Ange Nzihou

• B R Stanmore

B R Stanmore

The Formation of Aerosols During the Co-combustion of Coal and Biomass

Keywords: Aerosols, Biomass, Coal, Alkali metals, Co-combustion

The Formation of Aerosols During the Co-combustion of Coal and Biomass

Ange Nzihou, B. R.

Introduction

The substitution of biomass for coal is being promoted as a suitable response to the demand to lower mankind's release of carbon into the atmosphere. Many reports on the cocombustion of various forms of biomass comment favourably on the overall emission levels e.g. Baxter, Grammelis, Hartmann, Luts, but most do not take account of the ultrafine particles [START_REF] Baxter | Biomass-coal co-combustion: opportunity for affordable renewable energy[END_REF][START_REF] Grammelis | Effects of biomass cofiring with coal on ash properties[END_REF][START_REF] Hartmann | Electricity generation from solid biomass via co-combustion with coal: energy and emission balances from a German case study[END_REF][START_REF] Luts | Co-incineration of dried sewage sludge in coal-fired power plants: a case study[END_REF]. For example, in their assessment of the particle size distribution of ash, Grammelis report the particle size distribution (psd) of the ash to a minimum 'bin' size of \53 lm [START_REF] Grammelis | Effects of biomass cofiring with coal on ash properties[END_REF]. Hartmann et al. [START_REF] Hartmann | Electricity generation from solid biomass via co-combustion with coal: energy and emission balances from a German case study[END_REF] address greenhouse and acid gas emissions, but ignore particulates. In his review of the topic, Baxter [START_REF] Baxter | Biomass-coal co-combustion: opportunity for affordable renewable energy[END_REF] focusses on the properties of fly ash with regard to fouling, corrosion and use as a cement extender, but ignores its environmental effects.

The co-combustion of biomass with coal must take account of the different physical properties of the two fuels. The low density and lower specific energy of biomass restricts the extent of its application, as its overall energy density is approximately one tenth of that of coal. Consequently, co-firing biomass at a 10 % heat input rate results in volumetric coal and biomass flow rates of comparable magnitude [START_REF] Baxter | Biomass-coal co-combustion: opportunity for affordable renewable energy[END_REF]. Even though it may contribute only a fraction of the energy and mass input to a coal-fired boiler, it has the potential to significantly alter the physicochemical environment in the flame.

Airborne Aerosols

A sample of urban air may contain 10-100 9 10 3 particles per cm 3 , which on a mass basis may be 20-100 lg m -3 . The number concentration is dominated by fine particles, and the mass loading by large particles. It is the very small particles which are of most interest for air quality, because of their effects on human health. The term ''nanoparticles'' is applied to those\50 nm in diameter [START_REF] Hinds | Aerosol Technology: Properties, Behaviour and Measurement of Airborne Particles[END_REF]. The ''ultrafine'' category is generally defined as being smaller than 0.1 lm (100 nm), or PM 0.1 , while the legislation is specified in terms of PM 10 and PM 2.5 . It is the latter size category which is known to damage respiratory systems.

Airborne aerosols are comprised of material from a number of different sources, which is reflected in their composition. The primary particles may be originally siliceous or carbonaceous, or salt from an evaporated droplet of seawater, or be of biological origin. Subsequent secondary growth by adsorbed water, salts and organics produces a complex mixture of these components. Because of their small size, they can remain suspended in the atmosphere for long periods.

Urban aerosols begin life in the nucleation mode (1-2 nm), formed predominantly in combustion processes. Both gasoline and diesel motors are responsible for a large part of the nucleation mode particles in the built environment, with coal combustion also contributing [START_REF] Harrison | Observations of new particle production in the atmosphere of a moderately polluted site in eastern England[END_REF]. The particles then grow by coagulation when moisture, organic molecules and gases are adsorbed onto their surface to form ammonium, sulphate and nitrate radicals (accumulation mode). The large sizes (''coarse fraction'' [1 lm) settle quickly and are not present in high concentrations. Polyaromatic hydrocarbons (PAH) and their oxy and nitro derivatives are present on the surface.

The growth of fine particles by 'accumulation' is well illustrated by a program to follow the changes in a parcel of air originating above the Pacific Ocean off the coast of California [START_REF] Hughes | Evolution of atmospheric particles along trajectories crossing the Los Angeles basin[END_REF]. The particles were sampled at four sites, the first originally at sea i.e. with a salt nucleus, and then as the air passed over urban Los Angeles. The particles grew in size by the accretion of predominantly nitrogenous (ammonium, nitrate) and organic species adsorbed from the urban pollution background, Fig. 1. The quantity and composition of the particles measured at Long Beach on the coast, shown in Fig. 1 can be contrasted with those inland at Riverside, where the parcel of air arrived approximately 8 h later.

Since the combustion of both coal and biomass represents potential sources of nuclei for the formation of aerosols, attention has been focussed on their performance. The literature is well stocked with reports on the quantity of aerosol release in both cases, while the respective formation mechanisms have been extensively investigated. However, the adoption of co-combustion for these two fuels generates a different flame environment which has not been adequately examined. This paper explores the likely effects of co-combustion on ultrafine or fume emissions.

Aerosol Formation During Coal Combustion

The source of fine particles during coal combustion is the included minerals, such as pyrites and clays, and some organically-bound elements such as sulphur and metals. The minerals are oxidised and melt during combustion to form ash, sometimes losing water of hydration and decomposing e.g. carbonates. Commercial coals generally produce an ash yield of between 10 and 25 % of the dry fuel mass. Organically associated trace elements exposed to very high temperatures and a highly reducing environment will be released from the coal matrix when the associated bonds break. The morphology of submicron particles from pulverised coal (pc) combustion, like those in the micron range, is overwhelmingly spherical [START_REF] Yao | Studies on formation and control of combustion particulate matter in China: a review[END_REF].

Most of the mass of the mineral matter in coal is originally finely distributed in pulverized coal particles either as micron-size inclusions or organically bound to the coal. During combustion the mineral inclusions in coal fuse, adhere to the receding carbonaceous surface, and coalesce to form particles in the size range of 1-30 lm with typical mass mean diameters in the range of 5-10 lm [START_REF] Haynes | Factors governing the surface enrichment of fly ash in volatile trace species[END_REF]. The process of fume production described by Seames [START_REF] Seames | An initial study of the fine fragmentation fly ash particle mode generated during pulverized coal combustion[END_REF] is depicted in Fig. 2.

Coal combustion for power generation is generally carried out in pulverised fuel boilers at maximum temperatures around 1500-1600 °C, although fluidised beds, which operate at 900 °C are becoming more popular. An early report on ash behaviour during pc combustion was presented by Pueschel [START_REF] Pueschel | Aerosol formation during coal combustion: condensation of sulphates and chlorides on flyash[END_REF], who tracked particles in the downwind plume of a coal-fired power plant for 32 km. Micrographic analysis of the fine particles 0.05 \ d p \ 1.0 lm showed that at all locations silicon and aluminium were distributed evenly throughout their interior, while Fig. 1 Composition of total suspended particulates [START_REF] Hughes | Evolution of atmospheric particles along trajectories crossing the Los Angeles basin[END_REF] sulphur and chlorine were concentrated in an outer shell. This illustrated previous conclusions that sulphate and chloride species had condensed from the vapour phase onto larger ash particles while the fluegas/particles were still passing through the furnace flow system.

Volatile oxides or metals expelled at high temperatures to the gas phase can become supersaturated, and thus homogeneously nucleate to form a very large number of nano-particles (probably much smaller than 0.01 lm). For coal, aerosols in the 0.02-0.1 lm size range form initially by nucleation of Si, Ca and Al vapour species, with subsequent accumulation of other volatiles, such as sulphate and chloride, and to a lesser extent As, Sb and Zn. This results in a bimodal, or sometimes trimodal, psd in the product coal ash.

A recent review of the process by Xu et al. [START_REF] Xu | Coal combustiongenerated aerosols: formation and properties[END_REF] notes that vaporisation is favoured by high particle temperatures and a reducing environment. With coals, especially lignite, metals are present as salts at surface carboxylic and phenolic groups. The same applies to biomass, see [START_REF] Miller | The occurrence of inorganic elements in various biofuels and its effect on ash chemistry and behavior and use in combustion products[END_REF]. Metals contained in clays typically do not vaporise [START_REF] Zeng | Vaporisation of arsenic, selenium and antimony during coal combustion[END_REF]. When metal oxides in the gas phase nucleate to form small particles, these can grow by accumulation or coagulation mechanisms. In the former, further condensation takes place, while the latter represents collisions in which the particles coalesce. Xu et al. [START_REF] Xu | Coal combustiongenerated aerosols: formation and properties[END_REF] also identifies surface ash shedding, bubble bursting and the carryover of fine fuel particles as other nuclei sources for the accumulation of vapour species.

The presence of refractory oxides like silicon, calcium and magnesium in fume has been explained by Christensen et al. [START_REF] Christensen | The formation of submicron aerosol particles, HCl and SO 2 in straw-fired boilers[END_REF] as taking place in temperature ranging from 800 to 1000 °C in reducing atmosphere within the particle. The normal ash species SiO 2 , MgO and CaO can be reduced to the more volatile SiO, Mg and Ca. When these vapours leave the particle, they are re-oxidised to the standard oxides, some of which then homogeneously nucleate. The oxidation of SiO is slow, so that other oxides probably form first. More volatile species such as potassium, sulphate and chloride can later condense onto the surface of the nuclei as the gas cools. It was found in a pilot scale furnace [START_REF] Fix | The effect of combustion temperature on coal ash fine-fragmentation mode formation mechanisms[END_REF] that increasing the combustion temperature for a bituminous coal led to a decrease in the amount of micron size particles, but an increase in the ultrafine submicron range. This is consistent with increased volatilisation followed by condensation, see Fig. 2.

The amount of submicron ash (\0.54 lm) from a range of coals when burned at 1750 K in gas containing 0.2 atm of oxygen was found to lie between 0.54 and 9.6 % of the ASTM ash [START_REF] Quann | A laboratory study of the effect of coal selection on the amount and composition of combustion-generated submicron particles[END_REF]. This was equivalent to 7.2-65 mg of fume per kg of coal burned. Coals of lower rank produced more aerosol. A similar study by Buhre et al. [START_REF] Buhre | Submicron ash formation from coal combustion[END_REF] on five bituminous coals in a drop tube furnace found similar results, with fume accounting for between 0.12 and 1.6 % of the total ash formed. The coals with higher sulphur content tended to produce more aerosols, as illustrated in Fig. 3. Although Coal 240 contained more sulphur than Coal 272, it formed less aerosol, probably because it contained considerably less of the complementary alkali metals.

