international standard (IS) for EGF, with an assigned unitage of 2000 International Units/ampoule, and one of the preparations of EGF(1-52) (coded 91/550) as the first international reference reagent (IRR) for EGF (1-52) with a nominal mass content of 1.75 microg EGF(1-52)/ampoule. The IS for EGF and the IRR for EGF(1-52) may be obtained by writing to NIBSC, PO Box 1193, Potters Bar, EN6 3QH, UK.

The International Standard for Platelet-Derived Growth Factor-BB: Comparison of Candidate Preparations by In Vitro Bioassays and Immunoassays

In an international collaborative study, four preparations of recombinant, human sequence, platelet-derived growth factor-BB (PDGF-BB), two glycosylated and two nonglycosylated, were evaluated, using in vitro bioassays and immunoassays, by seven laboratories in three countries, for their suitability to serve as the international standard (IS) for PDGF-BB. The study shows that interlaboratory variation in estimates of PDGF-BB potency is reduced by use of a common standard. The bioassays, using various types of fibroblast, detected PDGF-BB and PDGF-AB as equally potent, while immunoassays discriminated between the two isoforms. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 94/728 as the first IS for PDGF-BB, with an assigned unitage of 3000 International Units per ampoule. The IS may be obtained by writing to NIBSC, PO Box 1193, Potters Bar, EN6 3QH, UK, or through web site http:¿¿www.nibsc.ac.uk.

The International Standard for Basic Fibroblast Growth Factor (FGF-2); Comparison of Candidate Preparations by In Vitro Bioassays and Immunoassays

Three preparations of recombinant basic fibroblast growth factor (bFGF, FGF-2) were evaluated, using a variety of in vitro bioassays and immunoassays, by 11 laboratories in six countries for their suitability to serve as international standards (IS) for bFGF. Two preparations of human sequence bFGF showed broadly similar behavior in different assays, but the relative activity of the third preparation, a modification of the human sequence, showed more variability. All three preparations were predicted to be sufficiently stable to serve as an IS. On the basis of the results reported here, the World Health Organization (WHO) established one of the preparations (coded 90/712) as the international standard for basic fibroblast growth factor, and assigned it a content of 1600 International Units (IU) per ampoule. The IS may be obtained for use in the calibration of local standards by writing to NIBSC, PO Box 1193, Potters Bar, EN6 3QH, UK.