Elemental metal variance in cell culture raw materials for process risk profiling.

Elemental metals are critical raw material attributes which can impact cell culture performance and associated therapeutic protein product quality profiles. Metals such as copper and manganese act as cofactors and reagents for numerous metabolic pathways which govern cell growth, protein expression, and glycosylation, thus mandating elemental monitoring. The growing complexity of modern cell culture media formulations adds additional opportunities for elemental variance and its associated impact risks. This article describes an analytical technique applying inductively coupled plasma mass spectrometry to characterize a list of common raw materials and media powders used in mammalian cell culture and therapeutic protein production. We aim to describe a method qualification approach suitable for biopharmaceutical raw materials. Furthermore, we present detailed profiles of many common raw materials and discuss trends in raw material subtypes. Finally, a case study demonstrating the impact of an unexpected source of raw material variation is presented along with recommendations for raw material elemental risk profiling and control.

Enabling Robust and Rapid Raw Material Identification and Release by Handheld Raman Spectroscopy.

A fast, reproducible, non-destructive method to confirm raw material identification in real-time upon material receipt within a warehouse environment is desired. Current practices in pharmaceutical manufacturing often employ compendia methods for raw material identification tests, which require sample preparation prior to time-consuming chemical analysis and often employ subjective spectral comparisons. We have developed, qualified, and validated a rapid objective identity method ("Rapid ID") by Raman spectroscopy using the Bruker BRAVO handheld Raman spectrometer for 46 common raw materials used in upstream and downstream biopharmaceutical cell culture-based processes. Materials in the Raman identification library include amino acids and other solid neat organic chemicals, liquid organics, polyatomic salts, polymers, emulsifiers, peptides, aqueous solutions, and buffers. Selection of reference spectra and hit quality index limit(s) was based upon a comprehensive spectral survey across multiple suppliers and lots to account for normal cause spectral variation. Method repeatability and reproducibility, selectivity, and robustness against various operational and environmental factors (e.g., instrumental variance, material packaging, and thermal effects) were evaluated. Benefits of a handheld Raman Rapid ID approach include significant reduction of the time for raw material quality release from weeks to minutes, enhanced objectivity, and robust data integrity via autonomous electronic reporting. In addition, routine collection of rich spectroscopic data on raw materials can be leveraged to support further continuous improvement initiatives, including routine monitoring of method performance, continuous improvement of the library, proactive detection of shifts in raw material properties, and provision of data for investigations focused on raw materials. Rapid ID methods are consistent with the move toward the principles of Pharma 4.0-high automated processes with continuous process verification and a holistic control strategy.LAY ABSTRACT: A fast, reproducible, non-destructive method is desired to confirm raw material identification in real time upon receipt within a warehouse environment. We have developed, qualified and validated a rapid objective identity method ("Rapid ID") by Raman spectroscopy using the Bruker BRAVO handheld Raman spectrometer for 46 common raw materials used in upstream and downstream biopharmaceutical cell culture-based processes. Benefits of a handheld Raman Rapid ID approach include significant time reduction of raw material quality release from weeks to minutes, enhanced objectivity, and robust data integrity via autonomous electronic reporting. Rapid ID methods are consistent with the move toward the principles of Pharma 4.0: high automated processes with continuous process verification and a holistic control strategy.

Glucose monitoring and adaptive feeding of mammalian cell culture in the presence of strong autofluorescence by near infrared Raman spectroscopy.

Raman spectroscopy offers an attractive platform for real-time monitoring and control of metabolites and feeds in cell culture processes, including mammalian cell culture for biopharmaceutical production. However, specific cell culture processes may generate substantial concentrations of chemical species and byproducts with high levels of autofluorescence when excited with the standard 785 nm wavelength. Shifting excitation further toward the near-infrared allows reduction or elimination of process autofluorescence. We demonstrate such a reduction in a highly autofluorescent mammalian cell culture process. Using the Kaiser RXN2-1000 platform, which utilizes excitation at 993 nm, we developed multivariate glucose models in a cell culture process which was previously impossible using 785 nm excitation. Additionally, the glucose level in the production bioreactor was controlled entirely by Raman adaptive feeding, allowing for maintenance of glucose levels at an arbitrary set point for the duration of the culture. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1574-1580, 2018.

Closed loop control of lactate concentration in mammalian cell culture by Raman spectroscopy leads to improved cell density, viability, and biopharmaceutical protein production.

