Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS||) and Synchronous Light Scattering (SyLS||) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS|| spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS|| measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.

Accurate size measurements of nanosized polymer chains in dilute solutions is important for understanding polymer behavior, however, these measurements can be challenging to implement accurately, and are technique dependent. Here we explore the use of Fluorescence Correlation Spectroscopy (FCS) to determine the size of single polymer chains in dilute solutions (<2 wt%). FCS, a technique with single molecule sensitivity, generally requires the use of covalently labelled polymers which can distort physicochemical behavior. Here, FCS based size measurements were based on the non-covalent interaction of fluorophores (Alexa 405, Atto 390, and Atto 425) with PNIPAm in water at 25°C (below the Lower Critical Solution Temperature). FCS estimated size (hydrodynamic radius) of three different MW PNIPAm samples in water were: 5.0±1.0 nm (28.5 kDa), 5.0±1.0 nm (38 kDa), 3.3±0.5 nm (55.5 kDa), in reasonable agreement with theoretical calculations. Accuracy was directly related to the fraction of PNIPAm-bound fluorophore, which were, for 1 wt% PNIPAm solutions in water: ∼11.5% (Atto 390), ∼8.1% (Atto 425), and 4% (Alexa 405). This method has several advantages in that it does not require a covalent labelling of PNIPAm, it can be implemented on very small sample volumes, and allows for in-situ measurements.

Yeastolate is often used as a media supplement in industrial mammalian cell culture or as a major media component for microbial fermentations. Yeastolate variability can significantly affect process performance, but analysis is technically challenging because of its compositional complexity. However, what may be adequate for manufacturing purposes is a fast, inexpensive screening method to identify molecular variance and provide sufficient information for quality control purposes, without characterizing all the molecular components. Here we used Size Exclusion Chromatography (SEC) and chemometrics as a relatively fast screening method for identifying lot-to-lot variance (with Principal Component Analysis, PCA) and investigated if Partial Least Squares, PLS, predictive models which correlated SEC data with process titer could be obtained. SEC provided a relatively fast measure of gross molecular size hydrolysate variability with minimal sample preparation and relatively simple data analysis. The sample set comprised of 18 samples from 12 unique source lots of an ultra-filtered yeastolate (10 kDa molecular weight cut-off) used in a mammalian cell culture process. SEC showed significant lot-to-lot variation, at 214 and 280 nm detection, with the most significant variation, that correlated with process performance, occurring at a retention time of ∼6 min. PCA and PLS regression correlation models provided fast identification of yeastolate variance and its process impact. The primary drawback is the limited column lifetime (<300 injections) caused by the complex nature of yeastolate and the presence of zinc. This limited long term reproducibility because these age-related, non-linear changes in chromatogram peak positions and shapes were very significant.

Antibody-drug conjugates (ADCs) are promising anticancer therapeutics, which offer important advantages compared to more classical therapies. There are a variety of ADC critical quality attributes (CQAs) such as the protein structure, aggregation, and drug-to-antibody ratio (DAR), which all impact on potency, stability, and toxicity. Production processes can destabilize antibodies via a variety of physical and chemical stresses, and or by increased aggregation after conjugation of hydrophobic drugs. Thus, a proper control strategy for handling, production, and storage is necessary to maintain CQA levels, which requires the use of in-process quality measurements to first identify, then understand, and control the variables which adversely affect ADC CQAs during manufacturing. Here, we show how polarized excitation emission matrix (pEEM) spectroscopy, a sensitive, nondestructive, and potentially fast technique, can be used for rapidly assessing aggregation and DAR in a single measurement. pEEM provides several sources of information for protein analysis: Rayleigh scatter for identifying aggregate/particle formation and fluorescence emission to assess chemical and structural changes induced by attachment of a linker and/or a small molecule drug payload. Here, we used a nontoxic ADC mimic (monoclonal antibody with linker molecule) to demonstrate efficacy of the measurement method. Emission changes caused via light absorption by the attached linker, allowed us to predict DAR with good accuracy using fluorescence signal from the final purified products (6% relative error of prediction [REP]) and also from unpurified alkylation intermediates (11% REP). pEEM changes could also be correlated with size (hydrodynamic radius, Rh ) and aggregate content parameters obtained from dynamic light scattering and size exclusion chromatography (SEC). For the starting material and purified product samples, pEEM correlated better with Rh (R2  = 0.99, 6% REP) than SEC determined aggregate content (18% REP). Combining both fluorescence and light scatter signals also enabled in-process size quantification (6% REP). Overall, combining polarized measurements with EEM and Rayleigh scatter provides a single measurement, multi-attribute test method for ADC manufacturing.