Modelling of fly ash-forming processes has been widely undertaken. For the aerosol component, a routine developed by Jokiniemi et al. [START_REF] Jokiniemi | Numerical simulation of vapour-aerosol dynamics in combustion processes[END_REF] develops mathematical Fig. 2 Mechanism for fume production during coal combustion [START_REF] Seames | An initial study of the fine fragmentation fly ash particle mode generated during pulverized coal combustion[END_REF] descriptions of nucleation, condensation, coagulation, chemical reactions and deposition. They found that the ash particle size distribution is formed early in the combustion sequence, and is virtually unchanged over the final section of the ducts leading to the ESP. The model indicates that it is important to know the volatilisation of sodium, potassium, chlorine and sulphur, and also the rate of formation of alkali sulphates in the gas phase. The initial ash size distribution determines the distribution of condensed alkalis between the sub-and super-micron particle modes, because much smaller saturation ratios are necessary for heterogeneous nucleation to take place than in the case of homogeneous nucleation. For homogeneous nucleation to occur, super-saturation of the vapour is necessary. However, if there is an existing nucleus, the required free energy is less and condensation readily takes place.

In summary, the literature identifies sulphur as the major ash component which drives the formation of aerosols. The alkali and alkaline earth elements provide the cationic balance for the sulphate products. Pyrites is the most common form of inorganic sulphur in coals, and is also the location of the bulk of arsenic, selenium and antimony. Silicates do not appear to contribute in a significant way, but phosphorus has been identified as active in some coals.

Fluidised bed combustion is operated at lower temperatures than pc furnaces, so that the extent of mineral devolatilisation tends to be lower. For example no significant vaporization was observed for Ca, Al, Fe, Mg, and Si in CFBC [START_REF] Xu | Coal combustiongenerated aerosols: formation and properties[END_REF]. The morphology of the ash particles is not spherical, but irregular, because complete fusion has not taken place [START_REF] Yao | Studies on formation and control of combustion particulate matter in China: a review[END_REF]. As a result the concentration of aerosols tends to be lower during this form of co-combustion.

Aerosol Formation During Biomass Combustion

Many investigations into aerosol formation during biomass combustion have been produced in Scandanavian countries, where the practice is widespread. Although biomass has a lower ash content than coal, typically 0.5-1 % for wood and 4-8 % for agricultural by-products, it is rich in volatile elements like potassium and chlorine. For example, in a range of straws the potassium content was as high as 2 % of the dry weight [START_REF] Christensen | The formation of submicron aerosol particles, HCl and SO 2 in straw-fired boilers[END_REF]. Their sulphur content was constant at 0.1 %, while the chlorine was approximately proportional to the potassium content at 40 % of its weight.

The extent of release into the vapour phase of K, Cl and S was measured for wood chips and corn stover (the leaf, husk and cob remaining after harvest) by Johansen et al. [START_REF] Johansen | Release of K, Cl and S during combustion and co-combustion with wood and highchlorine biomass in bench and pilot scale fuel beds[END_REF]. The effect of temperature for potassium is shown in Fig. 4; it rises from 40 % at 900 °C to 80 % at 1300 °C. Chlorine is even more volatile, as is sulphur. They found that the scale of operations i.e. lab, bench and pilot scale modifies their behaviour, and secondary reactions influence the amount and speciation of chlorine.

When potassium is in excess in relation to chlorine in a biomass feed, it is largely retained in the residual i.e. bottom ash under grate combustion conditions [START_REF] Fix | The effect of combustion temperature on coal ash fine-fragmentation mode formation mechanisms[END_REF]. With mixtures of wood chips and high Cl corn stover, the release of K per gram of fuel remained almost constant, regardless of the available amount of Cl. This occurred even though the maximum temperature varied by about 200 °C. Most of the Cl present was released as HCl rather than alkali chlorides.

Nussbaumer [START_REF] Nussbaumer | Combustion and co-combustion of biomass: fundamentals, technologies, and primary measures for emission reduction[END_REF] notes that biomass combustion leads to relatively high emissions of particulates i.e. well above 50 mg Nm -3 at 11 % O 2 . The majority of these are smaller than 10 lm (PM 10 ), with a high share of submicron particles (PM 1 ). The main constituents of biomass aerosols are K, Cl, S, Ca, Na, Si, P, Fe and Al. For a number of biomass types viz wood, demolition wood and coffee wastes, Fig. 4 Fractional release with temperature of potassium from corn stover [START_REF] Johansen | Release of K, Cl and S during combustion and co-combustion with wood and highchlorine biomass in bench and pilot scale fuel beds[END_REF] Becidan et al. [START_REF] Becidan | Ash related behaviour in staged and non-staged combustion of biomass fuels and fuel mixtures[END_REF] report that K, S, Cl and Na dominated the aerosol composition. It was shown by Gao and Wu [START_REF] Gao | Combustion of volatiles produced in situ from fast pyrolysis of woody biomass: direct evidence on its substantial contribution to submicrometer particle (PM1) emission[END_REF] that the ultrafines are produced mostly from the volatiles, and not during char combustion.

It is now common to regard biomass ash as composed of 'reactive' and 'non-reactive' components [START_REF] Hupa | Interaction of fuels in co-firing in FBC[END_REF]. The non-reactive constituents e.g. the silica-based mineral matter, is assumed to remain unchanged during FBC and is regarded as virtually inert. As noted below, this may not always be the case with pulverised fuel firing. The reactive fraction, typically alkali compounds, will be easily converted into new compounds during combustion, particularly by sulphur and chlorine. The formation of aerosols will be a function of these reactive components. Any reaction between alkali species and the silicate phase will predominantly take place in the condensed phase, as no significant increase in the retention of potassium in the ash has been observed with increasing residence time [START_REF] Nussbaumer | Combustion and co-combustion of biomass: fundamentals, technologies, and primary measures for emission reduction[END_REF].

An illuminating study on aerosols in a grate-type MSW furnace is reported by Brunner et al. [START_REF] Brunner | Investigations of aerosol formation pathways during MSW combustion based on high-temperature impactor measurements[END_REF]. Using a high temperature impactor, they sampled aerosols (0.09-1.26 lm) in the gas stream at four locations: the afterburner shaft (978 °C average), the downshaft (674 °C), the convective superheater duct (541 °C) and the economiser duct (363 °C). The quantities involved increased by a factor of SIX over the test path. At the high temperature point, the ultrafines were predominantly silicon and calcium, whereas once the gas had cooled to 670 °C, Na, K, S and Cl also appear in roughly equal and significant amounts. For the last two positions, the compositions are dominated by Na, K and Cl, with traces of other semi-volatile metals.

This result appears to confirm the proposition that two mechanisms can generate silicon, aluminium and calciumrich nuclei from fuel ash. Firstly, thermal stresses can fracture excluded minerals to generate submicron fragments. Secondly, at a high temperature and in a reducing atmosphere, such as that in a char particle, oxides of Si, Al, Mg and Ca embedded in the particles may be reduced to easily volatile suboxides such as SiO or Al 2 O, which can be released into the gas phase. These are subsequently reoxidised and being readily supersaturated, nucleate homogeneously.

A similar time-resolved study of wood and bark combustion [START_REF] Wiinikka | High temperature aerosol formation in wood pellets flames: spatially resolved measurements[END_REF][START_REF] Wiinikka | Influence of fuel ash composition on high temperature aerosol formation in fixed bed combustion of woody biomass pellets[END_REF] undertook sampling of the particles in the flue gas at nine positions in a vertical furnace, ranging in temperature from 1450 to 400 °C. Two size modes were present, a coarse mode located between 0.219 and 3.253 lm with a mode diameter of 0.523 lm, and a fine mode located between 0.033 and 0.219 lm with a mode diameter of 0.085 lm. Surprisingly, the mass contained in the fine mode was larger than that of the coarse mode.

Particles from incomplete combustion (soot and/or other hydrocarbon particles) dominated the fine mode composition early in the fuel-rich flame, but were rapidly oxidized in the hot oxygen-rich flow downstream. At the third sample point (975 °C) there were approximately 18 % of fines present as K, 12 % as S, 11 % as Na and 2 % as Zn. Thereafter the fine mode was dominated by the inorganic elements K, Na, S, Cl, O and Zn. The coarse mode particles were generally similar, but contained significant amounts of Ca and Mg, as well as lesser amounts of Si, Mn and Fe. A high alkali to silicon ratio (K ? Na)/Si increased the amount of vaporised aerosol.

The submicron ash fraction generated from orujillo (a waste from olive production) in a laboratory drop tube furnace over a range of temperatures amounted to about 3 % of the total ash [START_REF] Jime ´nez | Effect of co-firing on the properties of submicron aerosols from biomass combustion[END_REF][START_REF] Jime ´nez | Vaporisation of trace elements and their emission with submicrometer aerosols in biomass combustion[END_REF]. Many elements in the biomass were recovered in the PM 1 , with As, Pb, Cr, Cu, Ni, Zn, Co, Mn and Fe represented (in decreasing amounts as listed). Elements prior to cobalt in this list were enriched over the feed ash, indicating some vaporisation and subsequent condensation. They conclude that the amount of fine particles generated is essentially fixed by the amount of volatilised alkali, and the ratio [sulphate]/[chloride] has a minor effect on the total mass emitted. Pb was found to condense in the temperature range 900-560 °C, whereas Cu remained partly as vapour at 560 °C.

Glarborg and Marshall [START_REF] Glarborg | Mechanism and modelling of the formation of gaseous alkali sulphates[END_REF], and Hidiyarti et al. [START_REF] Hidiyarti | An exploratory study of alkali sulphate aerosol formation during biomass combustion[END_REF] identified the importance of alkali sulphation to aerosol formation, and developed models for the gaseous sulfation of alkali hydroxide and alkali chloride which rely on detailed chemical kinetic models for the high-temperature gas-phase interaction between alkali metals, the O/H radical pool, and chlorine/sulphur species. The role of sulphur oxidation as the limiting step in the formation of alkali sulphate was later confirmed [START_REF] Jime ´nez | Formation of alkali sulphate aerosols in biomass combustion[END_REF], and the importance of the gas cooling rate on determining the amount of sulphate formed and, as a consequence on the properties of fine particles.

