Low Temperature Thermal Dependent Filgrastim Adsorption Behavior

Nov 26, 2013 - Nicholas G. Welch , Robert M. T. Madiona , Judith A. Scoble , Benjamin W. Muir , and Paul J. Pigram. Langmuir 2016 32 (42), 10824-10834...
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Low Temperature Thermal Dependent Filgrastim Adsorption Behavior Detected with ToF-SIMS Ivan M. Kempson,*,† Patrick Chang,‡ Kristen Bremmell,‡ and Clive A. Prestidge† †

Ian Wark Research Institute, University of South Australia, Mawson Lakes, S.A. 5095, Australia School of Pharmacy and Medical Sciences, University of South Australia, S.A., Adelaide 5000, Australia



ABSTRACT: Time-of-flight secondary ion mass spectrometry (ToF-SIMS) detected changes in Filgrastim (granulocyte colony stimulating growth factor, G-CSF) adsorption behavior at a solid interface when exposed to temperatures as low as 35 °C, i.e., before thermal denaturation, was detected by circular dichroism (CD) or dynamic light scattering (DLS). Biopharmaceuticals rely on maintaining sufficient conformation to impart correct biological function in vivo. Stability of such molecules is critical during synthesis, storage, transport, and administration. CD analysis indicated loss of structure at temperatures greater than ∼60 °C, while DLS detected aggregation at ∼42 °C. Furthermore, we demonstrate the nature of G-CSF interaction with a surface was altered rapidly and at relatively low temperatures. Specifically, after 10 min thermal treatment, changes in adsorption behavior occurred at 35 °C indicated by principal component analysis of spectra as primarily due to increasing yields of methionine fragments. This was likely to be due to either altering the preferential protein orientation upon adsorption or greater denaturation exposing the hydrophobic core. This investigation demonstrates the sensitivity of ToF-SIMS in studying biopharmaceutical adsorption and conformational change and can assist with studies into promoting their stability.



INTRODUCTION With increasing biopharmaceutical prevalence in drug development and therapies, stability of protein solutions is becoming a critical aspect of efficacious delivery.1 Areas of research and development of nucleic acid and protein based products are rapidly growing and require improved characterization for quality control and monitoring. Interaction of the protein/ biomolecule with its surrounding environment (surfactants, solvents, container walls) to maintain a functional conformation is critical. Filgrastim is a granulocyte colony stimulating growth factor (G-CSF) that stimulates proliferation and differentiation of granulocytes (polymorphonuclear leukocytes) and in particular neutrophils (a category of white blood cells). Hence, it finds application in neutropenia prophylaxis following chemotherapies.2−4 Effective regimen implementation can enable increasing chemotherapeutic doses and improve cancer survival rates.5 Filgrastim’s function is ultimately mediated by interaction with a cell surface receptor. It is therefore imperative that the structure and behavior of the G-CSF protein remain functional for culminating in effect. The analysis here was conducted to assess subtle differences in surface interaction of G-CSF as a function of thermal exposure. This was motivated by the desire to explore alternative approaches in assessing biopharmaceutical stability and developing rapid screening of factors influencing biological function. Typically, biological assays are the surest approach to assess drug efficacy. However, recent work by Skrlin et al. on physicochemical characterization of G-CSF with liquid chromatography mass spectrometry (LCMS) has shown correlation with bioassay response.6 These data demonstrated that physicochemical behavior of G-CSF directly imparts an effect on biological interaction which can be © 2013 American Chemical Society

predicted by rapid screening and can act to assist in minimizing the number of more intensive biological assays. A more recent analytical technique for studying protein interactions and adsorbed states on surfaces is time-of-flight secondary ion mass spectrometry (ToF-SIMS)7−9 which provides mass spectral information principally from the first monolayer of molecules on a surface. This has been applied to characterizing complex mixtures of proteins,10 molecular orientation,11−14 imaging protein and functional distributions,15−18 and studying protein adsorption and interactions8,19,20 and denaturation.21,22 Typically, to handle the complexity in spectra, multivariate analysis is used that can provide powerful discrimination of very similar, complex, organic samples.23−26 Here, we demonstrate that ToF-SIMS is highly sensitive in probing surface bound G-CSF, as a model biomolecule, to detect subtle changes in interfacial interactions. Subsequently, this data further validate ToF-SIMS as a tool to develop for investigative screening of physicochemical parameters that can alter biomolecule interactions and function. As shown, ToFSIMS has sensitivity in the analysis of sub-attomol quantities in regimes where conventional circular dichroism (CD) and dynamic light scattering (DLS) do not detect any gross structural/morphological change. We have investigated aggregation and changes in conformation as a function of temperature using standard analytics of DLS and CD, respectively. These data present rather predictable effects of temperature on the structure and aggregation of G-CSF. Received: September 30, 2013 Revised: November 25, 2013 Published: November 26, 2013 15573

