ORIGINAL PAPER

Impact of residual moisture and formulation on Factor VIIIand Factor V recovery in lyophilized plasma referencematerials

Anthony Hubbard & Sally Bevan & Paul Matejtschuk

Received: 30 June 2006 /Revised: 8 September 2006 /Accepted: 12 September 2006 / Published online: 27 October 2006# Springer-Verlag 2006

Abstract Residual moisture content and formulation areimportant parameters when preparing lyophilized referencematerials containing labile proteins. The protection ofFactor VIII and Factor V activities were monitored in alyophilized plasma preparation following formulation witheither no additional excipient, 40 mM Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 10 mg/mLglycine or a combination of 40 mM Hepes and 10 mg/mLglycine. The preservation of Factor VIII activity duringfreeze-drying was improved by the addition of eitherstabiliser and improved most, amongst the options studied,by the addition of both glycine and Hepes. The predictedstability at −20 °C and 20 °C was estimated usingaccelerated degradation studies. Although for plasmalyophilized alone there was some benefit from furtherdesiccation over phosphorus pentoxide, resulting in verylow moistures, for suitably formulated samples the pre-dicted stability was as good for freeze-dried only samplesas for those with further desiccation. This study emphasisesthe importance of optimum formulation on the stability oflyophilized proteins.

Keywords Lyophilization . Human plasma . Factor VIII .

Factor V. Stability

Introduction

Residual moisture is a key quality control parameter forlyophilized biological materials, and typically reducing

moisture content to low levels improves the stability ofbiological molecules within the lyophilized state. However,the ideal moisture content may vary between referencematerials, some biological materials lose activity if they areover-dried, for instance viruses and bacteria. Some proteinsmay also be de-stabilised by over-drying [1].

The WHO guidelines for preparation of biologicalreference materials stipulate that the residual moisturecontent should be such as to assure adequate stability andsuitability for purpose. Though this may need to beidentified on a case by case basis a moisture of below 1%w/w has been shown to usually deliver suitable stability [2].

Haematological reference materials consisting of bloodplasma or purified therapeutic concentrates have historical-ly undergone extensive drying [3–6] and this has frequentlyrequired further desiccation post-lyophilization to achievevery low residual moisture content of ≤0.1% w/w. Modernlyophilizers can readily deliver low to medium moisturecontents of 0.1– 0.5% w/w and these levels may wellproduce adequately stable reference materials if suitablestabilisers are added.

Coagulation Factors V and VIII are labile plasma glyco-proteins which act as crucial cofactors in the procoagulantpathway [7–9]. When preparing reference materials for thediagnostic assessment of Factors V and VIII in humanplasma it is important that the activity in the referencematerial is stabilised. Lyophilization is a commonly usedmethod for stabilizing labile proteins as many principaldegradative pathways for proteins are water-catalysed.

To stabilise Factor VIII activity very low residualmoisture levels were required and this has previouslynecessitated further desiccation over phosphorus pentoxide.In order to assess the impact of optimising freeze dryingcycles and formulations to achieve suitable stability inFactor VIII reference plasma a study was set up to compare

Anal Bioanal Chem (2007) 387:2503–2507DOI 10.1007/s00216-006-0855-x

A. Hubbard : S. Bevan : P. Matejtschuk (*)National Institute for Biological Standards and Control,Blanche Lane, South Mimms, Potters Bar,Hertfordshire EN6 3QG, UKe-mail: pmatejt@nibsc.ac.uk

the effects of glycine and Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer as well as furtherdesiccation on Factor VIII stability.

Materials and methods

Plasma

Citrate phosphate dextrose anti-coagulated blood wascollected by the UK Blood Transfusion Service (NBSColindale), shown to be negative for anti-HIV, anti-HCVHBsAg and syphilis. Plasma was obtained by doublecentrifugation and held at −70 °C until used. Plasma unitswere thawed on the day of use by warming to 37 °C,pooled and dispensed immediately holding at 4 °C.Formulation with Hepes-free acid (H-3375, Sigma Aldrich,Poole Dorset, UK, final concentration 40 mM) or glycine(G-7126 Sigma) was compared to unformulated plasma.Dispensing was at 1 g per ampoule using Hamilton M-Lab(Hamilton, Bonaduz, Switzerland) with the product heldover wet ice during filling.