A simple plug flow model of aerosol formation in biomass boilers was developed by Jo ¨ller et al. [START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion in grate furnaces and comparison with measurements[END_REF][START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion for various furnaces and boiler types[END_REF], and verified in various furnaces of different size and type. An aerosol population balance was utilised, together with a gas phase mass balance. The appropriate growth mechanisms such as coagulation, nucleation and condensation were combined with various mass transport mechanisms. The routine was applied to clean wood chips, waste wood and coal-wood co-combustion. For clean wood chips, the aerosols are predominantly K 2 SO 4 and other alkali compounds (Na 2 SO 4 , KCl, NaCl). Thermodynamic considerations show that alkali sulphates are less volatile than the chlorides, beginning to condense around 950 °C. Alkali chlorides condense on these nuclei at a lower temperature, *600 °C. The use of higher air rates in a fixed bed furnace burning wood and bark produced increased quantities of aerosols, which was explained by increased combustion temperatures [START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion for various furnaces and boiler types[END_REF].

When waste wood was combusted by Jo ¨ller et al. [START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion in grate furnaces and comparison with measurements[END_REF], the feed was contaminated with metals which strongly influenced the result. The zinc present formed ZnO at high temperatures and began to condense around 1000 °C, thus providing sufficient nuclei for heterogeneous condensation of the alkali sulphates. Hence there were far more ultrafine particles present. With an abundance of sulphur in the fluegases, any KCl present, which had condensed earlier in such systems can be converted to K 2 SO 4 [START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion in grate furnaces and comparison with measurements[END_REF]. The cocombustion result will be discussed below.

Aerosol Formation During Co-combustion

The few papers found on the subject of coal-biomass aerosols will be reviewed. A relevant study of the topic was carried out by burning orujillo at 1300 °C in a laboratory furnace to identify the aerosol products [START_REF] Wiinikka | Influence of fuel ash composition on high temperature aerosol formation in fixed bed combustion of woody biomass pellets[END_REF]. With orujillo alone, these were found to be K 2 SO 4 and KCl in approximately equal amounts. In a second test the biomass was burned in a mixture of 2:1 mass ratio of 2.25 % sulphur coal. Under these conditions the mixture produced more submicron particles, and of a smaller size, than when orujillo was burned alone. With high sulphur present, as with the coal, coke or SO 2 addition, sulphate displaced chloride from the aerosol. A repeat run with orujillo-coal at 1450 °C also produced more material in the submicron range, as reproduced in Fig. 5.

The participation of sulphur in the process was confirmed by adding gaseous SO 2 at 180 ppm to the furnace burning only orujillo, where a similar, but even more pronounced effect, was found. When a 6.34 % sulphur petcoke was burned with the orujillo, the ultrafine component was increased but the micron range material was greatly reduced. This may have been because the coke contained only 2 % ash, compared to 21 % for the coal, which is consistent with preferred heterogeneous condensation in the model of Jokiniemi et al. [START_REF] Jokiniemi | Numerical simulation of vapour-aerosol dynamics in combustion processes[END_REF] mentioned above.

The work of Seames [START_REF] Seames | An initial study of the fine fragmentation fly ash particle mode generated during pulverized coal combustion[END_REF] in an entrainment flow furnace showed that the co-combustion of sewage sludge (SS) increased the emission of aerosols. The furnace was operated at a burner temperature of 1500 °C with bituminous coal, SS and a 50:50 mixture of the two on a heat-release basis. The supermicron size material distributions were similar for each of the three feeds, whereas the major difference in the psd was in the submicron range. The mixed feed displayed an enhanced concentration band below 0.3 lm, as depicted in Fig. 6.

In contrast, a laboratory study of sewage sludge burned with pulverised coal at 1200 °C in a laboratory furnace found that the amount of PM 10 actually decreased when compared to straight coal [START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion for various furnaces and boiler types[END_REF]. When burned independently, sewage sludge produced over ten times more \1 lm material than did the low ash, 0.5 % SO 3 content coal. However, even when burned in a 1:19 SS:coal mass ratio, the formation of PM 10 was suppressed to near the coal value. The authors propose that fine Si-Al minerals from the coal preferentially adsorb the P and S emitted from the sewage sludge, thus eliminating homogeneous nucleation/condensation.

Apparently the high phosphorus content of the SS played a significant role in the ash formation process. Phosphorus content in the sewage sludge is particularly high indeed in regards to the literature where the phosphorus content ranging from 1 to 6 % are reported [START_REF] Eckenfelder | Municipal sewage sludge management: a reference on processing[END_REF]. This is explained by the origin of the SS. The SS contained 15 % ash (db), of which 27 % was in the form of Fig. 5 Particle size distribution (mass basis) of ash formed during the co-combustion of coal and orujillo [START_REF] Jime ´nez | Effect of co-firing on the properties of submicron aerosols from biomass combustion[END_REF] Fig. 6 PSD of particles formed in a pilot furnace when burning SS only (natural gas assisted), coal only and a coal-SS blend [START_REF] Wiinikka | The influence of air distribution rate on particle emissions in fixed bed combustion of biomass[END_REF] phosphorus, and 12 % as sulphur. The concentration of potassium in the SS and coal ash was [1 %, and that of sodium about 1 % each. In the 1:9 mass ratio SS:coal test, the combined mass of alkalis was approximately equal to the combined mass of the P and S concentrations. Phosphorus was the dominant element in the PM 1 product, but was barely represented in the 1 \ d p \ 10 lm and[10 lm samples. It appears that in this case alkali phosphates have preferentially condensed onto existing coal ash particles. Phosphorus is a known fluxing agent [START_REF] Lindstro ¨m | Slagging characteristics during combustion of cereal grains rich in phosphorus[END_REF], and will lower the melting point of ash particles into which it becomes incorporated. Wang et al. [START_REF] Wang | Mineral interactions and their impact on the reduction of PM 10 emissions during co-combustion of coal with sewage sludge[END_REF] propose that larger melted Ca-Fe-Al-Si or Ca-Fe-P-Al-Si particles possess a large viscous surface which captures volatile elements like P and S, and toxic elements such as Zn.

It was found by Seames [10] that the addition of SS to coal can change the speciation of the arsenic and selenium compounds emitted. The As and Se were scavenged by calcium to form calcium arsenates and selenates, which are likely to be more benign than the oxides. Thus there is a trade-off, environmentally, for SS co-combustion with coal. More fine particles are generated, but these particles may decrease the emission of these semi-volatile trace elements as vapour and/or ultrafine particulate matter.

The tests by Jo ¨ller et al. [START_REF] Jo ¨ller | Modeling of aerosol formation during biomass combustion for various furnaces and boiler types[END_REF] in a 404 MWth pulverised fuel furnace indicate that particle formation over the submicron range during co-combustion of coal and sawdust is greater than for coal alone. The fines collected from the last four impactor stages ranged in median size from 0.09 to 0.70 lm. They contained between 30 and 50 % Si, with calcium and sulphur also well-represented. The concentrations of the latter two elements were higher in the fines, while Si showed the opposite trend. The mechanics are strongly influenced by the psd and the chemical composition of the submicron particles resulting from the defragmentation of coal ash (consisting mainly of Al, Ca, Fe, K and Si). The bulk of the nuclei appear to come from this source.

In the accompanying modelling exercise (see above), the Jo ¨ller routine gives a satisfactory simulation of the amount and psd of the ash formed, including the aerosols. This predicts that the formation processes are essentially complete by the time the gas reaches the stack. The release of Ca to the gas phase at high temperatures and the subsequent nucleation of CaO particles during cooling of the flue gas could be reproduced. Further growth may well take place after the ultrafines enter the atmosphere, as indicated by the experience of urban aerosol accumulation in Los Angles described in the introduction.

In considering the likely effects of co-firing, a complex physico-chemical system involving gas, liquid and solid phases results, with a large temperature range traversed under both localised reducing and oxidising conditions.

The type of combustion system will influence the reaction sequence and conditions. Also, it is apparent that the results are strongly influenced by the composition of the mineral components present in the ash. For example, the presence of high amounts of phosphorus in a sewage sludge modified the usual behaviour of the system.

When biomass is co-combusted with coal, the coal provides a high ash burden which can provide surface on which vapours can later condense. Excluded mineral matter can fragment to generate fine particles, and reducing conditions inside char particles at the high temperatures found early in the flame will add to the stock of nuclei. The latter will be based on Ca, Mg, Si and Al species. The smaller the particle, the larger the available surface area to mass, so that fines provide the bulk of the available area for heterogeneous deposition. The volatile species present, such as alkalis, sulphur and chlorine, and semi-volatile transition metals will remain in the gas phase at this stage.

As the gas stream cools, the semi-volatiles will begin to condense sequentially, followed by the more volatile species. This means that biomass, which is rich in aerosolforming species, will provide material for heterogeneous condensation onto the particle surface. When biomass is combusted alone, the overwhelming number of aerosol particles result from homogeneously-formed nuclei. During co-combustion, the minerals in the coal provide nuclei earlier in the combustion process, and are generally present in the order of 15-20 % of the coal mass.

The presence of the biomass with the coal means that extra volatiles are present to undergo homogeneous condensation. Hence there is the potential to form more particles i.e. PM 2.5 than when burning coal alone, and in some cases, than when burning the biomass alone. The latter is true in the case of Jime ´nez et al., Seames et al. and Wiinikka and Gebart [29,[START_REF] Glarborg | Mechanism and modelling of the formation of gaseous alkali sulphates[END_REF][START_REF] Wiinikka | The influence of air distribution rate on particle emissions in fixed bed combustion of biomass[END_REF]. The process will be exacerbated by the relatively high sulphur content of most coals. This is illustrated by the extent of K volatilisation being controlled by the K/Cl ratio as noted by Johansen [START_REF] Johansen | Release of K, Cl and S during combustion and co-combustion with wood and highchlorine biomass in bench and pilot scale fuel beds[END_REF], and the identification of a (K ? S)/Si ratio by Wiinikka et al. [START_REF] Wiinikka | Influence of fuel ash composition on high temperature aerosol formation in fixed bed combustion of woody biomass pellets[END_REF].

When biomass added to coal in BFB, the concentration of ultrafines increased out of proportion to the extremely low ash content of the added biomass [START_REF] Lopes | Particulate and PCDD/F emissions from coal co-firing with solid biofuels in a bubbling fluidised bed reactor[END_REF]. They conclude that ''the most important factor that could lead to the formation of very fine particles could be related with the presence of aerosol forming elements such as K, Na and Cl in biofuels, which even resulted in higher PM emissions when the ash content of fuels decreased, as was the case of straw and olive bagasse''.

