World Health
Organization
ii
EXPERT COhl31ITTEE ON BIOLOGICAL STAND,.1RDIZA'fIO%
Geneva, 24 to 28 October 2005
INTERNATIONAL COLLABORATIVE STUDY
TO EVALUATE A CANDIDATE INTERNATIONAL STANDARD
FOR IFIAEkIOPWILUS INKUENZAE TYPE B CABULAR POLYSACCWARIDE
National Institute for Biological Standards and Control. Blanche Lane. South Mimms, Potters Bar, Nerthrdshire, EN6 3QG, United Kingdom
dlJUet~zuphil~1~ inJrli~e/zcue b conjugate vaccines (Hib) are almost conlpletely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(l
--+
1)-P-D- ribose-3-phosphate) in final fomulations and bulk components, there is a need for an International PRP Standard to calibrate internal references used in the different laboratories. Ten laboratories from 8 different countries paflicipated in aO kVorld Health Organization 2005
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WWOIBS105,2018 Page
Z
collaborative itud] to a\5eb5 the suitabilit) and determine the PRP content of a candidate Irrternational Standllrd PRP preparation (0211081. On the hasii of the re$ult\ from thi\ 'tudy, it i \ recommended that the ,
cmclidate standard 021208 i' eitablished as an International PRP Standard u ith a content of 4.933 0.267 mgiampoule. a% determined by the riboie a l i a > i carried out by 7 oi the panicipating laboratories (e~panded uncertaint) calcutated uiing a cotrerage factor of 2-45 u hich cornspond\ to an approximate I 9 5 C ~ Izi el of confidence),
Current Hacu~oplzilrtr ir?$t~c.n:de b conjugate vaccines (Hih) are aln~ost completely erraluated by physico- chemical methods to ensure the
consistent!
of mmufacture of batches. and biological testing is carried out only to ensure safer) (WHO requirements 2000 and E P 1219: 20031. A wide variety of assajs are used for the quantification of the capsular polqsttccharide, PRP (polyribctsyl ribitol phosphate; 5-D-ribitol-( I + l)-P-~-ribose-3-phosph3te) content in purified polysacct~aride, bulk conjugates and final tot
vaccines. These include phosphorus assay, ribose detemination, high perfbmance anion exchange
a
chromatography-pulsed an~perornetric detection (HPAEC-PAD) for PRP and imrnunochemical assays. As a wide variety of assay methods are used for PRP quantifjcation. a meeting for discussion of WHO recommendations for the production and control of Hib vaccines (1996) and a WHOmlBSC (the National Institute for Biological Standards and Control) workshop on the use of physicocbemical methods for the characterisation of ff~iem~phillrs ir7flue~zrue type b conjugate vaccines ( 1998) recommended that a PRP reference preparation containing a known quantity of polysacchtltide that can be used to cross-calibrate various methods to quantify the PRP content of the bulk saccharide. bulk conjugate and final fomulations should be made available under the auspices of WHO to facilitate calibration of assays (Holliday and Jones. 1999). This is particularly required by National Control Laboratories (NCLs) and some manufacturers. The continuing development of new Hib vaccines, the establishment of pro, orarnmes to distribute Hib throughout the world and the likelihood that more NCLs will begin establishing methods to evaluate Hib vaccines means that a standard for the quantification of Hib PRP will be in demand,
The purpose of the collaborative study was to deternine the content of a candidate PRP standard in SI units (per weight ( W ) basis), and to evaluate its suitability for use as a standard for PRP quantification
assays (including ribose, phosphorus and HPAEC-PAD assays) in final fills and bulks of Hib vaccines.
a
PARTICIPANTS
Ten laboratories pa~icipated in the study including 5 manufacturers and 4 National Control Laboratories, following invirations sent to IS laboratories, The list of parlicipants is attached in Appendix A.
Participants were assigned a random code number. not corresponding to the order of listing.
WWOIBS/O5.2018 Page 3
MATERIALS AND RfETRODS
L The candidate standard
NIBSC received 900 rnl of liquid PRP unconjugated poI>itrtccharide from Wqeth-Lederle t'accinei and Pediatrici, on 3 1 January 3001. It wac itored at -30°C. It contained 6.93 i~-tg/ml PRP cl\ determined by the manufacturer using the orcinol as\ay for riboi,e determination. The material provided M a i iterile and the lbllowing content5 were provided by the manuftacturer: endororin content (0.41 Eli'iug: LAL assay).
protein content (CO. 1 or). nucleic acid contamination ( 0 % ) and Xa61 content (0.77 m ~ f m l ) . The WHO recommendations were h l l o s e d tits the prepartrtion of the candidate \tandard. Material !%as diluted ro 5 mg/n~l using sterile distilled water (DW) and 933 ampoule5 uere prepared a i lyophilised containing 5 mg1ampoule on 33 June 2003, at the Centre for Biological Reference Materials (CBRM, NIBSG). Freeze- drying was done at -30°C and secondary drying at 35OC for 24 hr. The candidate Standard u a s code- named 021208 and stored at -30'6 at CBKlf. Microbial anal!sis shotved no contamination. Twelce containers were used to meamre the precision and \%eight of the fill. CV of precision fill is 0.06G2 and the actual mean mass is 1.0059 g. The residual moisture content is 0.65 (wiw) with a CV 23.67%. Seven hundred ampoules are offered to N'HO.
Study materials
Participants in the collaborative study were sent t u o duplicate sets of 4 preparations coded A, B. C and D, where ampoules B and C were the candidate standard 02208. and A and D were Meningococcal serogroup Y polysaccharide (NLBSC code 0 11328; 1 mg/an-tpoule f and Meningococcal serogroup W 135 polysaccharide (NIBSC code 011426; 1 mg1ampoule). Upon receipt, participants uere asked to reconstitute the samples with Iml sterile distilled water and store at -20°C until further use. Prior to use in assays, samples would be thawed and kept at 4°C for up to 1 week, while completing the assay($). Further dilutions to gice test solutions in the correct concentration range for the assay sensitivity fiere to be made in the appropriate assay solution or buffer, according to the laboratory's protocol.