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Figure 1. Circular dichroism spectra of G-CSF as a function of temperature. Loss of definition of the peaks in (a) signifies loss of the α-helical structure. Monitoring the intensity at 222 nm identifies “melting” occurring above approximately 60 °C. area in positive ion mode utilizing a 30 kV Au+ liquid metal ion gun with a total primary ion dose less than 1010 ions cm−2. Spectra were acquired up to 1000 atomic mass units (amu) with peaks distinguishable up to approximately 300 amu. The highest mass peak utilized in the analysis was 159.092, corresponding to tryptophan. Mass resolution as determined for the Si+ peak and C5H12N+ was better than 6500 for each spectrum with peaks calibrated to within 0.01 milli-amu of their expected position. Data Analysis. TOF-SIMS spectra were calibrated using the Wincadence software (Physical Electronics Inc.), and peaks indicative of amino acid functional groups were selected as per Kempson et al.22 and integrated for statistical interrogation. Integrated peak values were normalized to the total selected secondary ion intensities to correct for differences in total yield between analyses and samples. Data were then processed with Statistica 7 software (Statsoft Inc.) utilizing the principal components and classification analysis module based on mean-centered correlations.

However, upon investigation of adsorption behavior during exposure to a silicon surface, subtle differences are observed with TOF-SIMS that indicate altered physicochemical behavior at temperatures substantially lower than indicated by changes in conformation or aggregation. This directly implies G-CSF’s affinity for surfaces occurs as a function of temperature even slightly above room temperature. Such information is important in many areas of understanding emerging biopharmaceuticals and biosimilars.27



EXPERIMENTAL SECTION

Human recombinant granulocyte colony stimulating factor (rhG-CSF) (Filgrastim) was supplied in a highly pure form by Hospira Inc., Australia, in 10 mM sodium acetate buffer at pH 4, with a concentration of 3.5 mg mL−1. For buffer preparation, acetic acid (glacial) was purchased from Ajax Chemicals (Australia) and sodium acetate from Sigma (Australia). Milli-Q water was used throughout. For characterization of adsorption behavior with ToF-SIMS, rhGSCF (as received) was heated by separate samples being placed in a water bath at the designated temperature (from 25 to 75 °C in 10 deg increments) for 10 min before removal and being allowed to cool. A drop of each solution was subsequently deposited onto a air-plasma cleaned Si wafer after approximately 15 min, rinsed thoroughly with a stream of double distilled, deionized water before drying in air, and immediately loaded for ToF-SIMS analysis. A Si surface was used due to its chemical simplicity and the ease with which its surface chemistry can be influenced. Oxidized Si materials are also of interest for drug delivery vehicles, and the functionality of the surface is important in influencing interactions with proteins for storage stability. Plasma cleaning of silicon is known for providing a hydrophilic native oxidized surface, free of organic contamination.28 Circular dichroism (CD) was used to characterize the extent of G-CSF denaturation in solution by changes in the secondary protein structure. CD spectra were obtained as a function of temperature (25, 35, 45, 50, 55, 60, 65, 70, and 75 °C) on a Jasco J-815, UK, instrument in the far-UV region from 250 to 190 nm, with 0.5 nm increments, a bandwidth of 1.5 nm, and an averaging time of 1 s, using a 1.0 mm cell and a protein concentration of ∼50 μg mL−1 (diluted with 10 mM acetate buffer, pH 4). For all spectra, an average of five scans was obtained. CD spectra of the appropriate reference were recorded and subtracted from the protein spectra. Dynamic light scattering (DLS) was used to assess protein size in solution during thermal exposure for denaturation sufficient to lead to aggregation. Size was assessed with increasing temperature from to 25 to 48 °C in 1 deg increments. The Nanosizer ZS (Malvern Instruments, U.K.) apparatus was equipped with a 4.0 mW He−Ne laser using a backscattering configuration with detection at a scattering angle of 173° using an avalanche photodiode. Time-of-f light secondary ion mass spectrometry (ToF-SIMS) was performed with a Physical Electronics Inc. PHI TRIFT V nanoTOF instrument (Physical Electronics, Inc., Chanhassen, MN). Ten spectra were acquired each for 1 min and each from different locations for each temperature point. Spectra were acquired from a 100 × 100 μm



