Joan C. May

Determination of residual moisture in freeze-dried viral vaccines: Karl Fischer, gravimetric and thermogravimetric methodologies*

This study establishes correlations between the gravimetric, Karl Fischer and thermogravimetric (TG) methods for the determination of residual moisture at levels of less than 3% in several measles, mumps and rubella viral vaccines freeze-dried in buffered glucitol (sorbitol)—gelatin matrices. The thermogravimetric balance was modified to eliminate static charge build-up which would interfere with its operation in the low humidity environment necessary for these determinations. TG/mass spectrometry verifies that for these products the loss of residual moisture coincides with the formation of the first weight loss transition on the temperature profile thermogram. Sample decomposition occurs above 150°C as evidenced by the evolution of CO 2 and H 2 O in the TG off-gas. Mass spectra were taken continuously while weight loss was recorded. The ion intensities of mass peaks m/e = 18 and m/e = 44 were monitored to show the changes in the amounts of water and carbon dioxide in the TG off-gases.

Optimization of parameters in protein nitrogen unit precipitation procedure for allergenic extracts

This work establishes the optimum conditions for the protein nitrogen unit (PNU) phosphotungstic acid (PTA) precipitation procedure for allergenic extracts. The volume of extract analyzed, the dilution of the extract prior to precipitation, the percent PTA in the precipitating solution, the washing of the precipitate, and the volume of concentrated HCl initially added to the extract were optimized, as they were found to be the factors which affected the amount of PTA precipitate obtained. Varying the digestion time, the digestion temperature, and the percent HCl in the 15% PTA precipitating solution did not cause detectable differences in the PNU values of the extracts studied.

The aluminum content of biological products containing aluminum adjuvants: Determination by atomic absorption spectrometry

Aluminum compounds are used as adjuvants in certain types of vaccines, toxoids and allergenic extracts for human use. The most common Al compounds used in biological products to enhance the immune response are aluminum potassium sulphate (alum), aluminum hydroxide and aluminum phosphate. This study describes an atomic absorption spectrometric method for the determination of the Al content of Al adsorbed toxoid preparations and allergenic extracts at levels of less than 0.85 mg of Al per half millilitre human dose. Aliquots of the samples which contained Al suspensions were acid digested with nitric and sulphuric acid and analysed in the nitrous oxide-acetylene flame of an atomic absorption spectrometer. The 396.2 nm Al line was used for analysis. The Al content of the National Bureau of Standards (NBS) Standard Reference Material No. 1075a aluminum 2- ethylhexanoate was determined to within 1% of the NBS certificate value by this method. Atomic absorption results for the Al content of tetanus toxoids containing aluminum potassium sulphate and aluminum phosphate were compared with polarographic and inductively coupled argon plasma (ICP) emission spectrometry results. Reproducibility and recovery data for Al are tabulated for a variety of biological products containing aluminum phosphate, aluminum potassium sulphate and aluminum hydroxide adjuvants. In addition, ICP has been used to characterize the Al and P compositions of the precipitates and supernatant solutions which resulted from centrifuging toxoid suspensions that contained the three different Al adjuvants.

TG/MS interface: applications to the determination of moisture in polysaccharides and freeze-dried biological products

Abstract A DuPont 1090 thermal analysis system has been interfaced to a Hewlett Packard 5995B quadrupole mass spectrometer with a turbo-molecular pumping system. The glass tubing interface connects the quartz combustion tube of the thermogravimetric analyzer (TG) to the jet separator of the mass spectrometer. This interface allows continuous monitoring of the ion intensities of mass peaks m/e = 18 (water) and m/e = 44 (carbon dioxide) for the determination of residual moisture in freeze-dried biological products such as Giant Ragweed Allergenic Extract and the moisture content of bulk polysaccharides such as Meningococcal Polysaccharides Groups A and C used in vaccine production.

