Stability of insulin lispro in insulin infusion systems.

OBJECTIVE: To test stability of insulin lispro in two insulin infusion systems over 48 h. RESEARCH DESIGN AND METHODS: We used reverse-phase and size-exclusion high-performance liquid chromatography (HPLC) to determine the purity, potency, and degree of polymerization of U100 insulin lispro (Humalog) after 24- and 48-h pump cycles conducted at 37 degrees C in five Disetronic H-TRON V100 and five MiniMed 504 pumps. Pumps were set to deliver a basal rate of 0.5 U/h and 6-U boluses at t = 0, 4, 8, 24, 24.5, 28.5, 32.5, and 48 h during each cycle. The effluent was collected into 1-ml vials, pooled at 24 or 48 h, and stored at 4 degrees C until assay. After each 48-h run period of insulin delivery, assays for potency, polymer, and purity were performed on the pooled samples from each individual cycle. m-cresol content and the pooled reservoir content were assayed in the 48-h pooled samples. RESULTS: Insulin lispro retained full HPLC potency (delta < or = 4%) at 48 h, with no degradation of insulin lispro to des-amidoinsulin forms (24 or 48 h). No increase in pumped insulin polymer concentration was observed following 24 h of pump flow. Nonsignificant increases of < or =0.09% (Disetronic) and < or =0.15% (MiniMed) from initial concentrations of 0.18% (polymer divided by total insulin) were detected in three of five pump cycles at 48 h when compared with 37 degrees C paired controls. Nonsignificant decreases (<5 and 10%, Disetronic and MiniMed, respectively) of m-cresol content occurred in both systems following 48 h storage in each device, but sterility was not compromised by this decrease (initial m-cresol concentration, 3.15 mg/ml). Pump performance was without mechanical or electrical fault throughout the study Basal and bolus insulin delivery was evaluated three times daily and remained as expected. Occlusion of catheters by insulin precipitation did not occur, and no change in pH was observed following delivery. CONCLUSIONS: We conclude that insulin lispro is suitable for prolonged infusion in these two medical devices when syringes and catheters are replaced at 48-h intervals.

Analytical strategies for the determination of protein modifications.

Assessment of recombinant protein product purity and consistency has been successfully accomplished by using a battery of quantitative analytical methods. The development of analytical methods for product control requires an understanding of potential changes in protein structure which could be caused by degradation and modification. This paper will review some of the common mechanisms of protein degradation and common modifications which can occur during the fermentation/cell culture and purification processes. It will also present examples of analytical techniques which have successfully elucidated some of these changes in protein structure.

Kinetic chromatographic sequential addition immunoassays using protein A affinity chromatography.

A new type of chromatographic immunoassay based on sequential addition is described. On a protein A column, the antibody, the sample containing the antigen, and then a known amount of antigen are sequentially injected. This assay is designed to shorten analysis times and reduce complexity of dual-column chromatographic immunoassays, circumvent desorption buffer interferences common to affinity chromatography, and eliminate the need for tagged molecules. This new technique is named kinetic immunochromatography sequential addition (KICQA). Because of its kinetic nature, flow rate will have a large effect on KICQA, and the impact of changing flow rate is studied extensively. By use of various amounts of antibody, the dynamic range of KICQA is shown to be selectable over 2.5 orders of magnitude. Finally, KICQA was used to determine transferrin and albumin in human serum. Both analytes show good agreement with their respective reference methods, and an albumin assay was performed in under 1 min.

Dual-column immunoassays using protein G affinity chromatography.

Tandem protein G affinity and reversed-phase chromatography (RPC) columns, coupled with a switching valve, were used for on-line immunoassays of antibodies and antigens. Columns with reversibly immobilized antibodies were prepared by adsorbing antibodies on the protein G column. Following antigen capture in the immunoaffinity column, antigen-antibody complexes were desorbed, dissociated, and transferred to the RPC column where they were separated and quantified. This system was used to determine the titer of a rabbit anti-human transferrin antibody sample with a precision of +/- 2%. Quantitation of human transferrin in human serum had a precision of +/- 6% and showed good agreement with rate nephelometry. The linear dynamic range for the transferrin, antigen immunoassay was 5 x 10(1) to 1 x 10(5) ng with a precision of +/- 3.5%.

Immunological-chromatographic analysis of lysozyme variants.

Immunological-chromatographic analysis (ICA) was used to evaluate the cross-reactivities of eight lysozyme variants with five different immobilized monoclonal anti-hen-egg-white lysozyme antibodies. ICA is a dual-column high-performance liquid chromatography-based method in which an immunoaffinity and a conventional analytical column are coupled with a switching valve. Antigens are first captured on the affinity column and then desorbed and concentrated on the second column, where they are separated further. This arrangement permits antigen-antibody interactions occurring on the affinity column to be monitored on-line with the second column. The ICA system was used to perform direct and competitive inhibition binding immunoassays with unlabeled antigens. Seven of the eight lysozymes tested bound to all five immobilized monoclonal antibodies. Competitive inhibition of binding of hen egg white lysozyme to the monoclonal antibody, HyHel-5, was measured by using the variants Japanese quail and bobwhite quail lysozymes as inhibitors. The ratio of the amount of bobwhite quail to Japanese quail lysozyme required to give 50% inhibition of binding of hen egg white determined by ICA compares well with the results obtained by other investigators who used an enzyme-linked immunosorbent assay plate binding assay.

Immunological-chromatographic analysis.

Tandem immunoaffinity and conventional high-performance liquid chromatography columns, coupled with a switching valve, were used for the analysis of single and multicomponent antigen samples. Immunoaffinity columns were prepared by hydrophobic adsorption or covalent immobilization of poly- or monoclonal antibodies on macroporous poly(styrene-divinylbenzene) packing materials. Capacities of these columns were constant through at least 77 cycles. Macromolecular antigens were analyzed at submicrogram levels. Antigens bound to the immunoaffinity column were desorbed and concentrated on a conventional analytical column. Gradient elution on the analytical column separated the desorbed antigen(s) from interfering species and permitted the analysis of all species which bound to the immunoaffinity column. Immunological-chromatographic analysis was useful for purification and discrimination of polypeptides of similar three dimensional structures, such as several lysozyme variants.