Upstream Processes III

• Lecture covers the environmental factors in protein biologics manufacturing as well as the parameters that are controlled within bioprocesses
• The learning outcome is to appreciate the considerations that must be made when planning for scaling up of a bioprocess

Need for Bioprocess Control

• Bioprocess control enables consistency in product yield
• Productivity and quality are better with control

Bioreactor Measurement Parameters

• Temperature is measured and controlled
• Osmolarity is measured and controlled
• pH is measured and controlled
• Oxygen/Dissolved O2 (DO) is measured and controlled
• Stirring speed/rate of mixing is measured and controlled
• Foaming is measured and controlled
• Flow rate is measured and controlled
• Cell concentration is measured and controlled
• Essential substrates and toxic products are measured and controlled

Bioreactor Measurements

• Measurements use sensors
• Discrete measurements are measured at periodic intervals
• Continuous measurements are measured constantly
• Measurements can be taken In situ i.e. directly in the culture medium
• Measurements can be taken Online where culture medium is moved to a special part for measurement via use of a bypass
• Measurements can be taken Ex situ when culture medium is removed from the bioreactor to the next process
• Measurements can be taken Offline when culture medium is removed from bioreactor to a lab away from the process

Bioreactor Sensors

• Physical sensors use changes in physical properties of materials (e.g. temperature sensor, foam detector)
• Chemical sensors measure changes in membrane properties through contact with sample (e.g. pH probes, DO probes)
• Biochemical sensors (biosensors) measure biochemical changes due to enzymatic conversion of substrate to product (e.g. glucose oxidase used in glucose measurement)
• Sensors should be stable and reliable for extended periods with straightforward calibration for each process
• Sensors should have good dynamic range, sensitivity, and response time, but be insensitive to interference
• In situ sensors should withstand steam sterilization/autoclave, with biochemical sensors as an exception
• Sensors should ideally not allow fouling

Simplified Overview of Bioreactor Elements

• Sensors control and monitor conditions such as Temperature, pH, DO, foam, Biomass and nutrients
• Base/acid adjust the pH via sensors
• Efficient transfer of feed is related to the growth rate
• Control feed rate by pump
• Efficient removal of metabolic heat via heat transfer
• Use cooling water and temperature sensors
• Avoid gradients of heat, nutrients, and additives via mixing
• Mixing is controlled by impeller number, design, and speed using a mechanical system
• Gas out prevents pressure buildup
• Gas transfer helps with High growth
• Gas transfer helps to Avoid O₂ starvation
• Gas transfer uses a gas flow rate controller and DO sensor
• Sampling ports are used for offline measurements
• Steam helps with In situ sterilization

Temperature Requirements

• Mammalian cell cultures require 37°C
• Yeast or fungi cultures require 24 - 30°C
• E. coli cultures require 37°C
• Insect cell cultures require 25 - 28°C

Temperature Sensors

• Resistance temperature devices (RTD) use a PT-100 probe
• They are most commonly used
• RTD correlates electrical property (resistance) to temperature changes
• RTD has a linear response over operating range (0-100°C)
• RTD is placed in deep well in bioreactor, not in direct contact with medium to prevent corrosion
• Thermocouples use two different electrical conductors
• Thermocouples measure voltage changes with temperature changes
• Thermocouples are less accurate than RTD

Temperature Control

• Jacketed vessels help control temperature with use of a water jacket
• Heating blanket helps control temperature
• Cooling coils help control temperature
• Cooling fingers help control temperature