Study design
a
Participants were requested to test the two sets of samples separately one after the other, preferably in different rveeks, using the in-house method commonly peri-ormed in their laboratories. Tests were to be carried out within 7 da> s of tbarving the reconstituted rample. A balue of 1-30 mg for the content of the preparations wa\ procided to participants as a guideline to help in preparation of dilutions. Details of all the assay methods pedormed by the participants are shown in Tables 1-4.Assays for evaluation of the candidate sstandard
The ribuse assay for measuring PRP content of the candidate standard was performed bq 7 out of the 10
e panicipating laboratories. Ribose comprises 40-41 5 by weight of the Hib PRP single repeating unit. In the BiaI reaction. ribo\e, as a pentose, reacts t\ith the arcinol reagent in a concentrated hydrochloric acid- fesric chloride solution ro produce a green coloration cAshwell, 1957; Kabat & Mayer 1971). The orcinol method can detect 3 nn-toles ribose (or PRP manomeric unit]. The method is not specific to pentoses.
ho\vever, and hexores and heprow5 such as those present as bulking agents in some lyophilized products, can c a w e intederence tree Appendix C f . D-Rihose was u5ed as a standard b5 the Iaborator~ei; Table 1 contains other details of rke assay used b j pafiicrpating labs.
\t'HOIBS/05.2018 Page 4
Phosphorus
(P)
dcrferrnifzufiorz \vas carried out by 5 laboratories. In this colorimetnc assay, samples are initially acidified. then oxidized with perchloric acid fChen r~lethod: Chen et al., 1956) or magnesium nitrate (Ames method: Ames, 1966) to activate and hydrolyze the polysaccharide prior to asking of the material at high temperature, In the second past of the assay, ascorbic acid acts to reduce a phosplilo- molybdate conlples to a blue-coloured compot~nd, P comprises 8.3-8.5% of PEP, and the assay can detect 5-10 nrnoles phosphorus, or PRP monorneric unit. The phosphorus assay is not specific to PRP, however, and satnples containing any phosphate salts or sugar phosphates woufd be expected to react. Sodium or potassium phosphare salts. or ribose-5-phosphate were used as srandards by the panicipants. Details of the phosphorus assay methods used by the Iaboratories are provided in Table I?,The HPAEC-MD assay was perfomled b> S laboratories. For PS hqdrolysis prior to chromatography, 5 laboratories wed the alkaline hydrofj\i\ 'Tsai method' iTsai et al., 1993; Sturgeis et al., 1999) mhich directly measures the PRP single repeating unit cai a mixture of ribitof-ribo\e-3-phosphate or ribitol- ribose-2-phosphate: Tsai et al., 1993) and one laboratory used the acid hydrolysis method (Ip et al.. 1992:
Bardotti et al.. 2000), which measure% ribitol content (41.3 % by d r j %eight of PRP). Chromatography i\
performed in a high pH, sodium hydroxide-based eluent so that hydroxg I groups become anionic, and may
a
be separated on an mion-exchange column. Neutral formulation sugars can. with care, be eluted before the negatibely-char,uctd P R P disaccharide. The use of a pulsed amperometric detector. makes this method highly sensitike (0.1-0.2 nmoles PRP) and specific. Laboratorq 9 obtained resultr using a PRP polysaccharide standard (lab code 9a) and also using a bulk conjugate standard (lab code 9b). Of the 0ate as a remaining laboratories. two used PRP polycaccharide as a standard, and three used bulk conjuz
standard. The WHO Recommendations cite HPAEC-PAD as a possible alternative to the calorimetric methods. Details frorn the laboratories performing the HPAEC-PAD assay are included in Table 3.
1 H-NMR was performed by 2 laboratories: one for quantification. and the other for a qualitative, diagnostic purpose (Table 4j.
The laboratories performing the ribose, phosphorus and HPAEC-PAD assays supplied conkersion factors used to convert the amount of ribose or phosphorus in the candidate standard to PRP. These factors are based on the proporrion of assayed group to total amount of PRP. It is assuined that their calculations correctly accounted for the counter-ion i.e. sodium or potassium present in the sample and diluent. As
specifications for PRP content are in rng units and results from assays are expressed in mg or molar units.
a
it &as deemed appropriate that S1 units be used for unitage rather than International Units.
STABILITY STUDY
To determine the stability of the reference standard. three studies of the candidate standard are currently undertvay at lab 10: real-time stability. accelerated degradation and stability of the reconstituted material.
The real-time stability ar -20°C. using a -70°C baseline sample. is being carried out on samples stored at - 30°C for 6 mo, I yr, 2.5 pr, 5 qr and 10 yr, and transferred to -70°C at each timepoint,
The accelerated degradation study of the candidate standard (at temperatures of 3. 20. 37 and 56°C). using
3 -?@'C baseline sample, is being carried out at 5 mo and 12 mo of samples from 1, 2, 3. 5 and 12 mo.
which have been transfened to -30°C at each timepoint.
WI-fO1fZS105.2018 Page 5
In addition. the stability of reconitituted material heid at 4'6 for 1, 2 and 4 weeks and tranrkrred to - 30"'C. and reconstituted iample held at -2fl"C for 1 nlonth is being determined. uling the -20°C I?ophilized
\ample as. baseline. Prior to the ltudq, arnpouIe% \tored at -20°C %ere recoiiitiruted uith 1 m1 s.terile ultrapure (Barnlread) u ater.
