Anthony B. Chen

Identification of multiple sources of charge heterogeneity in a recombinant antibody.

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.

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.

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.

Capillary isoelectric focusing and sodium dodecyl sulfate-capillary gel electrophoresis of recombinant humanized monoclonal antibody HER2

Capillary isoelectric focusing (cIEF) and IEF of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) show five charged isoforms with estimated pI values ranging from 8.6-9.1. The cIEF assay demonstrated good precision with relative standard deviations (R.S.D.) 0.7-3.7% and 0.4-4.2% for intra and interassay analysis, respectively. The method was linear for the area of the main peak over the concentration range 2-250 micrograms/ml with a Pearson correlation coefficient > 0.99. The limit of detection for the main peak was determined to be 2 ppm. With both sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and SDS-polyacrylamide gel electrophoresis, the nonreduced rhuMAbHER2 migrated as a single major peak with minor peaks in the aggregate and clip regions. After reduction, the electropherogram and the slab gel showed the expected heavy chain and light chain fragments with minor peaks in the aggregate and clip regions. The SDS-CGE assay showed good precision with R.S.D. values of 0.1-7.8% and 0.1-8.1% for intra and interassay analysis, respectively. The Pearson correlation coefficient for the area of the main peak was > 0.99 demonstrating linearity for the concentration range 0.5-500 micrograms/ml. The limit of detection for intact rhuMAbHER2 was determined to be 0.5 ppm. The data presented demonstrates the feasibility of replacing the slab gel techniques with capillary electrophoresis in a quality control environment.

Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8.

A capillary isoelectric focusing (cIEF) method has been developed for the purpose of determining the identity and charge distribution of mouse/human chimeric antibody to human CD20 antigen (C2B8). The assay was validated in accordance with ICH guidelines in order to demonstrate that it is suitable for its intended purpose and so that it may be performed as a lot release test for bulk and final product. As a result of the validation process the assay was found to be linear over the concentration range of 2-356 micrograms ml-1 with recovery of 125I-labeled C2B8 at the target sample concentration of 125 micrograms ml-1 equal to 99%. The repeatability and intermediate precision relative standard deviations of the four major peaks for migration time, peak area, and peak area percent ranged from 0.9-4.4%. The specificity of the assay was demonstrated by baseline resolution of the C2B8 main peak from product excipients, and other Genentech monoclonal antibodies. The results of this validation demonstrate that the cIEF assay for the determination of identity and charge distribution of C2B8 is accurate, precise, linear, and highly specific. The assay is rapid and suitably rugged.

Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator

The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.

Rapid one-step capillary isoelectric focusing method to monitor charged glycoforms of recombinant human tissue-type plasminogen activator.

A rapid (< 10 min) one-step capillary isoelectric focusing (cIEF) method was developed to monitor charged glycoforms of recombinant human tissue-type plasminogen activator (rt-PA). Focusing takes place between the detector and the anode and the electro-osmotic flow (EOF) sweeps the separated glycoforms past the detector, towards the cathode. The separation uses a neutral coated capillary and hydroxypropylmethylcellulose (HPMC) to reduce the EOF to a constant and reproducible value. The method uses an ampholyte mix with a 50:50 ratio of pH 5-8 and pH 3-10 ampholytes in 4 M urea and 0.1% HPMC to produce maximal resolution whilst maintaining protein solubility during focusing. The electropherograms were compared to isoelectric focusing (IEF) slab gels of samples of intact rt-PA. In both cases approximately ten charged species could be detected. Data analysis indicated that the intra-assay precision was < 5% for peak migration times and < 10% for normalized peak areas. The number of charged species detected by each of the two methods was consistent for samples of intact rt-PA, rt-PA types I and II and for neuraminidase-digested rt-PA. Overall the data indicate that the automated cIEF method can be an adjunct to slab-gel IEF in the characterization and routine analysis of recombinant glycoproteins.

Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins

Two immunoassays have been developed for the quantitation of part-per-million levels of contaminants likely to co-purify with monoclonal antibodies produced in tissue culture and purified by protein A affinity chromatography. These contaminants are bovine IgG originating from the fetal bovine serum used in cell culture, and protein A. Mouse IgG was shown not to interfere in the bovine IgG assay, where contamination levels of 0.2-0.7% bovine IgG were measured in the lots of monoclonal antibody tested. The protein A ELISA was developed with monoclonal antibody included in the standard, and in the preparations of monoclonal antibody tested, 64 parts per million (ppm) or less of protein A were demonstrated. An additional immunoassay was developed to quantitate monoclonal antibody contamination of two recombinant proteins, rHBsAg and rgp 120 from HIV, purified by affinity chromatography with such antibodies. Possible interference of monoclonal antibody quantitation by the respective antigens was examined in this ELISA, and contamination levels of less than 56 ppm of antibody were determined in the purified recombinant proteins. The three immunoassays were shown to be specific for the major protein contaminants in either monoclonal antibodies or the recombinant proteins and were necessary in demonstrating their purity.

Comparison of ampholytes used for slab gel and capillary isoelectric focusing of recombinant tissue-type plasminogen activator glycoforms

Four commercial ampholytes: Ampholine and Pharmalyte (Pharmacia Biotech), Bio-Lyte (Bio-Rad) and Servalyt (Serva) were evaluated for their ability to resolve recombinant tissue-type plasminogen activator (rt-PA) glycoforms by isoelectric focusing (IEF) and capillary IEF (cIEF). Each brand of ampholytes focused rt-PA into 3-4 major and 5-6 minor bands on slab gel electrophoresis. Visually, focused bands stained with Coomassie Blue appeared to be similarly resolved by all the ampholytes except for Ampholines, where the bands were closely grouped and more intensely stained. When cIEF was performed, Pharmalytes and Ampholines resolved rt-PA glycoforms consistent with the slab gels. No discernible peaks were detected during cIEF of rt-PA using Servalyts or Bio-Lytes. UV spectrophotometric scans of the components used for cIEF showed that Servalyts absorbed intensely over a range which overlapped the detector bandpass. Bio-Lytes showed absorption over a narrower UV range but still overlapped the detector bandpass, thus preventing the discernment of protein peaks. For this cIEF system the best ampholytes were Ampholines and Pharmalytes.

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.