This document outlines the process for laboratory generation of monoclonal antibodies. It discusses strategies such as fed-batch culture in bioreactors and downstream purification techniques including protein A affinity chromatography, ion exchange chromatography, diafiltration, and gel filtration. It also provides an economic assessment of the process and discusses various scenarios and optimizations that could improve yield and reduce costs, such as using membrane chromatography or exploring mixed mode resins for purification.

1. Samira Naderinezhad
s.naderinezhad@ut.ac.ir
Dr. Amoo-Abedini
Fall 2014
Monoclonal Antibodies

2. Outline
 Introduction
 Strategy to laboratory generate of
monoclonal antibodies
 Process Model
 Economic Assessment
 Different scenarios & Optimization
 Conclusions
 References
 Question

3. Introduction
• What is antibodies?
• Application
• Consumption of antibodies

4. A Brief Review of Antibody Structure, Operation
• The basic antibody is 2 heavy
chain-light chain pairs)
composed of repeats of a single
structural unit known as the
“immunoglobulin domain”
• Antibodies or are crucial
component of the immune
system, circulating in the blood
and lymphatic system, and
binding to foreign antigens
expressed on cells. Once bound,
the foreign cells are marked for
destruction by macrophages and
complement

5. V IDEO, Antibody
Others have been prepared in term paper.

6. Strategy to laboratory generate of
monoclonal antibodies

9. Fed-batch culture
 Fed-batch culture is one of modes of culture of microorganisms or
animal cells. In the broadest sense, defined as an operational technique
in biotechnological processes where one or more substrates are fed to
the bioreactor and in which the products remain in the bioreactor until
the end of the run.
 Bag bioreactor:

10. Bioseparation:

11. Protein A affinity chromatography
 Protein A affinity chromatography has come to be used as the
industry-wide standard for capture and purification of antibodies
and Fc-fusion proteins.
 Protein A itself is a naturally occurring protein found anchored in
the cell wall of Staphylococcus aureus.
 relies on the reversible interaction between a and a specific
ligand immobilized in a chromatographic matrix.
 Binding to the ligand as the result of electrostatic and
hydrophobic interactions, van der Waals' forces and/or hydrogen
bonding.
 After washing away the unbound material the bound protein is
recovered by changing the buffer conditions to those that favor
desorption.

12. Monoclonal antibodies Bioseparation, Protein A affinity
chromatography:
(1) Resin binding capacity is 15 mg of product per milliliter of resin
(2) The eluant is a 0.1 M solution of sodium citrate and its volume
is equal to 6 CVs;
(3) The product is recovered in 3 CVs of eluant buffer with a
recovery yield of 95%, and the pH is maintained near neutral to
ensure product stability
(4) The total volume of the solutions is 13 CVs (column
equilibration + wash and regeneration ), 15.7 h and a resin
volume of 24.5 liters.

13. Ion Exchange Chromatography:
Ion-exchange chromatography (or ion chromatography) is a
process that allows the separation of ions and polar molecules
based on their affinity to the ion exchanger. It can be used for
almost any kind of charged molecule including large proteins,
small nucleotides and amino acids. The solution to be injected is
usually called a sample, and the individually separated
components are called analytes. It is often used in protein
purification, water analysis, and quality control.

14. HIC chromatography
Miss. Pishbin, described about this chromatography in
more detail in her presentation.

15. Diafiltration
Miss. Pishbin, described about this
filtration in more detail in her
presentation.

16. Gel filtration:
• It is a chromatographic method in which molecules in solution are
separated by their size, and in some cases molecular weight.
• It is usually applied to large molecules or macromolecular complexes
such as proteins and industrial polymers.
• Typically, when an aqueous solution is used to transport the sample
through the column, the technique is known as gel-filtration
chromatography, which is used when an organic solvent is used as a
mobile phase.

17.  The annual production is 307 kg produced in 34 batches.
 The yield of the downstream recovery is 63%.
 There are 4560 tons of raw materials needed per year=260 kg/kg P
without considering water.
 Output of the fermentation: unused raw materials (medium, serum-free
medium), biomass, carbon dioxide, impurities, inorganic salts, and the
product

18. Economic Assessment
 PEC= $ 9.3
million (most
expensive:
bioreactors)
 bioreaction +
upstream sections
=55%,downstream
= 45%operating
cost
 Protein A resin
=75%
consumables cost.
depreciation (10 years, linear): $ 12 million
net profit: $ 143 million
ROI (payback time=1 year): 108%

19. Different scenarios & Optimization:
Sensitivity Analysis:
Reducing fermentation time & Increasing
product concentration

20. Optimization:
Membrane chromatography offers a
cost-effective alternative to traditional
chromatography in flow-through
operations, such as polishing for the
removal of viruses and contaminants in
antibody manufacture.

21. Optimization:
Using bioreactor with membrane
can improve the UCP and TCA.

22. Optimization:
In this study, the separation attributes of three
mixed-mode resins, Mercapto-Ethyl-Pyridine
(MEP) resin, Capto adhere multi-modal anion
exchange resin, and ceramic
hydroxyapatite/fluoroapatite (CHT/CFT) resins,
were investigated to define their roles in
monoclonal antibody purification processes.
Investigation purification powers, process
efficiency in industrial-scale mAb.
Using ProA-MEP-CHT resin.