The lnethods ured for analyiii of the stability iarnpls ineIude the ribose assay for PRP content, according to the European Pharmacopoeia 12.5.3 1 J and Table 1 (Lab 10). rnotecuiar sizing to detemline a GC eluting b> a Kn value detem~ined in the main peak of the -20°C sarrrple, and tveight-a\erags molar ma\\ (MW) b j SECMALLS (Size-exctuiion chromatography with Multi-angle laser light s;cattering); m d , pH determination. Prior to analysis, stability samples (ampoules) from the real-time and accelerated degradation study mere reconitituted with l m1 of sterile ultrapure (Barnstead) warer at room tempemture (23 "'C It 3°C) and stored at -1°C for up to 2. week, during the duration of the analysis. It u a s asiumed that each an~poule was filled with 5.0 nlg PRE" for the purposes of this stud>.
The molecular siring was carried out using a Dionen HPLC system with a SpectraSjstsem RI-150 refractometer. The columns used were Tosoh Bioscience T S K 6 0 0 0 ~ w x ~ phi, a T S K 5 0 0 0 ~ u . x ~ in series with a PWXL guard column. The sample was loaded onto the column using an AS-50 autosampler: 100 u1 of the reconstituted sample, containing 50 ug caccharide was injected, and then eluted with PBS (10 mM Na2HP01, 1.8 mM KH2P04. 17 1 IlfM NaCl. 3.4 mM KCI) pH 7.3 buffer. In addition, column calibration markers for the void volume (Vo) and total column volume (Vt) were salmon DNA (Sigma D- 1626) and tyrosine iSignla T-3754), respectively. The data was m a l j sed for 4 of material eluting at a Kr, of 0.45 wing the refractice index signal and Chromeleon ver 6.50 software.
The pH of the samples was determined to lt: 0.1 units using a Jenway 3305 pH meter calibrated with pH 7.0. 4.0 and 10.0 standard buffer solutions (Jencons).
RESULTS
Statistical analysis
a
All recults were returned to NIBSC and analjsed in a consistent manner. Indik~idual assay result5 for the candidate standard (ampoulec B and C ) from all assays are shown in Appendix B and for ampoules A and D in Appendix C. A summary of mean results ii, shown in Table 5 and F~gure 1. Results obtained in the firit ribose assay b j laboratory 5 and for ampoule B in the first HPXEC-PAD asia5 by laboratory 5 were determined to be outliers and mrere omitted from an) further calculations. Although some laboratories used standards with molesfnlolar unitage for the nbore (lab 3 ) , phosphoms (labs 3 and 9) or Nh4R (lab 3) as%aqs, the sample results \\ere conterced ro rng amounts. It was felt that the ufe of milligrams was more appropriate as PRP content is specified using a weight (i.e. pg/viali for final product) unitage.hnaljsis of varimce showed no significant differences in the results obtained bq the difkrent methods.
'An overall mean of 5.155 rng (95% confiderlce limits 3.586 to 5.424: CV I l 25%: n=201 was calculated.
I n t e r - l a b o m t o ~ variability n a s smaller for ribose assays (CV 4.2%; n=7) and phosphoms assaqc (CV 3.6%; n=5f when compared to HPAEC-PAD assays (CV 16.5%; n=7t uhich gave both the smallest 13.239 mg; lab 10) and Iarged (6.688 mg: lab 6) laboratory means;. As some of the HPAEC-PAL) results were calculated using a bulk conjugate as standard. the analysis of rrariance was repeated treating this as a separate assay method. The results from this method were noted as being cignificantly larger ( p d . 0 5 in
lVHOIBS105.3018 Page 6
Tuke>l'\ multiple compari\on test) than tho,e obtained uring the other method5 (ribore, pho,phoru\ and HPAEC-PAD with PRP \tandard). The HPAEC-PAD method rernained the mo\t lariable. even when splitttng the results tn thir may (CV 9.6'~: n=3 for lab., uring PRP 5tandard. C V I0.lC;: n=3 for jab\ using bulk conjugate standard).
For each laboratory and assay method. the nlean result obttiined for each of the four ampoules of candidate standard was calculated. Csing these results, analysis of variance was used to compare the variability between laboratories to that within laboratories (between ampoi~lesf. For all assay methods, this was highly significant (p<0.001), as indicated by a11 within-laboratory (between-atnpoule) CV's being less than the corresponding between-laboratory CV. Table 6 gives a summary of the results for ampoule C only. For each of the ampoule sets sent to parricipants, the mean result for ampoule C is shown, together with the same result expressed relative to ampoule B. Between-laboratory CV's are reduced from 3.0% to 1.7% for the ribose assays, 3.1% to l .S%% for the phosphorus assays and 18.3% to 8.2% for HPAEC-PAD.
For all methods, this illustrates that a reduction in between-laboratory variability could be achieved by use of a conimon reference preparation. Using the results from all methods. the between-laboratory CV was reduced from 12.3% to 5.0%.
Method Analysis
In addition to statistical analysis, the methodology was also compared to highlight any differences between methods and laboratories as a possible cause of variation in results. All participants tested the two sets of ampoules separately on different weeks, as requested. For the ribose assay, 317 ltlboratories repeated the assay 2 or 3 times on both sets of samples (Appendix B): for the phosphorus assay, 115 laboratories repeated the assay twice on both sets; and, the HPAEC-PAD assay was also repeated twice by 316 laboratories for both sets of samples.
The ribose assay gave a mean value of 3.933 mg?/ampoule from 7 laboratories. Methodological details d o not account for the variation in PRP content (95% confidence limits 3.733 to S. 123 mg). Most laboratories had good reproducibility b e t ~ e e n ausays, with the exception of lab 5. In this case. similar standard curTes were obtained for the two assay\. but samples B & C gave very much lower results on one day than the other. Lab 10 estimated lower PRP content (4.6 1 S mg PRPlampoule) than the rest. Comparison of final
orcinol-ferric chloride-HCI-EtOH concentrations did not highlight an! real differences between this
*
laboratory's method tn hen compared with thaw of labs 3 and 7, which measured PRP contents close to the mean. It is plausible that the differences in the actual ribose concentration in the standard preparations can account for differences in PRP contents quantified by this method, and in other methods such a5 HPAEC- PAD in which standards have been quantified bared on the orcinol assay. A11 the laboratories were using
>99% pure D-ribose as standard.
