Development of a production process for a candidate BSA reference material.

5-6th june, 2019

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INTI ´S MISSION

Promote industrial developmentvia innovation & technologytransference.

Strengthen the metrology capabilitiesfor setting up traceability & quality of measurements.

Protein Reference Material.

INDUSTRIAL BIOTECHNOLOGY

METROLOGY DIVISION

Activities of National Metrology Institutes (INTI)

• CRM Production.• Development of

primary methods.

Final Report from Boreau International Des Poids et Mesures (BIPM).

1. John Marriott, Gavin O’Connor, Helen Parkes- Final Report Study of Measurement Service and Comparison Needs for an InternationalMeasurement Infrastructure for the Biosciences and Biotechnology: Input for the BIPM Work Program. 04-03-2011. Rapport BIPM 2011/02.

Final Report from Boreau International Des Poids et mesures (BIPM).

90%

BIOANALYSIS

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2012 2013 2014 2015 2016

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Suma de Fob total en dólares

Suma de Kg. netos

Import CRM (in Argentine)

Credit: Fernando Zornada (special projects-INTI).

Total sum Dolars

Total sum Kilograms

•Final Report BIPM 2011, on bioscience “America is a big producer of Bio-CRM materials.There is gap in the protein CRMs field in our region.

Development of a Protein Certificate Reference Material (CRM), Bovine Serum Albumin (BSA).

This will be the first plataform for Protein CRM with high degree of purity inLatin America & Caribbean Countries.

Proteins Standard Productions

INDUSTRIAL BIOTECHNOLOGY CENTER•Modern center that started to work in 2009.

•INTI is 62 years in existence.

Purification

Processes

Biomass

Production

Development

processes &

CRM

Quality Control

Measurement

Training for

universities

Operations

INTI-BIOTECHNOLOGY

Production of BSA Products by Plasma Fractionation

http://marketingresearchbureau.com/plasma-industry/history-of-plasma-fractionation

Problems

Purchase of Bio-CRMs, several disadvantages arise such as:

•Shipping delays.

• Global market dependence.

•Risk integrity due to loss of the cold-chain by a longdistance.

• Expensive Reference material.

FPLC

BSA

95%

Raw

Material

“Development of BSA Certified Reference Material”

Idea

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Project “Developmentof BSA CertifiedReference Material”

June 2017.

INDUSTRIAL BIOTECHNOLOGY

METROLOGY DIVISION

“Strengthening National Metrology Institutes (NMIs) in the Hemisphere, in support of emerging technologies”

Develop of traceable preparations of BSA “Certified Reference Material”. Setup measurement traceability for total protein quantifications.

Introduce the LAC region to protein CRM production and certificationaccording to ISO standards 17.034 and ISO guides 30-35.

Objectives

Images: Word Protein Data Bank, http://www.rcsb.org/pdb/explore/explore.do?structureId=5IFO

It`s target application as a daily working standard is for “quantification oftotal serum proteins” and also proteins Biotechnology productions andClinics analysis, in colorimetric methods.

Target applications

Bovine Serum Albumin (BSA)

•Is the major serum protein in Bovine plasma.•MW 66,399 KDa (1), It has 583 aa (1). pI 4,7 - 5(2).• Is a universally accepted reference protein used for total protein quantification (3).• Protein Carrier new fusion-antibodies. Fab (alb)2 (4), can transport fatty acids, organic molecules, synthetic peptides, bacterial albumin binding domain, Fab. (5)

•Most of the mammalian cells media uses BSA to grow cells, and BSA can be a potential contaminant. •It is a cheap protein, easy to purify, and it has high stability.

Bos taurus

• Lowry

• Bradford.

• Biúret

• Amido Black

• Ponceau.S/TCA

(3) Wu Liqing, Yang Bin and Jing Wang 2011. Development of bovine serum albumin certified reference material. Anal Bioanal Chem 400:3443-3449.

(2) Andrea Salis, Mathias Boström et., al 2011. Measurements and theoretical interpretation of points of zero charge/Potential of BSA protein. Langmuir 2011,

27, 11597-11604.

(1) http://www.rcsb.org/pdb/explore/remediatedSequence.do?structureId=3V03.(4) Ralph Adams et al. 2016. Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation

into the correlation between affinity and serum half-life. MABS, Vol. 8 NO. 7, 1336-1346.

(5) Emma Dave et al. 2016. Fab-dsFV: A bispecific antibody format with extended serum half-life through albumin binding. MABS vol. 0, NO. 0, 1-17.

Methods for total protein concentration

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20,000

40,000

60,000

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100,000

120,000

140,000

2012 2013 2014 2015 2016

$ -

$ 200,000

$ 400,000

$ 600,000

$ 800,000

$ 1,000,000

$ 1,200,000

$ 1,400,000

Suma de Fob total en dólares

Suma de Kg. netos

Credit: Fernando Zornada (special projects - INTI).

