C. Jane Robinson

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.

Quality control and analytical techniques for biopharmaceuticals

Biopharmaceuticals are complex products and to ensure their batch-to-batch consistency and continuing quality the use of a combination of complementary analytical tests is required. Regulatory guidelines indicate quality attributes of different product classes to be included in the specifications for product release. Whilst the continuing development of sophisticated physicochemical techniques make them increasingly powerful for defining product identity, integrity, purity and the consistency of the manufacturing process, the results generated are not easily related to the biological activity. Consequently, a bioassay is normally required in the quality control to determine the potency, that is, the quantitative measure of the products ability to cause a specific biological effect in a defined biological system. A wide, and rapidly increasing, range of bioassay systems exist, each type with particular advantages and disadvantages.

An in vitro bioassay for nerve growth factor based on 24-hour survival of PC-12 cells.

We have developed an in vitro bioassay for nerve growth factor (NGF) which offers several advantages over currently used alternatives. The assay uses the commercially available PC-12 cell line, and requires only 24 hr incubation with NGF. The cells do not require priming, and are dosed as they are originally seeded in serum-free medium in 96-well cell culture plates coated with collagen. Concentrations between one and 20 ng/ml NGF can be measured, and only standard cell culture equipment and a microplate reader are required.

Quantitative RT-PCR as an alternative to late-stage bioassays for vascular endothelial growth factor

We have investigated the use of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as an alternative to a selection of late-stage functional bioassays for determination of the potency of preparations of vascular endothelial growth factor (VEGF). Responses were measured in cultures of human umbilical vein endothelial cells (HUVECs). Late-stage responses measured were cell survival and proliferation, and production of interleukin-8 (IL-8), interleukin-6 (IL-6), and tissue factor. The dose-response range was similar across the assays, increasing from 2 ng/mL VEGF and reaching a maximum between 30 ng/mL and 125 ng/mL VEGF. A number of VEGF-induced mRNA species demonstrated dose-response curves suitable for VEGF potency determination. IL-8 mRNA induction after 45 min incubation with VEGF, which showed maximal responses between 15.6 ng/mL and 62.5 ng/mL VEGF, was selected for further characterization. This gene-expression bioassay was robust across a range of cell seeding densities and could be used for samples processed immediately following incubation with VEGF and for cell lysates stored at -80 degrees C for 3 months. We also compared this gene-expression bioassay and the assays of late-stage responses in the potency measurement of the inhibitors of VEGF activity, anti-VEGF monoclonal antibody MAB293, and a VEGF soluble receptor VEGFsR1 preparation. We present a critical evaluation of the use of qRT-PCR in assaying the potency of VEGF and its inhibitors, and of the potential of this platform for measuring the potency of other biological therapeutics.

The World Health Organization reference reagent for vascular endothelial growth factor, VEGF165

Preparations of human sequence recombinant vascular endothelial growth factor-165 (VEGF165) synthesized in Escherichia coli were formulated and lyophilized at NIBSC. Following evaluation at NIBSC, the first preparation, 01/424, has been distributed since 2002 as a NIBSC research reagent, but shows variation between ampoules in the volume and crystalline appearance of the lyophilized plug. A second preparation, 02/286, was subsequently lyophilized in a different formulation. Preparation 02/286 has now been evaluated in a collaborative study for its suitability to serve as a reference standard, and compared with preparation 01/424, by five laboratories using in vitro bioassays or immunoassays. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 02/286 as the WHO reference reagent (RR) for human VEGF165, with an assigned unitage of 13,000 units per ampoule. Details on ordering the WHO RR can be found at www.nibsc.ac.uk.

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 World Health Organization reference reagent for keratinocyte growth factor, KGF

Preparations of human sequence recombinant keratinocyte growth factor (KGF), fibroblast growth factor-7 (FGF-7) synthesized in E. coli were formulated and lyophilized at National Institute for Biological Standards and Control (NIBSC) and evaluated in a collaborative study using in vitro bioassays and immunoassays. One candidate standard was the full-length mature KGF molecule and the two others were different formulations of a truncated form of the molecule, KGF (24-163). The study demonstrated the difficulty of performing interlaboratory comparison of assays without a common reference standard and differences in dose-response relationships between molecular variants. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 03/150 as the WHO reference reagent for human KGF, with an assigned unitage of 4000 units of KGF per ampoule and the preparation coded 03/148 as the WHO reference reagent for human KGF (24-163), with an assigned unitage of 9000 units of KGF(24-163) per ampoule. Details on ordering the reference reagents can be found at www.nibsc.ac.uk.

Polypeptide growth factors — a growth area for biotechnology

The interaction between the fields of biotechnology and growth factor research was illustrated at the Biological Councils 5th Annual Symposium on Biotechnology*. This highly informative meeting was not limited to consideration of the role of polypeptide growth factors, but they, together with their signal transduction pathways, figured in many of the topics presented, a few examples of which are reported briefly here

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.