Accumulation of lactate in mammalian cell culture often negatively impacts culture performance, impeding production of therapeutic proteins. Many efforts have been made to limit the accumulation of lactate in cell culture. Here, we describe a closed loop control scheme based on online spectroscopic measurements of glucose and lactate concentrations. A Raman spectroscopy probe was used to monitor a fed-batch mammalian cell culture and predict glucose and lactate concentrations via multivariate calibration using partial least squares regression (PLS). The PLS models had a root mean squared error of prediction (RMSEP) of 0.27 g/L for glucose and 0.20 g/L for lactate. All glucose feeding was controlled by the Raman PLS model predictions. Glucose was automatically fed when lactate levels were beneath a setpoint (either 4.0 or 2.5 g/L) and glucose was below its own setpoint (0.5 g/L). This control scheme was successful in maintaining lactate levels at an arbitrary setpoint throughout the culture, as compared to the eventual accumulate of lactate to 8.0 g/L in the historical process. Automated control of lactate by restricted glucose feeding led to improvements in culture duration, viability, productivity, and robustness. Culture duration was extended from 11 to 13 days, and harvest titer increased 85% over the historical process. Biotechnol. Bioeng. 2016;113: 2416-2424. © 2016 Wiley Periodicals, Inc.

Sensitivity of coded aperture Raman spectroscopy to analytes beneath turbid biological tissue and tissue-simulating phantoms.

Traditional slit-based spectrometers have an inherent trade-off between spectral resolution and throughput that can limit their performance when measuring diffuse sources such as light returned from highly scattering biological tissue. Recently, multielement fiber bundles have been used to effectively measure diffuse sources, e.g., in the field of spatially offset Raman spectroscopy, by remapping the source (or some region of the source) into a slit shape for delivery to the spectrometer. Another approach is to change the nature of the instrument by using a coded entrance aperture, which can increase throughput without sacrificing spectral resolution.In this study, two spectrometers, one with a slit-based entrance aperture and the other with a coded aperture, were used to measure Raman spectra of an analyte as a function of the optical properties of an overlying scattering medium. Power-law fits reveal that the analyte signal is approximately proportional to the number of transport mean free paths of the scattering medium raised to a power of -0.47 (coded aperture instrument) or -1.09 (slit-based instrument). These results demonstrate that the attenuation in signal intensity is more pronounced for the slit-based instrument and highlight the scattering regimes where coded aperture instruments can provide an advantage over traditional slit-based spectrometers.

A scattering phantom for observing long range order with two-dimensional angle-resolved Low-Coherence Interferometry.

Angle-resolved low coherence interferometry (a/LCI) is an approach for assessing tissue structure based on light scattering data. Recent advances in a/LCI have extended the analysis to study scattering distributions in two dimensions. In order to provide suitable scattering phantoms for 2D a/LCI, we have developed phantoms based on soft lithography which can provide a range of structures including long range order. Here we characterize these phantoms and demonstrate their utility for providing standardized multi-scale structural information for light scattering measurements.

Fourier domain multispectral multiple scattering low coherence interferometry.

We have implemented multispectral multiple scattering low coherence interferometry (ms2/LCI) with Fourier domain data collection. The ms2/LCI system is designed to localize features with spectroscopic contrast with millimeter resolution up to 1 cm deep in scattering samples by using photons that have undergone multiple low-angle (forward) scattering events. Fourier domain detection both increases the data acquisition speed of the system and gives access to rich spectroscopic information, compared to the previous single channel, time-domain implementation. Separate delivery and detection angular apertures reduce collection of the diffuse background signal in order to isolate localized spectral features from deeper in scattering samples than would be possible with traditional spectroscopic optical coherence tomography. Light from a supercontinuum source is used to acquire absorption spectra of chromophores in the visible range within a tissue-like scattering phantom. An intensity modulation and digital lock-in detection scheme is implemented to mitigate relative intensity and spectral noise inherent in supercontinuum sources. The technical parameters of the system and comparative analysis are presented.

High-efficiency diffuse Raman spectroscopy through a fiber bundle.

Conventional spectrometers are limited in the amount of light they accept because of the requirement for narrow input apertures. A trade-off must generally be made between spectral resolution and input aperture width. This is especially a problem for performing spectroscopy on diffuse sources, such as with tissue, from which signal light has a broad spatial distribution. We introduce a method for achieving good spectral resolution from a fiber bundle input. The image of a fiber bundle has a characteristic structure. By distorting this image optically, we generate a pseudo-orthogonal intensity mask at the input of the spectrometer. The pseudo-orthogonal properties of the mask then allow decoupling at the detector plane of wavelength from spatial position. As long as the distorted image of the fiber bundle is well known, a spectrum can be recovered with spectral resolution equivalent to that of a conventional slit-based spectrometer. We demonstrate successful recovery of narrowly spaced spectral features as well as Raman spectra from a highly scattering sample with this method. This method enables probes with much higher throughputs and add fiber bundle-based spectroscopy to endoscopic designs.

In vivo and ex vivo epi-mode pump-probe imaging of melanin and microvasculature.

We performed epi-mode pump-probe imaging of melanin in excised human pigmented lesions and both hemoglobin and melanin in live xenograft mouse melanoma models to depths greater than 100 µm. Eumelanin and pheomelanin images, which have been previously demonstrated to differentiate melanoma from benign lesions, were acquired at the dermal-epidermal junction with cellular resolution and modest optical powers (down to 15 mW). We imaged dermal microvasculature with the same wavelengths, allowing simultaneous acquisition of melanin, hemoglobin and multiphoton autofluorescence images. Molecular pump-probe imaging of melanocytes, skin structure and microvessels allows comprehensive, non-invasive characterization of pigmented lesions.

