Optimisation of preparative protein
chromatography as part of whole
bioprocess development
Kosma Jurlewicz
Supervisor: Prof. Ajoy Velayudhan
Group members: Dr. Spyridon Konstantinidis,
Nicholas Field, Chris Wayne
Background
• Education: MSc, Eng. in Biotechnology
(2005-2010, Technical University Wroclaw, Poland)
– Specialisation: Biotechnological Processes
• Work experience: 3 years experience in Industry
(2010-2013, Polytherics [Abzena], London, UK)
– Preparation, isolation and characterisation of new
therapeutic protein candidates (e.g. PEGylation of
proteins, conjugation of antibodies, ADC).
• Interests: Hiking, climbing, travelling, art
2
Research aim
• Develop a tool for rational optimisation of
polishing step for challenging protein mixtures
Mab processing from (Hanke & Ottens, 2014) based on (Shukla & Thömmes, 2010)
Rationale:
• Efficient removal of impurities in polishing step is crucial to
achieve high purity of product and material throughput
• There is a problem of HCP and product related impurities
(glycosylation, deamination, phosphorylation and aggregation)
• Lack of well characterised chromatographic profiles of complex
protein systems under overloading conditions
3
Objectives
• Find a model ternary protein system
(Hemoglobin, Ovalbumin, BSA)
• Generate preparative chromatograms
• Optimise separation of model proteins (algorithms)
• Gain more understanding in non-linear competitive
chromatography
• Apply this understanding to a real world mixtures
from cell cultures
4
Mimicking product related impurities
Protein profiles on Hb,Ova,BSA
GE Capto Q column
1200
product
Protein mix constrains:
– Available (Sigma, etc.)
– Cheap at g quantities (~ £ 20)
– Representative size (> 50 kDa)
– Binding to cation or anion exchanger
– Binding with small amounts of salt present
– Not fully separated / not fully coeluted on
resin
•
•
System: Hb, Ova, BSA
Chromatography mode: anion exchange
Hb
Ova
1000
UV at 280 nm [mAU]
•
BSA
weak
800
strong
600
400
200
0
4
6
8
10
Retention volume [CV]
System: GE ÄKTA
Run conditions:
Column: HiScreen Capto Q 7.7 × 100 mm (4.7 mL)
Binding: 50 mM Tris pH 9 + 50 mM NaCl
Elution: 0 - 500 mM NaCl in 10 CV
Protein load: 50 mg (10 mg protein/mL resin)
2 mL/min (260 cm/h)
Hb loaded onto HiScreen Capto Q
5
Scheme of optimisation process
6
Preparative run and analysis
Ternary mixture of Hb,Ova,BSA
GE Capto Q column
Recreated ternary mixture chromatogram
GE Capto Q column
1200
12
product
800
strong
weak
Hb
Ova
10
Quantification using
analytical methods
600
HPLC / UV-VIS
400
200
Concentration [mg/mL]
UV at 280 nm [mAU]
1000
2:6:2
BSA
8
6
4
2
0
0
4
6
8
10
12
Retention volume [CV]
14
4
6
8
10
12
Retention volume [CV]
14
As previously, apart from: Protein load: 120 mg (25 mg/mL resin)
•
Several conditions of model system tested, i.e ratio of weak/strong
impurities to product
•
Fractionation and quantification of individual proteins allows to
identify input parameters for optimisation
•
Mass balance was incorrect – suggesting inaccurate quantification
7
Challenges and relevance of the project
Challenges
• Accurate quantification of weak and strong impurities for the model
system is challenging
• Gaining high understanding of analytical problems and techniques
before working with real bioprocess mixtures is needed
Impact
• Studying artificial mixtures, developing high throughput analysis
methods and using fast optimisation algorithms will help improve
relevant bioprocess separations
• Rational optimisation of polishing step will reduce the cost of a
bioprocess and ultimately benefit patients
• Understanding of competition effects will contribute to the whole
bioprocess development for even more challenging separations
(mechanistic modelling)
8
Acknowledgments
• Prof. Ajoy Velayudhan
• Nicholas Field
• Spiros Konstantinidis
9
Thank You for listening…
…time for questions
10
Backup slide:
11
HPLC quantification method development
HPLC analysis of Hb, Ova and BSA
BIA CIMac™ QA column
Hb
Ova
UV at 280 nm [mAU]
8
BSA
6
4
2
0
0
2
4
6
Retention time [min]
8
Conditions: 20 mM Tris pH 9
Elution: 0-350 mM NaCl, 5mM NaCl/ CV,
1 mL/min (280 cm/h) Protein load: 10 µg
10
•
•
•
•
Hb, Ova, BSA
Loaded separately
Range of concentrations
Load plotted against AUC
12