The impact of protein concentration on mannitol and sodium chloride crystallinity and polymorphism...

The Impact of Protein Concentration on Mannitol andSodium Chloride Crystallinity and PolymorphismUpon Lyophilization

DANIEL DIXON, SERGUEI TCHESSALOV, ANTHONY BARRY, NICHOLAS WARNE

Wyeth BioPharma, Drug Product Development, 1 Burtt Rd, Andover, Massachusetts 01810

Received 10 June 2008; revised 10 July 2008; accepted 21 July 2008

Published online 9 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21537

CorrespondeFax: 978-247-42

Journal of Pharm

� 2008 Wiley-Liss

ABSTRACT: The effect of protein concentration, in the range of 0–26 mg/mL for two Fc-fusion proteins, on the crystallinity and polymorphism of mannitol and sodium chloridein a lyophilized model formulation was examined. Mannitol hydrate levels were quan-tified based on moisture data and correlated to the X-ray diffraction peak area. In allformulation conditions, sodium chloride did not crystallize in samples with >44% totalamorphous content. As protein concentration increased through the range of 1–5 mg/mLprior to lyophilization, b-mannitol decreased in amount, becoming undetectable atprotein concentrations above 5 mg/mL. Conversely, d-mannitol increased as a functionof protein concentration, reaching a maximum level at �5 mg/mL protein. Above 10 mg/mL protein, mannitol crystallization was increasingly inhibited. Sucrose control vialsshowed higher levels of mannitol hydrate than either model protein. Both proteinsbehaved comparably with respect to mannitol crystallinity and polymorphism despitesignificant differences in molecular weight. Because of the differences between proteinand sucrose control samples, protein concentration must be taken into considerationwhen assessing the lyophilization of mannitol containing solutions. � 2008 Wiley-Liss, Inc.

and the American Pharmacists Association J Pharm Sci 98:3419–3429, 2009

Keywords: lyophilization; polymorphis
m; excipients; freeze-drying; protein formula-tion; X-ray powder diffractometry
INTRODUCTION

Mannitol is commonly used in lyophilized phar-maceutical formulations, frequently as a crystal-lizing bulking agent.1,2 Depending on formulationcomposition and lyophilization conditions, man-nitol can crystallize in any one of four differentpolymorphs (a, b, d and hydrate), or remainamorphous.3–9 Much of the focus of mannitolresearch has been on the meta-stable hemi-hydrate polymorph, which may convert to ananhydrous form during storage, releasing water

nce to: Daniel Dixon (Telephone: 978-247-4262;22; E-mail: [email protected])

aceutical Sciences, Vol. 98, 3419–3429 (2009)

, Inc. and the American Pharmacists Association

JOURNAL OF PHA

into the product. Sodium chloride, at elevatedlevels, also crystallizes during lyophilization andhas been used as a bulking agent in Factor VIIIformulations.10 Typically a crystalline bulkingagent serves several purposes—(1) To increasethe mass of the cake, (2) to aid in sublimation andreconstitution by providing a crystalline scaffoldfor the amorphous phase to dry upon,11 and (3) toaid in formulation tonicity.12 The impact offormulation components on the crystallinity andpolymorphism of mannitol has been investigatedpreviously.13–23 With the elevated protein contentrequired for many therapeutic proteins to reachefficacious dose levels, the protein constitutes asubstantial portion of the dried cake, accountingfor a majority of the solid content in some cases.Therefore, the protein, particularly at high con-

RMACEUTICAL SCIENCES, VOL. 98, NO. 9, SEPTEMBER 2009 3419

3420 DIXON ET AL.

centration, may influence the overall properties ofthe formulation.

Process conditions during lyophilization havebeen shown to impact crystallization and poly-morphic behavior of mannitol. The freezing step ofmannitol containing solutions has been exten-sively investigated as a source of polymorph-ism.5,7,14,21,24,25 Mannitol has been identified tocrystallize below the glass transition temperatureðT0

gÞ under certain conditions, and during the pri-mary drying stage of lyophilization in thepresence of a noncrystallizing sugar.21 Mannitolhydrate levels decreased with increasing anneal-ing temperature from �30 to �208C in a mannitol/sucrose/sodium chloride formulation.14

Formulation components also alter the kineticsand extent of crystallization of mannitol. Sodiumchloride slowed the crystallization of mannitol atmoderate concentrations, inhibiting it all togetherat higher concentration.7 Polyvinylpirrolidone(PVP) and buffer salts have also been demon-strated to hinder mannitol crystallization.24

Mannitol crystallized at mass fractions greaterthan 50% from binary mixtures with sucrose inone study16 or at mass fractions greater thanabout 30% with either disaccharides or lyso-zyme.17 These studies have demonstrated thatformulation excipients impact mannitol crystal-lization behavior. Clearly, at sufficient concentra-tions, protein would be expected to also impactmannitol crystallization.

