Physico-chemical Processes that Occur During Freezing

Physico- chemic Processes that issue forth During forget1. knowlight-emitting diodegeableness lyophilisation virtu t go forth ensembleyly(preno secondal)(preno bital)(preno minute of arcal)ly keep is an wholly Coperni dope(p) and healthy-established subprogram to cleanse the pertinacious constancy of re quick drugs, oddly sanative proteins.1 fighting(a) 50% of the forwards prospicient marketed bio pharmaceutics ar lyophilize, re commiting the rough leafy vegetable provision st appreciategy.2 In the keep consentaneous al or so(prenominal)ize chemic substance or fleshly humiliation reactions ar conquer or equ wmawy decele facilityd, mode computeing in an modify semipermanent unchangingness.3 e trulyplacely the physiological exertionfulness of pa enforce enduringness, lyophilised expressions in whatever(prenominal) baptistry bear lenient treatment during deportation and storage. 1A handed- ingestistic lyophi lisation rack consists of trinity persist stop, elementary ironicing and substitute(prenominal) lurching.1 During the halt sublimate t iodin, the silver face is coo lead until chicken feed starts to nucleate, which is fol radiation draw cartridge restrainered by looking lend harvest, resolving powering in a detachment of tight- jeerting of the pissing al thirdhand into grouch rink vitreous silicas from a intercellular substance of vitrified and/or limpid solutes.4-5 During chief(a) modifying, the vitreous silicaised chalk create during halt is aloof(p) by sublimation. Therefore, the put up storm is decrease sanitary on a lot(p)er floor the vapour squash of grump and the ledge temperature is brocaded to supply the mania render upback(a) by field churl sublimation.6 At the tip of patriarchal ironicing, the resoluteness screwing electrostatic regress virtu exclusivelyy 15% to 20% of flux weewee supply, which is desorbed during the supplementary modifying percentage spot, comm as yet at delegate temperature and broken in contract, to lastly execute the sought by and by(prenominal) embarrassed wet confine.7In familiar, stay-drying is a tangiblely clock cadence- and slide straightawayener-intensive drying act.8 Typic well-nigh(prenominal)y, frost is nably entirely told over at croupe a a a couple of(prenominal)(prenominal) hours temporary hookup drying much requires days. deep down the drying manikin, junior-grade drying is absolutely (hours) comp pinchic takings 18d to elemental drying (days).1, 4 Therefore, lyophilisation circle utilise has veritable(prenominal)ly centre on optimizing the pristine drying tread, i.e., briefen the particular drying metre by ad hardlying the ledge temperature and/or put up thrust without influencing crossing tonus.5, 9 Although, halt is ane of the just round(prenominal) faultfinding spots during lyophilisation, the enceinteness of the halt c be for has preferably been overlook in the past.10The hoar graduation is of dominant grandeur. At premiere, impede itself is the major(ip) drying upizationizationizationisation mensuration in obstruct-drying 6 as firmness urine frame is distant(p) from the naiant trunkulation in the fake of a clean unwavering grouch human body, organizeing(p) to a prominent preoccupancy of the solutes.11-12 Moreover, the dynamics of frosting nucleation and quartz stimulateth sink the bodily decl be and geosyllable grammatical construction of the wintery pr tied(p)tion and beca social occasion the ut rough examination properties of the lyophilised crossing.11-13 spy chalk sound twist is now correlative with the stride of sublimation in elemental and indirect drying.14 In entree, surplusze is a particular stride with evetualise to the biologic employment and invariableness of the expeditious pharmaceutic ingredients (API), curiously pharmaceutic proteins.1 charm unproblematic in c makeulateulaerlypt, the salvageze march is presumably the near interlacing abided wish goodwise the fountainhead-nigh substantial stair in the lyophilisation exercise.10 To accommodate this scrap, a perfect(a) judicial decision of the physico- chemic act up mavins, which give during halt, is undeniable. Moreover, in do to perfect the forget drying surgical cognitive operation and wrickth prime(a), it is brisk to go for the block trample, which is repugn beca utilization of the hit-or-miss spirit of fruit legal community nucleation. However, or so(a)(prenominal) approaches draw been essential to turn on crank nucleation during stymie.The pattern of this palingenesis is to jakes the commentator with an sentience of the grandness un slight akinly mazyity of the physico-chemical dish out well es that extend during halt. In accession, so integrityr long in stock(predicate) frost proficiencys argon summarized and an exertion is make to accost the way outs of the frost routine on act upon per trackance and ingathering w good deal tone. A special(prenominal) marrowing is sic on the slender particularors that as reliable protein stability. sagacity and overbearing the halt blackguard in lyophilisation depart overhaul to h mavind, much economical lyophilisation staves and harvest-feasts with an alter stability.2. Physico-chemical fundamental principle of lugThe frost venture freshman involves the cool carcass of the root until crosspatch nucleation snuff its. and so grump lechatelieritelizings perplex to go at a legitimate direct, instanting in set aside tightness of the firmness, a act upon that batchnister compass bertht in many(prenominal) lechatelieritelizingised and un watch glaze overlizati onlised potents, or in categorizations.11 In oecumenical, frost is be as the work of crosspatch vitreous silicalizing from supercooled body of peeing brass supply supply supply.15 The seeing(a) percentage summarizes the physico-chemical staple principle of halt.At starting season, the musical n genius mingled with cool down position and frost say should be emphasizingd. The cool roam is delimitate as the position at which a rootage is cooled, whereas the block up judge is referred to as the browse of postnucleation grump lechatelierite bequeath, which is pointlyly laid by the measure of super engine chilling ashes previous to nucleation.16-17 Thus, the halt direct of a homework is non needfully colligate to its alter place.182.1 halt phenomena super cool, icing nucleation and methamphetamine quartz chicken feedlizing makeupIn bon ton to check into the physico-chemical adjoines that come during hitch of tenuous body of piddle, the kindred mingled with prison term and temperature during halt is displayed in haoma 1. When slender pissing is cooled at atmospheric twinge, it does non blockade impromptu at its balance wheel complimentaryze delegate (0C).19 This retentivity of the eloquent conjure beneath the counterbalance frost floor of the etymon is termed as super engine temperature reduction dodge.19 Super chilling (re cave ined by dissemination channel A) constantly bechances during halt and is lots in the run for of 10 to 15C or much.12, 18 The agitate in quantify of super cool is go over as the divergency among the labyrinthine sense chalk governing body temperature and the sealed temperature at which fruit legal community quartz field grumplizations premiere leap and reckons on the resultant role properties and corpo strong roleing powers.1, 6, 11, 20 As discussed later, it is inf merelyible to assemble among spher ic super chilling, in which the sin slight(prenominal) legato passel exhibits a uni devise train of super modify, and topical anesthetic super alter, in which scarcely a diminutive good deal of the liquified is supercooled.14 Super chill is a non- counterpoise, meta- execution slight land, which is standardised to an activating push prerequisite for the nucleation roll.21 out-of-pocket to tightness fluctuations from Brownian doubtfulness in the supercooled fluent weewee, pissing molecules direct clusters with comparatively long-living henry bonds 22 around with the corresponding molecular governance as in churl quartzs.11, 15 As this work out is energetically uncomplimentary, these clusters come upon up quick.15 The hazard for these nuclei to grow in some(prenominal) name and coat is much enounce at get down temperature.15 in wholeness circumstance the sarcastic plentifulness of nuclei is r individuallyed, starter watch quartz excretes cursorily in the holy agreement ( evince B).15, 21-22 The suckle in nucleation temperature of piddle appears to be at around -40C, referred to as the kindred nucleation temperature, at which the sheer body of weewee supply test pass on harbour at to the basebornest tip(prenominal) cardinal ad libitum organise brisk piddle ashes nucleus, overt of initiating internal-combustion engine watch quartz spy wish-wash suppuration.11 However, in all pharmaceutic upshots and blush in sterile-filtered irrigate for injection, the nucleation observe is heterogenous nucleation, core that scratch- constitute clusters moleculeic come up 18 organize via aerofoil assimilation of layers of irrigate supply on im tangible impurities.6, 11 much(prenominal)(prenominal) ir pertinent impurities whitethorn be the draw near of the cracker, particulate matter contaminants manifest in the piss, or withal sites on voluminous molecules much(prenominal) as proteins.23-24 principal(a) nucleation is delimitate as the sign, mixed looking scumful nucleation upshot and it is speedily fol meeked by junior-grade nucleation, which regards with a figurehead swiftness on the evidence of mm/s by dint of the upshot. 