The determination of residual moisture in several freeze-dried vaccines and a Honey Bee Venom Allergenic Extract by TG/MS

Thermogravimetry/mass spectrometry is used to identify TG transitions corresponding to loss of residual moisture in freeze-dried biological products with complex TG curves. While TG weight losses were recorded, ion intensities of mass peaksm/e=18 (water) andm/e=44 (carbon dioxide) were monitored continuously for Typhoid Vaccine U.S.P., Meningococcal Polysaccharide Vaccine Groups A and C Combined and Honey Bee Venom Allergenic Extract. MS ion intensities indicated the difference between evolution of residual moisture and moisture associated with product thermal decomposition. Residual moisture values calculated from TG weight losses indicated by mass spectral data agreed with Karl Fischer moisture data.ZusammenfassungThermogravimetrie/Massenspektrometrie wurde zur Identifizierung von TG-Effekten herangezogen, die auf die Abgabe des Restwassers von gefriergetrockneten biologischen Produkten zurückzuführen sind. Für Thyphoid Vaccine U.S.P., Meningococcal Polysaccharide Vaccine Groups A and B combined und Honey Bee Venom allergenic extract wurden gleichzeitig mit der thermogravimetrischen Registrierung des Gewichtsverlustes die Ionenintensitäten der Massenpeaksm/e=18 (Wasser) undm/e=44 (Kohlendioxid) kontinuierlich verfolgt. Der Verlauf der Ionenintensität ermöglicht eine Unterscheidung zwischen Restwasser und dem bei der thermischen Zersetzung des Produktes entstehenden Wasser. Der massenspektrometrisch als solcher erkannte und auf Grund der TG-Kurve quantifizierte Restwassergehalt stimmt mit dem Wert der nach Karl Fischer bestimmten Feuchtigkeit überein.РезюмеТермогравиметрия и м асс-спектрометрия бы ли использованы для иде нтификации ТГ переходов, соответст вующих потере остато чной влаги в биологических вещес твах, высушенных путем вымораживания. Одновременно с регис трацией потери веса, на монито рную систему выводились ионные ин тенсивности масс-пик овm/e=18 (вода) иm/e=44 (двуокись углеро да). Mace-спектрометрические данные ионных интенс ивностей показали различные м ежду выделением остаточной влаги и вл агой, выделяющейся в результате термичес кого разложения. Знач ения остаточной влаги, выч исленные из ТГ кривых потери веса и установленные на ос нове масс-спектромет рических данных, согласуются с данными, установленными по ме тоду Карла Фишера.

A survey of the concentrations of eleven metals in vaccines, allergenic extracts, toxoids, blood, blood derivatives and other biological products

Approximately 85 samples of injectable biological products regulated by the Center for Drugs and Biologics of the United States Food and Drug Administration were surveyed for the presence of 11 elements, namely aluminum, arsenic, barium, cadmium, chromium, lead, mercury, selenium, thallium and zinc, by flame and flameless methods of atomic absorption spectrometry and flame emission spectrometry. The range of products tested included whole blood, red cells, plasma, normal serum albumin, antihemophilic factor, and other products derived from blood; allergenic extracts including honey bee venom and house dust allergenic extracts; vaccines such as measles virus vaccine and typhoid vaccine; and tetanus toxoid. The metal concentrations found in the majority of these products were low or undetectable. The metal levels varied from manufacturer to manufacturer, product and lot-to-lot of the same manufacturers products. House dust allergenic extracts had the highest concentrations of arsenic (2.4 ppm), cadmium (0.28 ppm), chromium (0.6 ppm) and lead (1.5 ppm) found in the study. A high zinc concentration (24 ppm) in an immune serum globulin was attributed to the zinc-containing rubber stopper in contact with the product. A range of 0.36-3.30 ppm aluminum was found for seven 25% normal serum albumin samples from seven manufacturers. Values of 8.2, 17 and 18 ppm aluminum were found in one manufacturers 25% normal serum albumin. These aluminum values appeared to be the result of an anomaly in this manufacturers production that has not been repeated to date.

