Scaleup considerations

3.1.3.1 Development at small scale This section will discuss some of the adjustable parameters in method development that are critical to successful process development. As a general approach these parameters are best examined on analytical scale before increasing the scale to something approaching production size for several reasons One has the freedom to examine a multiplicity of variables without sacrificing large amounts of expensive product. The process can be scaled relatively quickly in...

Info

Between the stationary phase and the flowing mobile phase. A given molecule is transferred into the stationary phase at a given point along the column. After a time, it is returned to the mobile phase and swept down the column a little way to a new site where the process is repeated. The locations at which the analyte is transferred into the stationary phase are conceptualized as plates, and the distance between them is conceptualized as the plate height. The parameters of isocratic column...

Sugars and carbohydrates

Carbohydrates are found in simple sugars, as constituents of glycoproteins and glycolipids, and in polymeric form as structural features of bacterial and fungal cell walls, among other sources. Polymeric sugars include dextran, used as a blood expander, and starch. Sugars are most commonly analyzed by ion-exchange or other chromatographies, since they tend to be so hydro-philic as to elute rapidly from reversed phase columns. N-butyldeoxynojir-imycin, deoxynojirimycin, and their degradation...

Other compounds

A bisphosphonate, clodronate, was separated from its esters on an HPIC AS7 column (Dionex) using dilute nitric acid as the eluent.277 Thorium-EDTA-xylenol was used as a postcolumn complexation agent for optical detection at 550 nm. Polyphosphonates and polycarboxylates, used as color preservatives and spoilage retardants in food, were separated on a PRP -X100 anion-exchange column (Hamilton) using vacancy detection and conductivity detection.278 Alkylbenzenesulfonates, used as surfactants, were...

Background

This section describes the isolation of the protease hementin. Hementin has antithrombotic properties that could find application in biopharmaceutics. Found in extracts of the salivary glands of the giant South American leech Haementeria ghilianii,286 hementin digests fibrinogen to fragments resembling plasmic fragments Y, D, and E, rendering the proteolyzed fibrinogen non-clottable.287288 As such, this protease was of potential interest in pharmaceutical and biomedical applications,...

Biopharmaceutical materials

While proteins and peptides continue to be the biopharmaceuticals of most widespread interest, new drugs based on carbohydrates and synthetic DNA require industry to develop new separation strategies for process scale purification. Each type of molecule presents its own unique set of challenges in purification with regard to contaminants, breakdown products, altered sequences, misfolding, and, in the case of proteins, immunological variants. The following review highlights some of the more...

Lipids and surfactants

Simple lipids, or fatty acids, are n-alkyl carboxylic acids typically derived from the hydrolysis of the triglycerides of fats or of the phospholipids found in the cell wall. Many naturally occurring fatty acids contain one or more sites of unsaturation. Fatty acids are found in nature conjugated to such sugars as glycolipids or lipopolysaccharides, to amino acid residues of pep-tides and proteins, to glycerol to form the acylglycerols of fats, and to glyc-erophosphocholate and...

Budgeting

Cost-benefit analysis is a part of managing a laboratory that many laboratory managers dread, yet it can be made relatively painless. The first step in doing cost-benefit analysis for instrumental acquisition is to determine how many hours an analysis or an analysis step presently requires and how many hours would be saved by acquiring a new instrument. If the anticipated time savings over the lifetime of an instrument, calculated from salary plus overhead plus benefits, is greater than the...

A126 Electrospray ionization

In electrospray ionization mass spectroscopy, nebulized sample droplets are introduced directly into the mass spectromer, negative or positive charge is introduced typically by the effects of an electric field, and ions so generated are separated by a magnetic field.16The sample may be introduced alone or with a sheath liquid to modulate surface tension or a sheath gas to assist nebulization. The types of interfaces used in electrospray mass spectroscopy are called sheathless, liquid junction,...

References

L., Recombinant human insulin, Biotechnol. Prog., 8, 469, 1992. 2. Colin, H., Large-scale high-performance preparative liquid chromatography, in Preparative and Production Scale Chromatography, Ganetsos, G. and Barker, P. E. Eds., Marcel Dekker, New York, 1988, 11. 3. Leser, E. W. and Asenjo, J. A., Rational design of purification processes for recombinant proteins, J. Chromatogr., 584, 43, 1992. 4. Wheelwright, S. M., Designing downstream processes for...

