Venkat R. Mukku

A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody

A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose-response curves in the concentration range of 0.02-3.3 micrograms/ml. With intra-assay variability of 5-12%, interassay variability of 6-10% and spike recoveries of 101-109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb.

The Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

AbstractPurpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.

Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect

The treatment of growth hormone deficiency requires the daily administration of recombinant human growth hormone (rhGH). Long lasting formulations of rhGH have the potential to increase patient compliance, improve quality of life, and increase the efficacy of rhGH (lower total dose). One approach to these formulations is the use of biodegradable, injectable microspheres consisting of poly(lactic-co-glycolic acid) (PLGA). rhGH PLGA microspheres (12% w/w rhGH) were produced using a conventional double emulsion process. Initial in vitro studies of these microspheres indicated an initial release of 35% (1 day) and a continuous release for 21 days. A single administration of these microspheres (200 mg) in rhesus monkeys resulted in an initial elevation of serum hGH levels followed by a lag phase (return to baseline) until day 12 and then a sustained level of hGH greater than 5 ng/ml for 30 days. Development of improved in vitro release methods yielded release profiles comparable to those observed in vivo as determined from a detailed pharmacokinetic analysis of the hGH serum data. Serum hGH concentrations at or above 5 ng/ml provided a maximal response in two primary indicators of hGH biological activity, insulin-like growth factor-I (IGF-I) and IGF-binding protein 3. Further assessment of serum samples in an in vitro cell-based assay indicated that the rhGH released in vivo from the microspheres was bioactive. The rhGH PLGA formulations were well tolerated with mild to moderate inflammation and fibrosis. One of four animals developed a low titer anti-hGH antibody response, but transgenic mice expressing hGH did not develop an immune response to rhGH released from the PLGA microspheres. Therefore, this formulation had a low immunogenicity similar to the commercial rhGH formulation (Nutropin®). Overall, these studies demonstrated that a single administration of rhGH PLGA microspheres provide a prolonged release of bioactive rhGH that is well tolerated. With the improved in vitro release methods, future rhGH PLGA formulations may be designed without a lag phase to yield a one month continuous release of bioactive rhGH.

Validation of a rat pheochromocytoma (PC12)-based cell survival assay for determining biological potency of recombinant human nerve growth factor.

A method has been validated, according to the Guidelines of the International Conference on Harmonization (ICH), for precise quantitation of the biological activity of recombinant human nerve growth factor (rhNGF) for lot release testing. The assay is based on the survival of a subclone of rat pheochromocytoma PC12 cells (PC12-CF) in response to rhNGF. Cell survival is measured by monitoring the reduction, by living cells, of the alamarBlue dye into a red form which is highly fluorescent. The assay is simple, has high throughput (performed in 96-well microtiter plates) and shows reproducible dose-response curves in the concentration range of 0.2-50 ng/ml. The method was validated for its linearity, accuracy, precision, robustness, and to meet current regulatory requirements. The assay demonstrated good linearity, yielding a coefficient of determination of 0.9902. Sample recovery studies demonstrated an accuracy ranging from 96 to 98%. The repeatability of the assay and intermediate precision had coefficients of variation (CV) of <9%. The assay was stability-indicating since it was able to detect changes in rhNGF samples degraded by protease treatment and in a number of isolated rhNGF variants. Robustness was demonstrated by the relative insensitivity of the assay to small deliberate changes in key method parameters. The validation data, provided in this manuscript, indicate that the newly described bioassay for rhNGF is robust, accurate, precise, and suitable for lot release potency testing of rhNGF.