Osmolarity

• Number of moles of chemical compounds contributing to a solution's osmotic pressure per liter of solution (osmole per liter Osmol/L)
• Normal physiological osmolarity range is 280 – 310 mOsmol/L with the use of salts like Na+, Cl-
• Culture medium should be iso-osmotic to intracellular solutions
• High osmolarity of culture medium (hypertonic) causes water movement out of cells, and low osmolarity of culture medium (hypotonic) causes water movement into cells
• Both cases lead to cell injury/cell death
• Measured offline with vapor pressure or freezing point-based osmometer

pH

• Mammalian cell cultures pH should be 7.0 – 7.4
• Yeast or fungi cultures pH should be 4 - 6
• E. coli cultures pH should be 5.8 - 8
• Insect cell cultures pH should be 6.0 – 6.4
• Measure of hydrogen ion (H+) concentration in a solution (pH is temperature dependent)
• pH affects enzymatic activities which affects cell growth
• Excess lactate and CO2 production add acidity to culture medium
• Ammonium production of degradation of L-glutamine add alkalinity to culture medium

pH sensors

• Glass membrane pH electrode causes a change in pH which causes H+ to diffuse into or out of the glass membrane à causing a change in electrode potential
• good mixing is essential for reliable measurement with the use of calibration with pre-determined pH solutions (pH 4, 7, 10)
• glass membrane pH electrode can withstand repeated steam sterilization and can be used as in situ sensor in bioreactor
• Fluorescence-based optical pH sensor used of fluorescing dyes (e.g. 8-hydroxyl-1,3,6-pyrene trisulfonic acid)
• Fluorescence-based optical pH sensor usually used with miniaturized and disposable bioreactors
• Fluorescence-based optical pH sensor has a limited measuring range (3 pH units)

pH adjustment

• Alkaline solutions (NaOH, KOH, NH4OH) and acidic solutions (HCl, H2SO4) adjust pH
• CO2 released by growing cells dissolves partially in solution to form weak carbonic acid
• weak carbonic acid will lower pH, which can be exploited to adjust pH(H2O + CO2 <-> H2CO3 <-> H+ + HCO3-)
• Use buffers like PO43- and HCO3 with Caution because hydrogen peroxide production increased in the presence of light and HEPES (pH = 7.4 at 37°C)

Dissolved Oxygen

• Amount of oxygen dissolved in solution, defined as percentage of saturated dissolved O2 concentration at the temperature
• Unit for DO is either in mg/L or % i.e. 45% DOsaturated where DOsaturated at 37°C 1 atm = 6.8 mg/L in air
• Biopharmaceutical production using mammalian cells requires O2
• Oxygen transfer is usually limiting, in particular when cells are growing at maximal specific growth rate (culture has high cell density)
• Oxygen transfer rate (OTR) depends on operational conditions (e.g. temperature, mixing, sparging), geometrical parameter of bioreactor and physicochemical properties of culture (e.g. viscosity of culture)
• Physio chemical properties depend on the presence of oxygen-consuming cells
• Consumption rate varies with cell type and cell numbers with high values for exponentially growing cells and higher cell density
• CHO and bacterial cells can deplete O2 within hours and seconds if O2 supply/OTR is compromised

DO Sensors

• Clark-type electrode works with O2 diffusing through membrane and gets reduced on platinum electrode surface
• O2 + 4 e⁻ + 4 H⁺ → 2 H₂O
• Clark-type electrode generates a current to measure DO but O2 gets consumed during measuring process
• Optical oxygen sensors works on basis of fluorescence quenching by O2 using fluorescence dye immobilized and attached to one end of glass fiber
• Optical oxygen sensors can be miniaturized and can measure O2 level in both liquid and gas phase
• Optical oxygen sensors have limited lifespan due to photobleaching of fluorescence dye therefore is not for long-term usage

Aeration

• Provide gases to the culture medium, commonly O₂ to maintain maximal specific growth rate of aerobic cells
• Sometimes, bioreactors are designed with settings that allow provision of CO2 (for pH control or photosynthesis if plant cells are cultured).