During the scale-up of a lyophilization processfrom development scale to clinical or commercialmanufacturing scale and early commercial pro-cess validation work, material requirements maybe prohibitive for using the actual drug in aformulation to assess cycle scale-up, transfer, andexecute seeded stability studies. As a result,buffer runs are often used as a substitute, someof which may be seeded with protein containingvials. Often, these runs employ the drug formula-tion with the omission of the active protein forlyophilization. The information gleaned fromthese early runs is used to establish the lyophi-lization cycle for generating either clinical mate-rial or later validation batches, leading tocommercial conformance batches. Understandingthe impact of the protein on the product perfor-mance during lyophilization and the resultingproduct physical characteristics is critical toassessing the success and suitability of thesestudies.

Increasing protein concentration leads to anincreased contribution of protein to the physical

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 9, SEPTEMBER 2009

behavior of a formulation. This may include, but isnot limited to, the ability and rate of excipientssuch as mannitol to crystallize, the polymorphdistribution of crystalline phases, the pore sizes ofthe lyophilized cakes, the ease of reconstitution ofthe dried formulation, and the stability of theprotein in that particular formulation. At lowerprotein concentrations, the presence of proteinmay have less impact on the crystallization ofother formulation components, thus placebo vialscan be operationally representative of productcontaining active drug. At sufficiently highprotein concentrations, mannitol crystallizationmay be inhibited entirely. This study seeks toexamine the impact of 0–26 mg/mL of two fusionproteins on the crystallization attributes of amannitol and sodium chloride containing formu-lation.

MATERIALS AND METHODS

Materials

The proteins Soluble Interleukin-13 Receptor(sIL-13r) and P-Selectin Glycoprotein Ligand(rPSGL-Ig) are both constructs designed by thefusion of a receptor to the Fc fragment of a humanIgG1 antibody. These proteins are both expressedrecombinantly in Chinese Hamster Ovary cellsand purified at Wyeth BioPharma (Andover, MA).sIL-13r contains significant glycosilation and hasan average molecular weight of 135 kDa. rPSGL-Ig is a smaller protein at 89 kDa.

Model Formulation

The pre-lyophilization composition of the baseformulation is 2% (w/v) mannitol, 1% (w/v)sucrose, 0.23% (w/v) (40 mM) sodium chloride,0.12% (w/v) (10 mM) tris(hydroxymethyl)amino-methane (Tris), 0.01% (w/v) polysorbate-80, pH7.4. All excipients are of USP grade or higher.

Variation of Protein or Amorphous Content

All vials contain the base formulation. Addition-ally, protein vials contain either rPSGL-Ig or sIL-13r at various weight fractions. As a control,sucrose is spiked on a weight basis as a surrogateto protein to yield the same total dry content ateach protein level. This control served to examinewhether observed effects were due to protein

DOI 10.1002/jps

IMPACT OF PROTEIN ON MANNITOL CRYSTALLINITY UPON LYOPHILIZATION 3421

interaction with mannitol (or sodium chloride) orsimply due to increased levels of noncrystallizingsolute. Using a protein concentration of 10 mg/mL(1% (w/v) protein) plus 1% (w/v) sucrose as anexample, the corresponding sucrose control wouldcontain 2% (w/v) sucrose. Proteins were dialyzedinto the model formulation at the highest con-centration and diluted with buffer containingformulation components to the respective targets.

X-Ray Diffraction (XRD)

A Bruker AXS D8 Advance (Madison, WI)equipped with a temperature control stage andscintillation counter detector was used for all XRDmeasurements. For powder samples, a range of2Q¼ 9–378 was scanned with a step size of 0.018,and a step time of 1 s. The mass of powder sampleswas measured after each scan and was typically inthe 10–20 mg range.

Subambient liquid XRD can identify criticaltemperatures to ensure crystallization and thepolymorphs observed.24 Data from subambientXRD can help elucidate critical process tempera-tures during lyophilization. In these studies,135 mL of buffer was dispensed into the samplepan. Liquid samples skipped the region of2Q¼ 22–308 to conserve liquid nitrogen becausehexagonal ice peaks dominate this region (datanot shown). For frozen liquid samples, the stepsize was 0.028, with a step time of 2 s.

For phase identification and quantitation,the 2Q values for the peak maximum were:d-mannitol—9.68, b-mannitol—14.78, mannitolhydrate—17.98, a-mannitol—17.38, sodiumchloride—31.88

For quantification, the amorphous hump wassubtracted from each scan as a baseline. The areasof peaks centered at the 2Q values listed abovewere then integrated for estimation of concentra-tion of that particular phase. Mannitol hydrateshows a peak at 9.68, which overlaps with theselected d-mannitol peak, that has been reportedto be approximately 80% of the size of the peak at17.98.8,26 For d-mannitol, 80% of the peak area forthe hydrate peak at 17.98 was subtracted from thed-mannitol peak.

