Yeast extract is commonly used as an excipient in cell culture, providing a range of nutrients that the cells require to grow. However, as a raw material it may contain variable levels of sodium chloride (NaCl), depending on the purification process. That is a problem, as NaCl concentration must be carefully controlled during the fermentation process; therefore, osmotic pressure in the media will not vary and cell growth is not to be inhibited.

	The process of assessing salt levels in yeast extract can be challenging because of the complex nature of the mixture. Traditionally, titration-based methods are used to quantify exact sodium or chloride levels in inorganic salts, but in this case, they are unsuitable. There are several reasons for this, including the fact that the salt level might be too low to be determined accurately, and because the yeast extract itself can interfere with the reaction components, and therefore affect the colour reaction. Furthermore, sample sizes required for titration may be fairly large. While yeast extract is relatively inexpensive, smaller sample sizes are always preferred.

	A far more suitable option for measuring low salt levels in complex biological samples is to use ion chromatography. Salts usually readily dissolve in aqueous media, giving free cations and anions that can be measured with good accuracy, precision, and specificity via ion chromatography. Other methods, such as those based on the colour reaction for identification and ion-specific electrode titration, can be used for pure NaCl samples, but they are not suitable for quantifying residual salt in complex biological matrices, and therefore are not applicable in this case.

	For a successful analysis, the sample should be readily soluble in water in order that all the ions are available for liquid chromatography separation and detection. If the sample is not completely soluble, a small amount of organic solvent can be used to promote dissolution, but this should be minimized where possible for ion chromatography. In contrast to reverseâphase chromatography, ion chromatography separation is based on the column’s affinity to specific cations and anions. Ion exchange columns should not be used when high levels of organic solvents are present.

	Another potential problem arises from the fact that the sample material should be able to pass through the column without killing it. The method should be sufficiently robust that the column remains functional for multiple sample injections. 

	In this example, the client specification for NaCl was no more than 5.0% w/w of the sample. As it is just one component of the complex yeast extract matrix, traditional titration methods were clearly inappropriate, and so a method based on ion chromatography was developed instead. 

	The technique uses a Model ICS-2100 ion chromatography system (Dionex), equipped with a conductivity detector to separate and quantitate the level of sodium ions present in the yeast extract sample. It also has an injection valve, pump, guard column, column, chemical suppressor, and data collection system. The amount of NaCl in the sample was calculated based on a 1:1 stoichiometry for sodium and chloride ions, and the assumption that salt was the only source of free sodium ions within the sample.

	The separation was performed on a IonPac CS12 250×4 mm column (Dionex), with an IonPac GC12 50×4 mm guard column (Dionex), and using a flow rate of 1.0 mL/min of 10 mM methanesulfonic acid (MSA), generated by the system online using reagent-free ion chromatography (RFIC) technology. The injection volume was 25 µL; both the column temperature and the conductivity detector temperature were 35 °C. A 4 mm suppressor with 30 mA suppressor current were used. A sample size of 1 mg/mL of yeast extract was injected. Sample concentration was low enough to promote sample solubility, but sufficient enough to detect sodium ions with acceptable accuracy and precision.

	The validation design was based on International Council for Harmonization ICH Q2B (1) and 

) <1225> (2) recommendations. The validation study is protocol-driven, and typically includes testing of system suitability, specificity, linearity, limit of quantitation, precision, intermediate precision, and accuracy. Robustness of chromatographic conditions and solution stability give information about the method’s limitations, and give additional information if troubleshooting of the method will be required for future use.

	Method accuracy was evaluated on samples spiked at the limit of quantitation of 0.1% NaCl, 50% of the specification level (i.e., 2.5% NaCl, 100% of the specification level of 5.0%, and also 150% of specification, 7.5%). The recovery of sodium at the limit of quantitation level was within 67–106%, and at other levels was 93–105%. The stability of the standard and the sample was evaluated after three days at both refrigerated conditions of 2–8 °C and at room temperature. 
	