The phosphorus assay gar e a mean -c alue of 5.077 mglampoule from 5 laboratories. Despite differences in the phosphate standard (sodium salts, potassium salts and ribose-5-phosphate) and internal reference preparations (synthetic PRP. polysaccharide. conjugate), little variation between laboratory methods is obsemed for this assay (95% confidence limits 4.817 to 5.307 mg). The use of the Ames method flab 3) for liberating phosphorus, and use of an organic ribose-5-phosphate standard, may have been a reason for the P R P content (5.365 mg) slight11 higher than the mean of 5.077 mglampoule.
1VHOIBS/05.2OlS Page 7
The HPAEC-PAD method gatre the most variable results (95% confidence limits 3.636-6.290 me). Labs 1. S and 9 appeared not to have as n~tich control over the hydrolysis time and remperature, and had higher PRP values. Two of these labs. 1 and 8 , also used higher sodium hydroxide concentrations (0.3 M ) rhan the others (-0.1 M,, Labs I and 8. did howe~rer. in addition to lab 10, attetnpt to controI variabilitq.
through the use of an internal standard.
U
In rhe case of lab 9, the stitndard used (PS or bulk conjugate) i~ppeared to be a source of variation. M'hen using bulk conjugate from lab S as a srtmdard, the value from lab 9b agreed with that of 8 (also using bulk conjugate as a stttndard) and were amongst the higher values; when using PRP PS from lab 5 as rt standard, the value from lab 9a \vas amongst: the loiver values. The size and design of the collaborative study did not permit a statistical comparison of results obtained for the HPilrEC-PAD assay using PS or conj ugate standards.
The NMR method wa\ used by two laboratories but quantitatile values ere a ~ a i l a b l e for on14 lab 4 and therefore we are unable to determine uhether the d~fferences between the 33say for lab, 2 and 4 might result in quantitative differences. Lab 3 did identify that ampoules A and D contained meningococcal polysaccharides of the correct ~erogroups.
STABILITY
The PRP content did not change significantly (95% CI) when stored up to 5 months at 4, 20, 37 or 56°C.
\vhen compared with a -20VC sample. As the 5 month data from the accelerated degradation study cannot be used to estimate a rate of loss of PRP content. and the stability program will be continued as planned.
Although the amount of PRP in the ampoules did not change, the molecular size of the candidate standard was sensitive to elevated storage temperature, with an increase in the elution time of the main peak and % of nlaterial eluting by a K,, of 0.45 corresponding with increasing storage temperature (Table 8). The pH of the samples remained between 6.6 and 6.8, with no discernible trend.
In a separate experiment. the 6 mo real-time stability sample from 2 0 ° C was also analyzed for molecular size. No significant difference tvas seen between it and the -70°C baseline sample (Table 8).
DISCUSSION
Current Irluernoyhilus in-fl~lenxue b conjugate saccines (Hib). \+hi& are made of purified capsular polysaccharide (PRP) conjugated to a carrier protein, are almost complete11 evaluated by phl sico- chemical methods to ensure the consistency of manufacture of batches. The absence of a potency assay makes the quantification (in S1 units) of PRP in final fills or bulk components of Hib vaccine3 a critical release test for both manufacturers and national control authorities (WHO requirements, Ph. Eur. and OMCL guidelines for batch release of Hib vaccine\).
WHOllBS/05.2018 Page 8
Determination of PRP conteat
The presence of a reference preparation containing a dsternlined amount of PRP should hcilitate caiibration of various n~ethods and in-l~ouse references to quantify the PRP content of the bulk saccharide, bulk conjugate and final fills. This was highlighted in the %%IHOIPIJfBSC workshop i 1998) tvbere it was clear rhat differences in assaj8 results between laboratories were due mainly to the use of different reference materials in the different Ittboratories. The results from this study illustrate that a reductiorl in between-laboratory variability could be achieved by use of a common reference preparation. Data analysis showed no signitlcatlt differences in the values obtained by rhe different assays, suggesting the suitability of the proposed reference for use across these assays.
It was decided that it would be more appropriate to calibrate this preparation in S1 units rather than International Units as all vaccine manupacturers assay the content of active component of Hib vaccines by chemical analysis and express the results in S1 units. This practice is well-established and it would prove very difficult to obtain acceptance of a change to IU. Furthermore, as most control laboratories also use physico-chemical assays to check the PRP content, it is appropriate that the results should be expressed in S1 units. For this reason, the only rational course is to express the activity of the standard in S1 units. This may raise the issue as to why a biological standard should be required for this type of assay. The main reason for this is that the vaccines are prepared from biological materials and are subject to batch to batch variation as they do not contain a single molecular species but a mixture of conjugate molecules that can vary in size and extent and position of substitution. Experience shows that inter-laboratory variation is common when assaying these materials, even by chemical methods. As shown in this study, the use of the standtlrd helps considerably to harmonise these results.
