Characterization of adsorbed vaccines

Marina Kirkitadze 11 September 2018

Drug Delivery and Formulation Summit 2018

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Biologics

Purpose of the study ● To gain product knowledge about the adjuvant surfaces and antigen

protein distribution ● Linked to antigen adsorption capacity and possibly immunogenicity

● To examine the effect of antigen proteins on adjuvant morphology and density ● Important for product lot-to-lot consistency

● To examine the effect of adjuvants on protein antigen structure and stability ● Assist in determining product expiry date

● To develop lean techniques for drug product identification ● Sensitive and time-efficient method for final vaccine product

identification

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Outline

● Particle sizing by laser diffraction ● Particle morphology by SEM and composition by EDS and XPS

● Structure of adsorbed proteins by FTIR and nanoDSF

● Thermal stability of adsorbed proteins by nanoDSF

● Al and P concentration by NMR

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SEM characterization of size, shape and morphology of adjuvant and adsorbed proteins

SEM image of AlPO4

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Low vacuum SEM: Effects of antigen protein adsorption on adjuvant morphology

Diphtheria Toxoid (DT)

Tetanus Toxoid (TT)

Filamentous Haemagglutinin (FHA)

Conclusion: morphology of AlPO4 did not change after protein adsorption

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Low vacuum SEM: Effects of antigen protein adsorption on AlPO4 adjuvant morphology (higher magnification)

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PT

TET

FIM PRN

FHA DT

AlPO4 with strongly adsorbed proteins (DT, FHA, TET) appear to bigger in size Conclusion: strongly adsorbed proteins coat the surface of AlPO4 thus making the surface less conductive, and the images less sharp

Cl Na

Elemental analysis of DT adsorbed on AlPO4 by EDS

Overlay of all elements SEM image of DT adsorbed to AlPO4

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Al

P

O

P N C

Conclusion: detection of nitrogen confirms presence of the adsorbed protein Adsorbed DT samples have to be rinsed to remove NaCl and intensify N spectrum

Conclusions: particle structure, morphology, and composition

● Aluminum phosphate suspension consists of small submicron particles that form a continuous porous surface

● Overall AlPO4 morphology is not impacted by sterilization, however its texture appears more dense ● This may have an impact on antigen adsorption capacity

● Protein adsorption does not change the morphology of the adjuvant ● EDS elemental analysis showed the crystalline feature to be NaCl

● Therefore adjuvant and adsorbed protein samples need to be rinsed prior to analysis

● Detection of carbon and nitrogen confirms presence of the adsorbed protein

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PHI Quantera II Scanning XPS

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A diagram depicting the photoemission process for XPS and Auger secondary electron emission (AES)

Courtesy of Fredrick Seitz Research laboratory. Center of microanalysis of materials. University of Illinois

XPS spectra of AlPO4 adjuvant The XPS spectrum is plotted on a binding energy scale by subtracting the kinetic energy measured for the photoelectron from the energy of the excitation X-ray. The binding energy of analyte from a sample is obtained from the relation BE= hν-KE-ɸ Where hν= photon energy from the X-ray source, KE is the measured kinetic energy of the electron, and ɸ is the work function (energy required to remove electrons from the Fermi to vacuum level).

XPS spectra of DT adsorbed protein on AlPO4

(AlPO4)

(AlPO4)

High resolution spectra of Al and O in adsorbed DT

High resolution spectra of N in adsorbed DT

Elements Surface atomic Percent (%)

DT DT TET TET FHA FHA Al 8.57 8.52 8.66 9.38 5.82 5.97 O 47.39 49.16 46.67 49.25 40.32 40.81 P 8.28 8.48 8.26 8.31 6.24 6.16 Na 3.35 1.87 3.91 2.74 6.04 1.69 Cl 0.56 0 0.64 0 2.93 0 C 26.69 27.08 26.56 25.80 33.10 40.80 N 5.16 4.89 5.30 4.53 5.65 4.57 Al:P 1.02 1.00 1.04 1.12 0.93 0.97 P:N 1.60 1.73 1.56 1.83 1.10 1.35

Elements Surface atomic Percent (%) AlPO4 (1) Rinsed AlPO4 (1) AlPO4 (2) Rinsed AlPO4 (2) AlPO4 (2 treated) Rinsed AlPO4 (2 treated)