	System suitability was checked before each validation run, and validation solutions were not evaluated unless the system suitability criteria were met. This was done by first injecting the diluent, USP High Quality Water (E-pure), and then the working standard six times to check system precision. Each validation solution was then injected once, with bracketing every six validation injections with a working standard injection, and at the end of the run. Results are shown in 

. All system suitability requirements were met each day. An example chromatogram from a blank (diluent) is shown in

	Specificity and selectivity were then checked, using injections of USP High Purity Water (diluent), working standard, unspiked sample solution, and sample solution spiked at the 100% concentration level of sodium. These were evaluated for chromatographic interference and resolution from other peaks. No interference from the diluent was observed. The resolution between the sodium peak and the nearest peak in the sample solution was 2.6, whereas in the accuracy 100% spiked sample, it was 2.8, and specificity criteria were met. Example chromatograms for the sample and spiked sample are shown in 

	Linearity was assessed by injections of five standard solutions prepared at concentration levels ranging from 0.1% to 7.5% NaCl. Each standard was injected once, and linearity determined through linear regression using the peak areas of sodium peak. The correlation coefficient was 0.99999, and the acceptance criterion was met. The results are summarized in 

, and the linear regression plot shown in 

	The limit of quantitation (LOQ) was determined by injecting the linearity standard at lowest level six times. The signal-to-noise ratio calculated for the sodium peak was 75.6; the limit of quantitation has been established as 1 µg/mL, or 0.1% NaCl in regard to the sample amount (1 mg/mL). An example chromatogram is shown in 

	Accuracy was determined using a yeast extract sample that was spiked with sodium across the LOQ-150% range from the target concentration. The samples were prepared in triplicate at LOQ, 50% and 150% levels, and six samples at the 100% level. An unspiked sample was used as a control to determine the endogenous amount of sodium present in the yeast extract. The percentage recovery at each concentration level was determined, with recovery calculations based on the spike contribution alone. The average level of sodium in the unspiked samples was subtracted from the spiked ones. Results are summarized in 

, with example chromatograms for 50%, 150%, and LOQ spiked samples shown in 

, respectively. Example chromatogram for 100% spiked sample is shown as 

. Acceptance criteria for accuracy stated in the validation protocol (50–150% for LOQ level and 80–120% for other levels) were met.

	Precision was determined using individually prepared yeast extract samples spiked at the 100% level concentration. Six of these samples were prepared and run. The percentage recovery was determined for each spiked sample preparation using the approach that was described in the accuracy section. Precision was calculated based on percentage recovery. The results from the accuracy test on comparable samples were used for this evaluation. Sodium chloride levels were less than 5.0% in the unspiked solution, and the results are summarized in 

; the acceptance criterion for precision (% RSD for % Recovery NMT 5.0%) was met.

	Similarly, intermediate precision was assessed, with a second analyst repeating the precision test on a different day using a different column and guard column on the same ion chromatography system; these results are summarized in 

, and, again, the acceptance criteria were met.

	In conclusion, the test method for the determination of NaCl in yeast extract by ion chromatography was successfully validated. The method is specific, linear, precise, and accurate, with a limit of quantitation of 0.1% of sodium chloride in the sample. Instrument parameters and sample preparation can be used as a starting point for method development/validation for other requests. 

Validation of Analytical Procedures: Text and Methodology

 (ICH, November 2005.
	2. USP, General Information <1225> 

	When referring to this article, please cite it as N. Belikova, "Determination of Sodium Chloride in Yeast Extract by Ion Chromatography," 

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Features

Determination of sodium chloride level is critical for assessment of purity of yeast extracts. This case study demonstrates the validation of an ion chromatography method as a suitable analytical approach.

Determination of Sodium Chloride in Yeast Extract by Ion Chromatography

The tendency of proteins to aggregate or to change conformation under certain conditions poses a problem for protein therapeutics such as monoclonal antibodies. If their physical form alters, then it is likely that their activity will also be impacted, so stability must be ensured throughout the product’s shelf life.

	A key technique in building stability into a protein therapeutic is to include a surfactant in the formulation. A suitable surfactant will help to minimize the aggregation, unfolding, and denaturation that the protein might otherwise undergo, which would affect its activity.

	Various surfactants are approved for use in therapeutic parenteral dosage forms, and by far the most frequently used is polysorbate 80 (PS-80), the common name for polyoxyethylene (20) sorbitan monooleate (

). This amphipathic, non-ionic surfactant is a hydrophilic polyoxyethylene (POE) sorbitan with about 20 ethylene oxide sub units, attached to a hydrophobic fatty acid ester tail that is, for the most part, oleic acid.

	This surfactant is also often found in both food products and cosmetics as an emulsifier. As it forms micelles, it helps to minimize the surface tension at the liquid interface. For the purposes of formulation, it helps to suppress aggregation and additionally aids solubility of the protein in solution.

	Although polysorbate 80 is the most common, the related polysorbate 20-the lauric acid analogue-is also used. Other surfactants that are sometimes found include the polyethylene glycol derivative, Triton X100, and Pluronic F-68, a polyoxyethylene/polyoxypropylene copolymer. 