A328 Conventional ion exchange

The lysozyme adsorption characteristics of cation exchange phases were examined, using Toyopearl SP650C and SP550C, SP Sepharose FF, Fractogel EMD SO3, Bakerbond Carboxy-Sulfone and Spherodex M as the stationary phases.36 Batch adsorption analysis was used to compare the predictions of the pore diffusion model and the homogeneous diffusion model of stationary phases. The pore diffusion model describes the stationary phase as a pore in which adsorption takes place at the pore walls, so the...

Simple sugars and lowermolecularweight oligomers

Sugar determinations are important in the food industry, in analysis of carbohydrates and their polymers, and in pharmaceutical analysis of glyco-proteins. A number of column types have been used in the separation of sugars, particularly amino columns and strong anion exchange columns. Amperometric detection is widely used, but post-column detection schemes, UV detection, and refractive index detection are also useful. Sugars are capable of being linked together at a number of locations on the...

Carbohydrate polymers

Higher oligosaccharides occur as natural homopolymers, such as inulin, dextran, starch, and pullulan, and as heteropolymers, such as pectin and heparin. Complex oligosaccharides are found as N-linked or O-linked prosthetic groups of glycoproteins. As has been noted, analysis of the branching of polysaccharides is one of the most challenging problems in contemporary analysis. One means of approaching that problem is though enzymatic digestion with linkage-specific enzymes. In Figure 23 is shown...

Validation considerations

Validation activity is the crucial end-stage in the process development of a biopharmaceutical chromatographic purification and one which young companies often underestimate. Regarding process validation, the FDA issued a guideline in 1986 which states Process validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specification and quality attributes. Validation is not...

Giddings Jc In Hplc References

R., and Horvath, Cs, An Introduction to Separation Science, John Wiley amp Sons, New York, 1973, 121-167. 2. Snyder, L. R. and Kirkland, J. J., Introduction to Modern Liquid Chromatography, Second ed., John Wiley amp Sons, New York, 1979, chap. 2. 3. Glajch, J. L., Kirkland, J. J., Squire, K. M., and Minor, J. M., Optimization of solvent strength and selectivity for reversed-phase liquid chromatography using an interactive mixture-design statistical technique, J....

Crossover Of Peak Retention Times

0.0 0.2 0.4 0.6 0.8 1.0 Median Organic Composition Fraction Figure 4 Crossover of peak retention times as a function of gradient rate in the separation of peptides. Previously unpublished data are drawn from Swadesh, J. K., Tryptic fingerprinting on a poly styrene-divinyl benzene reversed phase column, J. Chromatogr, 512, 3215, 1990.92 0.0 0.2 0.4 0.6 0.8 1.0 Median Organic Composition Fraction Figure 4 Crossover of peak retention times as a function of gradient rate in the separation of...

Development of an RPLC method

Method development starts from an understanding of the physical and chemical properties of the sample molecule and or the impurities present i.e., the properties such as solubility, stability, polarity, dissociation constant, molecular weight, electroactivity, and the UV absorption spectrum . If the sample is of a low-molecular-weight lt 2000 and is soluble in the reversed phase water organic solvents, then RPLC is one of the methods of choice. Preliminary estimates of retention may be made on...

The Science

R-aud, complacency, and secrecy in the scientific establishment Figure 6 The Science Mob. Reproduced with permission from The New Republic and Mr. Robert Grossman.110 Figure 6 The Science Mob. Reproduced with permission from The New Republic and Mr. Robert Grossman.110 work discovered to be erroneous in a regulatory audit. The low rate of fraud or error in science is a powerful testament to the fundamental dedication and decency of the overwhelming majority of scientists. Still, some members of...

Cip

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any...

Homologous series of simple hydrocarbons aromatic hydrocarbons

The central issue in separations of homologous series is column selectivity. Separations of a homologous series of compounds differing by a single methylene are a common method of studying the separating power of a column. Compilations of methylene selectivity are available in the litera-ture.59-61 Applications have included the separation of alkyl benzenes, dialkyl phthalate, and alkylanthroquinone 62 alkanes 63 mesogenic esters of biphe-nyl-4,4'-dicarboxylic acid 64 carboxylic acids as the...

Drugs pharmaceuticals and pharmacologically active compounds

Many, if not most, pharmacologically active compounds are amines. For this reason, issues in the RPLC of substances of pharmaceutical interest tend to be similar to those encountered in the separation of amines. Incompletely endcapped silica-based phases may exhibit tailing. The use of ion pairing or ion suppression is common in the analysis of pharmacologically active substances. Also, derivatization of the amine functionality prior to analysis may be required. The RPLC retention indices of...