Formulation A No stabiliser added, plasma and deionisedwater combined in volumes of approxi-mately 10:1.

Formulation B Hepes added to 40 mM final concentration,with effective dilution of plasma to bufferof 10 volumes: 1 volume.

Formulation C Glycine added to 10 mg/mL final concen-tration, with effective dilution of plasma tobuffer of 10 volumes: 1 volume.

Formulation D Hepes added to 40 mM and glycine to 10mg/mL final concentrations, with effectivedilution of plasma to buffer of 10 volumes: 1volume.

Lyophilization

Lyophilization was performed in Serail CS-100 freeze dryer(shelf 4 m2, Serail SGD, Argentueil, France). A total of 150ampoules (5 mL DIN, Adelphi Tubes, Haywards Heath,UK) of each formulation were prepared, fitted with closures(partially inserted) and a specially designed adaptor toallow stoppering prior to flame sealing [10]. Product wasloaded at 4 °C and ramped freezing to −50 °C wasperformed at 0.2 °C/min followed by holding at −50 °Cfor 7.5 h. A vacuum of 10 Pa was applied and the shelftemperature raised to −35 °C. Primary drying conditionswere held for 48 h followed by a temperature ramp to firstly0 °C over 175 min followed by a 2-h hold and then a rampto 25 °C over 125 min. Secondary drying was continued for

48 h at 3 Pa. Following lyophilization, ampoules wereback-filled with nitrogen, closures were pressed fully homeand part of the batch of each formulation was furtherdesiccated over phosphorus pentoxide under hard vacuumfor 6 days. Ampoules were then sealed on the AF10/10(Bausch & Stroebel, Ilshofen, Germany) with the internalatmosphere maintained using two-part plug device whichminimised atmospheric intrusion.

Residual moisture analysis was performed using acoulometric Karl Fischer (Mitsubishi CA-06, Anachem,Luton, UK) on six samples of each formulation with orwithout further desiccation. The coulometer performancewas checked before assaying the products using a waterstandard (Solution P, Anachem) with a value of 180–210 μg water per 50 μL and triplicate injections with a CVof <5%.

Factor VIII assay

Factor VIII coagulant activity was assayed using a two-stage chromogenic method (Coatest VIII:C/4 kit, Chrom-genix SpA, Milan, Italy) following the kit manufacturer’srecommended methodology adapted for use on the ACLFutura Analyser (Instrumentation Laboratory, Warrington,UK). Assay dilutions were made in sample buffer supple-mented with human albumin to a final concentration of1% w/v. Potency estimates were made relative to thelyophilized −20 °C samples using an arbitrary potency of100% for this sample.

Factor V assay

Factor V coagulant activity was measured using a conven-tional one-stage clotting method based on the ability ofFactor V in the test sample to shorten the thromboplastin-induced clotting time of Factor V-deficient plasma [11].Potency estimates were made relative to the lyophilised−20 °C samples using an arbitrary potency of 100% forthis sample.

Accelerated degradation study

Results of both Factor VIII and Factor V activities wereexpressed as the mean of two separate estimationsperformed on different ampoules.

Stability of the lyophilised preparations was predictedfrom accelerated degradation studies. This approach hasbeen applied to lyophilised preparations of blood coagula-tion factors for over 30 years [12] and assumes thatdegradation is caused by unimolecular decay with theprobability of any intact molecule changing state in anyunit of time remaining constant. The degradation rate mustalso follow a fixed law of temperature dependency, as

2504 Anal Bioanal Chem (2007) 387:2503–2507

described in the Arrhenius equation; hence the degradationoccurring at higher temperatures only differs from thedegradation at lower temperatures in terms of rate. Hencethe relative loss of activity observed for samples stored atelevated temperatures (4, 20, 37, 45 °C) can be used topredict the degradation rate for samples stored at the bulkstorage temperature of −20 °C. Validity of the modelrequires that the observed loss must not be significantlydifferent to the predicted loss at the 5% level when fitted tothe Arrhenius equation.

Ampoules were laid down at temperatures from −20 to45 °C and held over a period of 10 months. Followingincubation ampoules were tested for Factor V and FactorVIII activities. The loss of activity during freeze-drying wasmeasured by comparison with pre-processing liquidsamples.