Al 10.32 11.34 10.02 10.98 10.31 10.22 O 55.26 58.92 54.13 59.12 57.61 57.96 P 9.05 10.81 9.39 9.62 10.16 10.39 Na 5.78 3.12 7.45 4.06 5.68 5.33 Cl 1.89 0 3.04 0.31 0.89 0.68 C 17.68 15.80 15.97 15.91 15.06 15.42 Al:P 1.14 1.05 1.07 1.14 1.01 0.98 O:Al 5.35 5.19 5.40 5.38 5.59 5.67

Summary

Conclusions

● Atomic percent ratio for Al and P is 1 for the adjuvant and drug substance

● There was significant decrease in Al and P atomic percent upon absorption of the proteins

● Difference in the chemical environment of the protein were observed for FHA compare to DT and TT

Fourier Transform Infrared spectroscopy (FTIR) analysis of adsorbed protein antigens

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Perkin Elmer Life and Analytical Sciences 2005.http://las.perkinelmer.com/content/TechnicalInfo/TCH_FTIRATR.pdf

FTIR is used to identify compounds based on their unique vibrational energies of molecular bonds, and to estimate secondary structure in proteins.

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FIM in AlPO4

FIM alone Effects of adjuvant on Fimbriae (FIM) antigen structure

Adsorption to AlPO4 caused a increase β-sheet content in FIM

Wavenumber cm-1

FTIR: Adjuvant effect on protein secondary structure

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FTIR spectra for DT (red), FHA (green), FIM (blue), PRN (pink) and TT (brown)

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FHA, FIM and PRN showed similar spectral features; Similar spectra were observed for DT and TT

4th derivative spectra: changes in β-sheet, turns and α-helices at ~ 1624, 1676 and 1654 cm-1.

The low frequency region between ~1076 cm-1 and 990 cm-1 consists mainly of contributions from adjuvant and buffer phosphate

FTIR spectra for Pediacel® (red), Pentacel® (blue) and QuadracelTM (black)

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Spectral features of the products are quite similar yet there were small but detectable differences.

• The peak for P-O stretch around 1079 cm-1 has higher absorbance in QuadracelTM versus Pentacel® or Pediacel®, the latter showing a peak shift to 1083 cm-1.

• Peak observed at 1420 cm-1with Pentacel and Quadracel both showing a broad shallow peak whereas Pediacel showed a sharper peak at 1414 cm-1.

Reference: Sasmit S. Deshmukh, Kristen N. Kalbfleisch, Kamaljit Singh Bhandal, Wayne A. Williams, Bruce W. Carpick, Marina D. Kirkitadze. Identification of Vaccine Products and Drug Substances by Fourier Transform Infrared (FTIR) Spectroscopy. Pharma Focus Asia, 2018, Knowledge Bank, July issue https://www.pharmafocusasia.com/articles/identification-of-vaccine-products-and-drug-substances .

https://www.pharmafocusasia.com/articles/identification-of-vaccine-products-and-drug-substances

nanoDifferential Scanning Fluorimetry (nanoDSF)

• Measures: • Intrinsic fluorescence intensity • F330, F350, F350/F330 • Aggregation

• Requires: • Small aqueous sample • One fluorescent residue • Change in environment around fluorophore

• Produces: • 20 data points/min • Tm of unfolding and refolding

events • ∆G

20

Tyr

Trp

The Prometheus NT.48

nanoDSF is a fluorescence based method dependent on presence and number of Trp and Tyr residues in the protein and where in the molecule they are localized nanoDSF is a probe for protein tertiary structure. As with DSC, nanoDSF reports transition temperature (Tm), however does not report enthalpy of transition

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nanoDSF as a probe for protein tertiary structure and effects of buffer, AlOOH adsorption

Thermal profiles of the pre-adsorbed (red trace) versus AlOOH-adsorbed (black trace) 2-protein vaccine antigen formulation

Conclusion: A 2-protein vaccine antigen formulation showed similar thermal stability (Tm) when adsorbed to AlOOH. However, the slightly broader thermal transition for the adsorbed formulation was indicative of a more polydisperse structure adopted by proteins in adsorbed form