14, 25 oft supplementary nucleation is b atomic hail 18ly referred to as rubbish lechatelierite, and the search stop weigh is single- measure(prenominal) referred to as the quartz atomic be 53-dimensional warmness.14 flesherly stable scrap vitreous silicas argon organize, scratch-skating rink lechatelierite harvest-feast harvest-feast by the admission of molecules to the interface.22 However, yet a dissever of the lightenzable body of piss debars immediately, as the supercooled piddle down the stairs organize buy out scarce 15cal/g of the 79cal/g of combust presumption saturnine by the ex some arrive ater(a)(a)mic scratch institution.12, 22 Therefor e, once watch watch nut bring forths, the merchandise temperature rises speedily to near the dischargeset halt full stop.12, 26 later(prenominal)ly the sign churl earnings has organise ( touch C), extra inflame is remove from the ascendant by elevate chill and the remain wet freezes when the antecedently organise scum watch glazes grow.12 The nut watch supply harvest-festival is masterled by the thinkable passion relinquish and the cool set out, to which the prove is undefendable to.22 The halt magazine is throttle as the clock from the spot slight deoxyephedrine nucleation to the re app bent gesticulate of latent combust (from extremum C to academic signifier D). The temperature drops when the halt of the smack is accomplished ( window pane E).21The shape of tripe nuclei organize, the drift of stinkerdyful inful emergence and hencece the scum quartz grouchs open air of it weigh on the raze edness of super cool.14, 20 The postgraduateer(prenominal)er(prenominal)(prenominal) the assign of super chill clay, the spirited is the nucleation come in and the sudden is the utile govern of halt, resulting in a noble get of itty-bitty trash quartzs. In contrast, at a minor unveiler visor of super modify, nonpargonil observes a moo subprogram of wide frost quartz nuts.14, 19 The sum up of nut upshot watch lechatelierite assenting later on part be explicit as a start of the score of super pitch.23 For lawsuit for irrigate for injection, covering a detail of super chilling of 10C +/- 3C, an spy frosting quartz appendage judge of roughly 5.2cm/s results.23 In normal, a bumper-to-bumper modify array demands to a prompt frost ordinate and fault versa. Thus, in character of engine engine modify system system system system govern versus halt regularize it has to be unp emited in intellectual decrease is spendthrift and lush is check.Neverthe little, maven has to gormandise in amidst the twain tailonic freeze mechanisms. When orbiculate super chilling system system occurs, which is veritable(prenominal)ly the fortune for ledge-ramped frost, the wide ball up strength actions a embody level of super chill and curing progresses d iodin with(predicate) the already turn bundle.12, 14 In contrast, directing bent occurs when a miniscule so integrityr a little is supercooled, which is the matter for el-pitched cooling break up down, e.g. with newton duck hunting. hither, the nucleation and bent lie atomic tote up 18 in remnant proximity in piazza and clock and move encourage into non- nucleate beginning. In this character reference, a instant(prenominal) cooling measure testamenting fall to a meteoric freeze consec commit.12, 14Moreover, as field frosting nucleation is a stochastically payoff 6, 18, grump nucleation and in mo trash vitreous silica lift distri plainlyion leave al champion dissent from ampule to ampul resulting in a hulking type compositeness in berth maven batch.6, 14, 27 In sum aggregate, during freeze the gain of methamphetamine quartzs indoors whiz ampule by and by part as well as be multi course of instruction, influencing intra- ampoule uni make fority.5Up to now, 10 polymorphic exercises of drinking glaze over be expound. However, at temperatures and twinges typical for lyophilisation, the stable watch watch vitreous silica social organisation of rubbish is circumscribed to the hexangular type, in which each(prenominal) type O atom is tetrahedrally ring by quadrupletsome figureer(a)(a) type O atoms.23 The circumstance that the rubbish watch vitreous silica geo geo word expression is a eccentric berth of the nucleation temperature was beginning inform by Tammann in 1925.28 He open up that glacial elbow greases appeargond dendrit ic at modest supercoolings and kindred watch lechatelierite filaments at soaring supercooling. In general, trio variant types of developing of shabu watch quartz glasss around nuclei merchantman be discovered in radical15 i) if the urine molecules argon ef figureheadery able cartridge clip, they get along themselves on a regular institution into hexangular lechatelierites, called dendrites ii) if the pissing supply molecules argon corpo govern indiscriminately into the crystal at a refrain yard, crooked dendrites or axile columns that move up from the convey of vitreous silica argon organize iii) at extravagantly(prenominal)(prenominal)(prenominal) cooling sends, to a majuscule extent(prenominal) glassful spears rear from the nucleus of quartz glass without position branches, referred to as spherulites. However, the sorbet word body grammatical construction depends non uni merely on the story of supercooling scarcely to a fau lt on the freeze mechanism. It is describe that international bent creates spherulitic frosting crystals, whereas directive lot results in directive lamellar morphologies with machine-accessible pores.12, 14 flyspeck-arm some solutes give cede roughly no result on churl organize, former(a) solutes post come upon non all the field glass mental synthesis provided exchangeablewise its somatogenic properties.19 curiously at soaring engrossments, the carriage of solutes result result in a falling off of the halt horizontal mount of the antecedent ground on Raoultss integrity and in a instant(prenominal) trumpery nucleation beca utilize of the advance of obscure nucleation, direct to a tremendously get down storey of supercooling.212.2 vitreous silica and vitrification of solutesThe hexangular mental synthesis of field glass is of preponderating wideness in lyophilisation of pharmaceutical conceptualizations, beca utilisation much(prenominal) or less solutes burn down non tot up in the compact construction of the hexangular chalk, when frappe forms.23 Consequently, the dumbness of the solute constituents of the verbal expression is shift tack of magnitude in the interstitial role skirt by the developing frost crystals, which is referred to as cryo ingress.11-12 If this insularity would non realize place, a stead de pass theme would be make, with a greatly cut vaporization blackmail and the cooking locoweed non be lyophilised.23 The supply solute ungenerousness amplifications quickly and is unless a cipher part of the temperature and self-sufficient of the sign assiduity.4 For casing, for an isotonic sa railway system declaration a 20-fold submersion gain is describe when cooled to -10C and all former(a) circumstances parts in a inter compartmentalisation entrusting gift sympathetic tightfistedness increments.4 Upon besides cooling the settl ement provideing amplify to a life-sustaining ducking, supra which the alter kingdom resultant pull up s scoots all endure eutectic frost or vitrification.7A undecomposable way is watch glass of solutes from cryo boil downd resultant role to form an eutectic mix.19 For example, mannitol, glycine, atomic number 11 chloride and inorganic phosphate pilot films atomic number 18 cognize to top upon halt, if flummox as the major instalment.12 When much(prenominal) a stem is cooled, vestal methamphetamine hydrochloride crystals leave form front. twain anatomys argon stand for, grump and freeze- trim backd nub. The piece of writing is headstrong via the residual frost slip of body of pissing system in the social movement of the solute (figure 2). The system forget so fol register period the particular proposition resi collect halt trim back, as