Reversed phase HPLC scale

If the hGH is exported to the culture medium the product can easily be collected by removal of the cells from the culture medium by centrifiigation. Purification of hGH from the culture medium is faciUtated by low amounts of contaminating proteins present. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase hplc column (43). Mammalian cells growing in tissue culture have also been used as hosts to produce hGH, which is exported into the culture media (44). 

When 13 and 17 were sialylated on a larger scale (2.0 and 3.5 mg, respectively) glycopeptides 20 and 21 could be isolated in 94 and 64% yields, respectively, after purihcation by reversed-phase HPLC. (From George et ah, 2001)... 

A clean-up process-scale reverse-phase HPLC (RP-HPLC) step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 801 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 8.7). Separation is achieved using an acidic (often acetic acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30% acetonitrile). 

The sample extracts that show either toxicity or no dose response on initial testing should be fractionated. An aliquot of the extract is solvent exchanged to acetonitrile, and an initial analytical scale separation is made to assess the distribution of constituents in the sample. This separation is accomplished by using a Qg reversed-phase system eluted for 45 min with a linear gradient of 0-100% acetonitrile in water. If >75% of the sample elutes after the solvent composition of 80% and 20% acetonitrile, then the fractions are isolated by preparative reversed-phase HPLC. Fraction A is eluted with 100% water fraction B is eluted with a linear mobile-phase gradient from 100% to 75% water and 25% acetonitrile fractions C, D, and E are eluted with gradients with final compositions of 50%, 75%, and 100% acetonitrile. 

Additional Preparative-Scale HPLC Separations. After mutagenesis assessment of the HPLC fractions from the initial preparative-scale separation just discussed, those fractions containing mutagenic constituents are further separated on HPLC by employing the following strategy For example, if the mutagenic constituents were found to be in Fraction D from an initial reverse-phase HPLC preparative-scale separation, that is, a mobile-phase composition of 25 water 75 acetonitrile, a... 

Analytical-Scale HPLC Separations. Reverse-phase HPLC chromatography favors the distribution of the semi- and nonpolar constituents of a sample of residue organics, whereas normal-phase HPLC chromatography favors the distribution of semipolar constituents (32). This approach is illustrated in Figure 2 by the chromatograms of residue organics from a waste water sample separated by both reverse-... 

Figure 2. Absorbance (254 nm) profile of the analytical-scale HPLC separation of 25 pg of residue organics isolated from an industrially impacted influent waste water. The separations were accomplished via reverse-phase HPLC (top) and normal-phase HPLC (bottom). Mobile phases used in these separations were water (fyO), acetonitrile (CH3CN)> methylene chloride (MECL), hexane (HX), and isopropyl alcohol (IPROH), as indicated.

Figure 3. Absorbance (254 nm) profile of the analytical-scale reverse-phase HPLC separation of 2.5-L equivalents of residue organics isolated from finished drinking water L...

HPLC and Isolation of Mutagenic Fractions. Analytical and semipreparative reverse-phase HPLC separations were performed by using a water-to-acetonitrile linear gradient (J2). Separations were carried out on a Hewlett Packard Model 10084 B equipped with an automatic sampling device, a solvent programmer, a variable absorbance detector, and an automatically steered fraction collector. The instrument was fitted with a 3.9-mm X 30-cm prepacked analytical column of 10-/zm silica particles bonded with octadecylsilane (Bondapack-Cis) for analytical scale. For semipreparative scale separations, the HPLC was fitted with a 7.8-mm X 30-cm prepacked column packed with 10-/xm silica particles bonded with octadecylsilane. Samples for HPLC were injected at volumes of 20 /xL (flow rate 1 mL/min) and 80 /zL (flow rate 4 mL/min), and the absorption was measured at 254 nm. Fractions... 

SCX column on H PLC), the eluates were directly transferred onto a reversed-phase HPLC column, and subsequently the eluates from the second column were validated by tandem MS. Of 78 existing proteins, 75 were correctly identified. The analysis of an enriched yeast ribosomal fraction identified over 80% of the subunits in the complex (Lui, 2002). According to the described method of validation and optimization, the potential of this method lies within practical application due to automation and up-scaling. 