Co-combustion of coal with biomass seemed to have the effect of moving the fly-ash in PM 2.1 to a larger size range, but increasing the number counts of the ultra-fine particles [START_REF] Chao | Co-firing coal with rice husk and bamboo and the impact on particulate matters and associated polycyclic aromatic hydrocarbon emissions[END_REF]. The count of the ultra-fine particles at baseline (coal combustion) was about 7 9 10 5 particles per cm 3 . The counts were increased to 3 9 10 7 and 8 9 10 7 particles per cm 3 for rice husk and bamboo respectively at 20 % biomass addition. For pure rice husk and bamboo combustion, the ultra-fine particle counts were further increased to 3 9 10 8 and 5 9 10 8 particles per cm 3 . The increase for biomass co-combustion is not linear, but increases sharply with only a modest addition.

A recent publication illustrates the complexity of the reaction environment. When a coal fly ash was blown into an entrainment-fired 800 MWth furnace burning wood pellets at coal ash-to-wood ash mass ratios of 2-4 [START_REF] Damoe | Impact of coal fly ash addition on combustion aerosols (PM 2.5 ) from full-scale suspension-firing of pulverised wood[END_REF], the quantity of submicron particles was greatly reduced. A spike in the psd plot around 0.2 lm was almost eliminated. The authors suggest that the potassium emerged as amorphous potassium silicate and aluminosilicate phases. This is consistent with the mechanisms outlined above, where ash formed from the coal particles appears to be responsible for the greater portion of the nuclei formed. When the coal ash is in far lower concentration, and is of alreadyfused morphology at low supermicron particle size, it will presumably not fragment and act to form the basis for K, Cl and S condensation.

It can be concluded that in general co-combustion operations based on coal as the primary fuel will produce an increase in sub-micron particles. The sulphates and HCl will interact to determine the final composition of the PM 2.5 . Other semi-volatile elements such as Zn, As, Sb and Se will also be incorporated into the ultrafines by vaporisation/condensation.

The Effect of Gas Cleaning on Aerosol Removal

Most of the data presented above has been collected from pilot-scale equipment, whereas any potential health effects from particulate matter will be the result of emissions from a commercial boiler installation. Since the furnace gases from a large plant will have been subjected to a gas cleaning operation, its effect on the concentration of aerosol particles needs to be considered. Either an electrostatic precipitator (ESP) or fabric filter (FF) would be employed. A search of the literature for ESP efficiencies shows that in all cases the least efficient collection is when the particle size is in the 0.1-1 lm range [START_REF] Strand | Fly ash penetration through electrostatic precipitator and flue gas condenser in a 6 MW biomass fired boiler[END_REF][START_REF] Lind | Electrostatic precipitator collection efficiency and trace element emissions from co-combustion of biomass and recovered fuel in fluidized-bed combustion[END_REF][START_REF] Senior | Emissions of mercury, trace elements, and fine particles from stationary combustion sources[END_REF][START_REF] Lind | Compositional penetration of pulverised coal combustion fly ash through the electrostatic precipitator[END_REF][START_REF] Sippula | Particle emissions from small wood-fired district heating units[END_REF], which is the very range identified here. For fabric filters, the result is similar, as the 0.1-1 range falls between two effects which determine FF performance i.e. diffusion and impaction/ interception [START_REF] Whitby | Mechanics of air cleaning[END_REF]. These particles are too large for collection by diffusion and too small for collection by impaction or interception. For a range of filter types, there is a maximum penetration of the filter at sizes around 0.5 lm.

As a result of the information presented here, one can conclude that the operation of the gas cleaning systems of large co-combustion boilers will probably function least effectively with the offending particles. Although gas cleaning will reduce the particulate emissions by over 99 %, the ultrafines in them will actually become more concentrated.

The Health Effects of Airborne Ultrafine Particles

There is conclusive evidence that PM 2.5 is associated with a range of health outcomes, including respiratory and cardiovascular diseases e.g. Kan et al. [START_REF] Kan | Differentiating the effects of fine and coarse particles on daily mortality in Shanghai, China[END_REF]. Their study examined the situation in Shanghai, an extremely polluted city, and found a close association between PM 2.5 concentration and daily mortality rates. In contrast, they do not report a significant effect of PM 10-2.5 on these rates. Even biologically-inert particles in micron sizes such as titanium dioxide and quartz have been shown to be active in submicron sizes [START_REF] D'alessio | Combustion generated nanoparticles[END_REF]. It is therefore important to focus on the ultrafines in assessing any risk.

A review of the situation by Valavenidis et al. [START_REF] Valavenidis | Airborne particulate matter and human health: toxicological assessment and importance of size and composition of particles for oxidative damage and carcinogenic mechanisms[END_REF] found that the smaller the size of the PM, the higher the toxicity through mechanisms of oxidative stress and inflammation. Extractable organic compounds e.g. on diesel exhaust particulates, contribute to various mechanisms. The water-soluble fraction (mainly transition metals with redox potential) plays an important role in the initiation of oxidative DNA damage and membrane lipid peroxidation [START_REF] Saffari | Global perspective on the oxidative potential of airborne particulate matter: a synthesis of research findings[END_REF]. Any associations between chemical composition and particle toxicity tend to be stronger for the fine and ultrafine size fractions. Children are especially susceptible to induced respiratory problems [START_REF] Salvi | Health effects of ambient air on children[END_REF].

When considering particles generated from biomass, it should be noted that some cautions have already been raised. The burning of biomass in a domestic appliance, even under excellent combustion conditions, gives reason to suspect that the PM 1 may be harmful [START_REF] Leskinen | Fine particle emissions in three different combustion conditions of a wood chip-fired appliance-particulate physico-chemical properties and induced cell death[END_REF]. This is because they consist mostly of ultrafine aerosols in which inorganics such as zinc are represented. Poor combustion conditions result in fewer, larger particles, but these are still respirable and likely to have adsorbed PAH on the surface.

Fernandez et al. [START_REF] Fernandez | Resuspension of coal and coal/municipal sewage sludge combustion generated fine particles for inhalation health effects studies[END_REF] found that pulmonary stress was created in mice exposed to coal/sewage sludge ash of nominal 3.5 lm size, but not to coal ash alone. This was attributed to the action of transition metals, particularly zinc, which was significantly more concentrated in the SS/coal ash than in the coal ash particles themselves. The natural pH of the two sets of particles was similar. The effect is illustrated in Fig. 7, where mice were subjected to aerosol ingestion. Coal aerosols produced only a slight effect on lung performance, whereas the material from cocombustion produced aggravated deterioration. Further work confirmed that it was the zinc from the SS, in conjunction with sulphur from the coal, which was responsible for the observed damage [START_REF] Fernandez | Health effects engineering of coal and biomass combustion particulates: influence of zinc, sulfur and process changes on potential lung injury from inhaled ash[END_REF].

Since aerosols from co-combustion are likely to be readily soluble due to high concentrations of alkali metals, sulphates and chlorides, their potential for lung damage must be significant. In addition, the adsorption onto their surface of trace metals with redox activity has been noted, which indicates that this will contribute to detrimental health effects.

The regulation of particulate emissions is practised worldwide in order to lessen the impact of these effects. The EU emission limits for PM from large combustion plants burning solid fuel as recommended by the European parliament are 50 mg Nm -3 for plants \100 MW e and 30 mg Nm -3 for those[100 MW e [START_REF] Villeneuve | A critical review of emission standards and regulations regarding biomass combustion in small scale units (\3 MW)[END_REF]. The latter value is currently applicable in the USA also. Since the particles are specified as PM 10 , the miniscule mass contributed by the fine particles discussed above means they are virtually unregulated.

Conclusion

It appears that the co-combustion of biomass with coal, although beneficial in most aspects, will generally involve the release of an increased amount of aerosols into the atmosphere when compared with coal-only combustion, and in some cases with biomass-only combustion. This brings an increased health risks as a result of two hazards. Firstly, these fine particles themselves are known to produce adverse pulmonary effects, whatever their composition. They can also act as nuclei for subsequent agglomeration in an urban environment. Secondly they represent a vehicle for the transport over a wide area of respirable particles containing harmful transition metals such as zinc.

Fig. 3

 3 Fig.3Composition of fume formed during the combustion of five bituminous coals[START_REF] Buhre | Submicron ash formation from coal combustion[END_REF]


MATHEMATICAL MORPHOLOGY ON TENSOR IMAGES

FOR FIBER ENHANCEMENT S. Blusseau 1,2 , I. Bloch 2 , Y. Gousseau 2 , S. Velasco-Forero 1 , J. Angulo We introduce morphological methods to analyse images of positive semi-definite matrices (ellipsoids), such as diffusion or structure tensors. Morphological operators cannot be directly defined on these objects because they lack a complete lattice structure. Therefore, we extend recent developments in morphological signal processing on graphs. Graphs are constructed based on all the available information, whereas a scalar signal (anisotropy) is processed on these graphs, successfully enhancing structures such as vessels and fibers.

Basics of Mathematical Morphology

Complete lattices and morphological operators

Complete lattice A complete lattice (L, ≤) is a partially ordered set for which every subset A ⊆ L has a supremum noted A and an infimum noted A.

In the following, (L, ≤) and (L , ≤ ) denote two complete lattices.

Dilation, Erosion

-A dilation is a mapping δ : L → L that commutes with the supremum: for any family (x k ) k∈K of L, δ( k∈K x k ) = k∈K δ(x k ), where K is an index set.

-An erosion is a mapping ε : L → L that commutes with the infimum:

ε( k∈K x k ) = k∈K ε(x k ).
Opening, Closing -A closing is a mapping φ : L → L that is increasing, extensive and idempotent.

-An opening is a mapping γ : L → L that is increasing, anti-extensive and idempotent.

Adjunction A pair of mappings (ε, δ), ε : L → L, δ : L → L , forms an adjunction if and only if ∀x ∈ L, y ∈ L , δ(x) ≤ y ⇐⇒ x ≤ ε(y).

Examples

Proposition 1. If (ε, δ) is an adjunction, then φ = εδ is a closing and γ = δε is an opening.

B ≤ A ⇐⇒ A -B ∈ S + n (Left)
. The example on the right shows that this is not a lattice ordering: if it were, then C would be the supremum of A and B, but D is another upper bound of A and B and yet is not comparable with (and therefore not larger than) C.