As the candidate standard is to be calibrated in S1 units, a single reference traceable method needed to be used. with assignment of unce~tainty derived frorn collaborative study data (WHO recommendations, 2004). The ribose assay was chosen for determination of the PRP content of the candidate standard 0211208. It is acknowledged that the ribose, phosphorus and HPAEC-PAD assays rely on the degradation of PRP. The hydrolysis methods chosen for the HPAEC-PAD assay. for example. are based on the optirnal hydrolysis/degradation ratio. That the value obtained from the single NMR assay, which is a non- degradative technique. is verq close to the values obtained from the other 3 techniques gives credence to
the use of one of these assays to assign the PRP content in the ampoules.
a
In our study, either the phosphoms or the ribose assay could be used, but not the HPAEC-PAD assaq as it is not traceable due to the use of in-house PRP or bulk conjugate references. Ho~vever. as the ribose assay is more specific than the phosphorus assay. it W ~ C , therefore used for the qumtification of PRP content of the candidate standard.
Suihbifity of the candidate standard 02/208 for use as a reference in various assays
F%-hile this study did not fomally compare the quantification of PRP polysaccharide, activated polysaccharide. bulk conju,oate and final formulation b> the candidate standard, ~t is clear from the ptuticipants' methods (Tables 1-3 and submitted protocols) that most laboratories use their method for measuPing the PRP content for bulk conjugates and pol>saccharide (including some derivatised sticcharide). Hotvever. depending on the fornulacion, the phosphoms and ribose assays Rere not alaays used for quantification of final fills, due to the possibility of interfering substance\.
1lrHO1BS105.201 S Page 9
The use of a standard or intemal standard that is close in structural composition to the biological being tested i s an advantage to the laboratories performing this assay. The use of the candidate standard 02f208.
PRP polysaccharide. for quantification of bulk conjugateifinal fills in HPAEC-PAD assay wilt reqtiire careful consideration and validation in each labonltory, as it will with the ribose and phosphorous assays as well. Taking this into consideration. the use of a potysaccharide standard offers the advantage that irs repeating unit composition is common between all Hib vaccines, with only chain length and end-group structure changing. A saccharide-protein conjugate standard reference preparation manufactured with a pafiicular conjugation chemistry and carrier protein will vary in vastly more ways between different xraccines, and may not be as useful to the com~nunity at Iarpe. Betcveen conjugates there are difirences in the carrier proteins, conjugation chemistry, saccharide-to-protein ratios. and size, cross-linking of the conjugates, and the difficulty of using a PRP-TT conjugate standard for a PRP-GRM197 conjugate or another PRP-TT conjugate may well be greater than the col~siderations necessary in using a PRP standard.
The advantage a PS standard would provide in reducing inter-lab variation would outweigh the drawback of not ha\.ing a precisely matching conjugate standard for each individual product on the market.
The great reduction in the betlveen-laboratory variation from the use of this candidate standard in all assays (from 12.3% to 5.0%) strongly suggests that it can be suitable for the evaluationlquantification of PRP content in the vaccine and components (WHO. 2000: Ph. Eur, 2002): purified polysaccharjde, bulk conjugate, and. providing there are no interfering substances, final product as an internal reference in the ribose and phosphorus assays. or as a standard in the HPAEC-PAD assaq. The suitability of the candidate standard for use in immunochemical assays has not been evaluated in the present study.
Stability of the candidate standard
The stability study of the candidate standard is underway and indicates that the PRP content of the sample will remain constant at the designated storage (-20°C) and shipping (on dry ice) temperature of the sample. The stability study will continue as planned in order to monitor PRP content and molecular size of the reconstituted and lyophilized material.
PROPOSAL
Baced on the results obtained from the ribose assays in thi< collaborative stud?, we propose that the PRP candidate standard 031208 is established as an International PRP Standard with a content of 3.933 k 0.267 mgampoule (expanded uncertaintq calculated using a coverage factor of 2.55 which coiresponds to an approximate 95% 7cer.eI of confidence), for potential use in Phosphorus, Ribose, HPAEC-PAD and KMR assays for quantification of PRP in bulk PS, bulk conjugate and final fills of Hib vaccines.
!VNOIBS/05.2018 Page 10
CO1g:C"INTS FROM PARTICIPANTS ON THE DRAFT REPORT
Nine of the ten participating laboratories agreed \\ith the recommendtttions of the draft report. and four participant\ had the foIInwing coixments:
Lab l felt thttt the variation in the HPAEC-PAD results between laborarories was unlikely to be due to difference in the depofymerisation time (as had been speculated by the authors of this repost), but rather to the use of different calibration standards.
Re,~por~.se to Lu13 I - The oh.ser\*~rtiitn tltut sot~lc. 1uh.s did rzot 1zut.e u iigfitly dqfinecl hyrlrt>lssi,r tirnc ~ t . ~ t s
sugested us u possible cum,ve of v u r i u t i ~ ~ ~ ~ . T i l i ~ ~ C I P S not prt7clzidc~ other salcrccJ.s ~>ilriation, . ~ ~ t c h U S t11e source reference nwtorial/culil~t~-~ttic~~? st~zf~d'irtk.
Lab 4 did not agree with the propo\al of rhe \tudy and wggested [a] thttt the phospbomc assay should be used for determination of the actual content of PRP in the candidate standard. ac, it seemed to be the 'mo\t
accurate' assay from the data in the studq.
a
Respoi?se to Lub 4(u] - Accumcs is u izzc.usLtre o$' closer?ess to 'uctuul' anipoule content. As tlzis is not kizott.iz, tlzcz uccuruc~ qf tlze ussir! met1zod.s carrnclt be ussessed fronz tlze resu1t.s ohtairzed in tlzis stud!.
Wlzet? ussessing yrc~cisioi~, (1 smaller hertveen-luborzltq CV I1.a.s obtuilzed for tlze phosylzonrs u s s u y (3.69;; n=5) ~t*he~z e o ~ u r e d to tlze ribo.re ussu!s (4.2%: rz=k). Hott*e\>er, these CV's ure bused on clifet-e~t lliborutories und shortld rzot provide the Ousis for ~letenninirzg tchich ~ i ~ s s u j ?nethod is used in the finit1 lietemziizutior? of P W content.