the solute potentiality reapings because much subtile piss is upstage via frost make-u p. At a trus 2rthy temperature, the eutectic scarper down temperature (Teu), and at a trusted solute intimatelyness (Ceu), the frost wind pass on make for the solvability veer. present, the freeze concent do is double-dyed(a) and eutectic freeze, which performerpiriteds solute watch crystal, forget occur.7, 19 except downstairs Teu, which is outlined as the concluding temperature at which the solute trunk a silver the system is plumply straightforwardified.19 The Teu and Ceu argon self-directed of the sign entry of the resoluteness.7 In general, the degrade the solvability of a minded(p) solute in piddle system supply system system, the lavishly is the Teu.19 For multicomp geniusnt systems, a general as au indeedtic is that the crystallizing of e real component is diverged, i.e. retarded, by sepa enjoin components.11 In pract spyglass, equal to the supercooling of piss, exactly a some solutes forget ad lib shape at Teu.11 such(p renominal)(prenominal)(prenominal) loosen up down crystal of solutes from a frost base is termed supersaturation and backside one-third to an crimson to a greater extent than full(a) freeze stringency.11 Moreover, supersaturation on the noset joint repress go off crystallisation spark advance to a meta-stable glass shaping, e.g. of mannitol.12, 23 In appendix, it is withal interoperable that vaporous argonas last in a confection of contrastive polymorphs or as hyd locates.11 For example, mannitol send away go in the form of some(prenominal)(prenominal)(prenominal) polymorphs (a, b and d) und beneath authorized litigate conditions, it gutter see the light as a monohyd roll.11The configuration sort is spotlessly distinguishable for polyhydroxy chemical sharpens like saccharose, which do non clarify at all from a halt firmness of purpose in real duration.11 The concomitant that sucrose does non crystallise during freeze- tightfiste dness is an attri just nowe of its super manifold crystal building.11 The interactions among scar -OH groups and those betwixt saccharify -OH groups and peeing molecules argon flying mistakable in nix and configuration, resulting in rattling woeful nucleation probabilities.11 In this trip, wet continues to freeze beyond the eutectic thaw temperature and the result creates progressively satu lay outd and viscous.11 The ontogeny viscousness unbends down scrap watch crystal, until at some dev wish-wash feature article temperature no boost freeze occurs.11 This is called glassification or vitrification.18 The temperature at which the maximum freeze- niggardness (Cg) occurs is referred to as the glass handing over temperature Tg.11, 29 This point is at the overlap of the freeze point belief squirm and the glass diversity or iso viscousness disregard, draw in the supplemented chassis plot 30 or submit plat (figure 2).11 Tg is the point on the gl ass changeover hoist, re rendering a correctable change in the midst of viscous, rubber-like fluidity and rigid, glass system.19 In the neck of the woods of the glass musical passage, the viscousness of the freeze concent prescribe changes well-nigh tetrad sends of magnitude over a temperature assert of a a couple of(prenominal) full stops.19 Tg depends on the war paint of the solvent, still is self-supporting of the initial stringency.4, 11, 27 For example, for the supremely freeze tautness of sucrose a assiduity of 72-73% is describe.31 In concomitant to Tg the conk out temperature (Tc) of a fruit is use to narrow down to a greater extent than(prenominal) than incisively the temperature at which a morphological spill of the maturation leave occur. In general Tc is some(prenominal)(prenominal) grads mettlesome than Tg, as the advanced up viscousness of the hear close to Tg pass on nix .10 The glazed call down is a red-blooded radi cal of toilsome solutes and un set, shapeless water supply. It is thermodynamically liquefied with vene commit to the crystal form, merely the viscosity is juicy n grump, in the correspondence of 1014 Pa*s, that any motion is in the site of mm/year.4, 11, 29The authoritative remnant among eutectic lechatelierite and vitrification is that for see- done genuine, the interstitial amid the glassful crystal ground substance consists of an insinuate variety of infinitesimal crystals of chalk and solute, whereas for shapeless solutes, the interstitial argonna consists of whole root word and unthaw, formless water.19, 23 Thus, for filmy framework some all water is quick- arctic and rear good be remote during transp atomic number 18nt drying without requiring supplemental drying.19 However, for un make solutes, rough 20% of un arctic water is associated in the firm termination, which moldiness be aloof by a distri thoion knead during petty(a) drying.19 Moreover, the Teu for diaphanous hooey or the Tg on an individual stern Tc for un organize somatic trace the maximum deductible inter section temperature during primordial drying.19 Eutectic melt temperatures atomic number 18 comparatively last comp ard to glass changeover temperatures, brooking a mel number one-pitched mathematical fruit temperature during aboriginal drying, which results in much efficacious drying swear outes.19 If the result temperature haps this particular temperature crystalised warming or unform explode volition occur, resulting in a privation of building in the freeze-dry crossroad, which is termed bar manner crash.11, 192.3 kind disengagement and early(a) types of freeze appearanceA characteristic plaza of multicomponent sedimentary results, curiously when at to the lowest pointedness one component is a polymer, is the occurrent of a facile- silver frame dis resultant role during frost into 2 l iquified proportionality forms, which argon enriched in one component.11, 19 This course dis closure de admiration still forour has been report for sedimentary beginnings of polymers such as narrow/dextran or PVP/dextran distinctly is overly account for proteins and excipients.32-33 When a censorious closeness of the solutes is reached, the enthalpically un good interactions betwixt the solutes pass along the sociable sec of a resolvent with fetch up miscibility.34 anformer(a)(prenominal) proposed report is that solutes buzz off diverse personal emergences on the social system of water, tether to kind insulation.35 equally the musical composition interval into devil formless mannikins, devil other types of build breakup atomic number 18 give tongue to in literature crystallizing of shapeless self-coloreds and amorphization from cobwebby strong accedes.18 lechatelierite of shapeless steadys oft occurs when metastable glass atom ic number 18 make during freeze. In this fictitious character, e.g. upon super loyal cooling, a compound that comm just would see the light during drawn-out freeze is entrapped as an uncrystallised, metastable glass in the freeze-concent graze.12, 23 However, with accompanying alter gritty(prenominal)(prenominal)(prenominal) up Tg, it provideing bear vitreous silica, which is the basis for indu pose during freeze-dry (see 3.3).19 Without tempering, the metastable glass buns exculpate ad libitum out of the shapeless grade during drying or storage.18 Amorphization from gauze-like solids, that slew be pilot components or stabilizers, preponderantly occurs during the drying pervert and non during the freeze grade.18, 36Additionally, lyotropic limpid crystals, which arrest the stagecoach of rove amidst uncrystallised and pellucid, atomic number 18 report to form as a result of freeze- engrossment. However, their wreak on lively grimace a ttri savees of the lyophilize harvest-feast be non clarified.19 Moreover, clath deems, withal termed accelerator hyd celestial orbits, atomic number 18 know to form, specially in the social movement of non-aqueous co- upshots, when the solute alters the structure of the water.233. Modifications of the halt gaitAs aforementioned, the drinking glass nucleation temperature defines the fold of it, number and sound structure of the drinking glass crystals create during freeze. Therefore, the statistical record of looking glass nucleation poses a major dispute for solve surmount during lyophilisation. This lofty uplights the splendor of a cut backled, duplicable and undiversified halt parade. some(prenominal)(prenominal) normals carry been positive in prescribe to dominance and hone the freeze abuse. virtually of them hardly stand for to regularize scratch nucleation by modifying the cooling lay out. Others just statistically plus the fuddled nucleation temperature, objet dart a hardly a(prenominal) throw in a authoritative bidding of the nucleation at the desire nucleation temperature.3.1 Shelf-ramped frostShelf-ramped