Structure tensors for greyscale 2D images

We consider a greyscale image f , whose pixels coordinates are noted (x, y).

Scale zero tensor T

0 : (x, y) → ∇f (x, y) • ∇f (x, y) T Scale σ > 0 tensor T σ = G σ * T 0 where G σ is the σ-scale Gaussian kernel. Anisotropy image A σ = λ 1 -λ 2 λ 1 +λ 2
, where 0 ≤ λ 2 (x, y) ≤ λ 1 (x, y) are the eigenvalues of T σ (x, y).

Morphological signal processing on graphs

Graph. G = (V, E), weighted and directed graph, containing n vertices.

Adjacency matrix. W ∈ M at n (R ∪ {-∞}) such that for any (i, j), 1 ≤ i, j ≤ n -∞ ≤ w ij ≤ 0, w ii = 0, and -∞ < w ij ⇐⇒ (i, j) ∈ E (called Conservative morphological weight matrix in [START_REF] Velasco-Forero | Nonlinear operators on graphs via stacks[END_REF]).

Lattice. We work with vectors x ∈ [0, 1] n supported by the graph G, so the considered complete lattice is L = ([0, 1] n , ≤) equipped with the usual product partial ordering (Pareto ordering):

x ≤ y ⇐⇒ x i ≤ y i ∀i ∈ {1, . . . , n}.

Dilations, erosions, openings. We define the dilation δ W and its adjoint erosion ε W on L by

δ W (x) i = max 1≤j≤n x j +w ij = max j∈N i x j +w ij , ε W (x) i = min 1≤j≤n
x j -w ji = min j,i∈N j x j -w ji [START_REF] Blusseau | Tropical and morphological operators for signal processing on graphs[END_REF] where N i = j ∈ {1, . . . , n}, (i, j) ∈ E is the set of neighbours of vertex i in G. We also consider their iterated erosions ε p W and dilations δ p W , from which we build the openings γ (p) W for p ∈ N:

ε p W = ε W • • • • • ε W , δ p W = δ W • • • • • δ W , γ (p)
W = δ p W ε p W .

Co-circularity graph

In our application the vertices are the pixels or voxels of an image (2D or 3D), thus characterised by their coordinates and a scalar value (the anisotropy) [START_REF] Blusseau | Tropical and morphological operators for signal processing on graphs[END_REF].

Measure of co-circularity

i j w ij =w ji =0
Coefficient w ij is computed based on the principal orientations of the ellipsoids in i and j, and their relative positions in the image. Given the angular error θ ij to cocircularity (see illustration), we distinguish two cases:

Binary case with threshold α:

w ij = 0 if cos(θ ij ) ≥ cos(α) -∞ otherwise
Non-binary case:

w ij = log(cos(θ ij )) i j w ij =w ji <0 j θ ij

Additional constraints

Ladder Spatial constraint: Only pairs of vertices whose coordinates lie in a limited spatial neighbourhood can be neighbours. Denoting u i and u j the coordinates of pixels (or voxels) i and j, we impose w ij > -∞ ⇐⇒ ||u i -u j || ∞ ≤ k, for a fixed integer k. Conic constraint: Co-circularity allows ladder configurations. We avoid these by an additional constraint. is the non-positive sum q-1 l=1 w i l i l+1 . The paths with large weights are those with weights close to 0, and which therefore have the strongest contribution in the dilations and erosions defined above. Contributions We introduced a framework that generalises mathematical morphology for signals on graphs. The operators we defined based on the co-circularity of structure tensors show interesting behaviours for the processing of anisotropic structures.

Property of the graph

Results and discussion

Future work

-multi-scale approach or an automatic scale determination, -comparison with other methods, such as path openings and other morphological connected filters, -exploration of the properties of non-binary co-circularity graphs, -links between these operators and the max-plus algebra [START_REF] Blusseau | Tropical and morphological operators for signal processing on graphs[END_REF].

1

 1

1 :

 1 CMM, Mines ParisTech, PSL Research University, France 2: LTCI, T él écom ParisTech, Universit é Paris-Saclay, France

Figure 1 :Figure 2 :

 12 Figure 1: Example of morphological operators depending on a structuring element. (a) Input image: binary image on the first row, greyscale image on the second row. (b) Structuring element (SE) B. (c) Erosion ε B . (d) Adjoint dilation δ B . (e) Opening δ B ε B . (f) Closing ε B δ B .

Figure 3 :

 3 Figure 3: Image of structure tensors T σ (obtained thanks to [2]) and the corresponding anisotropy image (σ = 3).

Figure 4 :

 4 Figure 4: Maximal weight paths between pairs of vertices in a binary co-circularity graph. The weight of a path i 1 , i 2 , . . . , i q is the non-positive sum

Figure 5 :

 5 Figure 5: Results of the presented method based on co-circularity graphs (except last column). (a) Input images, (b) Anisotropy images (first and third rows: σ = 3; second row: σ = 1), (c) erosions ε W , (d) dilations δ W (e) openings γ W , (f) openings γ (p) W (first row: p = 10, second and thrid rows: p = 5). Here the adjacency matrices W are binary, with angular threshold α = π 6 and spatial neighbourhood parameter k (first row: k = 7; second row: k = 3, third row: k = 5). (g) Results of directional openings by a family of straight lines structuring elements (classical method in MM).


C Vigreux
email: cvigreux@lpmc.univ-montp2.fr

M Barillot

E Barthelemy

Lionel Bastard

Jean-Emmanuel Broquin

V Kirschner

Sandie Ménard

G Parent

C Poinsot

A Pradel

S Zhang

X Zhang

All-telluride channel

The search for Earth-like exoplanets showing biological activity is a very challenging quest. Nulling interferometry from space, in the mid-infrared, seems to be a promising technique for a direct observation of these extra-solar planets. It is studied by ESA and NASA in the framework of the Darwin and TPF-I missions, respectively. Although the studies began about 10 years ago, nulling interferometry remains a technological challenge at different stages. One of the challenges is the development of a modal filter that allows the filtering of the wavefronts to such quality that the central star flux can be rejected with an efficiency of about 10 -5 . The modal filter may be based on either single-mode integrated optics or fiber optics. In this paper, we are addressing the integrated optics solution.

In the framework of the Integrated Optics project, we worked on the fabrication of light-guiding structures for both [6-11µm] and [10-20µm] spectral bands. Te 82 Ge 18 thick films of controlled thickness and refractive index were deposited by thermal co-evaporation on Te 75 Ge 15 Ga 10 bulk glass substrates. Their geometry was then modified by reactive ion etching using a gas mixture of 59.5% CHF 3 /10.5% O 2 /30% Ar. After polishing of the input/output facets of the manufactured RIB waveguides (Figure 1), light guiding was confirmed at = 10.6 µm (Figure 2). To end, the first experimental assessments of the modal filtering capability of these components were performed using a nulling interferometer operating at = 10.6 µm. We demonstrated light rejection efficiency of 6.10 -5 using a preliminary non-optimized component. This result is very encouraging since it is in line with ESA's requirements and provides a confirmation of the potential of integrated optics for nulling interferometry.

Fig. 1 .

 1 Fig. 1. RIB waveguides: a) for the [6-11 µm] spectral band; b) for the [10-20 µm] spectral band. In both cases, the film composition is Te82Ge18, in order to achieve a n=nlayer -nsubstrate=4.10 -2 , approximately.


Rabah Gahoual

Alicia Burr

Jean-Marc Busnel

Lauriane Kuhn

Phillipe Hammann

Alain Beck

Nicolas Yannis

François

Emmanuelle Leize-Wagner

Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Keywords: monoclonal antibody, sheathless capillary electrophoresis, mass spectrometry, CESI-MS/MS, peptide mapping, N-linked glycosylation, posttranslational modifications, microvariant, microheterogeneity mAb, monoclonal antibody, HC, heavy chain, LC, light chain, HT, heavy chain tryptic peptide, LT, light chain tryptic peptide, PTM, posttranslational modification, RPLC, reverse phase liquid chromatography, MS, mass spectrometry, MS/MS, tandem mass spectrometry, CESI, sheathless capillary electrophoresis, ESI, electrospray ionization, XIE, extracted ion electropherogram, DTT, dithiothereitol, FA, formic acid

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product -and time -consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) -tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.

Introduction

Since 1986 and the approbation of muromonab-CD3 by the US Food and Drug Administration (FDA), monoclonal antibodies (mAbs) have taken a major market share in the pharmaceutical industry and their development is constantly increasing. [START_REF] Beck | Strategies and challenges for the next generation of therapeutic antibodies[END_REF][START_REF] Reichert | Marketed therapeutic antibodies compendium[END_REF] MAbs are highly complex glycoproteins potentially displaying many naturally-occurring molecular micro-heterogeneities and those that are introduced through imperfect processing, physico-chemical and enzymatic changes that occur during their production and long-term conservation. As a consequence, there is a continuous need for improvement of analytical methods to enable fast and accurate characterization. Mass spectrometry (MS) plays a major role in the characterization of therapeutic mAbs because it provides specificity, sensitivity and may also provide structural information. Characterization using MS has helped product and process development [START_REF] Ambrogelly | Characterization of antibody variants during process development The tale of incomplete processing of N-terminal secretion peptide[END_REF] and batch consistency assessment. [START_REF] Damen | Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab[END_REF][START_REF]Guideline on development, production, characterization and specifications for monoclonal antibodies and related products[END_REF] Furthermore, recent raising importance of biosimilar and biobetter mAbs represents a major stake for characterization methodologies. Biosimilar mAbs are produced from different clones and processes. [START_REF] Mccamish | Worldwide experience with biosimilar development[END_REF] The European Medicines Agency (EMA) and the FDA have published draft guidelines concerning the approval pathway of biosimilar mAbs.

