Upstream Processing Overview mini BIOMAN 2017 Upstream Processing

Upstream Processing Overview mini. BIOMAN 2017

Upstream Processing Overview Main objective – to create the environment necessary for cells to make a protein product (recombinant protein or biologic) § Product of interest • Called API- Active Pharmaceutical Ingredient eg growth factor, enzyme, antibody • Produced by mammalian cell culture (Chinese Hamster Ovary - CHO cells, mouse myeloma, NSO cells), or bacterial or yeast culture § Mammalian cells • can modify proteins (post translation modifications required for Ab activity) • harvest can be made without cell lysis • CHO cells most commonly used § Bacterial cells • do not usually have the machinery to secrete the desired product into the media • cells must be lysed during harvest

Biomanufacturing Process Flow – Upstream Processing

Upstream Processing Overview § Upstream – Product formation § Downstream – Product purification § Quality Control – Product safety and efficacy

Production Process Flow Diagram

Upstream Processing - Cells used in upstream processing: – contain the transfected gene (s) that expresses the desired API (protein) – transfected gene is linked to a gene that imparts special survival abilities to the cells that have it • kept in cryovials in Dewars » Dewar – a specialized vessel that provides the environment to keep cell processes in temporary frozen suspension

Upstream Processing Areas, Equipment & Systems § § § CIP/SIP systems Media Preparation area Cell Culture Cell Banking Area Bioreactors Primary Recovery (harvest)

Air Quality & Flow Clean rooms – Air cleanliness classification – Class 100, 000 (FDA Class D) clean room, class 100 biological safety cabinet for initial inoculation – HVAC system maintains the room at a positive pressure with respect to surrounding rooms and corridors – Room pressures continuously monitored by building automation system which collects data to show that room is maintained in controlled state – Circulate 90% of airflow through pre-filter and HEPA filter bank – HEPA filters certified and calibrated periodically (generally every 6 months)

Cell Culture Media Provides all the nutrition cells need within a narrow window of environmental conditions for optimal expression of the target protein – Major media components • Carbohydrate energy source – glucose • Nitrogen source such as amino acids • Lipids – often in the form of fatty acid – Cells also require • Trace minerals in the form of electrolytes (salts) • fetal bovine serum supplements – no longer common due to risk of animal viruses • Chemically defined, serum free media commonly used – reduces threat of adventitious animal virus contaminants • Selective agents that cells require for optimal expression of the target protein

Cell Culture Cell Growth - 4 distinct phases • lag phase – cells adapt to the environment • log phase – exponential growth • plateau phase – growth rate slows- rate of proliferation equals rate of cell growth • death phase – rate of cell death exceeds proliferation rate and cells start to die § Generation – doubling of cell concentration from the original seeding cell concentration • Doubling time vary depending on the cell type » E. Coli - doubling time 20 minutes – culture batch ~ 24 hours » CHO - doubling time 14 -17 hours – culture batch ~ 620 days

Cell Culture Growth Phases

Monitoring cell growth § Parameters monitored – p. H- measures degree of acidity or alkalinity – Cell growth – viable cells/ml – Conductivity – measure of ions dissociated in the solution; purity of solutions used in media prep – Glucose - indicator of cell growth; main energy source for cells in growth phase – Lactate byproduct of glucose metabolism – Optical density – monitors growth

Upstream Processing Stages § Inoculum – Frozen vials of cells from cell bank thawed and added ( “inoculated”) to spinner flasks ( 100 – 500 m. L) or cell culture bags (50 -200 m. L) – Cell culture expanded to meet cell density and volume requirements to inoculate larger volume bioreactor § Bioreactor Seed – Cell cultures from spinner flasks or culture bags transferred “seeded” into larger volume bioreactors ( up to 20, 000 L) – Bioreactors are either stainless steel or disposable – Cells grow to high densities producing API in the culture media § Primary Recovery (Harvest) – main purpose is to separate the cells from the media containing the API • Centrifugation to separate cells from media • Filtration to remove large debris

Inoculum stage

Production Strategy § Batch: the culture generally goes through the growth cycle of lag, exponential, plateau, and death; the production cycle is short in this type of strategy; protein is harvested one time at the end of the production cycle § Fed batch: the culture goes through the growth cycle of lag and exponential phases; additional feed (glucose and other nutrients) is added before the cells reach plateau phase, allowing the cells to continue growing and producing protein; eventually the cells will reach the plateau and death phase, completing the production cycle; protein is harvested one time at the end of the production cycle. § Perfusion: the continuous growth of cells within a determined amount of time, with continuous harvest of product during cell growth achieved by a liquid-solids (cell) separation device within or adjacent to the production bioreactor; multiple harvests of protein are obtained from the bioreactor.