	Polysorbate 80 predominates largely because it has been in use for so long, and scientists have decades of experience in its application in drug formulation. The use of PS-80 is not without drawbacks; perhaps the biggest challenge arises from the fact that it is very heterogeneous, with multiple different polymer chain lengths. Ensuring the purity of PS-80 poses a problem, because it contains variable quantities of oleic acid in unreacted form, in addition to structurally related materials and other impurities. This variance and the impurities will affect protein stability in formulation. For all of its benefits, characterizing PS-80’s composition precisely remains a challenge.

	PS-80 is also prone to a low level of hydrolysis, and there are many factors that can lead to this, including changes in pH and temperature, and the presence of peroxides. The protein in the formulation may even catalyse enzymatic hydrolytic degradation. 

	Fundamentally, the presence of hydrolysis can run the risk of damaging the protein so that it loses its efficacy, by affecting its conformational structure and the way that the molecule binds. But perhaps an even bigger issue is immunogenicity that results from protein aggregation or a change in conformation. Analytically, high aggregation levels can be detected via stability studies such as size exclusion chromatography, but unknown modifications of the protein may also be immunogenic.

	However, it is difficult to establish the exact purity of the polysorbate and analytical laboratories typically assay against a specification, looking at all the different components in the mix. For polysorbates, there are standards for the presence of free fatty acids but, generally speaking, it is difficult to determine what is considered ‘pure’.

	Another challenge facing analytical scientists studying polysorbate purity and composition is the reference standards themselves. As there can be significant variation for the material from lot-to-lot, batch-to-batch, and manufacturer-to-manufacturer, it is difficult to establish a solid reference for comparison to other products and lots.

	Historically, the regulators have asked for only limited information about polysorbates in a formulation, but in recent years they have started to increase the detail they require drug formulators to provide. Analytics have, therefore, been racing to catch up. Much of the additional work now required comprises of higher analytics that are not necessarily part of the release specifications, but it is crucial to determine what the differences from batch-to-batch are, and far more importantly, whether those differences are significant.

	When developing a protein therapeutic formulation, a large amount of early screening of potential formulation is required, with varying levels of the different additives. These are subjected to different stress conditions, such as temperature and time, as well as light stress, agitation stress, and pH changes. These can all give an insight into the behaviour of the PS-80, whether it is being degraded or not, and how it may impact the protein during that accelerated stability study. 

	This information can then be used in the selection of different grades of polysorbate, which are then subjected to longer-term stability studies to evaluate the product. Importantly, these data must be tied back to the toxicology studies and clinical studies.

	Various analytical methods are available for the characterization of PS-80. However, typically, a chromatography-based assay is required to give an accurate insight into all the various components. The tests cited in the United States Pharmacopeia (USP) and European Pharmacopoeia (Ph. Eur.) really only give high-level percentages of the components, and do not allow impurities to be resolved, or components that degrade or otherwise change over time. 

	Both of the methods for PS-80 concentration determination and characterization that were developed at Catalent are based on high-performance liquid chromatography (HPLC), coupled with charged aerosol detection. This method of detection is suitable for both non-volatile and semi-volatile compounds that lack chromophores. The response is mass-dependent and independent of the chemical properties of the analytes. The quantitation method looks at the total amount of polysorbate present in the formulation; the characterization method gives more of an insight into the nature of the components in the mixture.

	To perform the experiments, PS-80 solution was subjected to various stress conditions to prepare degraded samples. A 0.01% w/v PS-80 solution in 25 mM histidine at pH 6.0 was variously exposed to thermal stress at 80 °C, base hydrolysis at pH 10 and 40 °C, oxidation with 2 mM 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) at room temperature, and UV/fluorescent light exposure according to International Council for Harmonization (ICH) guidelines (1). The degraded samples were then spiked with bovine serum albumins or IgG monoclonal antibody, giving a final concentration of 5 mg/mL. The samples were then analysed using each of the methods.

	This fast, sensitive analytical method is based on the diluteâandâshoot approach, coupled with mixed-mode chromatography for the online separation of therapeutics and PS-80 components. PS-80 content is determined using a Waters Oasis Max column and a step gradient. 

	The result is a trace with one large peak, and the area of this can be used for quantitation against an external reference standard curve of polysorbate. Assay linearity was established in the range 12.5–100 µg/mL, or 0.00125–0.01%, with an R2 value of 0.99 by log–log plot [see 

	Degradation of PS-80 was manifested as a decrease in concentration, and this was observed in stressed samples. Calculated concentrations were normalized to their respective controls, either T = 0 or protected samples. The final relative PS-80 contents were found to be between 48% and 94%. Protected samples under light exposures had negligible changes in concentration. 