Pump-probe imaging differentiates melanoma from melanocytic nevi.

Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. We have developed a multiphoton technique (based on pump-probe spectroscopy) that directly determines the microscopic distribution of eumelanin and pheomelanin in pigmented lesions of human skin. Our initial results showed a marked difference in the chemical variety of melanin between nonmalignant nevi and melanoma, as well as a number of substantial architectural differences. We examined slices from 42 pigmented lesions and found that melanomas had an increased eumelanin content compared to nonmalignant nevi. When used as a diagnostic criterion, the ratio of eumelanin to pheomelanin captured all investigated melanomas but excluded three-quarters of dysplastic nevi and all benign dermal nevi. Additional evaluation of architectural and cytological features revealed by multiphoton imaging, including the maturation of melanocytes, presence of pigmented melanocytes in the dermis, number and location of melanocytic nests, and confluency of pigmented cells in the epidermis, further increased specificity, allowing rejection of more than half of the remaining false-positive results. We then adapted this multiphoton imaging technique to hematoxylin and eosin (H&E)-stained slides. By adding melanin chemical contrast to H&E-stained slides, pathologists will gain complementary information to increase the ease and accuracy of melanoma diagnosis.

Probing near-infrared photorelaxation pathways in eumelanins and pheomelanins.

Ultraviolet-visible spectroscopy readily discerns the two types of melanin pigments (eumelanin and pheomelanin), although fundamental details regarding the optical properties and pigment heterogeneity are more difficult to disentangle via analysis of the broad featureless absorption spectrum alone. We employed nonlinear transient absorption spectroscopy to study different melanin pigments at near-infrared wavelengths. Excited-state absorption, ground-state depletion, and stimulated emission signal contributions were distinguished for natural and synthetic eumelanins and pheomelanins. A starker contrast among the pigments is observed in the nonlinear excitation regime because they all exhibit distinct transient absorptive amplitudes, phase shifts, and nonexponential population dynamics spanning the femtosecond-nanosecond range. In this manner, different pigments within the pheomelanin subclass were distinguished in synthetic and human hair samples. These results highlight the potential of nonlinear spectroscopies to deliver an in situ analysis of natural melanins in tissue that are otherwise difficult to extract and purify.

Estimation of molar absorptivities and pigment sizes for eumelanin and pheomelanin using femtosecond transient absorption spectroscopy.

Fundamental optical and structural properties of melanins are not well understood due to their poor solubility characteristics and the chemical disorder present during biomolecular synthesis. We apply nonlinear transient absorption spectroscopy to quantify molar absorptivities for eumelanin and pheomelanin and thereby get an estimate for their average pigment sizes. We determine that pheomelanin exhibits a larger molar absorptivity at near IR wavelengths (750 nm), which may be extended to shorter wavelengths. Using the molar absorptivities, we estimate that melanin pigments contain approximately 46 and 28 monomer units for eumelanin and pheomelanin, respectively. This is considerably larger than the oligomeric species that have been recently proposed to account for the absorption spectrum of eumelanin and illustrates that larger pigments comprise a significant fraction of the pigment distribution.

Label-free in vivo optical imaging of microvasculature and oxygenation level.

The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption (ESA)-based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxy- and deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique.

Probing skin pigmentation changes with transient absorption imaging of eumelanin and pheomelanin.

As some of the most ubiquitous and biologically important natural pigments, melanins play essential roles in the photoprotection of skin. Changes in melanin production could potentially be useful for clinical diagnosis of the progression stage of melanoma. Previously we demonstrated a new method for imaging melanin distribution in tissue with two-color transient absorption microscopy. Here we extend this study to longer wavelengths and show that we are able to image melanin in fixed thin skin slices with higher signal-to-noise ratios (SNRs) and demonstrate epimode imaging. We show that both photothermal effects and long-lived excited states can contribute to the long-lived signal. Eumelanin and pheomelanin exhibit markedly different long-lived excited state absorption. This difference should enable us to map out their respective distribution in tissue samples with subcellular resolution. This technique could provide valuable information in diagnosing the malignant transformation of melanocytes.

Two-color, two-photon, and excited-state absorption microscopy.

We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10(-6) absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.

High-resolution in vivo imaging of blood vessels without labeling.

We demonstrate that both oxyhemoglobin and deoxyhemoglobin have sequential two-color, two-photon absorption properties that can serve as endogenous contrasts in microvasculature imaging. Using a sensitive modulation transfer technique, we are able to image hemoglobin in red blood cells with micrometer resolution, both in vitro and in vivo. We show that excellent contrast from hemoglobin without any labeling can be obtained in tissue.