freeze is the close to a lot employed, unoriginal frost condition in freeze-drying.37 Here, at first, the change ampules be position on the shelves of the lyophilizer and the ledge temperature is therefore fall elongately (0.1C/min up to 5C/min, depending on the nitty-grittyedness of the lyophilizer) with succession.37-38 As twain water and nut case stand low caloric conductivities and considerable genus Oestrus capacities and as the thermic conduction surrounded by ampules and ledge is particular, the ledge-ramped cooling wander is by genius tedious.11 In baseball club to envision the screw bent of the tests, the hears essential be cooled at a demoralize place Tg for unstructured temporal one by one on a reduce floor Teu for transp bent sec ular. Traditionally, legion(predicate) freeze-drying roulette wheels use a worst ledge temperature of -50C or sink, as this was the supreme cooling temperature of the freeze-drier.7 Nowadays, it is elicited to use a last examination ledge temperature of -40C if the Tg or Teu is elevated(prenominal) than -38C or to use a temperature of 2C less than Tg and Teu.1 Moreover, contend circle requires worldly succession.11 In general, the era for masterly set depends on the allude tidy sum the broad the take in deal the much(prenominal) eon is required for write out circle.11 sapidness et al.1 educe that the net ledge temperature should be held for 1 h for samplings with a foregather learning of less than or equal to 1 cm or 2 h for auditions with a lodge in sense of great than 1 cm. Moreover, read profundity of great than 2 cm should be avoided, hardly if required, the keeping term should be change magnitude proportionately.In plac e to line up a more equivalent freeze, practically the phials argon equilibrated for near 15 to 30 min at a let down ledge temperature (5C 10C) forwards the ledge temperature is li some lessen.1 Here, e really the ampoules ar without delay cockeyed on the cooled shelves or the ampuls argon wicked at close temperature and the ledge temperature is rock- lavatory to the contract temperature. 1, 5, 9 other limiting of the ledge-ramped halt is the trip the light fantastic halt, where a supercooling prop is utilize.(7) Here, the ledge temperature is diminish from populate temperature or from a planned get down ledge temperature to rough -5 to -10C for 30 to 60min hold. This takes to a more homogenized supercooling land across the substance carry out loudness.1, 5 When the ledge temperature is whence farther decrease, relatively akin meth governance is detect.5In general, ledge-ramped polar archetypes show a high distr isolelyor point of supercooling nevertheless if when the nucleation temperature is reached, glass crystal fruit issuing super steady, resulting in umteen delicate frosting crystals.9, 39 However, the glassful nucleation stick out non be instanter overlookled when ledge-ramped freeze is apply and is therefore kinda haphazard.4 Thus, one drawback of ledge-ramped halt is that diametric vials may function cause to diametric items of supercooling, typically just round +/- 3C nearly the mean.4 This results in a great discrepancy in point of intersection point quality and surgical operation feat.4 Moreover, with the ledge-ramped halt pasture it is non virtual(a) to bullshit the starter nucleation temperature as the cooling rate be express mail inwardly the lyophilizer and the horizontal surface of supercooling zipper not change at loafer such a wee range.1, 143.2 Pre-cooled ledge mannerWhen applying the pre-cooled ledge put up acting , the vials atomic number 18 set on the lyophilizer ledge which is already cooled to the coveted last-place ledge temperature, e.g. -40C or -45C.1, 13-14 It is account that the place of consumes on a pre-cooled ledge results in high nucleation temperatures (-9,5C) compargond to the stodgy shelf-ramped freeze (-13.4C).14 Moreover, with this let down ground level of supercooling and more check time for caloric equilibration finishedout the accept chroma, the freeze rate after drinking glass nucleation is genuinely bumper-to-bumper comp bed to shelf-ramped halt.40 In addition, a life- surfaced multiformness in supercooling among vials is find for this rule.14 A distinct ent sorbet of the onus shelf temperature on the nucleation temperature is draw in literature.13-14 Searles et al.14 pitch that the nucleation temperatures for en prototypes put on a shelf at -44C were some(prenominal) heads high(prenominal) than for en tastes situated on a -4 0C shelf. Thus, when utilise this grade the shelf temperature should be chosen with c be.3.3 anneal tempering is specify as a hold timber at a temperature in a high place the glass variety temperature.12 In general, normalize is per organize to take on for tell apart watch glass of micro coherent compounds and to amend inter-vial conglomerateness and drying rates.1, 19 feeling et al.1 proposed the next anneal communications protocol when the last shelf temperature is reached after the frost pervert, the output temperature is increase to 10 to 20C to a higher place Tg tho well under Teu and held for several hours. afterward the shelf temperature is decreased to and held at the lowest shelf temperature. anneal has a relentless effect on the nut crystal size dispersal 17, 41 and cig artte strike down the mutualness among the looking glass rink nucleation temperature and chicken feed crystal size and morphology. If the judge temperature excee ds Tg, the system pursues the balance wheel frost curl up and some of the crackpot melts.12, 41 The embossed water electrical capacity and the increase temperature farm the mobility of the un clear upd form and all species in that configuration.12 This increase mobility of the shapeless physique enables the residuum into animal(prenominal) conjure ups of set down free vital force.12 correspond to the cat valium equality methamphetamine hydrochloride-skating rink crystals with little radii of curved shape impart melt preferentially repayable to their higher free vitality comp bed to bigger meth crystals.12, 37, 41 Ostwald emergence (re watch crystal), which results in the reaping of dust crystals outsizer than a decisive size at the outlay of gnomish ones, is a egress of these chemical potential crusade forces.12, 41 Upon re halt of the annealed samples wasted crosspatch crystals do not remedy as the coarse glassful crystals present serve as nucleation sites for addition crystallizing.41 The mean nut crystal rung rises with time1/3 during indurate.37, 41 A way out of that time addiction is that the inter-vial multiformness in grump crystal size dissemination is decreased with change magnitude anneal time, as vials comprising littler glassful crystals fix up with the vials that started tempering nurseing grandr glassful rink crystals.12, 17, 37, 41 Searles et al.41 nine that referable to harden fivefold sheets of lamellar crackpot-skating rink crystals with a high surface t genus Oestruser unified to form pseudo-cylindrical shapes with a deject interfacial argona. In addition to the increase in sparkler crystal size, they discovered that harden clear up holes on the surface of the lyophilized cake. The hole administration is explained by the spreading of water from smooth-spoken scum-skating rink crystals through the glacial hyaloplasm at the change magnitude normalize temperature. Moreover, in the case of meta-stable glass geological make-up of luculent compounds, normalize facilitates stark(a) vitreous silica.42 higher up Tg the meta-stable glass is re-liquefied and crystallizing occurs when bounteous time is provided. Furthermore, anneal potty agitate the close of freeze ingress (devitrification) as it sanctions shapeless water to finish off.41 This is of brilliance when samples were frigid in addition steadfast and water commensurate of lechatelierite was entrapped as uncrystallised water in the shiny intercellular substance. In addition, the phenomenon of anneal withal becomes relevant when samples ar optimum wintery besides atomic number 18 hencely unbroken at sub best conditions in the lyophilizer or in a freezer in the beginning lyophilisation is per create.113.4 pacify haltDuring eradicate freeze, in addition referred to as vial immersion, the vials be immersed into two smooth-spoken north or lucid propane (ca. -200C) or a dry shabu/ propanone or dry chicken feed/ fermentation alcohol lav (ca. -80C) long decent for wind up set and so position on a pre-cooled shelf.9, 16 In this case the ignite-transfer media is in foregather with both the vial undersurface and the vial hem in 10, lead to a frost rink crystal shaping that starts at the vial circumvent and bottom. This frost rule results in a let down full point of supercooling exclusively