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Prediction of cell culture media performance using fluorescence spectroscopy.

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Cell culture media used in industrial mammalian cell culture are complex aqueous solutions that are inherently difficult to analyze comprehensively. The analysis of media quality and variance is of utmost importance in efficient manufacturing. We are
  Page 1 of  19   P.W. Ryan, B. Li, M. Shanahan, K.J. Leister, A.G. Ryder.  Anal. Chem. , 82 (4), 1311-1317, (2010).   P REDICTION OF C ELL C ULTURE M EDIA P ERFORMANCEUSING F LUORESCENCE S PECTROSCOPY . Paul W. Ryan, 1,2 Boyan Li, 1,2 Michael Shanahan, 1,2 Kirk J. Leister, 3 and Alan G. Ryder. 1,2 * 1 Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland,Galway, Galway, Ireland. 2 Centre for Bioanalytical Sciences, School of Chemistry, National University of Ireland,Galway, Galway, Ireland. 3 Bristol-Myers Squibb, Process Analytical Sciences, Syracuse, New York, USA.* Corresponding author: Email: Phone: +353-91-492943. Published Citation: Prediction of Cell Culture Media performance using Fluorescence Spectroscopy.P.W. Ryan, B. Li, M. Shanahan, K.J. Leister, and A.G. Ryder.  Analytical Chemistry , 82 (4), 1311-1317,(2010).   DOI: Note: This was the final revised version as accepted, this version does not include any proofingerror correction. The definitive, final version of the paper is available on the AnalyticalChemistry website. Abstract:  Cell culture media used in industrial mammalian cell culture are complex aqueous solutionsthat are inherently difficult to analyze comprehensively. The analysis of media quality andvariance is of utmost importance in efficient manufacturing. We are exploring the use of rapid“holistic” analytical methods that can be used for routine screening of cell culture media used inindustrial biotechnology. The application of rapid fluorescence spectroscopic techniques to theroutine analysis of cell culture media (Chinese Hamster Ovary cell based manufacture) wasinvestigated. We have developed robust methods which can be used to identify compositionalchanges and ultimately predict the efficacy of individual fed batch media in terms of downstreamprotein product yield with an accuracy of  ± 0.13 g/L. This is achieved through theimplementation of chemometric methods such as multiway robust principal component analysis  Page 2 of  19   P.W. Ryan, B. Li, M. Shanahan, K.J. Leister, A.G. Ryder.  Anal. Chem. , 82 (4), 1311-1317, (2010).  (MROBPCA), n-way partial least squares-discriminant analysis and regression (NPLS-DA andNPLS). This ability to observe compositional changes and predict product yield before mediause has enormous potential and should permit the effective elimination of one of the majorprocess variables leading to more consistent product quality and improved yield. These robustand reliable methods have the potential to become an important part of upstreambiopharmaceutical quality control and analysis. Keywords: Fluorescence, cell culture, media, chemometrics.  INTRODUCTION The essential purpose of cell culture media is to produce the correct physiologicalenvironment to allow effective in vitro culturing of mammalian cells by providing survival andgrowth requirements. Since the physiological environments of most cells are not fully defined,this is a difficult task and formulation of appropriate media requires the careful selection andblending of a wide variety of components. The resulting media are highly complex mixtures,containing a variety of amino acids, carbohydrates, cofactors, and other materials. Theseaqueous based media must provide all of the cell nutrients required for growth as well asproviding an energy source, while maintaining pH and osmolarity.In mammalian cell fermentation there are two designated types of cell culture media used inthe fed-batch process: basal media and feed media. Examples of basal media include Dulbeccos'Modified Eagles Medium (DMEM), Roswell Park Memorial Institute (RPMI) Medium, Iscovemodified Dulbeccos' medium and Ham's Nutrient Mixture medium (F12). 1,2 Basal media plusimportant growth factors are added at the initial bioreactor inoculation step and provide specificcell nutrients. Cell proliferation occurs in the basal media until the desired population density isachieved. At this point feed media is added to promote the bioreactor cell mass, a productionphase of the manufacturing process whereby the cells shift energy to protein synthesis. Thisproduction phase is where the genetically engineered biotherapeutic protein product is made.Feed media are used in fed-batch processes and media supply is strictly controlled to optimisegrowth rate, product yield and quality, while preventing the formation of unwanted metabolites.Feed media are a complex , nutrient enriched formulation of inorganic salts, sugars, amino acids,vitamins, metals, and other nutrients (e.g., growth factors, yeast/soy extracts, etc.). Thecomposition of the feed media and feed rate vary according to cell type and product beingproduced. 