Results

The loss of Factor VIII activity across the lyophilizationprocess was measured (see Fig. 1). Without stabiliser over30% of Factor VIII activity was lost during processing,compared to less than 20% activity if glycine, Hepes orHepes and glycine were added. Further desiccation did notresult in any further loss in Factor VIII activity.

Initial accelerated degradation testing (15.5 weeks at37 °C or 45 °C) revealed significant loss of Factor VIIIactivity (approximately 70%) for plasma without stabiliserand for the 45 °C degradation sample of plasma stabilisedwith Hepes alone, though much smaller loss occurred at37 °C. The degradation of both Factor V and Factor VIIIactivities were studied after 25 weeks at either 37 °C or

45 °C and compared to samples held at −20 °C for the sametime (Fig. 2). The same trend was apparent with plasmaalone suffering from marked loss of activity for both factorsbut loss being reduced for preparations stabilised witheither glycine or Hepes and loss being minimal for thosestabilised with both glycine and Hepes. There seemed to belittle if any difference in terms of activity loss for eitherfactor between those preparations which were just freeze-dried and those which were further desiccated.

The residual moisture content (Table 1) was lower forpreparations with further desiccation as would be expected,being half those of the corresponding freeze-dried onlypreparations. The 37 and 45 °C samples were very difficultto reconstitute for assay for plasma alone, as was the 45 °Csample for plasma with Hepes only but 37 °C and 45 °Csamples of formulations with glycine and glycine andHepes were much easier to reconstitute.

The predicted rate of activity loss for Factor VIII, underboth long-term storage temperature of −20 °C and at 20 °C,is given for each formulation in Table 1, based on fitting ofthe accelerated degradation data to an Arrhenius model [13].As expected the rate of Factor VIII loss was greater forunbuffered freeze-dried only plasma than for plasma whichwas also further desiccated to lower moisture content.However, the degradation rate of the Factor VIII activitywas interestingly far better for formulations containing Hepesalthough the moistures were not significantly different.

Freeze-drying alone was sufficient to stabilise the Hepes-containing formulation to levels similar to or better thanthose of the unsupplemented yet further desiccated plasma.

The impact of adding glycine was similarly to stabilisethe Factor VIII activity to as good or better degradationrates than desiccated unformulated plasma, and the combi-

Recovery of Factor VIII activity during freeze drying

0.0

20.0

40.0

60.0

80.0

100.0

120.0

Liquid FreezeDried

FurtherDesiccated

Process stage

% F

acto

r V

III r

eco

very

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ith

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id +

40m

M H

epes

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0%)

A: Plasma unbuffered

Plasma + 40mM Hepes

Plasma + 10 mg/mlglycine

plasma + 10 mg/mlglycine & 40mM Hepes

Fig. 1 Loss of Factor VIIIactivity on processing. FactorVIII activity is expressed as apercentage of the activity foundin the pre-processing liquidplasma, containing Hepesbuffer. The effect on Factor Vwas not studied

Anal Bioanal Chem (2007) 387:2503–2507 2505

nation of the two excipient materials was also effective. Therate of loss of Factor VIII activity was less for both furtherdesiccated glycine and glycine and Hepes formulations thanfor either unformulated plasma or Hepes-formulated plasmaalone.

Conclusions

Addition of Hepes or of glycine improved the stability ofFactor VIII activity in lyophilized plasma with modest

moisture content to a degree previously achieved only byexhaustive further desiccation to very low moisture content.In fact over-drying of the glycine and mixed glycine/Hepesformulation by further desiccation may result in less stableFactor VIII activity.

The mechanism of stabilisation of Factor VIII activityhas been studied previously and Hepes has been reported asa stabiliser of plasma when establishing a lyophilizedreference plasma [14, 15] and glycine is a commonconstituent of the formulations used as stabilisers forlyophilized Factor VIII concentrates [16, 17].