RESULTS

Circular dichroism (CD) data from analysis of G-CSF as a function of temperature are shown in Figure 1. Subtle differences in the curves in Figure 1a were observed up to 60 °C; however, the overall structural shape of the protein was still intact, indicated by the two minima showing that the protein still adopted its α-helical structure. Progressing above 60 °C saw the distinct onset of “melting” indicated by loss of the protein α-helical structure and denaturation of the protein (Figure 1b), as also observed for rhG-CSF in Krishnan et al.29 While CD indicates no major loss of structure up to 60 °C, dynamic light scattering (DLS) indicates that aggregation occurs at a lower temperature dependent on concentration, i.e., at approximately 42 °C (Figure 2) for a 3.5 mg/mL solution. Consequently, G-CSF potency can be expected to be severely diminished once reaching this temperature. Corresponding ζpotential measurements indicated no change in molecular charge over this temperature range. Over the entire temperature range, the ζ-potential measured remained between ∼+2 and +5 mV, suggesting that electrostatics did not contribute to the protein solution stability. Because of the challenges in accurately conducting such measurements, these data need to be taken with caution. ToF-SIMS was used to investigate differences in G-CSF mass spectral fragmentation after adsorption onto a silicon wafer over the temperature range from ambient to that where a loss of the α-helical structure is achieved (Figure 3). Examples of spectra in Figure 3 for a mass range of 10−120 amu demonstrate trends in fragmentation pattern with temperature. The spectra have been presented overlaying each other to emphasize that there are only subtle differences between each 15574

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principal components). The scores for a specific component can be used for discriminating samples. The important variables from the original data contributing to the discrimination can then be identified. A plot of the first principal component (accounting for 68.13% of the variability between spectra) increasing in value as a function of temperature is shown in Figure 4a. A distinct trend is observed over the temperature range of 25−55 °C. This trend was largely influenced by peaks in the mass spectra contributed to by amino acid residue units of tyrosine (Tyr), methionine (Met), and phenylalanine (Phe)all of which possess hydrophobic functional groups. The trend in the intensity of the fragment for the Met functional group (104.053 amu) over this temperature range is indicated in Figure 4b. The data produced from the PCA over the low temperature range was used to identify if any change in spectra could be deduced below the temperature point leading to aggregation and hence detectable by DLS. Figure 4a in fact shows a distinct change over this low-temperature range and identified Met as contributing to this trend. The Met peak intensity for the whole temperature range has subsequently been reproduced in Figure 4b. Because of the substantial literature studying the Met residues (see Discussion) with regard to G-CSF synthesis, stability, and activity, the Discussion section emphasizes the role of Met in the ToF-SIMS data rather than Tyr and Phe. As seen in Figure 3, ToF-SIMS spectra contain a large amount of data with subtly varying peak intensities at each temperature point. Identification of the key mass fragments and how they relate to each other are dramatically simplified by extracting “principal components” as plotted in Figure 4a. In essence, the first component characterizes the data in a way to reveal the most influential discrimination in the data that enables distinction of each sample. From this, the key mass fragments and their positive or negative correlations with discrimination of the samples have been extracted. In this way, the key fragment for Met was identified to play a major role in spectral differences between samples as a function of temperature. The increase in the peak intensity with temperature shown in Figure 4b implies a structural or orientational change that imparts an effect on the secondary ion yield of the Met fragment. While the number of Met residues remains the same for each sample, changing energetics of how the G-CSF is associated with the surface results in a change in the peak intensity.

Figure 2. Hydrodynamic diameter of rhG-CSF (3.5 mg mL−1) in 10 mM acetate buffer at pH 4 as a function of temperature. The data indicate monodisperse molecules until aggregation at ∼42 °C substantially lower than the temperature where CD detected loss in αhelical structure.