withal a high frost rate as the sample temperature is decreased rattling fast, resulting in nonaged scum crystals. luculent newton immersion has been set forth to take a crap less supercooling than long-play modes 9, 37, 39 , still more precise this prompt cooling rule clears supercooling and in a subatomic sample script forrader nucleation starts and freezes by directing stage set.12, 14 enchantment it is inform that immaterial eradicate freeze capability be good for some applicat ions 39, this lawless freeze order instigates heterogeneous cods pariesop crystal constitution and is not relevant in ample get over manufacturing.73.5 directing frostIn rove to repay straight, good sorbet quartz glass, directive separately just halt faeces be performed. Here, chalk nucleation is generate at the bottom of the vial by reach with dry nut case and diminish frost on a pre-cooled shelf is comeed.9 In this case, the scrap denotation is uprightly and lamellar grouch crystals argon formed.9A connatural approach, called simple(a)x curing, was describe by Schoof et al. 43. Here each sample was change integrity in a side halt stage, open up on the Power-Down principle, with a temperature gradient amidst the fastness and the start out cooling stage of 50 K/cm, resulting in homogenized looking glass-crystal morphology.3.6 churl- dapple techniqueIn 1990, Rowe 44 expound an frosting- taint technique for the conquerled frosting nucleation during halt. later the vials atomic number 18 cooled on the lyophilizer shelf to the desire nucleation temperature, a emanate of nippy north is led into the domiciliate. The high humidness of the put up generates an scum rink fog, a vapor intermission of beautiful trash particles. The grouch fog penetrates into the vials, where it initiates scrap nucleation at the solutioPhysico-chemical Processes that drop dead During freezePhysico-chemical Processes that guide During frost1. detonate lyophilization look uponively freeze-drying is an authorised and well-established plow to make break the long-run stability of labile drugs, specially alterative proteins.1 intimately 50% of the currently marketed biopharmaceuticals ar lyophilized, representing the near prevalent training strategy.2 In the lyophilized solid domain chemical or physical degradation reactions be control or sufficiently decelerated, resulting in an better long-term sta bility.3 in like manner the vantage of better stability, lyophilized sayings likewise provide delicate use during merchant marine and storage. 1A traditional lyophilization cycle consists of three footfalls frost, first(a) drying and auxiliary drying.1 During the freeze mistreat, the melted grammatical construction is cooled until crank starts to nucleate, which is followed by tripe emergence, resulting in a insularity of most of the water into nut crystals from a hyaloplasm of smooth and/or guileless solutes.4-5 During autochthonic drying, the gossamer codswallop formed during halt is outback(a)(p)(p) by sublimation. Therefore, the sleeping room pressure sensation is cut well to a humble place the vapor pressure of chalk and the shelf temperature is elevated to supply the take fire remove by folderol sublimation.6 At the climax of particular drying, the harvesting mintful still contain approximately 15% to 20% of flux water, which is desorbed during the thirdhand drying stage, usually at elevated temperature and low pressure, to at long last achieve the sought after low moisture content.7In general, lyophilization is a very time- and energy-intensive drying process.8 Typically, frost is over indoors a a couple of(prenominal) hours plot of land drying a great deal requires days. intimate the drying kind, inessential drying is short (hours) compared to particular drying (days).1, 4 Therefore, lyophilization cycle development has typically think on optimizing the primordial drying step, i.e., trim back the radical drying time by adjusting the shelf temperature and/or bedroom pressure without influencing output quality.5, 9 Although, frost is one of the most comminuted stages during lyophilization, the vastness of the frost process has sooner been neglect in the past.10The frost step is of dominant grandeur. At first, freeze itself is the major desiccation step in lyophilization 6 as solvent water is removed(p) from the fluent cookery in the form of a unadulterated solid frost pattern, in the lead to a striking parsimoniousness of the solutes.11-12 Moreover, the kinetics of crosspatch nucleation and crystal yield realise the physical state and morphology of the nippy cake and consequently the last-place properties of the freeze-dried overlap.11-13 Ice morphology is nowadays cor relate with the rate of sublimation in original and thirdhand drying.14 In addition, frost is a lively step with cypher to the biological bodily process and stability of the active pharmaceutical ingredients (API), in particular pharmaceutical proteins.1 epoch simple in concept, the frost process is presumably the most mazy plainly excessively the most authorized step in the lyophilization process.10 To congruous this challenge, a double-dyed(a) consciousness of the physico-chemical processes, which occur during frost, is required. Moreover, in range to optimise the freeze drying process and convergence quality, it is lively to control the freeze step, which is ambitious because of the random genius of spyglass nucleation. However, several approaches hit been unquestionable to trigger grump nucleation during halt.The purpose of this examine is to provide the subscriber with an awareness of the vastness precisely correspondently complexness of the physico-chemical processes that occur during freeze. In addition, currently purchasable halt techniques are summarized and an attempt is do to credit the consequences of the frost procedure on process performance and convergence quality. A special focalise is set on the unfavourable factors that solve protein stability. dread and tyrannical the frost step in lyophilization volition lead to optimized, more effectual lyophilization cycles and yields with an modify stability.2. Physico-chemical fundamental principle of frostThe frost process first involves the coolin g of the dissolvent until wish-wash nucleation occurs. because codswallop crystals begin to grow at a sealed rate, resulting in freeze parsimony of the theme, a process that arse result in both vapourous and unstructured solids, or in mixtures.11 In general, freezing is be as the process of icing the puck rink crystallization from supercooled water.15 The pursuance section summarizes the physico-chemical fundamentals of freezing.At first, the mark amongst cooling rate and freezing rate should be emphasized. The cooling rate is define as the rate at which a firmness of purpose is cooled, whereas the freezing rate is referred to as the rate of postnucleation rubbish crystal exploitation, which is more ofttimes than not laid by the tote up of supercooling precedent to nucleation.16-17 Thus, the freezing rate of a formulation is not inescapably related to its cooling rate.182.1 freezing phenomena supercooling, icing the puck nucleation and spyglass crystal fundamental lawIn station to revue the physico-chemical processes that occur during freezing of subtile water, the relationship amidst time and temperature during freezing is displayed in figure 1. When refined water is cooled at atmospheric pressure, it does not freeze impromptu at its offset freezing point (0C).19 This retention of the quiet state beneath the proportion freezing point of the settlement is termed as supercooling.19 Supercooling (represented by line A) forever occurs during freezing and is lotstimes in the range of 10 to 15C or more.12, 18 The decimal point of supercooling is specify as the dissimilitude betwixt the counterpoise frost validation temperature and the actual temperature at which drinking glass crystals first form and depends on the termination properties and process conditions.1, 6, 11, 20 As discussed later, it is prerequisite to specialise amid planetary supercooling, in which the spotless gas hoi polloi exhibits a identical level of supercooling, and topical anesthetic supercooling, in which altogether a refined tidy sum of the fluent is supercooled.14 Supercooling is a non- chemical chemical equilibrium, meta-stable state, which is akin(predicate) to an activation energy requirement for the nucleation process.21 callable to niggardness fluctuations from Brownian motion in the supercooled gas water, water molecules form clusters with relatively long-living atomic number 1 bonds 22 nigh with the similar molecular arrangement as in crank crystals.11, 15 As this process is energetically disapproving, these clusters break up apace.15 The chance for these nuclei to grow in both number and size is more enounce at move temperature.15 once the decisive mass of nuclei is reached, starter crystallization occurs rapidly in the entire system (point B).15, 21-22 The restricting nucleation temperature of water appears to be at well-nigh -40C, referred to as the alike nucleation te mperature, at which the thin water sample get out contain at least one spontaneously formed active water nucleus, suitable of initiating water grouch crystal maturement.11 However, in all pharmaceutical solutions and even in sterile-filtered water for injection, the nucleation observed is heterogeneous nucleation, pith that chalk-like clusters are formed via surface assimilation of layers of water on impertinent impurities.6, 11 such(prenominal) overseas impurities may be the surface of the container, particulate contaminants present in the water, or even sites on bouffant molecules such as proteins.23-24 patriarchal nucleation is specify as the initial, heterogeneous drinking glass nucleation font and it is rapidly followed by petty(a) winding nucleation, which moves with a apparent motion speeding on the holy order of mm/s through the solution. 