2,3,4  As industrial cell culture becomes more widespread so too does the demand for rapid,reliable, robust and non-destructive analytical methods that can be integrated into process controlto improve end product yield and quality. The complexity of these mammalian cell culturemedia means that efficient analysis, using current separations based analytical methods, ischallenging, time consuming, and expensive. 1,5,6 Therefore, the development of analyticalmethods with a rapid holistic approach to sample evaluation is more appropriate than attemptingto determine every individual component (which is not feasible).  Page 3 of  19   P.W. Ryan, B. Li, M. Shanahan, K.J. Leister, A.G. Ryder.  Anal. Chem. , 82 (4), 1311-1317, (2010).  Fluorescence spectroscopy is widely used for the analysis of biogenic materials and thus is auseful option for the holistic analysis of complex materials. 7 Unfortunately, conventional singleexcitation wavelength spectroscopy does not yield sufficiently detailed spectra suitable forcomplex sample characterization. However, it is possible with most scanning fluorometers torapidly record both excitation and emission spectra, yielding an excitation-emission matrix(EEM) spectrum. The tri-parametric EEM data incorporates information on excitationwavelength, emission wavelength, and fluorescence intensity. With EEM, the spectralinformation content is increased making it easier to accurately characterize multicomponentsamples. Because the technique is non-contact, non-destructive, and optically based, it is idealfor on-line, in situ and in vivo measurements via fiber optical systems. Other advantagesinclude: high sensitivity, high signal to noise ratios, and relatively large linear ranges. EEM andother multidimensional fluorescence (MDF) spectroscopic methods have found wideapplication, 8-14 from dye analysis in heritage science, 15 to crude oil characterisation, 16 to beerprocess analysis, 17,18 to human tissue analysis, 19,20 and the characterization of dissolved organicmatter (DOM) in water. 21-26 The application of MDF methods to online process control in thecontext of monitoring metabolic changes and turbidity has also been implemented. 27  Since EEM spectra cover a wide range of excitation and emission wavelengths, it allows forthe simultaneous detection of a wide variety of biogenic fluorophores present in complex, turbid,media environments such as amino acids, proteins, coenzymes, and vitamins. To extractquantitative and qualitative data from EEM spectra, advanced mathematical treatment andanalysis of the information rich spectra is required. Chemometric analysis of EEM data is thekey factor in developing robust analytical methods. Methods such as partial least squaresregression (PLS) and parallel factor analysis (PARAFAC) have been used for quantitativedetermination of various pharmaceuticals during formulation 28 as well as in biological fluids, 29,30  and also for the characterization of DOM. 31,32  Our group has been focusing on developing innovative optical spectroscopy basedanalytical methods for the rapid analysis of complex cell culture media. By combiningfluorescence methods in conjunction with Raman spectroscopy, the majority of all commonlyused materials throughout an entire biopharmaceutical process can be monitored both rapidly andeffectively. Here we demonstrate the use of EEM and chemometrics to rapidly assess the qualityof blended cell culture media, discriminate different blend types, observe storage inducedchanges, and correlate spectral data with product yield. This ability to predict whether a feedmedia is “good” or “poor” (based on product yield) before addition to a bioreactor is a powerfulapplication of the developed techniques and should result in major cost savings for the industryby means of greatly improved protein product yield consistency. MATERIALS AND METHODS  Page 4 of  19   P.W. Ryan, B. Li, M. Shanahan, K.J. Leister, A.G. Ryder.  Anal. Chem. , 82 (4), 1311-1317, (2010).   