These documents discuss the physicochemical characterization steps required to assess biosimilarity. For example, EMA guidelines suggest that the biosimilar candidate should have the same amino acid sequence as the reference mAb and a comparable glycosylation profile. [START_REF] Reichert | European medicines agency workshop on biosimilar monoclonal antibodies[END_REF] The selection of a highly similar candidate to the innovator product is mandatory before performing pre-clinical and clinical biosimilarity studies. Obtaining suitable structural information rapidly, which allows quick elimination of candidates dissimilar to the reference protein, is therefore crucial. The methods used should cover a wide range of structural information, e.g. amino acid sequence, disulfide bonds, glycosylation, posttranslational modifications (PTM). [START_REF] Zhang | Mass spectrometry for structural characterization of therapeutic antibodies[END_REF] Additional to MS, mAbs characterization requires a panel of orthogonal methods to cover a full range of structural information, [START_REF] Harris | Analytical characterization of monoclonal antibodies: linking structure to function[END_REF] including reverse phase liquid chromatography (RP-LC), [START_REF] Wagner-Rousset | The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies[END_REF][START_REF] Beck | Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells[END_REF][START_REF] Xie | Rapid comparison of candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies[END_REF] ion exchange chromatography (IEC), size exclusion chromatography (SEC), [START_REF] Alvarez | On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format[END_REF] capillary isoelectric focusing (cIEF) [START_REF] Michels | Charge heterogeneity of monoclonal antibodies by multiplexed imaged capillary isoelectric focusing immunoassay with chemiluminescence detection[END_REF] or micellar electrokinetic chromatography (MEKC). [START_REF] Kaschak | Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry[END_REF][START_REF] Rustandi | Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress[END_REF] Peptide mapping of mAbs by MS is commonly performed using a bottom-up proteomic approach because it allows acquisition of information on the amino acid sequence and PTMs. In this approach, the protein undergoes enzymatic digestion, resulting in a peptide digest most often separated by liquid chromatography and detected by UV spectrophotometry and MS.

A novel sheathless CE-ESI-MS platform, referred as CESI, allowing hyphenation of capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS) has been developed by Beckman Coulter based on a design previously described by Moini. 17 This CESI prototype (also known as sheathless capillary electrophoresis) uses a bare fused silica capillary whose outlet has been etched using hydrofluoric acid, which makes it porous to the electrical transport of small ions without permitting significant matter transfer through the pores. The porous tip provides electrical contact via a second capillary. Detailed description of this interface has been given by Haselberg et al. [START_REF] Haselberg | Performance of a sheathless porous tip sprayer for capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins[END_REF] The sheathless interface has already been used to perform successful CE-ESI-MS experiments in proteomics, [START_REF] Faserl | Optimization and evaluation of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry platform for peptide analysis: comparison to liquid chromatography-electrospray ionization mass spectrometry[END_REF][START_REF] Busnel | High capacity capillary electrophoresis-electrospray ionization mass spectrometry: coupling a porous sheathless interface with transientisotachophoresis[END_REF] metabolomics, [START_REF] Ramautar | Enhancing the coverage of the urinary metabolome by sheathless capillary electrophoresis-mass spectrometry[END_REF] intact proteins [START_REF] Haselberg | Characterization of drug-lysozyme conjugates by sheathless capillary electrophoresistime-of-flight mass spectrometry[END_REF][START_REF] Whitmore | Capillary electrophoresis-mass spectrometry methods for tryptic mapping of therapeutic antibodies[END_REF] demonstrating drastically increased sensitivity compared to sheath-liquid CE-MS due to operating flow rates of well below 100 nL/min. Whitmore and Gennaro recently used the CESI prototype to performed the peptide mass fingerprinting (PMF) of a mAb. They demonstrated the successful use of PMF to cover the amino sequence of a mAb, including the hydrophilic peptides that are eluted in the void volume in RP-LC-MS [START_REF] Whitmore | Capillary electrophoresis-mass spectrometry methods for tryptic mapping of therapeutic antibodies[END_REF] .

In this work, we used the CESI-MS system to develop a CE-MS/MS method that allowed the fast and precise characterization of a mAb digest. The mAb selected was trastuzumab which was approved by FDA and EMA, in 1998 and 2000 respectively. Trastuzumab has been intensively studied and can be considered as a highly representative therapeutic mAb. [START_REF] Demeule | New methods allowing the detection of protein aggregates: a case study on trastuzumab[END_REF] It is a humanized immunoglobulin gamma 1 (HzIgG-1) directed against the HER2/neu receptor which is overexpressed in about 20-30% of invasive breast cancer patients. [START_REF] Hudis | Trastuzumab -mechanism of action and use in clinical practice[END_REF][START_REF] Carter | Humanization of an antip185 HER2 antibody for human cancer therapy[END_REF] Trastuzumab is composed of two heavy chains (HC) having 449 amino acids and two light chains (LC) having 214 amino acids. [START_REF] Harris | Identification of multiple sources of charge heterogeneity in a recombinant antibody[END_REF] The HC has an N-glycosylation consensus site on its asparagine (Asn) in position 300, and the glycoprotein has a total molecular mass of about 148 kDa [START_REF] Damen | Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab[END_REF] . A tryptic digest of trastuzumab was prepared using a conventional in-solution digestion protocol. 28 The digest was then studied at different levels. First a peptide mapping of the mAb was performed using a CESI-MS/MS analysis. The MS/MS spectra were automatically analyzed to identify the peptides using a search engine widely used in proteomic studies in order to hasten peptide identifications. PTMs were also characterized using CESI-MS/MS, particularly those known as degradation hot spots. These PTMs were identified by Mascot search algorithm and manually confirmed. At a final level, glycosylation were also investigated using CESI-MS/MS. Although glycosylation is a type of PTM, they are treated in this article separately from amino acid PTMs because they are not degradation hot spots. The glycans could be located on the amino acid sequence and the structure of the major glycoforms could be deduced from MS/MS data. Remarkably, the characterization of the mAb over these different described levels was realized using only one CESI-MS/MS analysis of the digest.

Results

Peptide mapping of trastuzumab using CESI-MS/MS. Peptide mapping is commonly used to determine protein amino acid sequence and to locate or quantify PTMs. This methodology is quite important in early development of therapeutic antibodies as well as during long term life cycle management of the biopharmaceutical products. As part of this work, one of the objective was to investigate the peptide mapping of trastuzumab by CESI-MS/MS. Tandem MS data interpretation and peptide identification was done automatically using Mascot to obtain fast and accurate data treatment.

A sample of trastuzumab was digested with trypsin enzyme. Tryptic digestion was performed using an in-solution digestion protocol applied on a routine basis in the laboratory. It includes reduction and alkylation of disulfide bonds. Prior to digestion, an unfolding step was performed using guanidine as a chaotropic agent. Prior to MS analysis, peptides were separated using the CESI prototype which allows direct coupling of CE with the nanospray source of the MS without using an additional liquid like in sheath-liquid CE-MS. [START_REF] Gennaro | Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins[END_REF] Figure 1 illustrates an example of the separation obtained for the tryptic digest of trastuzumab for 100 fmol of injected protein. The total duration of the separation was less than 50 min and the resulting electropherogram showed that all peptides were detected between 19 and 47 min.

Separation was performed at a voltage of +20 kV to maintain a balance between peptide migration speed and separation efficiency.

Digested peptides were identified from MS/MS data acquired using a Mascot search algorithm, although 100% sequence coverage of a mAb has already been reported using the prototype CESI-MS platform. [START_REF] Whitmore | Capillary electrophoresis-mass spectrometry methods for tryptic mapping of therapeutic antibodies[END_REF] In this work, peptide identification was automated and based on MS/MS data, meaning that identification was made on precursor ion mass measure and fragment identification. Results of Mascot search showed sequence coverage of 100% for both the HC and LC of the trastuzumab. The detailed sequence coverages for both chains are shown in Figure 2. Some peptides were selected and fragmented several times during the analysis leading to the same identification. Similarly, missed cleavages could be promptly detected for several peptides reinforcing the confidence of the identification by enabling peptide overlapping.

Tryptic digestion produces a heterogeneous mixture of peptides, especially with regard to their number of amino acids. Results show the identification during the analysis of small peptides having 4-5 amino acids such as HT05 or LT09 (Figure 3) as well as a 63 amino acids peptide having a molecular mass of 6715.26 Da (HT21). During its elution, the ion corresponding to HT21 peptide was selected several time for fragmentation and resulted in the same identification, which reinforced the confidence of this important identification.

It should be noted that such heavy peptides usually can attain higher charge state; therefore, the search algorithm must consider high charge state as well. In the CESI-MS/MS analysis of the mAb, peptides having charge state up to 6+ could be detected. Fragmentation of the peptides enables an important part of the amino acid sequence to be retraced reinforcing the high confidence in peptide identification.

As seen in Figure 4, the MS/MS spectra quality, has allowed the amino acids sequence composing the variable domain of the mAb HC for 109 of 120 amino acids to be retraced.

Similarly 98 of the 107 amino acids sequence composing the variable domain of the LC could be retraced using the MS/MS data. Thus, in addition to allowing the identification of the digested peptides of the mAb, the MS/MS spectra enabled characterization, without ambiguity, of most amino acids composing such a sensitive part of the mAb, i.e. the variable domain.

To evaluate the robustness of the method, the CESI-MS/MS analysis of the mAb digest was repeated six times consecutively. Results of these analysis demonstrated 100% sequence coverage for each run and the same detected peptides in each analysis.

Characterization of amino acids PTMs hot-spots.

Mabs are heterogeneous by nature because of structural modifications that the proteins may undergo during their lifetime. Those modifications may be Critical Quality Attributes (CQA) because they can modify the conformation of the protein and therefore influence the antibody activity. Posttranslational modifications and other physico-chemical modifications can be induced by intra or extra cellular processes, buffer, purification process, storage or stress conditions. [START_REF] Houde | Post-translational modifications differentially affect IgG1 conformation and receptor binding[END_REF][START_REF] Liu | Heterogeneity of monoclonal antibodies[END_REF] Among those PTMs, several referred to as "hot spots" must be carefully monitored to determine the stability of the mAb during pharmaceutical development. Known degradation hot spots potentially modified on the HC are cyclization of N-terminal glutamic acid (Glu) into pyroglutamic acid and deamidation of Asn 55 and Asn 387 but also oxidation of Met 255 and Met 431 . The main hot spot on the LC is the deamidation Asp [START_REF] Beck | Biosimilar, biobetter, and next generation antobidy characterization by mass spectrometry[END_REF] . For the characterization of trastuzumab PTMs, the same CESI-MS/MS data presented in the previous section to obtain the mAb peptide mapping was used. In this case again, the PTMs were automatically identified by the Mascot search algorithm. Mass spectra were further manually studied to confirm the presence of the modifications.