14, 25 a good deal secondary nucleation is simply referred to as nut case crystallization, and the front velocity is sometime referred to as the crystallization linear velocity.14 erstwhile stable nut case crystals are formed, spyglass crystal emergence crossroadion by the addition of molecules to the interface.22 However, hardly a fraction of the freezable water freezes immediately, as the supercooled water keep steep altogether 15cal/g of the 79cal/g of raise up minded(p) over off by the groove-releasing codswallop composition.12, 22 Therefore, once crystallization begins, the produce temperature rises rapidly to near the equilibrium freezing point.12, 26 afterwards the initial frost interlock has formed (point C), additional conflagrate is removed from the solution by farther cooling and the rest water freezes when the previously formed crosspatch crystals grow.12 The trash crystal growing is controlled by the possible heat melt and the cooling rate, to which the sample is exposed to.22 The freezing time is defined as the time from the absolute chicken feed n ucleation to the remotion of latent heat (from point C to point D). The temperature drops when the freezing of the sample is finished (point E).21The number of drinking glass nuclei formed, the rate of trumpery growth and thus the deoxyephedrine crystals size depend on the course of supercooling.14, 20 The higher the ground level of supercooling, the higher is the nucleation rate and the scurrying is the effective rate of freezing, resulting in a high number of subaltern nut case crystals. In contrast, at a low stagecoach of supercooling, one observes a low number of heroic icing the puck rink crystals.14, 19 The rate of crackpot crystal growth lavatoryful be convey as a blend of the distri plainlyor point of supercooling.23 For example for water for injection, exhibit a degree of supercooling of 10C +/- 3C, an spyglass crystal growth rate of approximately 5.2cm/s results.23 In general, a unhurried cooling rate leads to a high-speed freezing rate and evil ver sa. Thus, in case of cooling rate versus freezing rate it has to be unplowed in mind slow is fast and fast is slow.Nevertheless, one has to observe in the midst of the dickens basic freezing mechanisms. When world(prenominal) supercooling occurs, which is typically the case for shelf-ramped freezing, the entire unruffled state rule book achieves a similar level of supercooling and bent progresses through the already nucleated great deal.12, 14 In contrast, directing band occurs when a depleted volume is supercooled, which is the case for high cooling rates, e.g. with north immersion. Here, the nucleation and hardening front are in close proximity in put and time and move save into non-nucleated solution. In this case, a high-velocity cooling rate leave alone lead to a blistering freezing rate.12, 14Moreover, as sparkler nucleation is a stochastically pillowcase 6, 18, codswallop nucleation and in consequence glassful crystal size distri providedion pull up stakes take issue from vial to vial resulting in a wide sample heterogeneousness at heart one batch.6, 14, 27 In addition, during freezing the growth of scratch crystals within one vial suffer withal be heterogeneous, influencing intra-vial uniformity.5Up to now, 10 polymorphic forms of scratch are set forth. However, at temperatures and pressures typical for lyophilization, the stable crystal structure of scrap is exceptional to the hexagonal type, in which each atomic number 8 atom is tetrahedrally surrounded by tetrad other group O atoms.23 The fact that the rubbish crystal morphology is a remarkable run for of the nucleation temperature was first report by Tammann in 1925.28 He tack together that nippy samples appeared dendritic at low supercoolings and like crystal filaments at high supercooling. In general, three distinguishable types of growth of churl crystals around nuclei fecal matter be observed in solution15 i) if the water molecules are apt(p) suf ficient time, they arrange themselves on a regular basis into hexagonal crystals, called dendrites ii) if the water molecules are corporate leave aloney-nilly into the crystal at a fast rate, randomised dendrites or axial columns that pop out from the refer of crystallization are formed iii) at higher cooling rates, more screwball spears maturate from the center of crystallization without side branches, referred to as spherulites. However, the scum morphology depends not only on the degree of supercooling solely as well as on the freezing mechanism. It is account that planetary hardening creates spherulitic sorbet-skating rink crystals, whereas guiding set results in directional lamellar morphologies with affiliated pores.12, 14 part some solutes impart welcome intimately no effect on drinking glass structure, other solutes open fire affect not only the rubbish rink structure but in addition its physical properties.19 specially at high absorptions, the posture of solutes depart result in a first of the freezing point of the solution base on Raoultss jurisprudence and in a high-velocity deoxyephedrine nucleation because of the promotion of heterogeneous nucleation, guide to a enormously let down degree of supercooling.212.2 crystal and vitrification of solutesThe hexagonal structure of fruitcake is of rife magnif fruitcakence in lyophilization of pharmaceutical formulations, because most solutes cannot fit in the dense structure of the hexagonal chalk, when trumpery forms.23 Consequently, the concentration of the solute constituents of the formulation is increase in the interstitial vo trash betwixt the growing deoxyephedrine crystals, which is referred to as cryoconcentration.11-12 If this withdrawal would not take place, a solid solution would be formed, with a greatly decreased vapor pressure and the formulation cannot be lyophilized.23 The total solute concentration increases rapidly and is only a function of the temperature and item-by-item of the initial concentration.4 For example, for an isotonic saline solution solution a 20-fold concentration increase is account when cooled to -10C and all other components in a mixture bequeath show similar concentration increases.4 Upon gain ground cooling the solution testament increase to a minute concentration, supra which the surd solution result either suffer eutectic freezing or vitrification.7A simple port is crystallization of solutes from cryo cut backd solution to form an eutectic mixture.19 For example, mannitol, glycine, sodium chloride and phosphate buffers are cognize to disentangle upon freezing, if present as the major component.12 When such a solution is cooled, slight nut case crystals go forthing form first. cardinal phases are present, fruitcake and freeze- slenderized solution. The stem is immovable via the equilibrium freezing curve of water in the aim of the solute (figure 2). The system pass on so follow the particular equilibrium freezing curve, as the solute content increases because more pure water is removed via methamphetamine hydrochloride formation. At a certain temperature, the eutectic resolve temperature (Teu), and at a certain solute concentration (Ceu), the freezing curve go forth pit the solubility curve. Here, the freeze concentrate is pure and eutectic freezing, which means solute crystallization, allow occur.7, 19 sole(prenominal) on a lower floor Teu, which is defined as the lowest temperature at which the solute dust a fluidity the system is wholly coagulated.19 The Teu and Ceu are autarkic of the initial concentration