Differential Scanning Calorimetry (DSC)

A TA Instruments Q1000 m DSC (New Castle,DE) performed all modulated DSC scans. Themethod includes: modulation amplitude of�0.58C, modulation period of 100 s, heating ramp

DOI 10.1002/jps JOURN

from 0 to 1908C at 28C/min. To minimize absorp-tion of atmospheric moisture, DSC samples wereprepared in a nitrogen-purged glove box at arelative humidity below 2%.

Thermogravimetric Analysis (TGA)

A TA Instruments 2050 TGA measured moistureby heating from 25 to 1008C at 0.58C/min, thenholding isothermal for 120 min.

Lyophilization

A Virtis Genesis 25 EL (Gardiner, NY) was usedfor producing lyophilized vials. West 5 mL USP,Type I, clear glass tubing vials were rinsed withRODI water and depyrogenated prior to use. West4432/50 S-87J stoppers were autoclaved and driedprior to use. Each vial was filled with 2 mL ofsolution and partially stoppered in a biosafetycabinet. The lyophilization cycle consisted offreezing to �508C, annealing at �158C to promotemannitol crystallization, vacuum initiation at�408C, primary drying at �288C, and secondarydrying at 258C. Primary drying was consideredcomplete as the difference between the Piranipressure and capacitance manometer was lessthan 10 mTorr. At this point, the shelf ramp wasinitiated to secondary drying. Product tempera-tures remained near �408C during primarydrying, which is below any anticipated glasstransition temperatures of the product.

Sample Handling

Samples were lyophilized according to thedescribed lyophilization cycle. For each formula-tion and protein concentration, three vials werelyophilized. The contents of each vial weresufficient for analysis by XRD, DSC, and TGA.After lyophilization, samples were stored at 2–88Cuntil analysis could be performed.

RESULTS

Subambient XRD

Crystallization of excipients can occur at severalpoints during lyophilization—(1) the initial freezedown, (2) annealing, or (3) during primary orsecondary drying. To identify critical nucleationtemperatures, subambient XRD was performed on

AL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 9, SEPTEMBER 2009

Figure 1. Subambient XRD of model formulation buffer. From bottom to top, thescans show: (1) Freeze to �508C, (2) Raise to �158C for annealing of mannitol, (3) End ofhold at �158C, (4) Refreeze to �508C, (5) Raise to �358C to anneal sodium chloride.Crystalline mannitol is first observed at the start of the �158C hold, which is verified bysubambient DSC thermograms (data not shown). Annealing at �358C does not show anycrystalline sodium chloride. Data was not collected in the range of 2Q¼ 22–308 becauseof the presence of ice peaks and to save time. All measured phases could be identified bypeaks outside of the omitted region.

3422 DIXON ET AL.

protein-free formulation buffer (Fig. 1). Theseexperiments mimicked the ramp rates, holdtemperatures, and hold times utilized duringlyophilization. Mannitol crystallization was notobserved on the initial freeze down of the buffer.The model formulation buffer was selected for thesubambient XRD studies because it containedsodium chloride and mannitol at the highestrelative content in frozen solution (after the phaseseparation of pure ice), compared to proteincontaining formulations. These high concentra-tions were assumed to be the most favorable forobservation of mannitol and sodium chloridecrystallization. During the annealing step, amixture of d-mannitol and b-mannitol wasobserved. Subsequent runs have shown the pre-sence of mannitol hydrate during the initial freezeunder the same experimental conditions (datanot shown). In other studies, mannitol hydrate b,d-mannitol have been formed during freezingmeasured in vitro using Raman spectroscopy.25,27

These observations are consistent with the randomnature of mannitol nucleation during freezing.Freezing hold temperatures and cooling rates havebeen shown to impact polymorphism of mannitol,including the presence of mannitol hydrate inmannitol/sucrose/sodium chloride systems.14,23,25

Sodium chloride crystallization can be pre-vented until after mannitol crystallizationoccurs.7 In the model formulation for this study,any eutectic sodium chloride formed during the


initial freeze would melt during the annealingstep at �158C. To examine whether sodiumchloride would crystallize after mannitol crystal-lization, a second annealing step was performed at�358C.4,7,13 Crystalline sodium chloride was notseen during this hold. As such, any crystallinesodium chloride that is observed after lyophiliza-tion is concluded to have crystallized duringdrying (either primary drying or secondarydrying).