Results obtained showed glutamine cyclization of the N-terminal extremity of the HC. Also, the same peptide without the modification was identified, which suggests a partial modification of the protein. Use of the extracted ion electropherograms (XIE) for m/z ratio of 932.500 (2+) and 941.501 (2+) corresponding to the peptide HT01 with both N-terminal extremity forms allowed the determination of a migration time of 34.5 min for the intact form of HT01 and 45.6 min for the modified HT01. This charge difference influences the electrophoretic mobility of the modified peptide, leading to its separation.

In the same manner, several deamidation hot spots were also identified. Results presented in Figure 5 show the correct identification of the deamidation of Asn 55 (HT04). In this case again the parent peptide could be identified without the modification as well, and the fragmentation allowed confirmation of the deamidation of this specific amino acid. Another deamidation hot spot present on the HC on Asn 387 (HT41) was also characterized with the method. In this case again, the MS/MS spectra obtained for HT41 allowed the identification of the deamidation on this specific amino acid which is crucial because the digested peptide is composed of several Asn. Other micro-variants, such as methionine (Met) oxidation hot spots, were also identified by the algorithm using the CESI-MS/MS data. Results allowed the identification of the oxidations of Met 255 (HT25) and Met 431 (HT48) as a confirmation of the trend of a partial modification/degradation; both peptides could also be observed without any oxidation. The peptide HT25 was detected at a time of 23.8 min for the intact form and 24.0 min for the oxidized HT25. A similar results could be observed for peptide HT48 as the intact peptide could be detected at 25.2 min and respectively at a time of 25.5 min for the oxidized HT25. Concerning the LC, the main deamidation hot spot located on Asn 30 (LT03) was correctly identified by the algorithm. As for the other hot spots, the manual confirmation suggested a partial modification/degradation of the protein.

These results unambiguously demonstrate the possibility of using CESI-MS to perform simultaneous MS/MS peptide mapping and amino acid PTM characterization. The separation of the digested peptide mixture allowed the MS to fragment a large number of ions, which enables the detection of PTMs. The mass spectra quality obtained using the CESI interface allowed the algorithm to successfully identify each studied modification.

Characterization of major N-glycoforms. For most IgGs, glycosylation represents only 2 to 3% of the total mass, but it can significantly influence the protein's structure, immunogenicity and stability. [START_REF] Jefferis | Glycosylation of Recombinant Antibody Therapeutics[END_REF][START_REF] Beck | Marketing approval of mogamulizumab: a triumph for glycoengineering[END_REF] Glycosylation is also a class of mAb PTMs: here their study was described separately from amino acid PTMs because they are not considered degradation hot spots. To perform an advanced characterization of trastuzumab, an estimation of the ratio of the main glycoforms, as well as a structural determination is necessary. Trastuzumab is N-glycosylated on the Asn 300 in the CH2 region. [START_REF] Beck | Trends in glycosilation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins[END_REF][START_REF] Beck | GlycoFi's technology to control the glycosilation of recombinant therapeutic proteins[END_REF] G0F and G1F, the major glycoforms found on human and recombinant IgG produced in CHO, NS0 and SP2/0 cells, represent more than 80% of the glycoforms. [START_REF] Beck | Biosimilar, biobetter, and next generation antobidy characterization by mass spectrometry[END_REF][START_REF] Damen | Electrospray ionization quadrupole ion-mobility time-of-Flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab[END_REF] Considering that Mascot search algorithm is only intended to identify the peptide sequences it was necessary to manually study the CESI-MS/MS data to identify the glycosylations and determine their structures. Interestingly, in this case as well, no extra experiments needed to be performed to focus on glycopeptides. The same datasets used for peptide mapping and hot spots characterization were also used here.

In conventional protocols, a mAb is commonly treated with PNGase F before analysis to release the glycans and allow study of them while not attached to the peptide backbone. [START_REF] Damen | Electrospray ionization quadrupole ion-mobility time-of-Flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab[END_REF] In this work, the mAb was digested by trypsin only, and therefore we expected to detect glycans still linked to the corresponding peptide backbone of the HC. The extracted ion electropherograms (XIE) of the m/z ratio matching to the peptide TKPREEQYN 300 STYR bearing respectively the glycosylation G0F, G1F, G0F-GlcNac, G2F and G0 are shown in Figure 6 A-E, respectively. All the detected glycopeptides have a charge state of 3.

The glycopeptides were detected between 26.3 and 28.5 min. The electrophoretic resolution calculated between the peaks corresponding to the glycopeptides bearing G0F and G1F gave a value of 0.88, while the resolution between the peaks of glycopeptides G1F and G2F gave a value of 1.08. These results demonstrate the separation of the glycopeptides G0F, G1F and G2F glycan having only one galactose difference in each case. The glycopeptide bearing G0 could also be observed on its corresponding XIE; however, this glycopeptide co-migrated with glycopeptides G0F. Moreover, the sensitivity provided by the CESI prototype allows detection of these glycopeptides with significant intensities and the signal was sufficient to generate good quality MS/MS spectra of each glycopeptides. More generally, these results allowed us to accurately locate the position of the glycosylation on Asn 300 because it is the only potential glycosylation site on this peptide. The nature of the glycosylation, however, had to be confirmed using MS/MS data. MS/MS spectra (Figure 7) corresponding to the fragmentation of the different glycopeptides were subsequently extracted from the data. The precursor ion matches the mass of the glycopeptide being glycosylated by G0F with a precision of 8.66 ppm. As expected with low energy collision induced dissociation (CID), product ions observed in MS/MS mainly correspond to the fragmentation of the glycan moiety and the peptide backbone is usually preserved. Study of the fragmentation spectra allowed us to deduce the structure of the glycan G0F. In the same manner, the MS/MS spectra of the glycopeptides bearing G1F, G2F and G0 glycans were extracted from the data; in each case the mass measure precision on the precursor ion was inferior to 9.69 ppm. Similarly to the previous results, every ions corresponding to the different fragmentation of the glycan could be observed while the peptide backbone remained intact, allowing the structures of the studied glycans to be deduced.

Results presented here demonstrate the possibility of using CE to separate and identify glycopeptides obtained from the trastuzumab enzymatic digestion mainly due to the baseline separation efficiency of the five major glycopeptides provided by the CE. As an additional benefit, the sensitivity provided by the CESI interface and the microTOFQ-II MS allowed us to obtain the complete fragmentation of the glycan and thereby deduce the structure of the five major glycans (G0F, G1F, G2F, G0F-GlcNac and G0). The same data was used for MS/MS peptide mapping and hot-spot characterization without requiring additional experiments.

Discussion

Peptide mapping is essential for the characterization of therapeutic mAbs, which are complex glycoproteins that contain a plethora of micro-heterogeneities due to the production process. In comparison to PMF, MS/MS peptide identification is not only based on digested peptide m/z ratio measure, but also on the fragmentation of those peptides, increasing the confidence of the identification. Capillary zone electrophoresis (CZE) separation is based on the migration of species present in a background electrolyte (BGE) under an electrical field. Separation is obtained by differences in electrophoretic mobility among analytes depending on size and charge of the molecule at the BGE pH.

Peptide mapping results showed the identification of wide variety of peptides regarding their chemical composition (Figure 2). This result indicates clearly that CE separation driven by peptide migration phenomena is able to separate and transfer successfully to the MS every digested peptide regardless of its nature. In RP-LC, small peptides (from one to two amino acids [START_REF] Whitmore | Capillary electrophoresis-mass spectrometry methods for tryptic mapping of therapeutic antibodies[END_REF] ) and more generally hydrophilic peptides, are often not detected because they are not retained on the column and elute in the void volume. [START_REF] Faserl | Optimization and evaluation of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry platform for peptide analysis: comparison to liquid chromatography-electrospray ionization mass spectrometry[END_REF] The detection and identification of peptide such as LT09 or HT05 demonstrate the ability of CE to conserve and detect small peptides. The identification of the 63 amino acids peptide HT21 also demonstrates one of the advantages of using CE hyphenated to MS for peptide characterization. The presence of such large peptides is not surprising as trastuzumab HC does not have a tryptic cleavage site from amino acid 151 to 213. The elution and detection of such an imposing peptide may be difficult in RPLC-MS/MS because it can irreversibly interact with the stationary phase. Identification of very large peptides having no enzymatic digestion sites or corresponding to peptides or with several missed cleavages is also a very important asset of this method because 100% sequence coverage is obtainable in various cases. Indeed, some digestion sites in mAbs may not be accessible to trypsin due to the quaternary structure of the protein, which may cause missed cleavages during the digestion even when guanidine is present as a chaotropic reagent. In addition to the complete sequence coverage obtained of the HC and the LC, the sensitivity provided by the CESI prototype supported by the high resolution of the mass spectrometer allowed generation of high quality MS/MS spectra. This result is explained by a remarkable ionization efficiency of the peptides. The low BGE flow rate during the separation indeed enhances the ionization process and reduces ion suppression. [START_REF] Busnel | High capacity capillary electrophoresis-electrospray ionization mass spectrometry: coupling a porous sheathless interface with transientisotachophoresis[END_REF][START_REF] Wilm | Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last ? Inr[END_REF] The spectral quality is reflected by the possibility of retracing the major part of the amino acid sequence in the variable domain.

As the CESI-MS system is able to transfer efficiently an important number of ions, MS/MS spectra were of an excellent quality in terms of resolution and intensities (Figure 3). Therefore, a significant number of y/b fragments could be attributed, allowing retracing on the variable domain of the HC 109 of 120 amino acids and 98 of 107 on the LC. Understanding the variable domain of the mAb is crucial because it is directly involved in the antibody-antigen binding. It is noteworthy that such level of characterization could be attained with only one run of the CESI-MS/MS system.