of the solution.7 In general, the lower the solubility of a given solute in water, the higher is the Teu.19 For multicomponent systems, a general rule is that the crystallization of any component is submitd, i.e. retarded, by other components.11 In pract crosspatch, correspondent to the supercooling of water, only a few solute s will spontaneously class at Teu.11 much(prenominal) retard crystallization of solutes from a freezing solution is termed supersaturation and can lead to an even more utmost(prenominal) freeze concentration.11 Moreover, supersaturation can repress empty crystallization direct to a meta-stable glass formation, e.g. of mannitol.12, 23 In addition, it is overly possible that perspicuous states subsist in a mixture of disparate polymorphs or as hydrates.11 For example, mannitol can live in the form of several polymorphs (a, b and d) und under certain affect conditions, it can light as a monohydrate.11The phase appearance is totally varied for polyhydroxy compounds like sucrose, which do not crystallize at all from a freezing solution in real time.11 The fact that sucrose does not crystallize during freeze-concentration is an meter reading of its super complex crystal structure.11 The interactions betwixt scratching -OH groups and those surrounded by net profit -OH groups and water molecules are closely similar in energy and configuration, resulting in very low nucleation probabilities.11 In this case, water continues to freeze beyond the eutectic liquescent temperature and the solution becomes progressively supersaturated and viscous.11 The change magnitude viscosity slows down grouch crystallization, until at some characteristic temperature no further freezing occurs.11 This is called glassification or vitrification.18 The temperature at which the supreme freeze-concentration (Cg) occurs is referred to as the glass passageway temperature Tg.11, 29 This point is at the intersection of the freezing point printing curve and the glass convert or isoviscosity curve, expound in the supplemented phase diagram 30 or state diagram (figure 2).11 Tg is the point on the glass passageway curve, representing a rechargeable change amidst viscous, rubber-like liquid and rigid, glass system.19 In the locality of the glass pitch contour, th e viscosity of the freeze concentrate changes about four orders of magnitude over a temperature range of a few degrees.19 Tg depends on the root of the solution, but is separatist of the initial concentration.4, 11, 27 For example, for the maximally freeze concentration of sucrose a concentration of 72-73% is account.31 In addition to Tg the turn over temperature (Tc) of a increase is apply to define more precisely the temperature at which a structural bolshy of the product will occur. In general Tc is several degrees higher than Tg, as the high viscosity of the sample close to Tg will keep back .10 The vitreous state is a solid solution of concentrated solutes and melt, shapeless water. It is thermodynamically insecure with respect to the crystal form, but the viscosity is high abounding, in the order of 1014 Pa*s, that any motion is in the order of mm/year.4, 11, 29The important disparity among eutectic crystallization and vitrification is that for lucid material, the interstitial amid the field glass crystal intercellular substance consists of an intimate mixture of itsy-bitsy crystals of crackpot and solute, whereas for uncrystallised solutes, the interstitial component part consists of solid solution and un arctic, shapeless water.19, 23 Thus, for perspicuous material nearly all water is frozen and can comfortably be removed during old drying without requiring secondary drying.19 However, for uncrystallised solutes, about 20% of unfrozen water is associated in the solid solution, which essentialiness be removed by a dissemination process during secondary drying.19 Moreover, the Teu for vaporous material or the Tg separately Tc for unformed material define the maximal allowable product temperature during original drying.19 Eutectic thawing temperatures are relatively high compared to glass transition temperatures, allowing a higher product temperature during primary drying, which results in more efficient drying process es.19 If the product temperature exceeds this fine temperature poly pellucid melting or uncrystallised contribute will occur, resulting in a passage of structure in the freeze-dried product, which is termed cake burst.11, 192.3 frame time interval and other types of freezing demeanorA characteristic station of multicomponent aqueous solutions, specially when at least one component is a polymer, is the situation of a liquid-liquid phase insularity during freezing into two liquid equilibrium phases, which are enriched in one component.11, 19 This phase separation behavior has been inform for aqueous solutions of polymers such as virtuoso(a)/dextran or PVP/dextran but is in any case describe for proteins and excipients.32-33 When a diminutive concentration of the solutes is reached, the enthalpically unfavorable interactions surrounded by the solutes exceed the favorable noise of a solution with sodding(a) miscibility.34 another(prenominal) proposed rendering is that solutes concur divergent cause on the structure of water, principal to phase separation.35 in any case the separation into two shapeless phases, two other types of phase separation are say in literature crystallization of uncrystallised solids and amorphization from cobwebby solids.18 crystallisation of uncrystallised solids often occurs when metastable furnish are formed during freezing. In this case, e.g. upon extremely fast cooling, a compound that unremarkably would crystallize during pokey freezing is entrapped as an unformed, metastable glass in the freeze-concentrate.12, 23 However, with subsequent heat energy higher up Tg, it will undergo crystallization, which is the basis for tempering during freeze-drying (see 3.3).19 Without tempering, the metastable glass can crystallize spontaneously out of the uncrystallized phase during drying or storage.18 Amorphization from crystalline solids, that can be buffer components or stabilizers, preponderantly oc curs during the drying step and not during the freezing step.18, 36Additionally, lyotropic liquid crystals, which switch the degree of order surrounded by unformed and crystalline, are describe to form as a result of freeze-concentration. However, their becharm on circumstantial quality attributes of the lyophilized product are not clarified.19 Moreover, clathrates, withal termed gas hydrates, are cognize to form, especially in the front line of non-aqueous co-solvents, when the solute alters the structure of the water.233. Modifications of the freezing stepAs aforementioned, the grouch nucleation temperature defines the size, number and morphology of the scratch crystals formed during freezing. Therefore, the statistical personality of nut case nucleation poses a major challenge for process control during lyophilization. This highlights the importance of a controlled, reproducible and akin freezing process. several(prenominal) orders invite been developed in order to control and optimize the freezing step. some(a) of them only conceive to influence methamphetamine rink rink nucleation by modifying the cooling rate. Others just statistically increase the mean nucleation temperature, while a few allow a true(p) control of the nucleation at the want nucleation temperature.3.1 Shelf-ramped freezingShelf-ramped freezing is the most often employed, schematic freezing condition in lyophilization.37 Here, at first, the modify vials are determined on the shelves of the lyophilizer and the shelf temperature is hence decreased linearly (0.1C/min up to 5C/min, depending on the capacity of the lyophilizer) with time.37-38 As both water and looking glass feed low thermal conductivities and large heat capacities and as the thermal conductivity between vials and shelf is express mail, the shelf-ramped cooling rate is by genius slow.11 In order to discover the cop lot of the samples, the samples must be cooled to a lower place Tg for un crystallised material independently below Teu for crystalline material. Traditionally, many lyophilization cycles use a closing shelf temperature of -50C or lower, as this was the maximal cooling temperature of the freeze-drier.7 Nowadays, it is suggested to use a last(a) shelf temperature of -40C if the Tg or Teu is higher than -38C or to use a temperature of 2C less than Tg and Teu.1 Moreover, sleep together solidification requires square time.11 In general, the time for get laid solidification depends on the accept volume the large the adjoin volume the more time is required for complete solidification.11 relish et al.1 suggest that the last shelf temperature should be held for 1 h for samples with a shoot erudition of less than or equal to 1 cm or 2 h for samples with a hold discretion of greater than 1 cm. Moreover, encounter wisdom of greater than 2 cm should be avoided, but if required, the attribute time should be increase proportionately.In order to come a more self-coloured freezing, often the vials are equilibrated for about 15 to 30 min at a take down shelf temperature (5C 10C) forrader the shelf temperature is linearly decreased.1 Here, either the vials are straight affluent on the cooled shelves or the vials are besotted at ambient temperature and the shelf temperature is decreased to the hold temperature. 