Figure 3. Positive ion ToF-SIMS spectra of G-CSF after thermal exposure. Example spectra for each temperature point are overlaid with arbitrary color representation, indicating changes in fragmentation yields occur after thermal exposure. Note: multiple fragments can contribute to each peak when shown with unit mass resolution as given here.

temperature point. Various fragments’ yields change after thermal exposure in an increasing or decreasing manner with an increase in temperature. Because of the complex nature and inter-relation of fragment yields of ToF-SIMS spectra of proteins, a large number of peaks (mass fragments) are used and processed with multivariate statistics, specifically in this case, principal component analysis (PCA). This can represent high-dimensional data into just a few key dimensions (or

Figure 4. Analysis of ToF-SIMS spectra of adsorbed G-CSF after exposure to increasing temperature. An holistic analysis of the spectra is represented by the PCA (a) that indicates a trend in fragmentation with temperature. This analysis then identified a fragment containing the methionine functional group to increase in yield with increasing temperature. Error bars represent 95% confidence intervals. 15575

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DISCUSSION ToF-SIMS is extremely surface sensitive (i.e., to the first molecular monolayer) but limited in its mass range (up to ∼1000−2000 Da). While the molecular weight of G-CSF is 18.8 kDa, the fragmentation patterns resulting in ToF-SIMS analysis are highly sensitive to local binding environment; thus, minor changes such as molecular orientation and protein conformation can yield significant variation in fragment yields.22 The ToF-SIMS data here emphasize this sensitivity by confirming changes in G-CSF interfacial interactions in solution after exposure to reasonably low temperature (i.e., 35 °C). In comparison, Skrlin et al. compared physicochemical properties between Hospira and Amgen G-CSF.30 A degradation study monitored samples after 12 weeks storage at 40 °C with a RP-HPLC assay. Over this time the parent molecule concentration decreased, correlating with an increase in impurities of G-CSF derivatives. This correlated with a decrease in biologic activity; however, CD did not detect any changes in secondary structure. In the current study, CD also did not detect any appreciable difference in structure below and just above the temperature at which the DLS indicated aggregation had occurred. Our data have additionally indicated change in CSF adsorption behavior even after only 10 min exposure to 35 °C. Degradation of proteins can occur through a variety of inter- and intramolecular mechanisms including hydrolysis, deamination, oxidation, aspartate isomerization, and aggregation.31 The four methionine residues in G-CSF are prone to oxidation.32 As well as the oxidized form, G-CSF may also form dimers or aggregates which reduce potency.6 Lu et al. studied oxidation and reactivity of G-CSF methionine residues that reside as Met1, Met138, Met127, and Met122.32 Met1, residing at the N-terminal, is an initiator residue resulting from the synthesis approach of expression from Escherichia coli.33 The other three methionine residues play roles in biological activity. Oxidation of any, or all, methionine residues does not grossly effect conformation or cause aggregation but does reduce function.32 Lu et al. also demonstrated Met138 to be exposed in solution and the local environment plays a significant role in G-CSFs function. Their data indicate that functional change in G-CSF can occur while CD and DLS techniques would not detect change in conformation or aggregation, respectively. These observations are consistent with our data; i.e., we have shown a difference in how G-CSF interacts with a solid surface at a temperature below that which CD or DLS indicate any degradation/ alteration in the protein. Consequently, this reinforces the utility of ToF-SIMS in monitoring subtle changes in how proteins interact in their local environment. Met138 is an exposed functional component in solution while Met122 is contained within the hydrophobic core. The correlation of increasing influence of Met residues with other hydrophobic residues could indicate the increasing yield of peaks contributed to by Met is due to exposure of the hydrophobic Met122 region upon heating, adsorption, and drying. However, Met122,127,138 are all reasonably proximal in the protein sequence. The increase in Met yield could also be an indicator of protein orientation, with the Met-rich region distal from the interface. Lu et al. demonstrated low-temperature intolerance after oxidation of the G-CSF Met residues with melting beginning at temperatures as low as 38 °C.32 Our data indicate that even