Cake Appearance

After lyophilization, vials were photographed tocompare the cake appearance at varying protein(in this case rPSGL-Ig) and sucrose levels, shownin Figure 2. Below 13.5 mg/mL rPSGL-Ig (greaterthan �43% (w/w) mannitol in the dry cake), theprotein and sucrose control cakes were visuallycomparable. At higher levels of rPSGL-Ig, thecake appearance became more dense and smooth,rather than the more granular appearance atlower protein concentrations. At sucrose concen-trations above 3% (less than 37% (w/w) mannitolin the dry cake), cake collapse was observed at thebottom of the vial. This is most likely due to theinhibition of both sodium chloride and mannitolcrystallization at elevated amorphous contents,which significantly suppresses the glass transi-tion temperature of the mixture, decreasing the

DOI 10.1002/jps

Figure 2. Photographs of lyophilized cakes over concentration ranges where changeswere observed. The vials in the top row contain varying amounts of rPSGL-Ig (8–44% (w/w) protein or 55–33% (w/w) mannitol). The second row shows varying amounts of sucrose(60–32% (w/w) mannitol). Vertically, the compositions do not show the same amorphouscontent.


collapse temperature. During primary drying,these formulations likely lyophilized above theircollapse temperatures, resulting in the alteredcake appearance.

Increasing protein concentration did not lead tothe same cake appearance observations due to therelatively high T0

g and more rigid structure ofprotein relative to sucrose or mannitol. Theintention of the sucrose vials was to act as asubstitute for protein. The cake appearance showsthat from a lyophilization performance perspec-tive, sucrose is not a suitable substitute for proteinat elevated levels.

Figure 3. Moisture measured by thermogravimetricanalysis as a function of mannitol fraction in the driedstate. Sucrose only vials consistently show highermoisture levels. Each data point represents an indivi-dual vial.

Moisture

Moisture was measured by TGA for every vialthat was used in DSC and XRD. The resultingmoisture as a function of mannitol fraction isshown in Figure 3. At mannitol mass fractionsbelow approximately 57% (or the equivalent of5 mg/mL protein), sucrose samples exhibit highermoisture than protein samples. rPSGL-Ig orsIL-13r performed similarly with regard tomoisture at comparable protein levels. Thesucrose samples were also observed to contain asubstantial amount of mannitol hydrate by XRD.Graphing moisture against the area of themannitol hydrate peak clearly shows the relation-ship between mannitol hydrate and moisture(Fig. 4). Vials of sucrose that collapsed exhibitedhigher moisture due to inappropriate lyophiliza-tion conditions and were excluded from Figures 3


and 4. Trend lines are included in Figure 3 forrPSGL-Ig and sucrose formulations for readerreference. The elevated moisture and mannitolhydrate levels in sucrose-only formulationsdemonstrate the impact of protein, and not justamorphous content, on formulation performanceduring lyophilization.

Mannitol hydrate is undesirable in lyophilizedformulations due to the possibility of conversion toan anhydrous polymorph. Such a conversionwould release newly freed water into the amor-phous phase, with potential detrimental proteinstability effects. From a risk-based perspective,the ability to easily quantitate the amount of


Figure 4. Moisture measured by TGA as a function ofmannitol hydrate peak area of the peak centered at 2Qof 17.98. Each data point represents an individual vial.

3424 DIXON ET AL.

mannitol hydrate present can be useful inidentifying the potential exposure to moisturethe formulation may observe. This can beaccomplished by correlating the moisture, mea-sured with the TGA, to the hydrate peak area,measured with the XRD.

To calculate the mannitol hydrate level, abaseline moisture of 1% was attributed to theamorphous phase (representing the intercept ofthe trend-line shown in Fig. 4). The remainingwater was assigned to mannitol hydrate, whichhas been identified to be a hemi-hydrate (twomannitol molecules per water molecule).26 Basedon the hydrate stoichiometry (2:1 mannitol towater) and the relative molecular weights (182 g/mol: 18 g/mol mannitol to water), the calculatedamount of mannitol hydrate can be determined.By weight, the amount of mannitol incorporated

Figure 5. XRD Overlay of rPSGL-Ig scans.tion is shown to the right of the scans. Peaaccording to the key to the left. Samples offse


as hydrate will be 20 times the amount ofassociated water.

DSC Versus XRD Comparison

Mannitol polymorphism was examined primarilyvia XRD. Figure 5 shows the diffraction patternsat increasing mass fractions of rPSGL-Ig. Theconcentrations of protein prior to lyophilizationare shown to the right of each figure. The peaksintegrated for phase quantitation are indicated onthe individual graphs. The key for peak identifica-tion is included as a text box next to Figure 5. AsrPSGL-Ig concentration increased, the smallamounts of b-mannitol and sodium chlorideobserved disappeared, while the area of the d-mannitol peak initially increased. Between 8.7and 18.3 mg/mL, the overall degree of crystal-lization decreased as the peak areas reduced andthe size of the amorphous hump (immediately tothe right of the indicated mannitol hydrate peakin Fig. 5) increased. This indicates that, at thoseconcentrations, rPSGL-Ig is interfering withmannitol crystallization.