	Although it can give an insight into the degradation of polysorbate, in reality, substantial degradation will have to have occurred before it can be detected. It is, however, useful for routine product monitoring.

	The characterization method is broadly similar, as it also relies on HPLC with charged aerosol detection. However, in this case an off-line extraction is performed to remove the protein while preserving the hydrophilic POE sorbitan that elutes with protein.

	The polysorbate-containing sample is then injected straight onto an Agilent Zorbax SB-CN column, which has reverseâphase properties. This captures all the polysorbate in a long, shallow gradient, and allows all the different components to be separated. The result is a chromatographic profile or ‘fingerprint’ of the polysorbate and all its various components. This method is able to observe PS-80, POE sorbitan, various POE sorbitan esters, and oleic acid.

	The challenge is then to determine what all of those peaks are, and this can be achieved using mass spectrometry. One of the most important components to monitor is oleic acid, because of the stability problems it can cause. This method allows this individual component to be quantified. 

	Interestingly, there are noticeable differences in chromatographic profile between 7 and 16 minutes among PS-80 sourced from different vendors. This clearly shows that the chemical composition varies between the different products [

	Degradation affects all sorbitan POE esters. However, the POE sorbitan peak appeared to be unchanged under most conditions. The generation of the base hydrolysis products oleic acid and POE sorbitan was detected in the chromatographic profile (see 

	These two complementary techniques permit the quantitative and qualitative analysis of PS-80 in therapeutic products. Both methods have been shown to be sensitive and stability indicating. They are, therefore, suitable for the monitoring of the degradation of PS-80 in biopharmaceuticals as required by the regulators. Other applications for these methods include quality assessment of PS-80 raw material during manufacturing, and PS-80 content confirmation. They should also be applicable to the study of other polysorbates, notably PS-20. 

	There remain many unknowns about the behaviour of PS-80 in protein therapeutics, and future studies should include a detailed exploration of its degradation pathways, and the impact of PS-80 on the critical quality attributes of these important biopharmaceuticals.

Stability Testing: Photostability Testing of New Drug Substances and Products

	When referring to this article, please cite it as L. Deters, "Quantification and Characterization of Polysorbate-80 in Protein Formulations," 

HPLC coupled with charged aerosol detection is a suitable analytical technique to quantitate and characterize polysorbate-80 in therapeutic products; allowing quality assessment of raw material, content confirmation during manufacturing, and monitoring product stability.


Quantification and Characterization of Polysorbate-80 in Protein Formulations

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Analytical scientists in the pharmaceutical industry are tasked with ensuring the highâperformance liquid chromatography (HPLC) purity methods that they develop and validate provide complete separation of compounds, and this work can yield unexpected findings that lead to improvements in methods and manufacturing processes.

	In the following case study, peak spectral analysis was used as an analytical tool to determine the cause of peak purity failure during HPLC method validation. This analysis resulted in the separation and identification of a previously unknown impurity, and the development and validation of a new HPLC method that allowed this impurity to be separated and independently determined.

	Early phase HPLC purity/assay validations typically include the following components, based on International Council for Harmonization (ICH) Q2(R1) guidelines (1): 

		Specificity-the ability of a method to separate the analyte, or compound of interest, from other components expected to be present in the sample 

		Linearity-the ability of a method to provide results within a specified concentration range that are directly proportional to concentration of the analyte 

		Accuracy-the ability of a method to produce assay results near the true value 

		Precision-the ability of a method to give consistently reproducible results 

		Detection limit-the lowest concentration of analyte that can be detected 

		Quantitation limit-the lowest concentration of analyte that can be consistently measured 

		Forced degradation-exposing the drug substance to severe environmental and chemical stresses to rapidly evaluate material stability. 

	This final component is performed according to the ICH guideline Q1A(R2), Section 2.1.2, and will demonstrate that a method is stability-indicating (2).

	During a recent validation on an API that was being manufactured for a client, forced degradation studies were performed on the API using the client-supplied method. During the study, the HPLC-diode array detector (DAD) peak purity analysis of the analyte peak failed for all conditions, including the control. Failure was not observed during pre-validation exercises, which were performed on a different, previous batch of API. This indicated that a new, unknown impurity could be co-eluting with the analyte peak.

	Using Chemstation software (Agilent), the ultraviolet (UV) spectra of the analyte peak collected by the diode array are analysed for spectral purity, giving the purity factor. A purity factor of ≥990 generally indicates a peak is pure and suggests no other peaks co-elute.