without oxidation, low-temperature changes occur imparting an effect on how G-CSF interacts with local environments. These data are highly relevant to aspects of biopharmaceutical storage, stabilization, and transport for improving efficacy via conjugation of biomolecules.34,35 For instance, stabilization of G-CSF with poly(ethylene glycol) has significantly reduced incidents of rehospitalization, and associated costs, after chemotherapy.36 What is further required is an appreciation of how subtle changes in biopharmaceuticals, undetected by conventional means, may impart variability in ultimate efficacy. Many techniques are described as validated techniques in characterizing physicochemical properties of G-CSF;37 however, these methods may not fully represent a comprehensive characterization suite to include subtle phenomena. Advanced characterization by ToF-SIMS offers significant potential in detecting subtle differences in how biopharmaceuticals interact with surfaces which relates to stability, storage, and hence shelf life and efficacy. Degradation of proteins is often considered to be indicated by the extent by which a protein aggregates, being an easier parameter to monitor rather than functional assay techniques. However, the stability with regard to protein interactions and the nature of the interaction is demonstrated here to occur at temperatures significantly lower than that which aggregation occurs. This is also supported by the data of Lu et al.32 Extrapolating the observed trend to lower temperatures suggests variation may occur at and/or below 25 °C with respect to interfacial interactions.



CONCLUSIONS Alteration in physicochemical interactions of G-CSF as detected by ToF-SIMS has been shown to occur at temperatures lower than detectable by CD and DLS methods. Exposure to 25 °C for 7 days was previously concluded to impart no effect on G-CSF as determined by tests for aggregation and oxidation; however, from the data presented here exposure to 35 °C for only 10 min altered the nature of GCSFs interaction with a silicon surface. ToF-SIMS has been demonstrated for this example to exhibit exceptional sensitivity to detecting changes in protein interfacial interactions. Short periods of exposure of just a few minutes to temperatures greater than room temperature can alter G-CSFs interactions with interfaces.



AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS Hospira Inc., Adelaide, is acknowledged for supplying the rhGCSF. The authors also thank Dr. John Denman for assistance with the ToF-SIMS acquisition.



REFERENCES

(1) Frokjaer, S.; Otzen, D. E. Protein drug stability: A formulation challenge. Nat. Rev. Drug Discovery 2005, 4 (4), 298−306. (2) Di Lorenzo, G.; D’Aniello, C.; Buonerba, C.; Federico, P.; Rescigno, P.; Puglia, L.; Ferro, M.; Bosso, D.; Cavaliere, C.; Palmieri, G.; Sonpavde, G.; De Placido, S. Peg-filgrastim and cabazitaxel in prostate cancer patients. Anti-Cancer Drugs 2013, 24 (1), 84−89.