Polymorphic crystalline mannitol (a mixture ofmannitol hydrate, b and d-mannitol) converts to d-mannitol for sIL-13r between 0 and 9.2 mg/mLprotein (not shown). Comparable results havebeen identified for Human Serum Albumin(HSA).22 Mannitol hydrate is apparent in mostsamples, with a maximum appearing at 2.3 mg/mL sIL-13r. Mannitol hydrate behavior in rPSGL-Ig samples were comparable to sIL-13r results.Sodium chloride crystallization has been inhibitedalmost completely when the fraction of sodium

Protein concentration prior to lyophiliza-ks used for quantitation are indicatedt on the y-axis for clarity.

DOI 10.1002/jps

Figure 6. Differential Scanning Calorimetry overlay of reversible signals for sIL-13r.The sIL-13r concentrations are (from bottom to top): 0, 2.3, 4.4, 9.2 mg/mL.


chloride in the dried powder falls below 6.4% (at2.5 mg/mL protein concentration in the pre-lyophilization solution).

Figure 6 shows the reversible heat flow for sIL-13r formulations from 0 to 9.2 mg/mL pre-lyophilization. The glass transition temperature(seen as a step change in baseline of reversibleheat flow) increases as a function of proteinconcentration. That protein content increases theglass transition temperature of the mobile phaseis not surprising. The glass transition tempera-ture of amorphous mannitol has been measured as138C,17 meaning that mannitol would be expectedto reduce the overall glass transition temperatureof the model formulation in this study. The

Figure 7. X-ray diffraction of melt of sucroy-axis for clarity.


mannitol hydrate melt is large enough that itbleeds into the reversible heat flow at approxi-mately 708C as an endothermic transition (con-firmed by the nonreversible heat flow, data notshown). The larger contribution of mannitolhydrate in the 2.3 mg/mL sample relative to the0 mg/mL sample in Figure 6 is consistent withXRD results, which show an apparent maximumof mannitol hydrate at 2.3 mg/mL.

To identify the transitions observed on thenonreversible DSC signal, elevated temperatureX-ray diffraction was performed for a 1% sucrosecontrol vial at temperatures near DSC transitions(Fig. 7). From 25 to 558C, there were no changes tothe crystalline structure of the powder. Between

se control sample. Samples offset on the


Mannitol Weight Factor

¼ Sample Weight � 2% Mannitol

ð2% Mannitol þ 1% Sucrose þ 0:23% NaCl þ 0:12% Tris þ ððprotein concentrationðmg=mLÞÞ=10ÞÞ(1)

Mass Adjusted Peak Area

¼ Actual Peak Net Area

Mannitol Weight Factor(2)

3426 DIXON ET AL.

558C and 758C, mannitol hydrate disappeared andd-mannitol increased, while a-mannitol appeared.No further changes were observed until 1358C, atwhich point most crystalline content was lostexcept for small amounts of b-mannitol. Theseresults are consistent with those previouslyreported for pure D-mannitol,28 as well as theDSC results for this study. In 9.2 mg/mL sIL-13rsamples (46.9% (w/w) mannitol), sodium chloridecrystallization, which was inhibited duringlyophilization, was observed at 958C by hightemperature X-ray diffraction (data not shown).

Phase Quantitation of XRD Peaks

Mannitol and sodium chloride were the onlyexcipients that readily crystallize during lyophili-zation in these formulations. The addition of proteinor additional sucrose serves to dilute the amount ofmannitol or sodium chloride present in the driedstate. While qualitative trends were indicated fromthe data shown in Figure 5, a quantitativecomparison between proteins and sucrose providesinsight into formulation performance.

To compare across different samples and con-centrations, X-ray diffraction peak area wasnormalized to the mass of mannitol in the sample.A sample calculation for the weight fraction ofmannitol is shown below for protein samples. Foramorphous only samples, the sucrose value was

Figure 8. d-mannitol as a function of overall manni-tol mass fraction. Approximate pre-lyophilization pro-tein concentrations are annotated on the graph. Eachdata point represents an individual vial.


adjusted to the appropriate concentration. Forsodium chloride graphs, the fraction of mannitolin the numerator was replaced by the fraction ofsodium chloride.

Using Eqs. (1) and (2) to weight the designatedpeak areas by the mass of mannitol (or sodiumchloride) available for crystallization, hence pro-viding an assessment of the degree of crystal-lization of that particular phase, yields the resultsshown in Figures 8–10. While the raw area of aparticular peak is proportional to the concentra-tion of the phase(s) that constitute that peak,quantitation and comparison of different poly-morphs requires additional calibration.13

Graphs of d-mannitol (Fig. 8) and mannitolhydrate (Fig. 9) in terms of mannitol fraction inthe dried state are presented below. As proteincontent increases, the mannitol fractiondecreases. In these figures, approximate pre-lyophilization protein concentrations are shownfor reference. Trend lines are included in thesefigures for rPSGL-Ig and sucrose formulations forreader reference.

