Samira Naderinezhads.naderinezhad@ut.ac.ir Dr. Amoo-Abedini

Fall 2014

Monoclonal Antibodies

Outline IntroductionStrategy to laboratory generate of monoclonal antibodiesProcess ModelEconomic AssessmentDifferent scenarios & OptimizationConclusionsReferencesQuestion

Introduction

• What is antibodies?• Application• Consumption of antibodies

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

V IDEO, Antibody

Others have been prepared in term paper.

Strategy to laboratory generate of monoclonal antibodies

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:

Bioseparation:

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.

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.

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.

HIC chromatographyMiss. Pishbin, described about this

chromatography in more detail in her presentation.

DiafiltrationMiss. Pishbin, described about this

filtration in more detail in her presentation.

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.

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

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 millionnet profit: $ 143 millionROI (payback time=1 year): 108%

Different scenarios & Optimization:

Sensitivity Analysis:Reducing fermentation time & Increasing product concentration

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.

Optimization:

Using bioreactor with membrane can improve the

UCP and TCA.

Optimization:

In this study, the separation attributes of threemixed-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.

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.

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.

Question:محققان پژوهشکده مهندسTی علوم زیسTتی دانشگاه تهران، درصدد هسTتند تTا یک

، Mercapto-Ethyl-PyridineسTتون کروماتوگرافTی پروتئینTی بTه وسTیله رزین هایبTا ابعاد بزرگتTر نسTبت بTه نمونTه در دسTترس را طراحTی نماینTد بTه این منظور در حال افزایTش مقیاس ایTن سTتون کروماتوگرافTی پروتئینTی را مTی باشند. دستگاه

سTانتی متر 4 سTانتی متر برای طول، قطر داخلTی 22کروماتوگرافTی حاضر ابعاد گرم مونوکلونال آنتTی بادی را بTه خوبTی جداسTازی میکند 4.5دارد و در هر سیکل

ساعت زمان می برد.8و هر سیکل گرم در 20الTف) چنانچTه برای طراحی ستون کروماتوگرافTی جدید توان عملیاتی

سTاعت مTد نظTر باشTد؛ ابعاد سTتون جدیTد چگونTه خواهTد بود؟ ایTن محققیTن در حل مساله فرض می کنند سرعت خطی ثابت می ماند.

بازار قطTر سTانتی متTر داشته 30ب) چنانچTه سTتون های اسTتاندارد موجود در باشند، دبی حجمی و عمق بستر را برای این ستون ها بیابید.

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.

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/GELifeSciences/

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