The determination of low levels of aluminum in antihemophilic factor (human) preparations by flame atomic absorption spectrometry

Aluminum hydroxide is used to adsorb extraneous protein during the preparation of Antihemophilic Factor (Human) (AHF). Removal of Al(OH)3 is accomplished by centrifugation and filtration. This study describes a method for the determination of residual aluminum in AHF by flame atomic absorption spectrometry. Matrix interferences were minimized by employing sample digestion with HNO3 prior to nebulization in a nitrous oxide-acetylene flame. Under these conditions, the limit of detection of aluminum in an aqueous system was estimated to be approximately 0.13 micrograms Al ml-1, while the limit of quantitation was estimated to be approximately 0.56 micrograms Al ml-1. Residual aluminum levels in AHF determined by this method ranged from less than 0.20 micrograms ml-1 to 0.82 micrograms ml-1. Butyl alcohol was used to modify sample matrices after digestion to increase the sensitivity of the assay system. An increase in the aluminum absorption signal was demonstrated after the addition of butyl alcohol to an AHF digest.

Assay for mercurial preservatives in biological products by cold-vapor atomic absorption spectrophotometry.

The cold-vapor method of flameless atomic absorption spectrophotometry was applied to the rapid, accurate and precise assay for mercurial preservatives in vaccines and other biological products through the quantitation of mercury. Sample dilution or a microliter sample size permitted the use of an uncomplicated cold concentrated sulfuric acid-potassium permanganate digestion procedure. This digestion procedure freed the mercury from the organic material in the organomercury preservative compounds and from the sample matrices which ranged in complexity from diluents with preservatives to toxoids, influenza virus vaccines and globulins. Microgram amounts of mercury were detected in some rubber components of final container closure-systems in contact with liquid products containing mercurial preservatives.

Compositional analysis of drugs and injectable biological products by thermogravimetry

Thermogravimetry (TG) is specified in the U.S. Pharmacopeia for the compositional analysis of certain drugs. It is specified for use in determining the percentage of volatile substances in drugs such as vincristine sulfate and vinblastine sulfate. TG reveals interactions between a controlled atmosphere and a drug and between an active substance and excipients. TG is used in the compositional analysis of injectable biological products in various ways. For pneumococcal, Haemophilus b, and meningococcal bulk polysaccharides that are used in vaccine production, TG is used to determine the moisture content (approximately 5 to 25 percent) so that the content of protein, nucleic acid, phosphorus, and other constituents may be accurately determined on a dry weight basis to demonstrate that a sample meets the specifications for the product. TG is also used to determine residual moisture in freeze-dried final container biological products such as Blood Grouping Sera and Antibody to Hepatitis B Surface Antigen. TG is used to confirm or supplement information obtained from Karl Fischer or gravimetric (loss on drying) moisture analysis techniques. TG has been combined with mass spectrometry to verify moisture data for group Y meningococcal polysaccharide and freeze-dried antibody to hepatitis B surface antigen and to identify an impurity in a Haemophilus b polysaccharide.

Determination of phenol in injectable biological products by gas chromatography.

A gas chromatographic method for the determination of phenol in a wide variety of biological products has been developed by extending a USP procedure. The procedure and its extension were validated for preparations of cholera vaccine, typhoid vaccine, tuberculin purified protein derivative, pneumococcal polysaccharide vaccine, horse serum-derived products such as antivenins, several types of aqueous, glycerinated and alum precipitated allergenic extracts and others. The average recovery of phenol at the 0·3–0·5% phenol level was 99·6%. The average precision was measured as a relative standard deviation of 0·43%. Glycerin contained in glycerinated allergenic extracts and tuberculin purified protein derivative did not interfere with quantitation. Horse serum-derived antitoxins, antivenins and antirabies serum produced a precipitate upon the addition of the internal standard solution, benzyl alcohol in 100% methanol. This precipitate was eliminated by the use of 5% (or 0%) methanol in water as diluent in the internal standard solution. Two aqueous allergenic extracts were encountered that produced peaks that were not adequately resolved from the phenol and benzyl alcohol peaks. Lowering the injection temperature to 185 °C and the column temperature to 125 °C produced satisfactory separations. Gas chromatography/mass spectrometry was used to determine the purity of peaks representing benzyl alcohol and phenol in tuberculin, cholera vaccine, typhoid vaccine and several allergenic extracts.