Manufacture of Adcetris Upstream processing Downstream processing

Manufacture of Adcetris

Production bioreactor adcetris

Emerging trends in upstream processing A. Single use technology § Single use manufacturing technologies, like wave bags and platforms for scale-up § 2, 000 L max disposables for production § Sometimes 6 x 2000 L bioreactors for production § Modular construction of facility that can be assembled together rapidly

Emerging trends in upstream processing B. Increase in cell titers § m. Abs can be expressed at titers of >5 g/L in a 14 day fed-batch production § Due to advances in : - Cell line expression vectors - Clone selection - Cell culture medium § This allows the use of smaller scale bioreactors for production runs

Emerging trends in upstream processing C. Continuous production

From Traditional to Integrated Continuous Bioprocessing

Maintaining & Monitoring Culture Cell growth and viability monitored during culture by counting cells methods Cell counting: – tallies the number of viable (living) and non-viable (dead) cells • calculate the % viability (viable/viable + nonviable)*100 • calculate viable cell concentration • Accurate and consistent cell counts essential to robust production process • Viable cell concentration & % viability used as Forward Processing Criteria (FPC) & Critical Process Parameter (CPP)

Cell counting § Automated cell counting: use image analysis to automate the Trypan Blue exclusion method, Examples include Vi-Cell, Cedex, and Nova units. § Trypan Blue/ Dye Exclusion Method. : trypan blue passes through the leaky membrane of a dead cell. Hence dead cells exhibit a distinctive blue color under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method. -This method is time sensitive, and the cells must be counted as quickly as possible after staining. The Trypan Blue will begin to kill the cells over time. This method is among the most commonly used cell counting methods. For manual counting, these are used in conjunction with a manual hemocytometer § Biomass sensor: these sensors operate based on cells with intact plasma membranes (living, viable) having different capacitance (ability to store a charge) than dead cells with disrupted plasma membranes. The cells are put through an electric field; and the capacitance can be directly related to cell concentration.

Manual counting

CHO DP 12 anti IL-8 Mab

Monoclonal Antibody Therapeutics More than 50 m. Abs have been approved and sales of m. Abs are expected to reach $125 billion by 2020, with over 300 m. Abs currently in clinical development. Explosive growth of this class of biopharmaceutical in the last 20 years due to: § ability to bind to specific targets with high specificity and affinity § ease of developing human or humanized sequences to a target m. Abs have been approved for a wide range of indications covering oncology and auto- immune disorders and rare disease indications

Monoclonal antibody therapeutics

Cell line § CHO DP-12 clone#1933 [CHO DP-12, clone#1933 a. IL 8. 92 NB 28605/14]ATCC® CRL-12445™ § https: //www. atcc. org/en/Products/Cells_and _Microorganisms/By_Tissue/Ovary/CRL 12444. aspx#characteristics

CHO DP 12 § The clone#1933 cell line was derived by cotransfecting the CHO cell line DP-12 using Lipofection with the vector p 6 G 4 V 11 N 35 A. cho. SD. 9 designed to coexpress variable light and heavy regions of the murine 6 G 4. 2. 5 monoclonal antibody. Clones were selected in methotrexate.

CHO DP 12 cells § The cells are reported to produce 150 mg/L recombinant human anti-IL-8. § Antibodies inhibit IL-8 binding to human neutrophils. They also show equivalent neutralizing capabilities to inhibiting IL-8 mediated human neutrophil chemotaxis Therapeutic anti IL-8 m. Ab § Hu. Max®-IL 8 is a high affinity fully human antibody directed towards IL-8. Hu. Max-IL 8 is in development for the treatment of solid tumors under an agreement with Bristol-Myers Squibb. An IND for a Phase Ib study of Hu. Max-IL 8 for the treatment of metastatic solid tumors was filed in June 2015.

CHO DP 12 Growth requirements § DMEM, high glucose § 10% superlow Ig. G serum (or FBS if stopping after upstream) § Insulin/transferrin § methotrexate

IL-8 § IL-8 is a small 72 amino acid peptide § Molecular weight 7. 8 k. Da

CHO DP 12 Upstream Processing § Inoculum - 1 ml from cell bank to inoculate 100 ml spinner flask - Grow for 5 days Monitor growth § Scale-up - Inoculate bioreactor 100 ml to 1 L medium Monitor growth § Harvest - Centrifuge cell culture filter

3 week experiment in Biomanufacturing course 2/7 & 2/9 Quality Assurance (Chap 7) Guest speaker Quality Assurance Upstream Processing Chapter 7 Chapter 10 Popcorn GMP activity 2/21 & 2/23 Upstream Processing CHO-DP 12 batch culture production of recombinant anti IL-8 m. Ab Inoculation, spinner flask culture, sampling and QC Chapter 10 Batch Culture SOP and Batch Record Spinner flasks media hold Inoculate spinner flasks (day 0 -day 4 testing) Bioreactor Sterilization, p. H probe calibration and installation 2/28 & 3/2 Bioreactor operation, inoculation Bioreactor Operation SOP Applikon bioreactor operation, spinner flask day 6 testing prepare bioreactor-set temp, stirrer, DO control inoculate bioreactor and day 0 -day 3 testing 3/7 & 3/9 Bioreactor harvest Chapter 10 and chapter 11 TFF SOP bioreactor harvest, CIP, centrifugation, filtration and TFF 3/14 & 3/16 No Class SPRING BREAK 2/14 & 2/16 FDA group activitiy ENJOY!