Cell culture media samples: The feed media lots were manufactured for two differentmammalian cell culture processes (A- and L-processes), on a large scale (5,000 L+) by Bristol-Myers Squibb (Syracuse, NY). Each lot was manufactured in a single batch and stored for 20+days at 2-8 ºC and used as required during the bioprocess. Feed media were blended withappropriate nutrient and growth factors at approximately T=0. Aliquots from 33 different lotswere removed for analysis at four different timepoints over the 20 day storage period (Table 1).The exact composition of the media is a commercial trade secret and as such was unknown to theanalysts in Ireland. All media were sampled and aliquotted under sterile conditions and shippedto Ireland at low temperature, under monitored conditions. These samples were then aliquottedto smaller volumes and stored frozen at -70°C in order to limit further changes in mediacomposition. For analysis, the samples were randomly removed from cold storage and defrostedat room temperature. Once defrosted the samples were handled under aseptic conditions anddiluted (50 µL in 1 mL) with ultrapure water before being pipetted into cuvettes and sealed, allmeasurements were made within 6 hours of defrosting. Instrumentation and data collection:   Fluorescence spectra were measured at 25 °C using aCary Eclipse Fluorescence Spectrometer fitted with a thermostatted (Peltier) 4-position multi-cellholder (Varian). EEM data were collected with spectral ranges of 230 to 520 nm for excitationand 270 to 600 nm emission, a data interval of 5 nm, and measurement typically took ~7minutes. Semi micro quartz cuvettes (Lightpath Optical Ltd., UK) with a long path length of 10mm (excitation axis) and a short path length of 2 mm (emission axis) were employed for allmeasurements. A water background spectrum was measured at the beginning and end of eachsample collection run. Three EEM spectra were collected for every sample on different randomdates over a 6-month period. Data pre-processing and analysis:   Rayleigh and Raman scatter are largely unrelated to thechemical properties of the sample and the scatter peaks do not behave linearly (or trilinearly).This may complicate and bias fluorescence data modelling, 33 and so Raleigh and Raman bandswere removed prior to analysis. The bands were removed by replacement with a linear fit,connecting points either side of the peak using imputation. 34,35 All calculations were performedusing MATLAB  ®  (ver. 7.4) on a standard PC. 37 For the majority of the analyses undertaken,PLS_Toolbox 4.0  ®  , supplemented by in-house-written codes was used. 36,37   Methodology:   A range of chemometric methods have to be employed for the robust quantitativeanalysis of cell culture media. First the spectral quality and sample outliers must be assessed andidentified using multiway robust principal component analysis (MROBPCA). Second, changesin media composition with respect to storage time are evaluated using n-way partial leastsquares-discriminant analysis (NPLS-DA). Finally the quality of the cell culture media ismeasured by using n-way partial least squares (NPLS)   to correlate with protein product yield.  Page 5 of  19   P.W. Ryan, B. Li, M. Shanahan, K.J. Leister, A.G. Ryder.  Anal. Chem. , 82 (4), 1311-1317, (2010).  Brief descriptions of the chemometric methods employed, and the requisite references areprovided in the supplemental information. 38-58   RESULTS AND DISCUSSIONEEM Spectra: The media samples are reasonably fluorescent (Figure 1) when illuminatedby UV-blue light, with the Rayleigh scattering band being about twice the intensity of themaximum fluorescence signal. The complexity of the EEM spectra of all samples (Figure 1)indicates that fluorescence srcinates from a variety of fluorophores, the most significant of which are tryptophan and tyrosine, which are present in appreciable quantities: ~1.0 and ~4.8mg/L respectively. The interplay of energy transfer (ET) and quenching between all of thevarious components produces a unique EEM profile which can be used to characterize thesecomplex media. Small variances in media composition can cause changes in tryptophan ortyrosine (or both) emission, causing changes in the spectral profile which can be observed.Knowing the exact identity (or concentrations) of the individual absorbing and emitting speciesis not required (or possible in this case) for the use of EEM data for media characterization.There are six distinct local maxima within these EEM landscapes and Table 1 gives detailsof the mean intensity values of the maxima as a function of storage time (Figure 1F). While it isdifficult to assign these peaks only to specific fluorophores due to the complex nature of thesamples, peaks with 305 nm emission probably srcinate from tyrosine while emission peaks at355 nm are likely to result from tryptophan. The emission peaks at 600 nm are second ordertyrosine emission (validated by experiment, data not shown). While there are no significantspectral changes evident in the samples with storage time, there does appear to be a slightreduction in the intensity of the tyrosine bands as a function of storage time which isaccompanied by a slight increase in the intensity of peaks with emission at 355 nm. This trendwould appear to correlate well with a change in ET efficiency from tyrosine to tryptophan withstorage time due to compositional changes of the media. It is also worth noting that the majorityof this shift in intensity occurs within the first five days of media storage.
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