Our results demonstrated the successful characterization of every single degradation hot spots on HC and LC and we observed in each case the partial modification of the protein because the modified and the intact form of the peptide could be identified (Figure 5). In the case of the glutamine cyclization of the N-terminal extremity on the HC, the CE separation mechanism allowed the separation of the modified and intact forms of the peptide based on conformational changes induced by glutamine cyclization. Such separation is explained by the fact that even if the mass difference between the parent and the modified peptide is only 17.02 Da, Glu cyclization involves the loss of the amine function on its lateral chain; therefore, there is a difference of one charge between glutamic acid and pyroglutamic acid because the amine is protonated in 10% acetic acid (pH 2.2). The possibility of separate the peptides prior to MS analysis, allows the identification of both forms. Indeed as the number of precursor ions selected for fragmentation per cycle is fairly limited on the microTOF-Q II, and taking into account the complexity of the sample, it would have been difficult for the MS to automatically select the m/z ratio corresponding to both forms of this peptide during their detection window if they had comigrated. In the case of the deamidation hot spots present on Asn 55 and Asn 387 on the HC and Asn 30 on the LC, the MS was able to fragment both the modified and the intact form for each peptide. Unlike in the glutamic acid cyclization, the XIE corresponding to those peptides (HT04, HT41 and LT03) showed in each case the complete co-migration between the intact peptide and its deamidated homologue. This is explained by the fact that the mass difference between asparagine and aspartic acid is only 0.98 Da. Regarding deamidation, the amide functional group located on asparagine lateral chain, is replaced by an alcohol function. Most importantly, there is thus no significant charge difference between the deamidated peptide and the intact peptide because those functions are protonated at BGE pH. In this case of oxidized Met 255 (HT25) and Met 431 (HT48), even if the detection times were not exactly the same for the intact peptide and its oxidized homologous, differences in electrophoretic mobilities could not lead to the baseline separation of those peptides. The sensitivity provided by the system allowed unambiguous characterization of each studied degradation hot spot, in addition to the low represented to PTMs and degradations present in small amounts.

Using the CESI-MS/MS data, the complete structural characterization of the glycosylation G0F, G1F, G2F, G0F-GlcNac and G0 could be achieved (Figure 7). Because trypsin is not able to cleave glycosylation from the peptide, the glycan was observed on the corresponding digested peptide. The XIE corresponding to the glycopeptides bearing different glycans (Figure 6) demonstrated the separation of several glycopeptides in a reduced time. This result demonstrated the ability of the CE migration mechanism to completely separate glycopeptides having a difference of only one galactose ( e.g. between G0F and G1F). Such levels of separation could be explained by the fact that one more galactose leads to a difference between the two glycopeptides in term of size (+ 162 Da). This difference affects their relative electrophoretic mobilities leading to their separation. The efficiency of CE in the glycopeptides separation has allowed the separation to be obtained in ~27 min. A difference of a fucose, however, did not lead to the separation of the glycopeptides G0F and G0, suggesting that the size of the glycopeptides is not significantly affected by the fucosylation at the BGE pH.

To evaluate the robustness of this method, the analysis was consecutively repeated six times using the same mAb digest sample. Each analysis resulted in 100% sequence coverage in term of MS/MS peptide mapping. Similarly, the degradation hot spots identified were the same in each case and the five same major N-glycans could also be structurally characterized in the six analyses. Intensity ratios between the different glycopeptides were calculated for each analysis; the results exhibited a standard deviations ranging from 6 to 13%. Based on this results, glycopeptide intensities appear to be related to glycan distribution. It is therefore it is possible to consider profiling the mAb glycan using this very same CESI-MS/MS method, which would enriching the amount of information obtain from only one analysis.

In conclusion, this work presents the development of a rapid and reliable method for performing a multi-level characterization of trastuzumab, which was used as reference therapeutic antibody in a single run. The mAb was digested using a conventional digestion protocol and the digest was analyzed by CESI hyphenated to a high resolution tandem MS. The CESI-MS/MS fragmentation obtained in only one acquisition allowed the complete amino acid sequence peptide map to be recorded. A Mascot search engine was used for peptide identification to provide a faster protocol. Also, the characterization of every PTM hot spot could be obtained within the same single run. Finally, separation of the major glycosylated peptides and their structural characterization could be obtained using the same CESI-MS/MS analysis dataset. Such advanced characterization using only a single injection of 100 fmol of digested mAb peptide mixture in a 45 min analysis demonstrates the potential of CESI-MS as a fast and sensitive method in mAb characterization and more generally in bottom-up proteomic approach.

Materials and methods

Materials. All chemicals used were of analytical grade or high purity grade. Acetic acid was provided by VWR (Fontenay sous Bois, France). Bicarbonate ammonium, guanidine hydrochloride, iodoacetamide (IAM), dithiothreitol (DTT), trifluoroacetic acid, acetonitrile (ACN) and trypsin enzyme were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Water used to prepare buffers and sample solutions was obtained using an ELGA purelab UHQ PS water purification system (Bucks, UK). Desalting steps were performed using C 8 spin tips purchased from Protea Biosciences (Morgantown, WV, USA).

Tryptic digestion procedure. Tryptic digests of antibodies were prepared using sequencegrade trypsin (1:10 w/w) . Before digestion, sample was diluted to a concentration of 10 mg/mL. One mg of protein was mixed with 155 μL of denaturing reagent (guanidine HCl 6M in 50 mM ammonium bicarbonate) and 15 μL of DTT 100 mM were added. The mixture was incubated for 5 minutes at 95°C, then cooled to ambient temperature. Then, 30 μL of IAM 100 mM was added and the mixture was placed in the dark for 20 minutes. Ten μL of trypsin was added and the mixture was incubated at room temperature for 3 hours; additional 10 μL of trypsin were added to the sample for overnight digestion at room temperature. Buffer exchange and concentration were performed using a C 8 spin tips. Four hundred μL of mAb tryptic digest in BGE could be obtained. A volume of ten μL of the obtained digest was used to perform all CESI-MS/MS experiments.

Capillary Electrophoresis. The CE experiments were carried out with a PA 800 plus capillary electrophoresis (CE) system from Beckman Coulter (Brea, CA) equipped with a temperature controlled autosampler and a power supply able to deliver up to 30 kV. Hyphenation was realized using a CESI prototype made available by Beckman Coulter. Prototype of bare fusedsilica capillaries (total length 100 cm ; 30 μm i.d.) with characteristic porous tip on its final 3 cm supplied by Beckman Coulter (Brea, CA, USA), a second capillary (total length 80cm ; 50μm i.d.) filled during experiments with BGE allows electric contact. New capillaries were flushed for 10 min at 75 psi (5.17 bar) with methanol, then 10 min with 0.1 M sodium hydroxide, followed 10 min with 0.1 M hydrochloric acid and water for 20 min also at 75 psi. Finally, the capillary was flushed 10 min at 75 psi with BGEn which was acetic acid 10%. Hydrodynamic injection (69 mbar for 1 min) corresponding to a total volume of 11 nL of sample injected was used.

Separations were performed using a voltage of +20 kV.

Mass Spectrometry. For antibody characterization, the CESI system was coupled to a microTOF-Q II mass spectrometer (Bruker Daltonics, Bremen, Germany). The microTOF-Q II MS is equipped with a hybrid analyzer composed of a quadrupole followed by a time-of-flight (TOF) analyzer. Positive mode acquisition was used to detect precursor ions (MS) and fragmented product ions (MS/MS). Concerning the ESI source parameters, capillary voltage was set to -1.3 kV. Nebullizer gas was deactivated, the dry gas was set to 1.5 L/min and temperature of the source was set at 160 °C. Spectra were collected at a data acquisition frequency of 2 Hz; for fragmentation spectra, collision energy ranged from 0 to 45 V depending on the m/z ratio and charge state of the precursor ion. For each MS scan, 3 precursor ions were selected for fragmentation, and total duty cycle was therefore 2 sec. Mass range was 50 -3000 for MS as well as MS/MS scans. MS/MS data analysis. Data obtained from CESI-MS/MS experiments were processed using Mascot search algorithm developed by Matrix Science (Boston, MA, USA). Tryptic cleavage rules were applied for both HC and LC sequences of the mAb. Carbamidomethylation of cysteine (+57.02 Da) was selected as a fixed modification, N-deamidation of aspartic/isoaspartic acid (+0.985 Da) or succinimide intermediate (-17.03 Da) were selected as a variable modifications. Methionine oxidation (+15.99 Da) and N-terminal glutamic acid cyclization (-17.02 Da) were also selected as variable modifications. The mass tolerance for precursor ions was set to ± 25 ppm and to ± 0.5 Da for fragments. A maximum of 3 missed cleavages was tolerated. MS/MS N-glycan identification and structural characterization were done manually.

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Base Peak Electropherogram corresponding to the analysis by CESI-MS/MS of trastuzumab tryptic digest. Experimental conditions: bare fused silica capillary with porous tip, total length 95 cm (30 μm i.d., 150 μm o.d.); CE voltage +20 kV; BGE 10% acetic acid; sample trastuzumab tryptic digest 2.5 mg/mL in BGE (11nL injected). MS capillary voltage: -1.3 kV, m/z range : 50 -3000 Figure 2. Sequence coverage obtained by CESI-MS/MS for trastuzumab HC (left-hand side) and LC (right-hand side) based on the identified peptides. Experimental conditions: see materials and methods Figure 3. List of trastuzumab digested peptides identified by the CESI-MS/MS analysis. Experimental conditions: see material and methods

Figure 4 .

 4 Figure 4. MS/MS spectra of peptide HT01 and HT02 illustrating the amino acid sequence retracing in the variable domain. Experimental conditions: see material and methods.

Figure 5 .

 5 Figure 5. MS/MS spectra of peptide HT04 (LLIYSASFLYSGVPSR) A) without modification and B) with a deamidation on Asn 55 illustrating the partial modification of the mAb and the characterization of both forms by CESI-MS/MS. Experimental conditions: see material and methods.

Figure 6 .

 6 Figure 6. Extracted Ion Electropherogram of m/z ratios A) 1039.80 (HT29 + G0F), B) 1093.78 (HT29 + G1F), C) 972.09 (HT29 + G0), D) 1147.82 (HT29 + G2F), E) 991.10 (HT29 + G0). Experimental conditions: see material and methods.

Figure 7 .

 7 Figure 7. MS/MS fragmentation spectra of A) HT29 -G0F (precursor m/z 1039.78; charge state 3+), B) HT29 -G1F (precursor ion 1093.80; charge state 3+), C) HT29 -G2F (precursor ion 1147.82; charge state 3+), D) HT29 -G0F-GlcNac (precursor ion 972.10; 3+), D) HT29 -G0 (precursor ion 991.09; charge state 3+). Experimental conditions described in material and methods section.

ACKNOWLEDGMENT

Rabah Gahoual would like to thank the MRT for funding his Ph.D work. LSMIS would like to thank Beckman Coulter Inc. for lending a CESI prototype and Bruker Daltonics lending the microTOF-Q II. The authors would also thanks Dr. E. Wagner-Rousset, Dr. D. Ayoub, MC. Janin-Bussat and O. Colas (Centre d'Immunologie Pierre Fabre, Saint-Julien en Genevois, France) for discussions around sample preparation and antibody LC-MS characterization.