: 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 exploring the use of rapid “holistic” analytical methods that can be used for routine screening of cell culture media used in industrial biotechnology. The application of rapid fluorescence spectroscopic techniques to the routine analysis of cell culture media (Chinese Hamster Ovary cell based manufacture) was investigated. 1311-1317, (MROBPCA), n-way partial least squares-discriminant analysis and regression (NPLS-DA and NPLS). This ability to observe compositional changes and predict product yield before media use has enormous potential and should permit the effective elimination of one of the major process variables leading to more consistent product quality and improved yield. These robust and reliable methods have the potential to become an important part of upstream biopharmaceutical quality control and analysis.

Studying the interaction between plasma proteins and liposomes is critical, particularly for their use as drug delivery systems. Here, the efficacy of anisotropy resolved multidimensional emission spectroscopy (ARMES) for investigating the interaction of human serum albumin (HSA) with liposomes was explored and compared to conventional spectroscopic techniques. Dynamic Light Scattering (DLS) and absorbance spectroscopy (with Multivariate Curve Resolution (MCR) modeling) indicated that the highest degree of liposome rupturing, and aggregation occurred in water, with less in ammonium bicarbonate buffer (ABC) and phosphate buffered saline (PBS). Fluorescence emission spectra of HSA-liposome mixtures revealed significant hypsochromic shifts for water and ABC, but much less in PBS, where the data suggests a non-penetrating protein layer was formed. Average fluorescence lifetimes decreased upon liposome interaction in water (6.2→5.2 ns) and ABC buffer (6.3→5.6 ns) but increased slightly for PBS (5.6→5.8 ns). ARMES using polarized Total Synchronous Fluorescence Scan measurements with parallel factor (PARAFAC) analysis resolved intrinsic HSA fluorescence into two components for interactions in water and ABC buffer, but only one component for PBS. These components, in water and ABC buffer, corresponded to two different HSA populations, one blue-shifted and penetrating the liposomes (λex/em = ~ 280/320 nm) and a second, similar to free HSA in solution (λex/em = ~ 282/356 nm). PARAFAC scores for water and ABC buffer suggested that a large proportion of HSA interacted in an end on configuration. ARMES provides a new way for investigating protein-liposome interactions that exploits the full intrinsic emission space of the protein and thus avoids the use of extrinsic labels. The use of multivariate data analysis provided a comprehensive and structured framework to extract a variety of useful information (resolving different fluorescent species, quantifying their signal contribution, and extracting light scatter signals) all of which can be used to discriminate between interaction mechanisms.

Fluorescence Correlation Spectroscopy (FCS) studies of the interaction of polymers or proteins in solution are strongly affected by the viscosity and refractive index of the medium, and the effects are likely to be more significant with the use of short wavelength excitation (e.g., 405 nm diode lasers). Failing to account for these issues can lead to incorrect measurement of average size, conformational changes, and dynamic behaviour of polymers and proteins. Steady-state, time-resolved, and FCS measurements of Alexa 405 in glycerol:water mixtures were performed to determine its suitability for FCS measurements with 405 nm excitation. The effects of the refractive index and viscosity on the diffusion coefficient and photophysical parameters (lifetime and relative quantum yield) of the fluorophore were determined. Alexa 405 lifetime decreased from 3.55 ns in water to 3.25 ns in a 50:50 glycerol:water mixture, while its diffusion coefficient dropped from 333 ± 16 to 44 ± 1 µm2s− 1. Lifetime data collected from micromolar solutions of Alexa 405 did however also suggest that as solvent polarity decreased, aggregates (excimers) were formed as evidenced by the appearance of a rising edge in the decay plots. The interdependence between lifetime, refractive index, and diffusion coefficient could be accurately fitted by a simple polynomial function indicating that the probe is well behaved and predictable in the glycerol:water model system. Overall, Alexa 405 is a most promising and reliable probe for FCS measurement using violet laser diode excitation sources.