Thermal profiles of P1 and P2 in two different buffer conditions. shows the sensitivity of DSF to detect protein conformational changes due to buffer matrix. (a) ratio of fluorescence at 350nm and 330nm. Tm is marked for each protein (dashed line). P1 had a Tm of 50.7°C in Buffer 1 (green) and 52.0°C in Buffer 2 (red). P2 had a Tm of 58.2°C in Buffer 1 (black) and 55.5°C in Buffer 2 (blue). (b) first derivative of the fluorescence ratio is shown to highlight and determine Tm.

nanoDSF: Assessing the thermal stability of AlPO4 adsorbed protein antigens

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Tetanus Toxoid Pertactin

● nanoDSF can be performed on adsorbed protein antigen samples, as an alternative to DSC, which is not amenable to these samples due to lower sensitivity for low protein concentrations and potential instrument fouling.

● nanoDSF results show evidence of degradation (protein conformational changes) upon incubation at elevated temperature. The degradation profiles for the adsorbed samples are similar to those observed suing DSC (not shown) for the same pre-adsorbed antigens.

Reference: Kristen Kalbfleisch, Marina Kirkitadze. NanoDSF – A lean technology to test thermal stability of vaccines. BioPharma Asia, November/December 6(6): 38-41. https://biopharma-asia.com/magazine/novemberdecember-2017/ .

https://biopharma-asia.com/magazine/novemberdecember-2017/https://biopharma-asia.com/magazine/novemberdecember-2017/

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• Aluminum phosphate adjuvant suspension consists of small submicron particles that form continuous porous surfaces

• AlPO4 morphology is unchanged after sterilization; however its surface texture appears more dense, which may impact antigen adsorption

• Protein adsorption does not change the overall morphology of the adjuvant • Adsorption to aluminum adjuvant increases secondary structure content of

protein antigens compared to their soluble form; however their thermal stability is unaffected

• Adsorbed Tetanus Toxoid and Pertactin antigens had thermal stability profiles similar to that of their pre-adsorbed stages

Conclusions: particle size, compositional and antigen structure analyses

Sanofi Pasteur team: Ibrahim Durowoju Kristen Kalbfleisch Liliana Sampaleanu Danielle Johnson Neil Blackburn Bruce Carpick

York University team:

Moriam Ore Sylvie Morin

Acknowledgements

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27Al and 31P NMR spectroscopy for Quantitation of Al and PO4 Present in Vaccine Formulations

Reference: Rahima Khatun, Howard Hunter, Yi Sheng, Bruce Carpick, Marina Kirkitadze “27Al and 31P NMR Spectroscopy Method Development to Quantify Aluminum Phosphate in Adjuvanted Vaccine Formulations” – Journal of Pharmaceutical and Biomedical Analysis, 2018, 159:166-172.

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1H, 15N, 19F, 13C, 31P, 27Al

Karlik et al (1982): pH: 2 • Interaction of Al with

Phospholigands

• Binding affinities of 27Al to the ligands

• 27Al is a quadrupole nucleus with Spin 5/2

• Mol Wt: > 20 kDa

Quadrupole Nuclei & Challenges of 27Al NMR

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27Al NMR Detectable Al Salts

27Al NMR study for Product 1 at low pH using HCl

3.96 7.1

1.67

0.83 1.21

pH Aluminum adsorbed to vaccine antigens in Physiological pH

pH Titration in Product 1: 27Al NMR

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3.96 7.1

1.67

0.83 1.21

pH

31P NMR study for Product 1 at low pH using HCl

Phosphate adsorbed to vaccine antigens in physiological pH

H3PO4

pH Titration in Product 1: 31P NMR

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PresenterPresentation NotesProton nucleus is the most nmr sensitive nucleus and yields sharp signals compared to other nucleusQuadrupole Nuclei has > ½ spin quantum number, their energies split into multiple levels in magnetic field, thus producing wider spectra compared to ½ spin nuclei

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Stacked NMR (27Al and 31P) spectra of AlPO4 solution (30 x 10-3 M) at different pH. Each adjacent spectrum of a series is offset slightly to give a perspective view of the resonances.

27Al NMR and 31P NMR study of AlPO4 adjuvant at low pH

27Al and 31P quantitation: calibration using equimolar and variable stoichiometric AlPO4 solutions

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Calibration curves for the determination of total PO43- using AlPO4 solutions at pH 0.80. Graph A (top) 27Al:31P ratio of 1:1; Graph B (bottom) variable phosphate with constant 27Al concentration of 35 x 10-3 M. In both calibrations R2 = 0.99.