23. Optimization:
The purification of monoclonal antibodies anti-CD34 produced in hybridoma cells
was accomplished by aqueous two phase extraction, using an integrated process that allowed
to clarify and partially purify the produced mAb in just one step. The feasibility of using
polyethylene-glycol (PEG)/dextran systems was studied at different ionic strengths (0-300 mM
NaCl) and at different pH values (pH 3, 4 and 7). The effect of molecular weight (MW) of PEG
(3350 and 6000 Da) was also evaluated. For all the conditions studied, it was observed that
antibodies partition preferentially to the PEG-rich phase, cells to the interface and soluble
proteins to the bottom dextran-rich phase. The best recovery yield was obtain with an ATPS
composed by 7% PEG 6000 Da, 5% dextran 500 000 Da, 150 mM NaCl at pH 3. In this system, it
was possible to recover 84±6.5% IgG with 0.1±0.2 % of cells in the top phase.

24. Conclusions
 ROI and net profit are affected by selling price.
 Environmental performance: large volume of waste & low pollution potential.
 Improvements of process: increasing productivity in the fermenter, higher Mab
concentration (2 g/L).
 Decrease quantity of impurities.
 Improve resin chromatography.

25. Question:
‫ستون‬ ‫یک‬ ‫تا‬ ‫هستند‬ ‫درصدد‬ ،‫تهران‬ ‫دانشگاه‬ ‫زیستی‬ ‫علوم‬ ‫مهندسی‬ ‫پژوهشکده‬ ‫محققان‬
‫های‬ ‫رزیین‬ ‫وسییله‬ ‫بیه‬ ‫پروتئینیی‬ ‫کروماتوگرافیی‬Mercapto-Ethyl-Pyridine‫ابعاد‬ ‫با‬ ،
‫افزایش‬ ‫حال‬ ‫در‬ ‫منظور‬ ‫این‬ ‫به‬ ‫نمایند‬ ‫طراحیی‬ ‫را‬ ‫دسیترس‬ ‫در‬ ‫نمونه‬ ‫به‬ ‫نسبت‬ ‫بزرگتر‬
‫حاضر‬ ‫کروماتوگرافی‬ ‫دستگاه‬ .‫باشند‬ ‫می‬ ‫را‬ ‫پروتئینی‬ ‫کروماتوگرافی‬ ‫ستون‬ ‫این‬ ‫مقیاس‬
‫ابعاد‬22‫داخلی‬ ‫قطر‬ ،‫طول‬ ‫برای‬ ‫متر‬ ‫سانتی‬4‫سیکل‬ ‫هر‬ ‫در‬ ‫و‬ ‫دارد‬ ‫متر‬ ‫سانتی‬4.5‫گرم‬
‫سیکل‬ ‫هر‬ ‫و‬ ‫میکند‬ ‫جداسازی‬ ‫خوبی‬ ‫به‬ ‫را‬ ‫بادی‬ ‫آنتی‬ ‫مونوکلونال‬8.‫برد‬ ‫می‬ ‫زمان‬ ‫ساعت‬
‫عملیاتی‬ ‫توان‬ ‫جدید‬ ‫کروماتوگرافی‬ ‫ستون‬ ‫طراحی‬ ‫برای‬ ‫چنانچه‬ (‫الف‬20‫ساعت‬ ‫در‬ ‫گرم‬
‫فرض‬ ‫مساله‬ ‫حل‬ ‫در‬ ‫محققین‬ ‫این‬ ‫بود؟‬ ‫خواهد‬ ‫چگونه‬ ‫جدید‬ ‫ستون‬ ‫ابعاد‬ ‫باشد؛‬ ‫نظر‬ ‫مد‬
.‫ماند‬ ‫می‬ ‫ثابت‬ ‫خطی‬ ‫سرعت‬ ‫کنند‬ ‫می‬
‫قطر‬ ‫بازار‬ ‫در‬ ‫موجود‬ ‫استاندارد‬ ‫های‬ ‫ستون‬ ‫چنانچه‬ (‫ب‬30‫دبی‬ ،‫باشند‬ ‫داشته‬ ‫متر‬ ‫سانتی‬
.‫بیابید‬ ‫ها‬ ‫ستون‬ ‫این‬ ‫برای‬ ‫را‬ ‫بستر‬ ‫عمق‬ ‫و‬ ‫حجمی‬

26. References:
 HEINZLE, E; BIWER,A,P; COONEY, C,L. (2006). Development
of Sustainable Bioprocesses Modeling and Assessment. John
Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester,West
Sussex PO19 8SQ, England
 Belter,P; Cussler,E.L; Shou Hu,W. (1988).Bioseparation
downstream process for biotechnology
 A. Shukla . (2007). PROCESS SCALE BIOSEPARATIONS FOR
THE BIOPHARMACEUTICAL INDUSTRY
 Richard Tran.(2011).Evaluation of Challenges to the Ubiquitous
Nature of Chromatography
 Grodzki AC1, Berenstein E.(2010).Antibody purification: affinity
chromatography - protein A and protein G Sepharose.
 Uwe Gottschalk. (2008). Bioseparation in Antibody
Manufacturing: The Good, The Bad and The Ugly.

27. References:
 M.Rodrigues et al. (2010).Technological Progresses in Monoclonal Antibody
Production Systems.
 Silva et al. (2014).Integrated purification of monoclonal antibodies directly from
cell culture medium with aqueous two-phase systems
 Chen et al. (2010).The distinctive separation attributes of mixed-mode resins and
their application.
 Wang et al. (2013). A novel ‘pipeline’ system for downstream preparation of
therapeutic monoclonal antibodies
 in monoclonal antibody downstream purification process
 Website:http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GE
LifeSciences/products/AlternativeProductStructure_17370/
 Website:http://www.ncbi.nlm.nih.gov/pubmed/20012816
 Website:http://amrita.vlab.co.in/?sub=3&brch=70&sim=1099&cnt=1
 Website: Wikipedia
 Website: http://www.slideshare.net/imaginarybiologist/affinity-chromatography
 Weihua Wu. Monoclonal antibodies Anticancer therapy. Presentation