Biotechnology and BioengineeringVolume 118, Issue 5 p. 1757-1761 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 118, Number 5, May 2021 First published: 14 April 2021 https://doi.org/10.1002/bit.27401AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Volume118, Issue5May 2021Pages 1757-1761 RelatedInformation

: The accurate fluorescence analysis of complex, multi-fluorophore containing proteins requires the use of multi-dimensional measurement techniques. For the measurement of intrinsic fluorescence from tyrosine (Tyr) and tryptophan (Trp) one needs tuneable UV excitation and for steady-state measurements like Excitation Emission Matrix (EEM) simple pulsed Xe lamps are commonly used. Unfortunately, simultaneous multi-dimensional wavelength and time resolved measurement of intrinsic protein fluorescence in the 260 to 400 nm spectral range are challenging and typically required the use of very complex tuneable laser systems or multiple single excitation wavelength sources. Here we have assembled and validated a novel Excitation Emission Fluorescence Lifetime Spectrometer (EEFLS) using a pulsed, frequency doubled, SuperContinuum Laser (SCL) source coupled with a 16 channel multi-anode Time Correlated Single Photon Counting (TCSPC) measurement system. This EEFLS enabled the collection of near complete lifetime and intensity maps over the most important intrinsic protein fluorescence spectral range (λ ex = 260–350 / λ em =300–500 nm). The 4-dimensional (λ ex /λ em /I (t) /τ) Excitation Emission Fluorescence Lifetime Matrix (EEFLM) data produced can be used to better characterize the complex intrinsic emission from proteins. The system was capable of measuring fluorescence emission data with high spectral (1-2 nm) resolution and had an Instrument Response Function (IRF) of ~650 ps for accurate measurement of nanosecond lifetimes. UV power output was stable after a warm up period, with variations of <2% over 9 hours and reproducible (relative standard deviation RSD <1.5%). This enabled the collection of accurate EEFLM data at low resolution (~12 nm in excitation and emission) in 1-2 hours or high resolution (4 nm) in ~17 hours. EEFLS performance in the UV was compared with a conventional commercial TCSPC system using pulsed LED excitation and validated using solutions of p-terphenyl and tryptophan.

Protein quantification during bioprocess monitoring is essential for biopharmaceutical manufacturing and is complicated by the complex chemical composition of the bioreactor broth. Here we present the early‐stage development and optimization of a polarized total synchronous fluorescence spectroscopy (pTSFS) method for protein quantification in a hydrolysate‐protein model (mimics clarified bioreactor broth samples) using a standard benchtop laboratory fluorometer. We used UV transmitting polarizers to provide wider range pTSFS spectra for screening of the four different TSFS spectra generated by the measurement: parallel (||), perpendicular (⊥), unpolarized (T) intensity spectra and anisotropy maps. TSFS|| (parallel polarized) measurements were the best for protein quantification compared to standard unpolarized measurements and the Bradford assay. This was because TSFS|| spectra had a better analyte signal to noise ratio (SNR), due to the anisotropy of protein emission. This meant that protein signals were better resolved from the background emission of small molecule fluorophores in the cell culture media. SNR of >5000 was achieved for concentrations of bovine serum albumin/yeastolate 1.2/10 g L–1 with TSFS||. Optimization using genetic algorithm and interval partial least squares based variable selection enabled reduction of spectral resolution and number of excitation wavelengths required without degrading performance. This enables fast (<3.5 min) online/at‐line measurements, and the method had an LOD of 0.18 g L–1 and high accuracy with a predictive error of <9%.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

Being able to measure the size and distribution of oligomers in solution is a critical issue in the manufacture and stability of insulin and other protein formulations. Measuring oligomers reliably can however be complicated, due to their fragile self-assembled structures, which are held together by weak forces. This can cause issues in chromatographic based methods, where dissociation or re-equilibration of oligomer populations can occur e.g. upon dilution in a different eluting buffer, but also for light scattering based methods like dynamic light scattering (DLS) where the size difference involved (often less than a factor 3) does not allow mixtures of oligomers to be resolved. Intrinsic fluorescence offers an attractive alternative as it is non-invasive, sensitive but also because it contains scattered light when implemented via excitation emission matrix (EEM) measurements, that is sensitive to changes in particle size. Here, using insulin at formulation level concentrations, we show for the first time how EEM can both discriminate and quantify the proportion of oligomeric states in solution. This was achieved by using the Rayleigh scatter (RS) band and the fluorescence signal contained in EEM. After validating size changes with DLS, we show in particular how the volume under the RS band correlated linearly with protein/oligomer molecular weight, in agreement with the Debye-Zimm relationship. This was true for the RS data from both EEM and polarized EEM (pEEM) measurements, the latter providing a stronger scatter signal, more sensitive to particle size changes. The fluorescence signal was then used with multivariate curve resolution (MCR) to quantify more precisely the soluble oligomer composition of insulin solutions. In conditions that promoted the formation of mainly one type of oligomer (monomer, dimer, or hexamer), pEEM-MCR helped identify the presence of small amounts of other oligomeric forms, while in conditions that were previously said to favour the insulin tetramer, we show that in the presence of zinc, these insulin samples were instead a heterogenous mixture composed of mostly dimers and hexamers. These MCR results correlated in all cases with the observed discrimination by principal component analysis (PCA), and deviations observed in the RS data. In conclusion, using pEEM scatter and emission components with chemometric data analysis provides a unique analytical method for characterising and monitoring changes in the soluble oligomeric state of proteins.