15576

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aluminum oxide membranes. Angew. Chem., Int. Ed. 2010, 49 (43), 7933−7937. (19) Tejero, R.; Rossbach, P.; Keller, B.; Anitua, E.; Reviakine, I. Time-of-flight secondary ion mass spectrometry with principal component analysis of titania-blood plasma interfaces. Langmuir 2013, 29 (3), 902−912. (20) Zheng, L.; McQuaw, C. M.; Baker, M. J.; Lockyer, N. P.; Vickerman, J. C.; Ewing, A. G.; Winograd, N. Investigating lipid-lipid and lipid-protein interactions in model membranes by ToF-SIMS. Appl. Surf. Sci. 2008, 255 (4), 1190−1192. (21) Killian, M. S.; Krebs, H. M.; Schmuki, P. Protein denaturation detected by time-of-flight secondary ion mass spectrometry. Langmuir 2011, 27 (12), 7510−7515. (22) Kempson, I. M.; Martin, A. L.; Denman, J. A.; French, P. W.; Prestidge, C. A.; Barnes, T. J. Detecting the presence of denatured human serum albumin in an adsorbed protein monolayer using TOFSIMS. Langmuir 2010, 26 (14), 12075−12080. (23) Graham, D. J.; Castner, D. G. Multivariate analysis of ToF-SIMS data from multicomponent systems: the why, when, and how. Biointerphases 2012, 7 (1−4), 49. (24) Denman, J. A.; Skinner, W. M.; Kirkbride, K. P.; Kempson, I. M. Organic and inorganic discrimination of ballpoint pen inks by ToFSIMS and multivariate statistics. Appl. Surf. Sci. 2010, 256 (7), 2155− 2163. (25) Muramoto, S.; Graham, D. J.; Wagner, M. S.; Lee, T. G.; Moon, D. W.; Castner, D. G. ToF-SIMS analysis of adsorbed proteins: Principal component analysis of the primary ion species effect on the protein fragmentation patterns. J. Phys. Chem. C 2011, 115 (49), 24247−24255. (26) Aoyagi, S. Review of TOF-SIMS bioanalysis using mutual information. Surf. Interface Anal. 2009, 41 (2), 136−142. (27) Kresse, G. B. Biosimilars - Science, status, and strategic perspective. Eur. J. Pharm. Biopharm. 2009, 72 (3), 479−486. (28) Sirghi, L.; Kylián, O.; Gilliland, D.; Ceccone, G.; Rossi, F. Cleaning and hydrophilization of atomic force microscopy silicon probes. J. Phys. Chem. B 2006, 110 (51), 25975−25981. (29) Krishnan, S.; Chi, E. Y.; Webb, J. N.; Chang, B. S.; Shan, D.; Goldenberg, M.; Manning, M. C.; Randolph, T. W.; Carpenter, J. F. Aggregation of granulocyte colony stimulating factor under physiological conditions: Characterization and thermodynamic inhibition. Biochemistry (Moscow) 2002, 41 (20), 6422−6431. (30) Skrlin, A.; Radic, I.; Vuletic, M.; Schwinke, D.; Runac, D.; Kusalic, T.; Paskvan, I.; Krsic, M.; Bratos, M.; Marinc, S. Comparison of the physicochemical properties of a biosimilar filgrastim with those of reference filgrastim. Biologicals 2010, 38 (5), 557−566. (31) Shire, S. J.; Shahrokh, Z.; Liu, J. Challenges in the development of high protein concentration formulations. J. Pharm. Sci. 2004, 93 (6), 1390−1402. (32) Lu, H. S.; Fausset, P. R.; Narhi, L. O.; Horan, T.; Shinagawa, K.; Shimamoto, G.; Boone, T. C. Chemical modification and site-directed mutagenesis of methionine residues in recombinant human granulocyte colony-stimulating factor. Effect on stability and biological activity. Arch. Biochem. Biophys. 1999, 362 (1), 1−11. (33) Young, D. C.; Zhan, H.; Cheng, Q. I. L.; Hou, J.; Matthews, D. J. Characterization of the receptor binding determinants of granulocyte colony stimulating factor. Protein Sci. 1997, 6 (6), 1228−1236. (34) Almenar Cubells, D.; Bosch Roig, C.; Jiménez Orozco, E.; Á lvarez, R.; Cuervo, J. M.; Díaz Fernández, N.; Sánchez Heras, A.; Galán Brotons, A.; Giner Marco, V.; Codes M. De Villena, M. Effectiveness of daily versus non-daily granulocyte colony-stimulating factors in patients with solid tumours undergoing chemotherapy: A multivariate analysis of data from current practice. Eur. J. Cancer Care 2013, 22 (3), 400−412. (35) Naeim, A.; Henk, H. J.; Becker, L.; Chia, V.; Badre, S.; Li, X.; Deeter, R. Pegfilgrastim prophylaxis is associated with a lower risk of hospitalization of cancer patients than filgrastim prophylaxis: A retrospective United States claims analysis of granulocyte colonystimulating factors (G-CSF). BMC Cancer 2013, 13.