Peak purity analysis results for an older lot are shown in 

, where the upper-left quadrant shows the chromatogram, with its baseline enlarged for detail. The green bar under the peak indicates the peak has a purity factor above the threshold of 990, with an observed result of 999.433, which is shown in the upper-right quadrant. The lower-right quadrant shows the analysis of spectral purity within the peak, as indicated by the wide line that remains within the green bar, and the lower-left quadrant includes the peak spectral results taken from five points across the peak. 

 show the new, failing, lot of API, which has a purity factor of 835.895. The wide line beneath the peak in the lower-right quadrant indicates the presence of a spectral impurity on the tail of the peak. A careful review of the spectra in the lowerâleft quadrant indicates that one of the spectral plots differs from the others, as it is shifted to the right, and this corresponds to the co-eluting impurity. This shift pattern would be useful in developing a method for the new impurity, as it implied that the impurity responded better than the analyte at a wavelength of about 290 nm.

	The three-dimensional spectral results of the analyte peak were also modelled, and the area around the baseline enlarged for detail. The spectral map of the pure analyte peak obtained from the older lot is shown in 

, which plots wavelengths between 240 and 310 nm against time (11.6 minutes to 10.9 minutes, right to left). Note that the peak is reversed from the normal chromatogram to allow the spectral details to be observed. The spectral map of analyte peak from the impure lot is shown in 

 (using the same axis scale), and the impurity shows up as a ‘bulge’ on the main peak.

	The method that was being validated was performed at a wavelength of 260 nm, but when the impure analyte peak was evaluated at 290 nm, it became clear that there were two distinct compounds. Overlays of the impure analyte peak at 260 nm and 290 nm wavelengths were created, as shown in 

. The later peak (right, red, 290 nm) shows the location of the bulge in the three-dimensional model in 

 indicated that it might be possible to separate the later-eluting new compound from the analyte, based on the differences in properties of the two molecules. As the identity of the new impurity was unknown, data from the manufacturing campaign of the impure batch were reviewed to check for information that might point to the source of the impurity. HPLC data from the manufacturing batch record indicated that the impurity was introduced early in the manufacturing. The data also revealed that the method used for in-process checks was able to separate out the impurity from an intermediate material with the use of a different column and mobile phase.

	Isolated intermediate material from the manufacturing campaign that produced the impurityâcontaining API was analysed using the in-process HPLC method, column, and mobile phase. Although the method was not optimized for this separation, the impurity appeared as a peak before the main intermediate peak, and was resolved from the intermediate peak. Additional analysis showed a spectral profile similar to the impurity in the final compound. The HPLC results are shown in 

. Spectral results are shown in an overlay in 

, where the impurity has a spectral maximum of approximately 274 nm, compared to the intermediate, which has a spectral maximum of 260 nm. 

	Additional method development was performed on the API, leading to a final set of conditions that successfully separated out the impurity. Although the intermediate and the API have a similar structure (the intermediate molecule contributes a significant structural fragment to the API), the API is less polar, and elutes much later than the intermediate shown in the previous figures. Using the final method conditions, the impurity elutes first, as shown in the example chromatogram in 

, and the API peak was determined to be spectrally pure. 

	Spectral results from the peaks in the example chromatogram are shown in 

, where the impurity peak has a spectral maximum at 274 nm. Because the impurity’s spectral maximum was different from the API’s maximum at 260 nm, a response factor correction for the impurity was empirically determined and added to the new method. This new method was subsequently validated and implemented prior to release of the next batch (a clean-up of the impure batch of API), demonstrating the versatility and utility of spectral analysis as an analytical tool. 

, (ICH, November 2005).
	2. ICH, Q1A (R2) 

Stability Testing of New Drug Substances and Products

	When referring to this article, please cite it as D. Hayes, " Utilizing Spectral Analysis in HPLC Diode Array to Discover Impurities,"

Investigation of peak purity failure during HPLC method validation led to discovery of a co-eluting impurity under the main peak. Spectral analysis, including three-dimensional modelling, was used to characterize the peak, leading to the development of a new HPLC method for analysing impurity content.

Pharmaceutical Technology Europe-03-02-2019

Determination of Sodium Chloride in Yeast Extract by Ion Chromatography

Quantification and Characterization of Polysorbate-80 in Protein Formulations

Pharmaceutical Technology Europe 2019 Outsourcing Resources Supplement (PDF)

Utilizing Spectral Analysis in HPLC Diode Array to Discover Impurities