The level of d-mannitol initially increases withincreasing protein concentration up to 5 mg/mLpre-lyophilization (or 55% (w/w) mannitol on a dry

Figure 9. Mannitol hydrate as a function of overallmannitol mass fraction. Approximate pre-lyophilizationprotein concentrations are annotated on the graph.Each data point represents an individual vial.

DOI 10.1002/jps

Figure 10. Sodium chloride crystallinity as a func-tion of sodium chloride mass fraction. Each data pointrepresents an individual vial.

Figure 11. Overall fraction crystallinity of the for-mulation as a function of fraction crystallizing excipi-ents. For reference, a line of slope¼ 1 is shown. Eachdata point represents an individual vial.


basis). For protein samples, this increase occurssimultaneously with a decrease in mannitolhydrate (Fig. 9) and b-mannitol (not shown).When the mannitol level falls below 45% of the dryweight of the cake (greater than 10 mg/mL proteinpre-lyophilization), crystallization was hindered,as shown by the decrease in the level of d-mannitoland mannitol hydrate in rPSGL-Ig samples. Thisconcentration dependent inhibition of mannitolcrystallization is consistent with observations offormulations containing a monoclonal antibody23

and other excipients.17

Sucrose control vials behaved differently thanprotein vials in their propensity to form mannitolhydrate rather than d-mannitol. The lower level ofd-mannitol for sucrose control vials supports theconclusion that mannitol hydrate is a moresignificant contributor to the crystallinity of thesevials. This suggests that the protein itself mayimpact the mechanism of mannitol nucleation andcrystal growth during freezing and annealing(either through viscosity effects or actual inter-action with mannitol on a molecular level). Amonoclonal antibody has been shown to favorcrystallization of mannitol in the d polymorphover the mannitol hemihydrate.9

Using the model formulation and lyophilizationcycle, the a and b polymorphs of mannitol appearin fewer samples than the d and hydratepolymorphs (a-mannitol was not observed insIL-13r samples, b-mannitol was not observedin sucrose-only samples). The magnitudes of thepeaks used for quantitation of a and b-mannitolwere significantly smaller in total area. The peakfor a-mannitol was very difficult to separate frombaseline, even at the highest observed levels.

Attempts to quantify the amount of b and d-mannitol crystalline by comparing against physi-


cal mixtures of pure crystalline mannitol andamorphous sucrose phases were unsuccessful.

Sodium chloride crystallization is quickly inhib-ited during lyophilization when sodium chloridefalls below 6.4% of the total dried content of thecake, shown in Figure 10. There is no differencebetween sucrose and protein on the inhibition ofsodium chloride crystallization in this formulation.

Overall degree of crystallinity can be approxi-mated by integrating the area under a baselinecorrected curve, where the baseline incorporatesthe amorphous hump in the diffraction pattern.Taking the ratio of this number to the total area(amorphous content included) indicates the frac-tion of total crystalline content.29 Dividing thecalculated crystalline area by the known fractionsof crystallizing excipients (in this case, mannitoland sodium chloride) yields an estimate of thecompleteness of crystallization upon lyophiliza-tion. Figure 11 shows that increasing the amor-phous content, either by adding sucrose orprotein, decreases the completeness of crystal-lization roughly linearly. The x-axis in Figure 11shows the combined weight fraction of mannitoland sodium chloride.

In principle, XRD analysis shown in Figure 11could be orthongonally compared to DSC resultsfor melting of mannitol. High temperature XRDindicates that sodium chloride, in this formula-tion, has significant crystalline phase transitionsin the temperature range of 100–1508C (data notshown), which also includes the primary manni-tol-melting peak. The DSC method utilized wasunable to de-convolute mannitol from sodiumchloride thermal events. As a result, the apparentdegree of crystallization by DSC did not agreewith the results obtained from XRD.


Figure 12. Crystalline content not incorporated asmannitol hydrate. At lower mannitol fractions, thisrepresents anhydrous crystalline mannitol. Each datapoint represents an individual vial.

3428 DIXON ET AL.

Increasing protein weight fraction inhibits cry-stallization more than increasing sucrose weightfraction, also shown in Figure 11. Two potentialexplanations for this observation are: (1) differ-ences in relative viscosities between protein andsucrose solutions, and (2) stronger protein/manni-tol molecular interactions than sucrose/mannitol.sIL-13r has been shown to increase viscosity moreat 208C than sucrose alone (data not shown). Whilethis difference is relatively small at 208C, it maybe magnified as the cooled, concentrated solutionapproaches the T0

g. Additionally, the impact of sIL-13r and rPSGL-Ig (on a weight basis) seem to besimilar to one-another with respect to degree ofcrystallinity, despite a 45 kDa (roughly 50% of theweight of rPSGL-Ig) difference in molecular weightbetween sIL-13r and rPSGL-Ig.

