Rapid Analysis of RNA in Virus Like Particle

by CZE

Yan He

Pfizer Inc

-1-

Outline

1. Structure of Virus Like Particle

2. RNA size profile by chip CGE

3. RNA quantitation by CZE

4. CZE method performance

5. Conclusions

Virus-like Particle (VLP)

• Capsids of E. coli RNA phage Qbeta

• Consisting of 180 monomers ( 20 hexagons

and 12 pentagons)

• Monomer: 14kDa

• Genomic RNAs:40-4000 nt

• MW = 2500 kDa

Diameter = 30 nm

Virus-like particle

VLP-based Vaccines

Vaccine Company Engerix (hepatitis B virus) GSK Cervarix(papillomavirus) GSK

recombivax HB(hepatitis B virus) Merck Gardasil(papillomavirus) Merck

RNA

VLP-antigen Conjugate

Repetitive

protein shell

Linker

RNA

Antigen Antigen : • Conjugated Peptide

Characterization of RNA

---- RNA size profile by CGE

---- RNA concentration by CZE

Size Profile of RNA by Chip CGE

-5-

RNA ladder

RNA from VLP

Why CZE for Quantitation of RNA ?

CGE: size

CIEF: charge

MEKC: charge, mass and hydrophobicity,

CZE: charge to size ratio, Z/m

RNA: RNA of different mass has similar Z/M --- ca. -1/330

A T G U T

1. Standard selection

E.coli RNA std, two sizes, (1500 and 3000 nt)

2. Internal standard selection

Phthalic acid

3. Release RNA from VLP

4. Separation condition optimization

Capillary: uncoated capillary

Buffer: Hepes, HPMC, pH

CZE Method Development

Release of RNA from Virus Like Particle

RNA Release Time Course

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25

Heating Time (min)

No

rmalized

Sig

nal

Release buffer: 8 M urea, 30 mM Borate, pH 9.6

Incubation temperature: 95 C

CGE of DNA ladder in uncoated capillary

Z.K. Shihabi, J. Chromatogr. A 853 (1999) 349-354

50 mM Hepes, 65 mM boric acid, pH 8.1, HPMC 0.5% HPMC , 70 bp to 1400 bp

Hepes

Tris

Hepes

-10-

CGE of DNA ladder with Different HPMC Con.

HPMC concentration: (A) 0.25%, (B) 0.5%, (C) 0.75%

Uncoated capillary, effective length, 20 cm

Size of DNA ladder: 6 to 10 K bp

CGE of DNA ladder with different capillary length

-11-

Effective capillary length (A) 20 cm (B) 40 cm

CGE CZE

CZE of Internal Standard and RNA released from VLP

-12-

mV

100.00

102.00

104.00

106.00

108.00

110.00

112.00

114.00

116.00

118.00

120.00

122.00

124.00

126.00

128.00

130.00

132.00

Minutes0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80

Buffer: 50 mM Hepes -60 mM boric, pH 7.2, HPMC 0.05%

Capillary: (10+20) cm x 75 um id, injected from short end

Detection: 254 nm, Vinj:0.2 psi x 5 sec. Voltage: 12 kV.

IS

RNA

(A) RNA std

(B) RNA from VLP

(C) RNA from VLP-conj

IS

RNA

RNA

Separation done within 3 min!

Specificity by UV spectrum

RNA std

RNA released

RNA std: 260/280=2.16

RNA released: 260/280=2.13

The similar ratio of RNA std and RNA released suggest that released RNA peak

is pure.

Recovery of RNA

Recovery is evaluated by spiking VLPs with 53 ug/ml RNA standard before

released treatment. The original RNA inside VLP is subtracted.

VLP Spiked RNA (ug/ml) Measured RNA (ug/ml) Recovery%

VLP 53.3 59.7 112.0

VLP-conj 1 53.3 56.4 105.9

VLP-conj 2 53.3 50.1 93.8

Intermediate Precision

Instances TCA of IS TCA of RNA TCA Ratio (RNA/IS) RNA (ug/mg)

Analyst 1 Inst 1 Cap 1 4568.99 10014.33 2.19 228.5

Analyst 1 Inst 1 Cap 2 6362.17 12763.87 2.01 193.3

Analyst 2 Inst 2 Cap 2 5168.06 13192.46 2.55 241.1

Analyst 2 Inst 2 Cap 1 4405.94 10667.19 2.42 232.1

Average 5126.29 11659.46 223.8

Std Dev 886.69 1555.73 21.0

% RSD 17.3 13.3 9.4

15

Test of RNA in VLP and VLP Conjugate

VLP VLP (ug/ml) Released RNA (ug/ml) RNA%

VLP 500 156.3 31.3

VLP conj-1 500 113.4 22.7

VLP conj-2 500 131.0 26.2

Summary

17

1. RNA in VLP can be characterized by CGE for size

profile and CZE for concentration

2. The use of Hepes buffer, low con HPMC enable the use of

short uncoated capillary for fast CZE separation of RNA.

3. CZE method demonstrate adequate specificity, linearity

and accuracy. Improvement of reproducibility is needed.

Acknowledgements

18

Vicky Hou, Margaret Ruesch

Helen Sato Michael Jones

Heidi Holovics John Amery

Qian Wang Rachel Robert

AU

-0.028

-0.026

-0.024

-0.022

-0.020

-0.018

-0.016

-0.014

-0.012

-0.010

-0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00

RNA by HPLC (AEX)

RNA std (brown), RNA std stressed (blue), PVLP stressed (red), water (black)

Backup Slides

mV

100.80

101.00

101.20

101.40

101.60

101.80

102.00

102.20

102.40

102.60

102.80

103.00

103.20

103.40

103.60

103.80

Minutes1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80

Stability sample 120999-026 T13 YA -70c (black), 5 c (red)

RNA

protein

Linearity

y = 0.025x - 0.0386

R2 = 0.9995

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250 300 350

Concentration (ug/ml)

RN

A-I

S r

atio