Bio-manufacturing Overview Hyuncheol Kim, PhD Drug Delivery & Tissue Engineering Laboratory Sogang University

Manufacturing Overview Manufacturing Steps - Upstream - Recovery - Downstream The Rules = Compliance!!! - FDA Guidelines - cGMP - Batch Records, SOPs

Process Flow Diagrams Process flow diagrams (PFD): describe a specific product’s manufacturing processes indicates the general flow of the process through a manufacturing facility contains only important process details (i.e. temperature, pH, and cell densities) and steps shows major equipment in the facility PFDs are used to: assist in the transfer of a process to manufacturing from development write process batch records describe the process to the FDA in regulatory documents

Upstream PFD Example

Downstream PFD Example

Detail of One Step Example

Upstream PFD Example

Upstream – Seed Thaw & Expansion in Shake Flasks Purpose: To aseptically produce an inoculum of sufficient volume, cell density, and viability for inoculation of the first bioreactor Procedure: Thaw a Cell Bank Vial & continually passage into subsequently larger flasks to increase the total cell culture volume Maintain healthy cells Replace the nutrients previously consumed by cells with nutrients from fresh media Remove potentially toxic by-products of metabolism, ie. ammonia and lactic acid Increase the total volume of cell culture in stages This allows you to start with a cell bank vial containing 1.6-mL of cell culture and end with 20,000-L of cell culture Usually, batch phase

Upstream – Seed Thaw & Expansion in Shake Flasks

Seed Build-up Terminology Inoculation – The introduction of cells to the cell culture medium Passage – The act of diluting cells in new medium from a high density to a low density Seed Culture – The culture to passage. The culture used to inoculate a new flask Viable Cell Density (VCD) – The total number of cells per milliliter of culture that are alive Viability – The percentage of live cells in culture Inoculation VCD – The density of the new culture after the seed culture is passaged (typically low) Final VCD – The density of the seed culture prior to passaging Split Ratio – the ratio of the volume differences between subsequent stages

Seed Build-up

How to Passage Cells Sample the cell culture to be passaged aseptically Make sure the culture is well mixed before removing a sample Perform a cell count of seed culture on an automated cell counter machine (ViCell) Using the following equation to determine the volume of seed cell culture needed to inoculate a new flask. C1 x V1 = C2 x V2 C1 = Cell Density of Seed Culture V1 = Volume of Seed Culture Needed (Unknown) C2 = Inoculation Cell Density of New Culture V2 = Volume of New Culture

Seed Build-up Controls Incubator Agitation (oxygenation) CO2 (help aid in pH stabilization) Temperature Humidity Bioreactors Agitation pH control DO control

Bioreactors / Shake Flasks It is best to transfer the cell culture into a bioreactor as soon as possible because cells grow better in a bioreactor than they do in a shake flask. Advantages to seed build-up in bioreactors: Provide a controlled environment Easier to maintain sterility No hood work in a biological safety cabinet required Build-up cells to higher volumes Less likely chance for contamination Disadvantages to seed build-up in bioreactors: Cost of operation is greater Requires longer time to set up (CIP, SIP, etc) Some companies start the in vessels as small as a 5 L bioreactor

Designing a See Build-up Process When designing a seed build-up process, it is desired to make it as short as possible. It is not desired for the cells to produce protein, thus the goal is to quickly grow the culture to the necessary volume and cell density required to inoculate the production bioreactor. In order to design a seed build-up process, the following information is required: The maximum VCD the cells can grow to in the batch media. The time required for the culture to reach the maximum VCD. The minimum VCD allowed for inoculation. Cells like to be near other cells; if the inoculation VCD is too low, the culture will not grow. The maximum split ratio the culture can withstand. You also need to know the following about the facility’s bioreactors / equipment: The largest size shake flask, spinner flask, or roller bottle that is available The working volume of the seed build-up bioreactors. The working volume of the production bioreactor. The minimum and maximum working volumes in each bioreactor. Once you have all the information, the optimal process can be designed!