Subtracting the amount of mannitol hydrate(calculated earlier) from the overall crystallinecontent in Figure 11 yields an estimate ofanhydrous crystalline mannitol and sodium chlor-ide, shown in Figure 12. Because sodium chloridewas not observed to crystallize below 6.4% sodiumchloride (Fig. 10), points below 0.65 on the x-axis ofFigure 11 represent anhydrous mannitol only. Athigher mannitol and sodium chloride levels,mannitol hydrate accounted for the bulk of thecrystallinity in sucrose control vials. For proteincontaining formulations, the bulk of the crystallinecontent is anhydrous at all levels.

CONCLUSIONS

Protein concentration in the range of 1–5 mg/mLalters the crystallization polymorphism of man-nitol favoring the formation of d-mannitol over b-mannitol in the examined model formulation. A


protein concentration range of 1–5 mg/mL corre-sponds to a mannitol fraction of 55–60% (w/w) on adry basis. Below 1 mg/mL, protein concentrationdoes not have a significant influence on observeddegree of crystallization or polymorphism. At lowprotein concentrations (1–5 mg/mL), this formu-lation generated a mixture of b-mannitol, d-mannitol and mannitol hydrate. a-mannitol wasnot observed in any significant quantities in thisstudy. This trend applied to both of the two Fc-fusion proteins examined. Protein impact isstronger than that of pure amorphous phase onmannitol crystallization, evidenced by absence ofb-mannitol and increase in mannitol hydrate ofsucrose formulations relative to protein formula-tions and the diminished impact of sucrosecontent on the crystallinity of mannitol whencompared to protein content.

At mannitol levels below 46% on a dry basis(greater than 10 mg/mL protein pre-lyophiliza-tion), the observed crystallization, when correctedfor mannitol mass, decreased, indicating thatincreasing amorphous content was inhibitingmannitol crystallization. Sodium Chloride crys-tallization during lyophilization is inhibited com-pletely by amorphous material at sodium chloridelevels below 6.4% in the dried state (equivalent to2.5 mg/mL protein). In this formulation, thiscorresponds to 44% amorphous content (assuming100% crystallinity of mannitol). This is true forboth sucrose and protein samples.

Both proteins were shown to alter the behaviorof the lyophilized powder with respect to moisture.Sucrose-only samples retained more water afterlyophilization, most of which was incorporated asmannitol hydrate. From a formulation perfor-mance perspective, these studies suggest thatprotein does impact the crystalline content of thefinal product at all but the lowest proteinconcentrations. As a result, protein concentrationmust be taken into account when developingdosage forms or evaluating lyophilization cycleperformance at larger scale.

REFERENCES

1. Passot S, Fonseca F, Alarcon-Lorca M, Rolland D,Marin M. 2005. Physical characterization of formu-lations for the development of two stable freeze-dried proteins during both dried and liquid storage.Eur J Pharm Biopharm 60:335–348.

2. Johnson RE, Kirchoff CF, Gaud HT. 2002. Manni-tol-sucrose mixtures—Versatile formulations forprotein lyophilization. J Pharm Sci 91:914–922.

DOI 10.1002/jps


3. Torrado S, Torrado S. 2002. Characterization ofphysical state of mannitol after freeze-drying:Effect of acetylsalicylic acid as a second crystallinecosolute. Chem Pharm Bull (Tokyo) 50:567–570.

4. Telang C, Suryanarayanan R, Yu L. 2003. Crystal-lization of D-mannitol in binary mixtures withNaCl: Phase diagram and polymorphism. PharmRes 20:1939–1945.

5. Martini A, Kume S, Crivellente M, Artico R. 1997.Use of subambient differential scanning calorime-try to monitor the frozen-state behavior of blendsof excipients for freeze-drying. PDA J Pharm SciTechnol 51:62–66.

6. Cannon AJ, Trappler EH. 2000. The influence oflyophilization on the polymorphic behavior of man-nitol. PDA J Pharm Sci Technol 54:13–22.

7. Telang C, Yu L, Suryanarayanan R. 2003. Effectiveinhibition of mannitol crystallization in frozen solu-tions by sodium chloride. Pharm Res 20:660–667.

8. Yu L, Milton N, Groleau EG, Mishra DS, VansickleRE. 1999. Existence of a mannitol hydrate duringfreeze-drying and practical implications. J PharmSci 88:196–198.

9. Liao X, Krishnamurthy R, Suryanarayanan R.2007. Influence of processing conditions on thephysical state of mannitol—Implications infreeze-drying. Pharm Res 24:370–376.