Lab 4 [b] also suggested that the only use of the candidate standard would be in the HPAEC-PAD assays which rely upon PRP rekrence material.
Re.~ponse to Lub 4(b] - Tlze ucitlzo~r disagree thut the stundurd 1t.oul0 orzly be clselfill for HPAEC-PAD.
For the rihcise ussuy, 6 oz-lt of 7 1aborutorie.s are 1i.sii~g in-C~ouse PRP or co~ljrtgute refei-ences. Sevei-ul orcqfcrzisutiorzs huve requested the use ofthis nzutericcl (is u sturzdurd-fir rzon-HPAEC-PL4D ussuys PRP.
Lab 4 [c] also commented that the methodology for estimation of the error range did not allow for
systematic errors in the methodology within laboratories. or allow for errors due to the purity of the ribose
a
reference material.
Re,rpor~sc to Lab 4(c] - h respoizse to this cor?tnzef?t, ccll Iuhot-utories pe@?)i-n?ing the rihose ussus ~t'et-e uskezl to esrintute the u~zcei-tuir~t~ of wteusurenzerzt for tlzeir ussuy, Or~e /ribor-utors (Lub 10) estinzutecf tlze uncertair~tx o(ff.r?eusureinent to be rt: 3.8% tt9ith tlze ~1~1~7rintlrztjC;rc~or urisirzg fronz the ~veighirzg cf the ribnse .~tundtrrci! In uccordur~ce ~ ' i t l z ELTM CHEiM gitiilelir?e.s, corzzbitzed stundanl ~#ncertczinty nf 0.109 us culculutedfr~?m stu~zdtlrd L I I Z C C ~ P - ~ C ~ ~ ~ ~ ~ ~ . C of 0.078 (.~tuf~duI%I deriutiot? ofr~zeun of ribose asscl! I-esul'ts) und 0.076 ;G(uncertuitzts cofrzporzeizt due to udtfition~rl so5trces of error such us tt.eiglzir?g; uss~lming u triallgulur distribrrtion of 4.933 rt: 3.8%). ~kf~cltiplicufion by 2.35 !Sftldent9s t @r 95% conjfiifeizce (2-t1iilecl] on 6 degrees of fi-eedomj gi1.e~ un q i t i z ~ l e c l urzcertuint\* cif rt: 0.267 corresponrfirzg to un ~rppl-o~virnute 95% le~.eI of catzj2den ce.
iVHOnBS105.201 S Page 11
Lab 4 id] Related to [c]. lab 4 felt that the candidate standard n 3 s a chel~lical rather than ri biologicat rtandard which required tl different approact~ from the one u\ed in the stud).
Rr,sport.re to Luh 3 j d j - This is 0 1 7 1 ~ i~hc7mii,i-r( sti~n~ftir-~l ir? tlrc scizse thirt the PRP C O ~ E ~ C I I I i.s u s ~ c l l l s Liet~riilir?t%l hs chemicu1 u.r.suys. t j O l t ' ~ ~ , e r , t h ~ t.(;lcci~z~5 arc3 b i ~ l o ~ q i ( ' ~ t l p f - o d ~ i ~ , f ~ ~ rt*l?iel? .rliolt li7~ttcI? to b ~ i t c h
~,uri~~tinlr. f i c l ~ ntui?~tfi~rtut-er r4.r.e.s their- o t t ~ it? Izou.~c rt<ic;?~-(~~c.llcc 1.t*lric'lz i.s j7i*o~I~c*t-.spocfii.. E-iytrief?i'e I?UJ slictit*~~ t l z ~ t t12i.s C'LIFI lecid to ii7ter-luholrrtc~rs rl(#2~r~ltzi.c~s ~vlirefz hutchrs urr t<..rted c~lsei+*ht~re, Tltc? present
. Y ~ C I L / F sl~ott's thut tlw useJ ~lf'tl?~' j 3 r o p o ~ ~ d ,rtirndur~l rc>d~fccs i n t e r - l ~ ~ b o r u t c f ~ ; ~ vuric~tiorz. \+'lt~tl~~r it is culled
i r hiologicul or cl~c~i?zicul ,sturzcfurd is I~irgelx qfsei?zurztic sigtziJ"icsurtcr. B'P believe it sizotrld he ~ ~ ' g t l r d e d U S
u hiologic~~l stund~ird bec,ilu.sc cg'its uyplicutic~rz fo U b i ~ > I o g i c ~ ~ l preld~icf.
Lab S commented that different Hib vaccines. with specific molemlar weight. different carrier protein conjugated to the PS. combined with other anrigens or excipients are present in the market. When ucing a PS reference for analy\is of conjugated purified bulk, form~~lated and h a 1 products any potential interference related to conjugation, formulation. pre-treatment of the vaccines should be taken into consideration.
Response to Lub 5 - Tlzc. uutlzorc ure ir-t Llgrecutlcfzt ivitl? Lub 5 lrnd ud\*iw c~lref~il ~ o i ~ ~ i d e r u t i ~ ~ z of t l z e ~ c jhctor.5 (see ulso D ~ J c ' L I J ~ ~ o ~ z ) .