The growing use of therapeutic proteins requires accurate analytical techniques for measuring biophysical and structural changes during manufacturing. This is particularly true for the PEGylation of proteins, because characterization of PEGylation reactions and products can often be difficult due to the relatively small impact on protein structure, the lack of an accessible polyethylene glycol (PEG) chromophore, and the heterogeneous final product mixtures. Intrinsic fluorescence spectroscopy is one potential solution due to its relatively high sensitivity to small changes in protein structure and its suitability for online or atline measurements. In this study, we use the PEGylation of lysozyme as a model system to determine the efficacy of polarized excitation-emission matrix (pEEM) spectroscopy as a rapid tool for characterizing the structural variability of the lysozyme (LZ) starting materials and PEGylated products with varying PEG-to-protein ratios (PPR). Dynamic light scattering showed that as PPR increased from 0 to 2.8, the hydrodynamic radius increased from ∼2.2 to 4.8 nm. pEEM measurements provided several sources of information: Rayleigh scattering to identify size changes and aggregate/particle formation, and fluorescence emission to assess chemical and structural changes. PEGylation induced sufficient physicochemical changes in LZ, which produced changes in the pEEM spectra, largely due to variations in the hydrophobic environments of tryptophan residues close to a PEG attachment site. These significant spectral changes when modeled using conventional multivariate analysis methods were able to easily discriminate the raw product solutions according to the degree of PEGylation and were also able to predict PPR with reasonable accuracy (root mean square error for calibration ∼10%, relative error of prediction < 20%), considering the reference size exclusion chromatography method error of ∼7.2%. The variable selection of the pEEM data suggests that equivalent predictions could be obtained with faster and simpler two-dimensional spectra, making the method a more viable online measurement method.

Background: Forster Resonance Energy Transfer (FRET) is widely used to study the structure and dynamics of biomolecular systems and also causes the non-linear fluorescence response observed in multi-fluorophore proteins. Accurate FRET analysis, in terms of measuring changes in donor and acceptor spectra and energy transfer efficiency is therefore critical. Methods: We demonstrate a novel quantitative FRET analysis using anisotropy resolved multidimensional emission spectroscopy (ARMES) in a Human Serum Albumin (HSA) and 1,8-anilinonaphathalene sulfonate (ANS) model. ARMES combines 4D measurement of polarized excitation emission matrices (pEEM) with multivariate data analysis to spectrally resolve contributing fluorophores. Multivariate analysis (Parallel Factor, PARAFAC and restricted Tucker3) was used to resolve fluorophore contributions and for modelling the quenching of HSA emission and the HSA-ANS interactions. Results: pEEM spectra were modelled using Tucker3 which accommodates non-linearities introduced by FRET and a priori chemical knowledge was used to optimise the solution, thus resolving three components: HSA emission, ANS emission from indirect FRET excitation, and ANS emission from direct excitation. Perpendicular emission measurements were more sensitive to indirectly excited acceptor emission. PARAFAC modelling of HSA, donor emission, separated ANS FRET interacting (Tryptophan) and non-interacting (Tyrosine) components. This enabled a new way of calculating quenching constants using the multi-dimensional emission of individual donor fluorophores. Conclusions: FRET efficiency could be calculated using the multi-dimensional, resolved emission of the interacting donor fluorophores only which yielded higher ET efficiencies compared to conventional methods. General significance: Shows the potential of multidimensional fluorescence measurements and data analysis for more accurate FRET modelling in proteins.