0

10

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ADT 37°C ADT 45°C ADT 37°C ADT 45°C

Factor VIII Factor V

Freeze dried only

Ac

tiv

ity

(% o

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0oC

sa

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les

)

Plasma control

Plasma+ Hepes

Plasma+ glycine

Plasma +glycine + Hepes

0

10

20

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ADT 37°C ADT 45°C ADT 37 °C ADT 45°C

Factor VIII Factor V

Freeze dried and further desiccated

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0 C

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mp

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Plasma +glycine + Hepes

a

b

o

Fig. 2 The impact on Factor Vand Factor VIII activities ofstorage at 37 °C or 45 °C for25 weeks, expressed as apercentage of the −20 °C storedsample (processed without(a) or with (b) furtherdesiccation)

2506 Anal Bioanal Chem (2007) 387:2503–2507

For the preparation of lyophilized Factor VIII referenceplasma further desiccation can be avoided, halving processtime and simplifying the production process, providing thatsuitable stabilisers are included. Hepes is routinely added tobatches of reference materials prepared at NIBSC for FactorVIII activity. Indeed the addition of glycine or glycine andHepes may offer even greater protection of Factor VIIIactivity than does Hepes alone.

The addition of Hepes and/or glycine to the plasma didnot present any major commutability issues as demonstrat-ed by the similarity of Factor V and Factor VIII estimates inthe liquid plasma samples after buffering and before freeze-drying (data not shown). Moreover, the assessment ofstability based on accelerated degradation relies entirely oncomparisons of the preparations against themselves(e.g. +45 °C vials vs. −20 °C vials) and so the effectsdemonstrated allow for the differences that addition of theextra excipients might potentially have caused.

Losses of Factor V activity showed a similar trend interms of the impact of stabiliser to that seen for Factor VIIIactivities, with the addition of both excipients givinggreatest stabilisation of both Factor V and Factor VIIIactivities. The use of further desiccation to further dry thesamples did not seem to affect the stability of Factor V orFactor VIII over 25 weeks incubation at either 37 °C or45 °C. It was also observed that samples incubated at 37 °Cor 45 °C for 25 weeks were difficult to reconstitute for thecontrol formulation and for that with Hepes only but not forthose preparations which contained glycine. Samples whichwere formulated with glycine alone showed a change in

colour to a deeper yellow/orange when stored for 25 weeksat 45 °C irrespective of whether processed with furtherdesiccation or not; however, those formulated with glycineand Hepes did not show this colour change.

Studies on the lyophilization of purified Factor VIIIconcentrate have been published [16–18] and the appear-ance of a product (collapsed or pharmaceutically elegant)and its stability has been reported recently [17]. In thatstudy the collapsed material had similar stability to the non-collapsed product and the higher moisture content productwas no less stable.

In this study we have shown that for Factor VIII andFactor V activities in lyophilized reference plasma thechoice of stabiliser is as important as the level of residualmoisture achieved by the lyophilization process. Bychoosing suitable stabilisers the process time can bereduced and simplified without any loss in the stability interms of Factor VIII activity. The best results were obtainedfor those formulations tested with a combination of 40 mMHepes and 10 mg/mL glycine. This approach illustrateshow formulation optimisation and lyophilization processingcan be combined to deliver suitable procedures for thepreparation of stable biological reference materials forlabile biomolecules.

Acknowledgements We thank the staff of the Standards Division,NIBSC for technical assistance with processing the plasma.

References

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Table 1 Residual moisture content and predicted loss of Factor VIIIactivity from accelerated degradation testing

Formulation ResidualMoisture(% w/w) withCV (%) inparentheses(n=6)

Mean % lossper year at−20 °C (and95% upperconfidencelimit)

Mean % lossper year at20 °C (and95% upperconfidencelimit)

Plasma FD 0.47 (5.8) 0.75 (1.00) 36.4 (38.7)Plasma FD & SD 0.15 (9.9) 0.12 (0.30) 20.7 (27.8)Plasma+Hepes FD 0.38 (15.4) 0.08 (0.09) 22.0 (22.4)Plasma+Hepes FD &SD

0.10 (5) 0.07 (0.14) 16.3 (20.4)

Plasma+glycine FD 0.37 (15.9) 0.03 (0.04) 8.8 (9.7)Plasma+glycine FD& SD

0.13 (16.5) 0.13 (0.26) 10.33 (12.9)

Plasma+glycine/Hepes FD

0.61 (14.5) 0.03 (0.08) 6.3 (8.3)

Plasma+glycine/Hepes FD & SD

0.09 (10.3) 0.06 (0.06) 7.54 (7.6)

-FD freeze-dried only, FD & SD freeze-dried and secondarydesiccated

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