Lab 8 commented on how the nature of the reference product may have an impact on the HPIZEC-PAD response, especially between PRP and bulk conjugates because the sugar units on which the coupling with the protein occurred do not behave as free sugars anymore, thus not parricipating to the HPAEC-PAD response. Therefore. in their experience. the dose-response obtained with a bulk conjugate reference is generally below the dose-response obtained with a PRP reference: then. the samples quantified against a bulk conjugate reference appear higher than when analysed against a PRP reference. Hence. lab 8 d o not recommend the use of the candidate standard for use in FIPAEC-PAD for determination of P W content in
a
Hib conjugates (bulks or final products) and suggest that this needs further investigation.Re~yon~re ro Lab 8 - L u ~ 8 ruisc un irirere~ting urzd important poi~zt ubtlur on eflect of corzjugutiorz on the 1z~dr-ol~sis of tl?e temzinal scicchuride cqr~~.tp. Tlzere i f , ho~vever, no rcientijfiic cl-iilencc. rlzur cot~jtc,q~/tiorz of PS to protein r-l*ill re'~1~1ee the poIvtizeri:urior? of the ~uccharidc? ~lte~ieties. TIzt ~zuinber of non-reacti~~e er~dgroups/totcil PRP repecitiu-tg unzt.5 i~ ~?zinirirlcllr?zinim~rl (- 1-4 %, bused on tlze rucclzurille: proteirz r~itior
of nr o tetut?us roxotd co~?julugcife r (UZHO, 2000).
The poir~t ubottr the use of LI PS s t ~ ~ r ~ d u r t f itz the HP'rlEG-PAD iir5u.r. for guur?tift.irzg PRP irz u bulk corzjugate ir ttjorth notir~g. Three of rife 6 purticiputing Iubs jor tlze HPAEC-PAD usruy rrwd PRP PS.
Aithougl? the authors do ~lcknort*Eedge ahut there crnrz b t diflereizcer bett.t.een the resztlts depending on PRP or corzjmgate stunthrds, tlwv feel thut t l ~ e adi,untcige (;l PS SLUI~LJLILZI ~t'nztld provide ill redlicing inter-klrb
X. turitltior? it,ouW oufiveiglz the dr-uit.buck cf'tznf Izu~.ing u precirels mutchitzg c ~ ~ t j u g ~ ~ t e sfuftdut-dJbr each individt4ul prod~lct orz tlze market.
%fTWOIBS/05.2018 Page 12
Special thanks are due to the late Dr. G. Lung Hsieh and Ml'beth-Lederle Vacsinex and Pediarriciz for preparing, cl~tiiracreri\ing and supplying the Wib PRP potysaccharide for the WHO.
At NIBSC, grateful thanhi are owed to Dr. Paul Marejt5chuL and Miii Rilichelle -Anderion fi-orn the Centre for Biological Reference Materials h r the careful preparation of the freeze-dried ampoule., and trial ,rudjes, to itliis\ Manofq~t Saydam for her valuable help throughout the m d y , to Dr. Chris Jonei h r his many contributio~is throughout the btud~, and to R4r. Xarier Lernercinier for hi\ help in preliminaq NhilR characteritation ctudie.1.
Acknowledgements are due to the Laboratotjes and Scientists who panicipzted in the study: man3 perfomed more than one type of assaq. In addition to those listed in Appendix A, the following desefile acknowledgement: Dr. Vicente Verez Bencomo. Mr. Felix Cardoso SanJorge, Dr. Volher Ocppling. Dr.
K Suresh. Sumeet Pendharkar, Pankaj Sharmn, Franz Lukas Sehnqder, Prof. A Gaslone, Ms. Carherine Poty. Dr. Xavier Lefebvre, Mr. Perret. Dr. Sundro D'Ascenzi. Dr. G. Pozzebon and Dr. Franceso Beai.
REFERENCES
WHOIDSf05.2018 Page 13
BN .4me\. Assay of inorganic phosphate, total phosphate and phosphataies. Method Enzymol 8: 1 15- 1 18.
G Ashttell. Calorimetric analysi., of iugart. 1957. Method Enzymol 3: '73-105.
X Bardotti. N Raven\croft, S Ricci. S D'Ascenzi, V Guarnieri, G Aksrani & P Constantino. Quantitatice drrerrninarion of saccharide in firer~zc~jjhil~r, i~lf;uu~>rr;~rc. t5pe b gl~coconjugate caccine, alone and in con~bination ~ i t h DPT, by use of high-pedormance anion-exchange chromatography ttcith pulied ampesometric detection. 20110. Vaccine 18: 1982- 1993.
PAn"H/OMCL r 04) 91 ; OCABR: Haemophiluil influenztle type b conjugate vaccines (2003).
PS Ghen, Jr., TY Toribara & H \i\'tlrner. Microdetermination of phosphorous. 1956. Anal. Ghem 28: 1756- 1755.
a
Ph. E u r 12 19: Hciefiioj,hiii~.s i12flueti:ue b vaccine (2002).MR Holliday & C Jones. WHONIBSC-co-sponsored informal workshop on the use of physico-chemical methods for the characterisation of Htlet~zoplii1~t.s ii2j7ucn:ue type b conjugate vaccines. 1999. Biologicals, 27: 51-53.
CC Ip. V Manam. R Hepler 8L JP Hennessey, Jr. Carbohydrate con~position analysis of bacterial polysaccharides: optimized acid hydrolysis conditions for HPAEC-PAD. 1992. Anal. Biochem. 142: 58- 67.
EA Kabat & M Mayer. Carbohydrate estimation. 1961. In Expermental Immunochemistry (pp 526-537).
Springfield, IL: C Thomas.
AW Sturgess, K Rush, RJ Charbonneau, JI Lee. DJ West. RD Sitrin & JP Hennessey. Jr. H<~ernophitus iqfluen:tre type b conjugate vaccine stability: catalytic depolymerization of PRP in the presence of aluminium hydroxide. 1999. Vaccine 17: 1 169-1 178.
C-M Tsai, X-X Gu & RA Byrd Quantification of polysaccharide in Hrrc>~?arphiii4.s ii!jli,cii:iie type b conjugate and polysaccharide vaccines bp high-pedomance anion-exchange chromatography with pulsed amperometric detection. 1993, Vaccine 12: 700-706.
WHO Recommendations for the production and control of Hue??zoplzilw i~~$zlencae type b conjugate vaccines iTRS 897, 20001.
EURACHEAM. Quantifying Cncertaint~, in AnaIytical Measurement (Second Edition, 2000). ISBN 0 918926 15 5.
