An improved size exclusion-HPLC method for molecular size distribution analysis of immunoglobulin G using sodium perchlorate in the eluent.

Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular size distribution (MSD) analyses of various therapeutic proteins. We report development and validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a TSKgel SuperSW3000 column and eluting it with 0.4M NaClO4, a chaotropic salt, in 40mM phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run time (12min), better peak resolution, improved peak shape and more stable base-line compared to HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A comparison of MSD analysis results between our method and the EP method shows interactions between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the stationary phase, when the latter method is used. Thus, the EP method shows lower percent of aggregates and tetramer than are actually present in the products. In view of the fact that aggregates have been attributed to playing a critical role in adverse reactions due to IgG products, our observation raises a major concern regarding the actual aggregate content in these products since the EP method is widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the interactions between the proteins and stationary phase as well as the adsorption of proteins onto the column. Our results also show that NaClO4 in the eluent is more effective in overcoming the protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO4 is shown not to affect the molecular size and relative distribution of different molecular forms of IgG. The method validated as per ICH Q2(R1) guideline using IgG products, shows good specificity, accuracy, precision and a linear concentration dependence of peak areas for different molecular forms. In summary, our method gives more reliable results than the SE-HPLC methods for MSD analyses of IgG reported in the literature, including the EP, particularly for aggregates and tetramer. The results are interpreted in terms of ionic (polar) and hydrophobic interactions between the stationary phase and the IgG protein.

Protein Nitrogen Determination by Kjeldahl Digestion and Ion Chromatography.

We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 µg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 µM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71>) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex(®) Detection System [RMDS]), and 2) by CO(2) monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iAST and iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1CFU than the compendial methods (p<0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p<0.05). RMDS detected all microorganisms significantly faster than the compendial method (p<0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p<0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.