10. Osterberg T, Fatouros A, Mikaelsson M. 1997.Development of a freeze-dried albumin-free formu-lation of recombinant factor VIII SQ. Pharm Res14:892–898.

11. Kasrain K, Spitznagel TM, Juneau JA, Yim K.1998. Characterization of the sucrose/glycine/watersystem by differential scanning calorimetry andfreeze-drying microscopy. Pharm Dev Technol 3:233–239.

12. Carpenter JF, Pikal MJ, Chang BS, Randolph TW.1997. Rational design of stable lyophilized proteinformulations: Some practical advice. Pharm Res14:969–975.

13. Lu X, Pikal MJ. 2004. Freeze-drying of mannitol-trehalose-sodium chloride-based formulations: Theimpact of annealing on dry layer resistance to masstransfer and cake structure. Pharm Dev Technol9:85–95.

14. Hawe A, Friess W. 2006. Impact of freezing proce-dure and annealing on the physico-chemical proper-ties and the formation of mannitol hydrate inmannitol-sucrose-NaCl formulations. Eur J PharmBiopharm 64:316–325.

15. Yoshinari T, Forbes RT, York P, Kawashima Y.2002. Moisture induced polymorphic transition ofmannitol and its morphological transformation. IntJ Pharm 247:69–77.

16. Lueckel B, Bodmer D, Helk B, Leuenberger H.1998. Formulations of Sugars with amino acids ormannitol—Influence of concentration ratio on the


properties of the freeze-concentrate and the lyophi-lizate. Pharm Dev Technol 3:325–336.

17. Kim AI, Akers MJ, Nail SL. 1998. The Physicalstate of mannitol after freeze-drying: Effects ofmannitol concentration, freezing Rate, and a non-crystallizing cosolute. J Pharm Sci 87:931–935.

18. Haikala R, Eerola R, Tanninen VP, Yliruusi J.1997. Polymorphic changes of mannitol duringfreeze-drying: Effect of suface-active agents. PDAJ Pharm Sci Technol 51:96–101.

19. Fakes MG, Dali MV, Haby TA, Morris KR, VariaSA, Serajuddin ATM. 2000. Moisture sorption beha-vior of selected bulking agents used in lyophilizedproducts. PDA J Pharm Sci Technol 54:144–149.

20. Cao W, Mao C, Chen W, Lin H, Krishnan S,Cauchon N. 2006. Differentiation and quantitativedetermination of surface and hydrate water in lyo-philized mannitol using NIR spectroscopy. J PharmSci 95:2077–2086.

21. Pyne A, Surana R, Suryanarayanan R. 2002. Crys-tallization of mannitol below during freeze-dryingin binary and ternary aqueous systems. Pharm Res19:901–908.

22. Hawe A, Friess W. 2006. Physico-chemical lyophli-zation behavior of mannitol, human serum albuminformulations. Eur J Pharm Sci 28:224–232.

23. Liao X, Krishnamurthy R, Suryanarayanan R.2005. Influence of the active pharmaceutical ingre-dient concentration on the physical state of man-nitol—Implications in freeze-drying. Pharm Res22:1978–1985.

24. Cavatur RK, Vemuri NM, Pyne A, Chrzan Z,Toledo-Velasquez D, Suryanarayanan R. 2002.Crystallization behavior of mannitol in frozen aqu-eous solutions. Pharm Res 19:894–900.

25. Romero-Torres S, Wikstrom H, Grant ER, TaylorLS. 2007. Monitoring of mannitol phase behaviorduring freeze-drying using non-invasive Ramanspectroscopy. PDA J Pharm Sci Technol 61:131–145.

26. Nunes C, Suryanarayanan R, Botez CE, StephensPW. 2004. Characterization and crystal structure ofD-mannitol hemihydrate. J Pharm Sci 93:2800–2808.

27. Beattie JR, Barrett LJ, Malone JF, McGarvey JJ,Nieuwenhuyzen M, Kett VL. 2007. Investigationinto the subambient behavior of aqueous mannitolsolutions using temperature-controlled Ramanmicroscopy. Eur J Pharm Biopharm 67:569–578.

28. Burger A, Henck JO, Hetz S, Rollinger JM, Weiss-nicht AA, Stottner H. 2000. Energy/temperaturediagram and compression behavior of the poly-morphs of D-mannitol. J Pharm Sci 89:457–468.

29. Rani M, Ramprakash R, Surana R, Suryanaraya-nan R. 2006. Structure in dehydrated trehalosedihydrate—Evaluation of the concept of partialcrystallinity. Pharm Res 23:2356–2367.


The impact of protein concentration on mannitol and sodium chloride crystallinity and polymorphism...

Documents

Transcript of The impact of protein concentration on mannitol and sodium chloride crystallinity and polymorphism...