Aeration Methods

• The process of Surface aeration is suitable for batch cultures and small scale cultures, but Oxygen supply and transfer becomes limited when cultivation is scaled up to larger volumes
• The process of Sparging or Bubbling utilizes a sparger placed below the impeller
• Membrane aeration, A multihole (macro)sparger creates larger bubbles of 6-8 mm with minimal foaming, but the gas transfer rate is lower because it needs to increase the gas flow rate to compensate for the lower transfer rate
• A porous metal (micro)sparger creates smaller bubbles with a larger gas-liquid interface area for more efficient gas transfer, however, foaming can easily happen

Straightforward Setups

• Sparging has a straightforward setup that can be used even for large scale bioreactors (scale-up)
• Surface aeration requires specialized tubings (up to ~ 500 L bioreactors) with a risk of fouling, and the process is complicated to replace or clean

Bubbling

• Bubbles are produced via Sparging where large energy dissipation occurs when bubbles break at their surfaces
• risk of shear stress happens to cells proximal to bubble breakup, leading to higher risk of cell death, especially for shear-sensitive cells
• Sparging has a risk of foaming therefore Shear stress should be counteracted by addition of shear protecting agents (e.g. pluronics, serum, polyethylene glycol (PEG), bovine serum albumin (BSA), dextran) working be either reducing liquid-air surface tension so that cells adhere to bubbles gently or agents get adsorbed on cell membranes to strengthen membranes against breakup due to high shear stress
• Foaming can be counteracted by addition of antifoams which can potentially inhibit cell growth, and may cause fouling at filtration membranes
• With Surface aeration, no bubbles are formed, so it is usually used for mammalian cell cultures leading to low risk of foaming and antifoams are not required
• For suspension cultures where there is risk of shear stress, shear protecting agents can be added

Mixing

• Purpose: to achieve and maintain homogenous culture environment, especially important in large scale bioreactors
• Goal: to ensure even distribution of nutrients, gases, heat, etc so that dead space and the local environment that has suboptimal culture conditions that leads to sub-optimal cell growth/cell death are prevented/minimized
• Mechanical mixing is usually achieved by impellers where important considerations are number of impellers to be fitted in bioreactor, impeller diameter, impeller speed
• Types of impellers: radial flow impellers circulate liquid flow along radius of the tank, and axial flow impellers circulate liquid flow in the direction of the axis of the tank towards the base of the bioreactor
• Mechanical mixing causes shear stress, which makes shear-sensitive cells susceptible leading to retarded cell growth, reduced protein production and even cell death.

Foaming

• Rate of protein denaturation is accelerated
• There is a risk of contamination if foam reaches the neck of culture vessel
• Blockage of air exit filters create pressure build-up in bioreactors
• Breaking up of bubbles contribute to shear stress
• Suspension cultures in stirred tank bioreactor (STR) are usually susceptible to foaming where the process of adding a non-ionic surfactant like Pluronic F68 is added as antifoam to possibly increase viscosity of culture
• Conductivity switch is used for foam detection

Flow Rate

• Rotameters are most commonly used, comprised of a "float" in a tapered sight glass that will be suspended by fluid flowing through, but the simple setup cannot be adapted for automation
• Other meters used: magnetic, venture and orifice meters

Bioreactor Parameters

• Temperature is essential
• Osmolarity is essential
• pH is essential
• Oxygen/Dissolved O2 (DO) is essential
• Stirring speed/rate of mixing is essential
• Foaming is essential
• Flow rate is essential
• Molecules monitored for animal cell culture are glucose, glutamine, lactate, ammonia
• Few in situ sensors are available, mostly using online aseptic sampling devices and biochemical sensors/analyzers

Process Control

• Objective: to maintain operating conditions at design values
• Rate of change implementation is dependent on system size (small scale versus large scale bioreactors)
• Some changes can only be made in one direction (e.g. glucose concentration only to be increased but not decreased)
• Feed-forward control (open loop control) uses predictive control
• Process modifications are implemented at predetermined times
• Extensive knowledge of the system is required, but is difficult to implement directly for biological systems
• Feedback control (closed loop control) uses correction control (negative feedback)
• Measurement of the process is compared to a set-point
• Feedback is sent to the controller before the process happens again