Import BSA (in Argentine)

Total sum Dolars

Total sum Kilograms

BSA Certified reference Materials

Production of lyophilized BSA CRM

BSA 7 % solution USA SRM#297

BSA 7% solution as CRMc (under development)

Reference Certified Material: Reference material, characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability.

The beginning of the project Workshop: “Protein CRM and Biometrology”

•INMETRO, Brazil, 27-29 June 2017.

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LOREM IPSUMLOREM IPSUM FEDERALEnero 2019

General map

AnalitycalLine

Production Line ISO Standard

17.034

CertificationLine

CMCs forCertification

Design of Stabilitystudy & Homogeneity

study according to ISO guides-30-35

Protein Standar plus Certification of Analysis = BSA CRM

2018 2019 2020

Scale-up & shipping

Certification process

Lab Scale Prod.

Act

ion

pla

ns

INTI will produce 1 lot of 1000 vials, each one with 2 ml of BSA 7% solution, 150 mg

of protein each vial. 154 grams of protein.

Characterization & Certification: will be carried out by NMIs participants.

These activities are challenging to overcome for inexperienced National Institutes of Metrology in

Protein CRMs production.

Strategy

Development of Analytic Certification Techniques

2

3

1

Value-Asignament- Amino-acid analysis- Mass spectrometry-Labelled amino-acids. INMETRO.- Amino-acid analysis- Mass spectrometry-Labelled amino-acids. CENAM.

Stability StudyAmino-acid analysis- Mass spectrometry-Labelled amino-acids.

Homogeneity Study Purity by HPLC-UV(ISO GUIDE 35). INTI

4Additional StudiesPurity by CE/NMR-IDENTITY BY Botton-up proteomic-Mass balance study.

Analytic & Certification Study ISO standard 17.034; ISO guide 30-35

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Development of the ProductionJune-2017- April 2018

FPLC

BSA

95%

Raw

Material

Process Selection

Different combinations tested during the development of CRM candidate .

R.M. 1

R.M. 2

R.M. 3

Purity around

95%Purity ≥

98,5%

Four different processes and products were tested during the development of Candidate RM.

Pre-purified BSA serums. Fraction V or similar Fractions.

We analyzed the purification processes.

Process N° HPLC Purity Yield (gP/lR) cost (x)

BSA% RSD%

N°11 BSA 98,82 0,03 63,42 1,00 x

N°12 BSA 99,10 0,18 65,22 1,50 x

N°22 BSA 99,25 0,32 20,02 9,91 x

N°21 BSA 99,13 0,17 39,08 1,01 x

Process Selection

References: R.M.: Raw material. E, F, G: Purification fractions.

Blue Graph: Absorbance 280 nm detected. Red arrows point: SDS-PAGE profile of the purified

fractions. Brown line: Conductivity detected. Green Graph: Salt % added for elution.

Black striped area: Fraction selected as CRMc. Black arrow: point BSA on SDS-PAGE profile.

FPLC Chromatogram of the purification process 21

66,2 KDa

01M2-A EQ

05 M2-E EQ

06 M2-F

EQ

07M2-G

EQ

Can we find purified impurities for this process?

05

M2-E

EQ

06

M2-F

EQ

07

M2-G

EQ

N° Peakname

R.T. (min)

Rel. area

1 7,290 5,002 7,490 2,623 BSA 7,910 92,38

total 100

First Third of BSA main peak

02

B

01

A

03

C

04

D

05

E

06

F

07

G

08

H

09

I 10

J

FPLC Chromatogram profile of F.“G” Lot#1

Correlation with production process

Sample HPLC-UV Purity

BSA % RSD%

R.M.2 97,73 1,72

Cand. (LOT#1) 99,13 0,17

Cand. (LOT#2) 99,23 0,13

BSA-CRM NIM 99,26 0,01

HPLC-UV Purity comparison

Has the product been purified?

200 KDa116 KDa97,5 KDa

66,2 KDa

45 KDa

31 KDa

6,5 KDa

21,5 KDa

14,4 KDa

02

NIM

7 µg

03

G Lot# 1

7µg

05

G Lot#1

15 µg

04

NIM

15 µg

Ref. SDS-PAGE 12%, dried with colloidal coomasie.

01: molecular weigh marker,

02,04: BSA CRM NIM 7 and 15 µg per well

respectively,

03 and 05: Fraction G lot#1 7 and 15µg per well

respectively.

Protein profile of fraction G lot#1 and BSA CRM NIM

Sample MS Purity

O.P.P BSA % RSD%

G Lot# 1 99,49 0,02 28

BSA-CRM 99,96 0,01 13

Mass spectrometry comparison between BSA CRMc and BSA CRM NIM

These results were provided by the University of Buenos Aires.

01

MWM

Has the product been purified?

MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA

BSA coverage sequence detected 87,52 %

Bottom-up proteomic, HPLC-ESI-Orbitrap:Hydrolysis: BSA-Trypsin (1:200) over nightHPLC: Gradient acetonitrile:water-TFA 0,1%Column: Phenomenex C18, 90A. Detector: Orbitrap (resolution 60.000)Analysis software: Skyline.Missed cleavages: 0Precursors charge: 1, 2.Ion type looked: Precursors.

DETECTED FRAGMENTS

NO DETECTED FRAGMENTS

SIGNAL FRAGMENTS NOT INCLUDED IN THE MATURE PROTEIN

SEQUENCE USED FOR BSA QUANTIFICATION BY ISOTOPIC DILUTION

Identity by Bottom-up proteomic

Amino-acid analysis by Isotopic dilution Mass spectrometry

Mol.

BSA 14 61 27 28 36

Isoleucine ValineProlineLeucine

HPLC/MS

CM Ref M spike(Probe) R Ref - R M(Ref) R spike - R M(Probe)

C = ------------ x -------------------- x ------------------------- x ------------------------------

M Probe M spike(Ref) R M(Ref) - R spike R M(Probe) - R Probe

Amino-acids calculation

Probe Spike SpikeRef.

175.33nmol/g 769.23nmol/g 341.33nmol/g 350.58nmol/g 461.74nmol/g

Pheylalanin Leucine Isoleucine Proline Valine

BSA cc. BSA BSA BSA BSA

12.52 nmol/g 12.61 nmol/g 12.64 nmol/g 12.52 nmol/g 12.83 nmol/g

BSA conc. RSD%

0.99 17.61 mg/g

Phenylalanine

Hydrolyzed amino-acids Reference amino-acids

Labelled amino-acids

Amino-acids

HCL6M+ Phenol+

160°C, 3 days

Phenylalanine

Leucine

Isoleucine Valine

Proline

Chromatogram lines

Sample Reference ValueExp.

Uncertainty

BSAcandidate

17,98 mg/g 2.9 %

Time

Inte

nsity

162 mg BSA candidate were produced

amino-acids

Labelled amino-acids

Does this result works?

Number of analysis: Six.

Uncertainty: Intermediate

precision ongoing.

MRC NIM 68,75MRC NIM; 68,35

MRC NIM 64,00

45

50

55

60

65

70

75

80

Colorimetric methods -calibrated with BSA Candidate-

Results Bradford Weight NIM Results Lowry

2018 2019 2020

Scale-up & shipping

Certification process

Lab Scale.Prod.

These activities are challenging to overcome for inexperienced National Institutes of Metrology in

Protein CRMs production.

Ongoing project

Analytic Certification Techniques

20 ml Resin

0,405 grs. 960 ml Resin

19,4 grs. (7-9 cycles need to reach 154 gr)

Scale-up & shipping

Conclusion & Impact of the Project

In all cases, the candidate showed purity ≥ 99,1%, regardless of the method used.

In addition to the amino acid analysis, a low standard deviation was found between

5 different amino acids. Therefore, this Candidate under-development has

been highly purified.

This project also seeks to create an international working group in order to develop

different protein CRMs, and Strengthen National Metrology Institutes in

Biometrology.

This CRM will help clinical, scientific and industry labs to improve their

measurements. However, to achieve the use of the reference material, a

commutability study with colorimetric methods should be performed.

Acknowledgements

A. Henrion, R. Ohlendorf, C. Arsene, G. O`Connor from Physikalisch-Technische Bundesanstalt, PTB, thank you for your help on the analytic section.

H. Laiz from SIM & Biotechnology Pilot plant team from INTI, thank you for your support on the production section.

W. Liqing From National metrology Institute from China (NIM) and S. Moreno from help on the comparison University of Buenos Aires (UBA), thank you for your section.

In order to compare amino-acid analysis results (ID-MS), we are interested on performing a comparison between others NMIs, during 2019.

Inter- comparison

Patricia Gatti, Maria de los Angeles Cappa, Fabian Nigro. From INTI, thank you for your support in this project.

Many thanksMuchas gracias

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Lic. Hugo Amedei: Amedei@inti.gob.ar: Hugo Alejandro Amedei

Escherichia coli

- B. Mabs Proteins

- GFP Protein

- Anti-inflamatory

- protein acellular

Vaccines.

Kluveromyces lactis

- Quimosin.

Aspergillus niger

Lactobacillus casei

. Blood Fractionation

E.coli (High Density)

Picchia pastoris

Saccharomyces

cerevisiae.

Mamalian Cells (CHO)

Yarrowia lipolytica

Candida vini.

Rodothorula sp

Purification

Processes

Biomass

Production

Enviroment

Bio Cleanup

Cell types

LA

PFMCC

Biotechnology Pilot Plant

NETWORK FOR TECHNOLOGICAL INNOVATION

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