Simple modification of the Yoshikawa phosphorylation allows the synthesis of (1-thio) triphosphates (a-thiotriphosphates) (Protocol 12). The initial phosphorylation with thiophosphoryl chloride is slower than that using phosphoryl chloride and requires the addition of 2,4,6-collidine as a catalyst for the pyrimidine nucleosides.7 Protocol 12 is intended for pyrimidine nucleosides on a 0.2 mmol scale and should be modified where indicated for purine nucleosides. The diaste-reoisomers of nucleoside (l-thio)triphosphates can be distinguished by both 31P NMR (the signal for the a-phosphate of the Sp diastereoisomer is down-field relative to that of the Rp diastereoisomer) and reversed-phase HPLC (the Sp diastereoisomer elutes first). Preparative separation can also be achieved using the latter method (see Protocols 21-23).17... 

Fritz and co-workers (F5) described the use of reversed-phase HPLC for the isolation of protected oligonucleotide intermediates in the stepwise synthesis of deoxyribonucleotides. They report that the use of reversed-phase preparative scale HPLC reduces the time required for synthesis of oligonucleotides by 30%. In addition, they report on the RPLC separation of unprotected S -hydroxylated oligonucleotides. 

A comprehensive semi-empirical description of reversed-phase HPLC systems, for predicting the relative retention and selectivity within a series of analytes, has been developed by Jandera and co-workers [72,73]. The approach consists of determining the interaction indices and the structural lipophilic and polar indices. A suitable set of standard reference analytes is necessaiy to calibrate the retention (or selectivity) scale. 

Figure 5. Reverse phase HPLC of in gel tryptic digests of 25 pmol transferrin (A) and 10 pmol bovine serum albumin (B) and of the corresponding digests carried out on blank sections of gels (lower profiles shown in above two figures). In each instance 90% of the digest was subjected to HPLC on a 1.0 mm ID Vydac C-18 coluttm eluted at 50 pl/min. The respective full scale deflections were 18.9 mV for panel A and 4.4 mV for panel B with 0.5 volt corresponding to an absorbance of 1.0 at 210 nm.

In terms of impact on the project, the N-oxide structural elucidation allowed for an appropriate specification of the degradant, and clinical time lines were not impacted. It is advised in all preparative HPLC isolations that an analytical or preparative scale reinjection is performed to clean up the analyte of interest from the salt. This can include washing the analyte by reversed-phase HPLC (preparative or analytical scale depending on isolate amount) with aqueous phase and ramping up the organic phase to elute the desalted analyte of interest. 

With normal-phase HPLC, oil samples were analyzed as is by simple dilution in n-hexane. A Du Pont Zorbax amino-bonded phase column, 25 cm x 0.46 cm ID, was used, with n-hexane and dichloromethane as solvents. For reversed-phase HPLC, Vydac 201TP5 columns were used (25 cm x 0.46 cm ID for analytical scale and 25 cm x 1 cm ID for preparative scale). Samples for reversed-phase HPLC were fractionated in order to remove the saturated hydrocarbons which can interfere with the separation mechanism. The samples dissolved in n-hexane were passed Baker silica solid-phase extraction cartridges. The PAH fraction was then collected by eluting with a 1 1 mixture of dichloromethane and methanol. Acetonitrile and dichloromethane were used in the HPLC gradient. 

Figure 1. Reversed-phase HPLC chromatogram of a bench-scale refold mixture. The refold procedure was as described for the gram amount production lots for preliminary animal testing. Active rSLPI is at 13 min. retention, while the fully unfolded protein is at 18-19 min. and the partial or incorrectly folded species are found in between.

In summary, we have demonstrated the potential utility of preparative reversed phase HPLC as a tool to purify fermentation products in multigram scale on laboratory instruments. Secondly, due to its intrinsic selectivity and compatibility with the use of high flow rates, RPHLC is a useful tool for rapidly purifying crude fermentation extracts to a level of purity equivalent to that obtained by multistep time consuming procedures. We... 

Equipment for preparative HPLC is relatively expensive. To maximize its utility, it should be specified with autoinjection to run unattended and collect previously identified peaks. A 30-min cycle is typical so that overnight operation can process over 30 portions of feed solution. This appears to be the best option for maximizing output with bench-scale apparatus. There is also the advantage that the separation can be fine-tuned during the course of the operation. A number of manufacturers supply equipment based on columns of 15— 50 mm diameter packed with stationary phases of 5-15 pm. Retention times in reverse phase HPLC can be influenced by temperature. It is recommended that a column used for repetitive injection be housed in an oven set at a temperature slightly higher than ambient, e.g., 30°C. 

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