Calibrations for the determination of total 27Al present in solution at pH 0.80. 27Al calibrations at pH 0.80 show: Graph A (top) 27Al:31P ratio of 1:1; Graph B (bottom) variable aluminum with constant 31P concentration of 35 x 10-3 M. In both calibrations R2 = 0.99.

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Sample Information Free PO4 Al & PO4 associated with vaccines

No Samples Sample ID (mg/ml) Al (mg/ml) PO4 (mg/ml)

1 DT Lot # 1 nd 1.39 1.38

2 DT Lot # 2 nd 1.26 1.26

3 TT Lot # 3 0.06 1.44 1.51

4 FHA Lot # 4 0.20 0.58 0.88

5 PT Lot # 5 0.21 0.55 0.82

6 FIM Lot # 6 0.22 0.74 1.04

7 PT Lot # 7 0.21 0.56 0.82

P Atomic Wt: 30.973 Al Atomic Wt: 26.981

Detection of Al3+ and PO43- in drug substances adsorbed to AlPO4

Detection of Al3+ and PO43- in Quadracel® lots

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# Sample information Free PO4

Al & PO4 associated with AlPO4 or vaccines

Samples Sample ID (mg/ml) Al (mg/ml) PO4 (mg/ml)

1 Quadracel Lot 1 0.07 0.66 0.8

2 Quadracel Lot 2 0.09 0.65 0.81

3 Quadracel Lot 3 0.07 0.64 0.78

4 Quadracel Lot 4 0.09 0.64 0.81

5 Quadracel Lot 5 0.09 0.65 0.86

6 Quadracel Lot 6 0.08 0.59 0.77

7 Quadracel Lot 7 0.07 0.65 (0.63)a 0.84

(0.82)a

8 Quadracel Lot 8 0.06 0.61

(0.62)a

0.83 (0.83)a

9 Quadracel Lot 9 nab 0.61 nab

a: 5mM of AlPO4 was added to the sample to verify concentration. The values in brackets indicates concentration calculated from the spiked sample b: data are not available

• NMR is a direct and fast method to identify and quantify Al3+ and PO43- from adjuvant to the final bulk product stages

• Can be applied for consistency and comparability

studies of adjuvant, drug substances and vaccine products

Conclusions: NMR Quantitation

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Sanofi Pasteur team: Liliana Sampaleanu Danielle Johnson Kamaljit Bhandal Ibrahim Durowoju Sasmit Deshmukh Webster Magcalas Wayne Williams Bruce Carpick

York University team:

Howard Hunter Yi Sheng

Acknowledgements

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Characterization of adsorbed vaccinesPurpose of the studyOutlineSEM characterization of size, shape and morphology of adjuvant and adsorbed proteins�Low vacuum SEM: Effects of antigen protein adsorption on adjuvant morphologyLow vacuum SEM: Effects of antigen protein adsorption on AlPO4 adjuvant morphology (higher magnification) Elemental analysis of DT adsorbed on AlPO4 by EDSConclusions: particle structure, morphology, and compositionPHI Quantera II Scanning XPSXPS spectra of AlPO4 adjuvantXPS spectra of DT adsorbed protein on AlPO4High resolution spectra of Al and O in adsorbed DTHigh resolution spectra of N in adsorbed DTSummaryConclusionsFourier Transform Infrared spectroscopy (FTIR) analysis of adsorbed protein antigens FTIR: Adjuvant effect on protein secondary structureFTIR spectra for DT (red), FHA (green), FIM (blue), PRN (pink) and TT (brown)FTIR spectra for Pediacel® (red), Pentacel® (blue) and QuadracelTM (black)nanoDifferential Scanning Fluorimetry (nanoDSF)nanoDSF as a probe for protein tertiary structure and effects of buffer, AlOOH adsorptionnanoDSF: Assessing the thermal stability of AlPO4 adsorbed protein antigensSlide Number 23Slide Number 24Slide Number 25�Quadrupole Nuclei & Challenges of 27Al NMR27Al NMR Detectable Al SaltspH Titration in Product 1: 27Al NMRpH Titration in Product 1: 31P NMRSlide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35