Table
1. L)et;tils of the ribose assay conditions used by participants in the collaborative study
Standard
Standard curve range
D-Ribose Sigma R-7500
Tetmj of incubation
-
Spectrophotorneter Waveten$%
X irversion factor
1 Mean PRP content in mg was taken fromTable 5.
D-Ribose Fluka
D-Ribose Acros Chimica
132360250
D- Ribose Merck 1.07605,
stored -20C
Ribose D- Ribose
Merck
D-R~bose Sigrna R-7500
Table 2. Dctnils of t l ~ e pllosphorus assay conditions used by participants i11 the collaborative study
Labaratoryl 2 3 7 8
----"-.p"-
Standards N&3P04. 121-120 Rib-5-P, 2H20
Fluka Lot 430900/1 Fluka 83875 K2HP04, anhydrous KH2PO4, pure
Standard curve range
--
I f levels
P-
---
Incubation Method lrrcubatio!?
-.- p--
---
Chen
blocW Water batch
-"
180C. 90 min/
37% 120 mm
--
S~ectraMax PLUS.
Molecular Devices
-,-.----F.p-.-
820 nm
-
1 Mean PRP content in rng was taken from Table 5.
Table 3. Hetails af tllc EIPAEC-PAD assay eoaditioi~s used by participants in t l ~ e eollal>orative sti~dy
1
Standard 81 references I ---.-
0.1 M NaQti p-
-p
.- -.
12 h, 21 C, then 4C
PA10. Arn117o Tr=
PRP, 18 t~iin. G-I-P, 10
G-I-P, 10 mirr n/a 4,239 1 Not applicable.
%lean PRP content in mg was taken from Table 5.
Table 4, Details of the NMR assay condition used by participants in the collaborative study,
1
LaboratoryI
41
2 1I Standard & references l
i
I
d
StandardQther references I
l
l
I j
l -i l
1
Temp/
25CI
Software / Varian vnmr 6.lc / Bruker XWIMNMR v2.6I
Conversion factor
1
100% of Na counter-ion1
not applicablel l
Data points Spectral Window
/ Mean PRP content / 4.986 mu
1
not determinedI
64k 5000 Hz
16k 4000 Hz
Table 5. S u m a r y of resuits (mean PRP content in mg) for the candidate standard
HPAEC-PAD
Overall Mean
m o o r - - c l - - r - m , *;tyc)r-.g\
ro? Or":?
- 0 - - 0 0
1.YH01BS105.20 18 Page 20
Table 7. hlean PRP content (mg) of candidate standard after storage at elevated temperatures (results determined by lab 10, ribose assay)
Fb'HOIBS105.2018 Page 21
Table 8. klolecular sizing chromatographic eiution of samples of Hib PRP frorn accelerated degradation and real-time stability studies
i
I Study Timepoint l Storage
/
Perrent eluting by1
1 temperature i KI, = 0.45 ( 5%
1
("C) l
!
Accelerutsd 5 nro. baseline
1
-70l
48.3Accelerated
1
5 rno. blticline1
-30 46.1Accelerated
1
5 mo i l 4-4 44.0hcceterated 1 5 mo
1
+30 33.3lccelerntrd
1
5 tno +37 I 31.8m P - m
CL. CCCL.
I
0 W Q CL.
-
14THOIBS/05.2018 Page 23
APPENDIX -4: Participating laboratories Dr Violeta Fcmandez Santmtl
Centre for- the Stud1 of Srnrhetic Antigens, Facult: of Chemistr?
e University of Har, ana, I&\ 3133 IO301f Cuba Dr Ghris Jones
Laborrrtory of Molecular S t ~ c t u r e NIBSC. South. Ilil-irnms. Porters Bar.
Herts. ENS 3QG. CK Dr Stefan Christians
Paul-Ehrlich Institut. Paul-Ehrlich Str. 5 1-59 D-63225 Langen, Gemuny
Dr Suresh S Jadhav
Serum Institute of India Ltd.
2 1212 Wadapsar. Pune-3 1 1 028, India Dr Juerg Stadler,
Head Laboratory Unit Vaccines, OMCL Biologika Hallerstrasse 7
PO Box
CH-3000 Bern 9 Switzerland
Dr. Ghristina von Hunolstein, Unit of Bacteria1 Vaccines.
Dept. of Infectious, Parasitic & Immune-mediated Diseases, lstituto Superiore di Sanjta.
a
Viale Regina Elena, 299,I-00 1 S 1 Roma, ItalyDr Benoft Thirion, QC Physicochemistr-y, ClaxoSn~ithKIine Biologicals, Rue de I'fnstitut 89, B-1330 Rixensarr, Belgium
Dr. Paolo Costmtino, Head, Technology Development Department. Chiron S.r.1.
Via Fiorentina, I , 53 100 Siena, Italy hlr Dennis Crane,
Bacteriologj Division
B NIBSC, South h"Iimms. Potters Bar, f-lerts, EN6 3QG. UK
P
Dr. Sylvie Uhtrich. Sanofi Pttsteur. Campus hlerieux. QCdev Physicochemistq Department 1541, avenue iMarcel Merieux. F-68280 Marcy I'Etoile, France
WH01BS/05.2018 Page 26
APPENDIX G : Results (PRP content in mg) for ampoules A and L) Ribose Assavs
A
L3h
1
iZs5a) ; I 1 D1 1 I l
I 2 1
l
Not dctecttthlel
i
Not detectabl~Phosphorus Assavs
D
<0.018 Lab
1
Assay2 l ,,
17
A
~ 0 . 0 1 X
2 l
h
0.0006
Not detectable
0.0003
Not detectable
1;C'"HOnBS/05.20 18 Page 27
HPAEC-PAD hssavs
NMR Assays