(3) Yang, B. B.; Savin, M. A.; Green, M. Prevention of chemotherapyinduced neutropenia with pegfilgrastim: Pharmacokinetics and patient outcomes. Chemotherapy 2013, 58 (5), 387−398. (4) Freyer, G.; Jovenin, N.; Yazbek, G.; Villanueva, C.; Hussain, A.; Berthune, A.; Rotarski, M.; Simon, H.; Boulanger, V.; Hummelsberger, M.; Falandry, C. Granocyte-colony stimulating factor (G-CSF) has significant efficacy as secondary prophylaxis of chemotherapy-induced neutropenia in patients with solid tumors: Results of a prospective study. Anticancer Res. 2013, 33 (1), 301−308. (5) Livingston, R. B.; Ellis, G. K.; Gralow, J. R.; Williams, M. A.; White, R.; McGuirt, C.; Adamkiewicz, B. B.; Long, C. A. Doseintensive vinorelbine with concurrent granulocyte colony-stimulating factor support in paclitaxel-refractory metastatic breast cancer. J. Clin. Oncol. 1997, 15 (4), 1395−1400. (6) Skrlin, A.; Krnic, E. K.; Gosak, D.; Prester, B.; Mrsa, V.; Vuletic, M.; Runac, D. Correlation of liquid chromatographic and biological assay for potency assessment of filgrastim and related impurities. J. Pharm. Biomed. Anal. 2010, 53 (3), 262−268. (7) Kempson, I. M.; Hwu, Y.; Prestidge, C. A., Probing protein association with nano- and micro-scale structures with ToF-SIMS. In Proteins at Interfaces III; Horbett, T. A., Brash, J. L., Norde, W., Eds.; American Chemical Society: Washington, DC, 2012; Vol. 1120, pp 709−729. (8) Awsiuk, K.; Budkowski, A.; Psarouli, A.; Petrou, P.; Bernasik, A.; Kakabakos, S.; Rysz, J.; Raptis, I. Protein adsorption and covalent bonding to silicon nitride surfaces modified with organo-silanes: Comparison using AFM, angle-resolved XPS and multivariate ToFSIMS analysis. Colloids Surf., B 2013, 110, 217−224. (9) Menzies, D. J.; Jasieniak, M.; Griesser, H. J.; Forsythe, J. S.; Johnson, G.; McFarland, G. A.; Muir, B. W. A ToF-SIMS and XPS study of protein adsorption and cell attachment across PEG-like plasma polymer films with lateral compositional gradients. Surf. Sci. 2012, 606 (23−24), 1798−1807. (10) Berman, E. S. F.; Wu, L.; Fortson, S. L.; Kulp, K. S.; Nelson, D. O.; Wu, K. J. Chemometric and statistical analyses of ToF-SIMS spectra of increasingly complex biological samples. Surf. Interface Anal. 2009, 41 (2), 97−104. (11) Lebec, V.; Landoulsi, J.; Boujday, S.; Poleunis, C.; Pradier, C. M.; Delcorte, A. Probing the orientation of β-lactoglobulin on gold surfaces modified by alkyl thiol self-assembled monolayers. J. Phys. Chem. C 2013, 117 (22), 11569−11577. (12) Baugh, L.; Weidner, T.; Baio, J. E.; Nguyen, P. C. T.; Gamble, L. J.; Stayton, P. S.; Castner, D. G. Probing the orientation of surfaceimmobilized protein G B1 using ToF-SIMS, sum frequency generation, and NEXAFS spectroscopy. Langmuir 2010, 26 (21), 16434−16441. (13) Baio, J. E.; Cheng, F.; Ratner, D. M.; Stayton, P. S.; Castner, D. G. Probing orientation of immobilized humanized anti-lysozyme variable fragment by time-of-flight secondary-ion mass spectrometry. J. Biomed. Mater. Res., Part A 2011, 97 A (1), 1−7. (14) Aoyagi, S.; Inoue, M. An orientation analysis method for protein immobilized on quantum dot particles. Appl. Surf. Sci. 2009, 256 (4), 995−997. (15) Wu, L.; Lu, X.; Kulp, K. S.; Knize, M. G.; Berman, E. S. F.; Nelson, E. J.; Felton, J. S.; Wu, K. J. J. Imaging and differentiation of mouse embryo tissues by ToF-SIMS. Int. J. Mass Spectrom. 2007, 260, 137−145. (16) Shon, H. K.; Son, J. G.; Lee, K. B.; Kim, J.; Kim, M. S.; Choi, I. S.; Lee, T. G. Chemical imaging analysis of the micropatterns of proteins and cells using cluster ion beam-based time-of flight secondary ion mass spectrometry and principal component analysis. Bull. Korean Chem. Soc. 2013, 34 (3), 815−819. (17) Kempson, I. M.; Barnes, T. J.; Prestidge, C. A. Use of ToF-SIMS to study adsorption and loading behaviour of methylene blue and papain in a nano-porous silicon layer. J. Am. Soc. Mass. Spectrom. 2010, 21 (2), 254−260. (18) Jani, A. M. M.; Kempson, I. M.; Losic, D.; Voelcker, N. H. Dressing in layers: Layering surface functionalities in nanoporous 15577

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Article

(36) Herbert, K. E.; Gambell, P.; Link, E. K.; Mouminoglu, A.; Wall, D. M.; Harrison, S. J.; Ritchie, D. S.; Seymour, J. F.; Prince, H. M. Pegfilgrastim compared with filgrastim for cytokine-Alone mobilization of autologous haematopoietic stem and progenitor cells. Bone Marrow Transplant. 2013, 48 (3), 351−356. (37) European directorate for the quality of medicines. filgrastim concentrated solution (2206). Eur. Pharmacopoeia 2009, 6 (3), 4142e4.

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