Bioreactor Harvest miniBIOMAN 2017

Production Process Flow Diagram

Monitoring Glucose Levels Cells use the energy source to grow and make the target protein. Measurements are taken of dissolved glucose. If no additional glucose is added, the amount will decrease with increasing time. the relationship between glucose concentration and cell density

Harvest separate the cells from the media containing the API (active pharmaceutical ingredient) Each cell line has been studied, and depending on the batch record, there are certain triggers on the proper time to end the cell culture/fermentation process and initiate harvest When cells are lysed enzymes are released that could digest the product, plus the amount of cell proteins will be increasing in the conditioned media; this could put downstream processing and the entire batch at risk.

Centrifugation The first step in mammalian harvest is usually centrifugation - rapid spinning of the culture from the bioreactor

Continuous Centrifugation Disc stack centrifuge – used to remove solids from liquids by means of a high centrifugal force  the denser solids which are subjected to these forces move outwards towards the rotating bowl wall while the less dense fluids moves towards the center the special plates (known as disc stacks) increase the surface settling area which speeds up the separation process

Filtration The first filtration step in harvest is depth filtration The next step in cell harvest is the filtration step to remove large debris. Particles too large to pass through the filter membrane are either trapped on the surface or within pore channels The first filtration step in harvest is depth filtration The large particles that do not fit through the pores are called the retentate-the substances small enough to pass through are found in the filtrate The API product passes through as the filtrate, along with other proteins and cell constituents small enough to pass through the filter Cells and cell debris stick to the ceramic encrusted fibers in pads and remain in the retentate

Depth Filtration: Equipment

Depth Filtration: Cells and Cellular Debris Stick to Ceramic Encrusted Fibers in Pads PROTEIN of INTEREST

Filtration The next and often last step in harvesting is to perform sterile grade membrane filtration. Membrane filtration is used to remove smaller particles and potential microbial contamination. Pore size is most often measured in micrometers (μm or microns) Sterile grade filtration, usually rated as 0.22 μm or smaller, removes most any bacteria The product now has a translucent appearance and is ready to be purified.

Downstream Process Unit Operations Filtration types based on flow directions: Direct Flow Filtration – allows the fluid to cross the membrane in perpendicular flow direction. It causes plugging or build up of reject elements on the membrane surface. Often used for particulate removal, sterile filtration, and virus removal/clearance filtration Tangential Flow Filtration (TFF) – allows the fluid to sweep across (tangentially) the membrane filtration surface. It minimizes pore plugging and surface fouling by its design.

Tangential Flow Filtration vs. Normal Flow Filtration 12

Ultrafiltration using tangential flow for protein concentration the protein of interest can be separated from other proteins and small molecules in the clarified medium To make sure that the protein of interest is retained, a 10KD cut-off filter is used the API is concentrated by ultrafiltration by cycling the retentate through the filter continuously, water and small molecules pass through the filter until the desired volume is obtained Diafiltration is achieved by introducing the appropriate buffer for the chromatography step to the concentrated sample and continuing with the ultrafiltration UF/DF

How TFF Concentrates and Purifies a Protein of Interest

Basic TFF System Basic Components: Membranes Pump Tank Piping Initial Feed Basic Components: Membranes Pump Tank Piping Diafiltration Buffer Retentate Tank Pump Membrane Feed Permeate

Diafiltration using TFF

DF

Downstream Processing Equipment Lab-Scale TFF System Large-Scale TFF System

mAb Downstream Process Flow Chart – Bioreactor: End of Run – conditioned medium Centrifugation to pellet cells Supplementation with protease inhibitors (aprotinin, leupeptin, PMSF) & Tween 80 (0.1%) 0.22u filtration Concentration by tangential flow filtration; final volume ~20 ml + 10 ml rinse Continue to capture chromatography with protein A Sepharose on AKTA system Analysis of fractions by ELISA.

Downstream learning module Mab purification process http://faculty.mc3.edu/downstreamprocessing/story.html