: The non-contact, measurement of water uptake in micro-scale (1-100 µ m), thermoresponsive Poly(N-isopropylacrylamide) thin films is challenging. We assessed the efficacy of three different 3-Hydroxyflavone (3-HF) based fluorophores; to monitor water uptake in pNIPAM thin films close to the Lower Critical Solution Temperature (LCST) at 25 and 37 ºC. These 3-HF fluorophores undergo excited state intramolecular proton transfer, yielding emission from normal (N*) and tautomeric (T*) excited-state forms. The emission intensity ratio, log(I N* /I T* ) and N* band position are environmentally sensitive. Water adsorption in pNIPAM thin films follows a non-linear, two phase process: at low relative humidity, the adsorbed water disrupts polymer-fluorophore hydrogen bonding, yielding small changes in log(I N* /I T* ) ratios, and overall intensity, at higher relative humidity, these intensity parameters (but not fluorescence lifetime) change dramatically, indicating a larger change in local polarity. These probes are thus sensitive indicators of water uptake in pNIPAM.

The effect of milling on the solid-state transitions of sulfathiazole polymorphs in the absence and presence of solvent and excipients was monitored by X-ray powder diffraction (XRPD), attenuated total reflectance infrared (ATR-IR) and near-infrared (NIR) spectroscopy. Sulfathiazole forms FII-FV undergo a transformation toward the metastable FI, which involves an intermediate amorphous stage upon milling at ambient temperature. Milling FIII with catalytic amounts of solvent converts FIII to FIV or to mixtures of FI and FIV depending on the solvent used. Pure FIV can be easily prepared from FIII by this method. The physical stability of pure sulfathiazole forms in the presence of different levels of relative humidity (RH) was also investigated. At low RH all sulfathiazole forms are stable but at RH levels above 70% FII, FIII and FIV remain stable while FI and FV transform to mixtures of FII and FIV without any apparent change in the external form of the crystals. Co-milling FIII with a range of excipients gave results which depended on the excipient used and co-milling with cellulose gave samples which had an amorphous content that was stable at 10% RH for at least nine months at ambient temperature.

Immunoglobulin G (IgG) is often used as a starting material for the production of functionalised antibodies, like Antibody Drug Conjugates (ADCs), PEGlyated-conjugates, or radioimmunoconjugates. The gross structural quality of the protein starting material is, therefore, an important factor in determining final product composition, purity, and quality. In terms of structural quality, one needs to know both the aggregation content and the tertiary structure of the protein. The measurement of structural quality in solution can thus be difficult, but the use of intrinsic fluorescence measurements might offer a solution because of its high sensitivity, ease of use, and when implemented in via multi-dimensional techniques like polarized Excitation Emission Matrix (pEEM) spectroscopy, its high information content. Here we demonstrate how pEEM measurements can be used as a multi-attribute screening method for protein quality using a polyclonal rabbit immunoglobulin (rIgG) model system. By using both Rayleigh scatter and fluorescence emission in combination with simple chemometric data analysis methods like Principal Component analysis (PCA) and unfolded partial least squares (U-PLS) one can simultaneously measure protein concentration, structural variance, and particle/aggregate composition. Furthermore, one can generate quantitative prediction models for non-reversible aggregation content as described by size exclusion chromatography (SEC) and obtain qualitative information about reversible aggregate content, which cannot be obtained from SEC measurements. In conclusion, the pEEM measurement approach is a potentially useful Process Analytical Technology (PAT) method for downstream processing operations in biopharmaceutical manufacturing.

The synthesis of a series of PPV derivative polymers by the Wittig-Horner reaction is described. The structure of each polymer is varied and the effects of these variations on the optical properties is explored. The effects of alkyloxy side chains is observed between the PPV derivatives Poly(-p-phenylvinylene-co-2,5-bis-octyloxy phenylvinylene.) PPV-OPV and Poly (para-2,5-bis-(n-octyloxy)phenylvinylene) POPV. The phenyl units of the soluble PPV derivative POPV are replaced by alternate naphthyl units in the polymer Poly(2,5-bis(n-octyloxy)-1,4-phenylene vinylene-1,5-bis(n-octyloxy)-2,6-naphthylene vinylene) POPN-ONV and then fully by naphthyl units in Poly(2,6-bis-(n-octyloxy)-1,5-NaphthyleneVinylene) PONV. The addition of alkyloxy sidechains served to red shift the fluorescence emission as expected. The systematic conversion of phenyl to naphthyl units blue shifted the emission considerably while reducing the Stokes shift. There is evidence to suggest some localization of the π electrons over the aromatic units of the polymer backbone. PONV is shown to have greater stability towards photo-oxidation then either POPV or PPV-OPV.