Proportional-Integral-Derivative (PID) Controller

• Error (e) is calculated as the formula = Ysp-Y, where Ysp is the set point while Y is the process output
• U is the control variable, N is the noise, and X is the process variable
• Proportional to magnitude and duration of error where positive error leads to increase in control signal and negative error leads to decrease in control signal
• "Gain” (high gain → system unstable; small gain → less responsive controller with a small response to a large error)
• When T₁ (value of integral time) ↑, response slowly creeps towards setpoints (less oscillatory)
• To improve closed-loop stability, the process should be Proportional to the rate of change of the process error with respect to time
• Damping increases with increasing derivative time, then decreases again when derivative time becomes too large

Bioprocess Development

• Processes are generally initiated at laboratory scale, then progressively scaled up to larger volumes at the pilot plant level, and finally to production scale (can take a few months)

Scaling Fermentation

• This allows for more products to be produced from growing more cells because there is an increasing amount of products needed to go from research (1 – 100 L), to clinical trials (100 – 500 L), to product manufacture for market (10,000 to 20,000 L)
• Cost of goods is lowered when scaling up the process by Pooling products together into a single process allows for greater consistency within the bioprocess and allows more feasible quality testing
• Example, From > $10,000/g at research laboratory scale to < $500/g at manufacturing scale since One 10 L process gives more consistency than ten 1 L processes and Testing product from 1 process versus those from 10 processes is more easier
• Limitation: Scale-up is typically used for suspension cells that able to grow to high volumetric cell densities, however can be problematic for anchorage-dependent cells
• For anchorage-dependent cells, you can use microcarriers or Grow cells in roller bottles or cell factories, but it can be difficult to maintain homogeneity in large systems with a change in surface to volume ratio
• Small Scale processes are limited by rate of cellular reactions while Large scale processes are limited by transport of materials (e.g. oxygen transfer to cells) and a Change of scale leads to changes in physical environment of cells

Scale-Up Methodology

• This should be maintained constant upon scaling up by being a specific physical or mechanical property that is most critical to process performance such as oxygen transfer rate, mixing, shear stress, flow regime.
• This can be carried out according to the principle of geometric similarity between the large and small scales.
• Constant geometry such as Typical height to diameter ratio – 2 to 1 or 3 to 1 relies on the same aspects in the vessel and internals scales which maintains similar environment control factors, and means there is a change of scale that result in changes in the physical environment
• A change of scale must be an assay for process and product consistency

Oxygen Transfer Rate

• Oxygen transfer from bubbles to bulk liquid medium, and from bulk liquid medium to cell
• Oxygen transfer is described by the formula OTR = k₁a (C* - C₁) where k₁ is the oxygen transfer coefficient, a is used to measure the gas-liquid interfacial area, C* is the dissolved concentration and CL is actual concentration in medium broth
• This can be achieved by scaling up aerobic bioprocesses
• Impeller tip speed is measured by Impeller diameter times N (rotational speed of impeller)
• In order to use constant geometry ,there should be ND; describes shear stress at the impeller and should proportional to shear stress exerted on the cells where ↑N↑ average and maximum shear rate with ↑Di ↑ maximum shear rate - So when an In scaling up increases with D₁, then problem can happen to the cells without cell walls
• Energy Input = P x N³D₁⁵, where P is Energy input, and will results to affect the size of cell aggregates in aggregate suspension cultivation and can affect attachment of dependent cultures

Reynold's Number

• It is a dimensionless number defined measure the flow regime and turbulence
• Re = measure of inertial forces is to viscous forces = VpD₁/µ = pND2/μ
• V: mean fluid velocity (m/s) = ND₁ ⍴: Density of fluid (kg/m³)
• D₁: Diameter of impeller
• Quantifies the relative importance of the two forces under given conditions
• If small Re, then flow is laminar with Viscous forces are dominant and flow In parallel layers with no disruption between layers causing Less mixing and less shear (for the same fluid density and viscosity)
• Conversely, If Re is big (> 10,000) flow is turbulent with dominant inertial forces and More mixing and more shear