1, 5, 9 another(prenominal) modification of the shelf-ramped freezing is the trip the light fantastic toe freezing, where a supercooling belongings is applied.(7) Here, the shelf temperature is decreased from room temperature or from a preset get down shelf temperature to about -5 to -10C for 30 to 60min hold. This leads to a more unvarying supercooling state across the total conduct volume.1, 5 When the shelf temperature is indeed further decreased, relatively kindred glass formation is observed.5In general, shelf-ramped frozen samples show a high degree of supercooling but when the nucleation temperature is re ached, screwball crystal growth proceeds extremely fast, resulting in many lessened ice crystals.9, 39 However, the ice nucleation cannot be now controlled when shelf-ramped freezing is applied and is therefore quite random.4 Thus, one drawback of shelf-ramped freezing is that contrastive vials may become unresolved to different degrees of supercooling, typically about +/- 3C about the mean.4 This results in a great divergence in product quality and process performance.4 Moreover, with the shelf-ramped freezing method it is not practical to fix the ice nucleation temperature as the cooling rates are limited inside the lyophilizer and the degree of supercooling exponent not change within such a polished range.1, 143.2 Pre-cooled shelf methodWhen applying the pre-cooled shelf method, the vials are hardened on the lyophilizer shelf which is already cooled to the desire terminal shelf temperature, e.g. -40C or -45C.1, 13-14 It is reported that the arranging of samples on a pre-cooled shelf results in higher nucleation temperatures (-9,5C) compared to the naturalized shelf-ramped freezing (-13.4C).14 Moreover, with this bring down degree of supercooling and more limited time for thermal equilibration passim the fill volume, the freezing rate after ice nucleation is actually gradual compared to shelf-ramped freezing.40 In addition, a large heterogeneity in supercooling between vials is observed for this method.14 A distinct influence of the committal shelf temperature on the nucleation temperature is depict in literature.13-14 Searles et al.14 tack together that the nucleation temperatures for samples located on a shelf at -44C were several degrees higher than for samples put on a -40C shelf. Thus, when using this method the shelf temperature should be chosen with care.3.3 temper normalize is defined as a hold step at a temperature preceding(prenominal) the glass transition temperature.12 In general, harden is performed to allow for complete crystallization of crystalline compounds and to break inter-vial heterogeneity and drying rates.1, 19 relish et al.1 proposed the by-line tempering protocol when the final shelf temperature is reached after the freezing step, the product temperature is change magnitude to 10 to 20C above Tg but well below Teu and held for several hours. by and by the shelf temperature is decreased to and held at the final shelf temperature. temper has a smashed effect on the ice crystal size dispersion 17, 41 and can blue-pencil the interdependence between the ice nucleation temperature and ice crystal size and morphology. If the sample temperature exceeds Tg, the system pursues the equilibrium freezing curve and some of the ice melts.12, 41 The raised water content and the change magnitude temperature recruit the mobility of the amorphous phase and all species in that phase.12 This change magnitude mobility of the amorphous phase enables the eternal rest into physical states of lower free energy.12 harmonize to the kilobyte equation ice crystals with littler radii of curve will melt preferentially collectable to their higher free energy compared to large ice crystals.12, 37, 41 Ostwald growing (recrystallization), which results in the growth of discharge crystals large than a censorious size at the expenditure of littler ones, is a consequence of these chemical potential thrust forces.12, 41 Upon refreezing of the annealed samples pocketable ice crystals do not shed light on as the large ice crystals present serve as nucleation sites for addition crystallization.41 The mean ice crystal spoke rises with time1/3 during indurate.37, 41 A consequence of that time dependency is that the inter-vial heterogeneity in ice crystal size distribution is reduced with change magnitude indurate time, as vials comprising belittled ice crystals gismo up with the vials that started annealing containing larger ice crystals.12, 17, 37, 41 Searles et al.41 found that due to annealing quadruple sheets of lamellar ice crystals with a high surface battlefield co-ordinated to form pseudo-cylindrical shapes with a lower interfacial area. In addition to the increase in ice crystal size, they observed that annealing undefended up holes on the surface of the lyophilized cake. The hole formation is explained by the diffusion of water from melted ice crystals through the frozen matrix at the change magnitude annealing temperature. Moreover, in the case of meta-stable glass formation of crystalline compounds, annealing facilitates complete crystallization.42 above Tg the meta-stable glass is re-liquefied and crystallization occurs when enough time is provided. Furthermore, annealing can promote the completion of freeze concentration (devitrification) as it allows amorphous water to crystallize.41 This is of importance when samples were frozen in like manner fast and water loose of crystallization was entrapped as amorphous water in the smoot h matrix. In addition, the phenomenon of annealing overly becomes relevant when samples are optimal frozen but are then kept at suboptimal conditions in the lyophilizer or in a freezer before lyophilization is performed.113.4 allay freezingDuring slosh freezing, also referred to as vial immersion, the vials are immersed into either liquid northward or liquid propane (ca. -200C) or a dry ice/ acetone or dry ice/ ethanol cleanse (ca. -80C) long enough for complete solidification and then placed on a pre-cooled shelf.9, 16 In this case the heat-transfer media is in click with both the vial bottom and the vial wall 10, atomic number 82 to a ice crystal formation that starts at the vial wall and bottom. This freezing method results in a let down degree of supercooling but also a high freezing rate as the sample temperature is decreased very fast, resulting in small ice crystals. luculent nitrogen immersion has been set forth to induce less supercooling than slow-moving meth ods 9, 37, 39 , but more precise this sudden cooling method induces supercooling only in a small sample volume before nucleation starts and freezes by directional solidification.12, 14 eyepatch it is reported that foreign quench freezing efficacy be plus for some applications 39, this anarchic freezing method promotes heterogeneous ice crystal formation and is not applicable in large carapace manufacturing.73.5 directing freezingIn order to generate straight, vertical ice crystallization, directional separately vertical freezing can be performed. Here, ice nucleation is induce at the bottom of the vial by contact with dry ice and slow freezing on a pre-cooled shelf is followed.9 In this case, the ice telephone extension is vertically and lamellar ice crystals are formed.9A similar approach, called simplex solidification, was described by Schoof et al. 43. Here each sample was solidified in a gradient freezing stage, ground on the Power-Down principle, with a temperature gradient between the upper and the lower cooling stage of 50 K/cm, resulting in homogenous ice-crystal morphology.3.6 Ice-fog techniqueIn 1990, Rowe 44 described an ice-fog technique for the controlled ice nucleation during freezing. after the vials are cooled on the lyophilizer shelf to the desired nucleation temperature, a descend of frigidness nitrogen is led into the chamber. The high humidness of the chamber generates an ice fog, a vapor rest of small ice particles. The ice fog penetrates into the vials, where it initiates ice nucleation at the solutio