Batch Cell Growth

Upstream PFD Example

Production Bioreactor Purpose: to produce the desired product! Production bioreactors can be: Batch, Fed-batch, or Perfusion Mammalian processes are on average 7 – 14 days

Upstream PFD Example

Recovery Purpose First stage of purification Remove the final product from the cellular material Most products are extracellular Remove cells from the liquid containing the product Some products are intracellular First must break the cell membrane to get the product into solution (mechanical presses, detergents) Next remove the cell debris

Recovery Consists of a series of steps ending with a final sterile filtration of the material Common methods include Centrifugation Homogenization / Microfluidization Depth Filtration Microfiltration Tangential Flow Filtration Process is designed based on the type of starting culture Cell density Cell type

Downstream PFD example

Purification A series of filtration and chromatography steps designed to purify the product while maintaining its bioactivity Need to remove viruses, host cell proteins, and exchange the buffer in order to get the product into the formulation buffer Purification steps are not sterile processes, however sterile filtration between steps is required Also, require viral filtration – utilizes very small micro filters; expensive

Downstream PFD Example

Types of Chromatography Flow-Through Mode Is when the target molecule does not bind to the column So the resin is selected to capture contaminants rather than the target protein Capture Mode Is when the target molecule binds to the column. The contaminants may either flow through or bind to the column So the resin is selected to capture the target protein

Stationary phase (resin) Mobile phase (buffer) Mobile phase Molecules spend different amounts of time between the two phases When a mixture of molecules is loaded on to a column; Molecules that spend 100% of the time in the stationary phase are “bound” Molecules that remain 100% in the mobile phase are in the “flow through”

Protein Capture

Step Gradient/ Step Elution: Gradient Elution: Modify solute interaction with the stationary phase by changing the composition of the mobile phase. Step Gradient/ Step Elution: Change mobile phase composition in a stepwise or instantaneous manner time Linear Gradient: Change mobile phase composition in a continuous (linear) manner

Chromatography Terms Equilibration When buffer is pumped though the column to adjust the pH and conductivity in preparation for loading the protein Load This is the step where the protein solution is pumped into the chromatography column. Wash The wash step can have several purposes, it can be used to finish pushing the load material though the column, it can be used to remove contaminates from the column or it can be used to begin separating molecules on the column prior to elution Elution This step is where the pH and conductivity of the buffer are adjusted so that the target protein migrates to the mobile phase and flows out of the column into a collection container

Chromatography Terms Strip / Regeneration A solution that is flowed though the column to remove remaining protein / contaminants and return the column to its original state Cycle 1 cycle is when the series of steps (Equilibration, load, wash, elution….) is completed one time. Multiple cycles per batch may be used on the column as needed. Cleaning is done when the batch is finished and not between each cycle. Cleaning / Sanitization This step is used to remove any remaining contaminates on the column and to eliminate any microbes that may have entered the column Storage After cleaning, a storage solution is pumped into the column to ensure the column resin is maintained in stable conditions until the next run. Storage solution is usually bacteriostatic (inhibits bacterial growth)

Chromatography Diagram

Downstream PFD Example

Bulk Product Fill Bulk = Product in bags Bulk product is the final product from the purification step (prior to filling) This product goes on to be filled into syringes / vials, lyophilized and either placed in vials or pressed into tablets Bulk = Product in bags

FDA Compliance

Compliance Compliance - Act according to the standards set by the regulations and guidelines

There are three GXPs: 1. Current Good Manufacturing Practices (CGMP): 21 CFR Part 210 & 211 2. Good Clinical Practices (GCP): Law that governs the action and environment of those working in clinical testing of drugs and medical devices on human beings. 21 CFR Parts 56, 312, and 314 3. Good Laboratory Practices (GLP): Enforced code of practice to reduce accidents affecting research projects or manufactured products. 21 CFR Part 58

Overall Purpose of cGMPs Establish Industry Standards Insure Traceability Insure Accountability Promote Uniformity Install Quality It’s the Law! Title 21 of U.S. Code of Federal Regulations - “21 CFR” ...To assure that all pharmaceutical, biologic, diagnostic and medical device or drug products meet all the requirements of the Federal Food, Drug and Cosmetic Act as to safety, and have the identity and strength and meet the quality and purity characteristics which they purport to have... Parts 210.1, 210.2

How do Companies Comply Standard operating procedures (SOPs) must be followed Manufacturing Processes must be validated Equipment must be validated Staff must be properly trained

Standard Operating Procedures Used for routine steps, equipment setup, procedures, tests Ensure that the procedure is performed the same way every single time, no matter who performs it People must be trained on the SOP Examples – taking a pH measurement; installing filters on the bioreactor; operation of the chromatography skid

SOP

Batch Records Used to record all the data coming from on batch for a process Batch records would be created for each step Raw data collected about the process Entries must be verified by additional person Examples – The Production Bioreactor for Protein X Collect the daily cell counts Mention if you needed to change the pH or agitation

Batch Records

Similarity and Difference between SOPs and BRs Version tracked to keep track of changes Reviewed and approved by multiple people / depts Batch Records are issued for a particular batch Given an assigned number for the document Only used for a particular step for a particular product Version tracked to keep track of changes Reviewed and approved by multiple people / depts Used across products and production sites Stored in the lab as a reference

In the compliance world documentation is KEY If you didn’t write it down it never happened!!

Thank You