Biopharmaceutical Manufacturing Introduction


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A brief overview of the manufacture of biopharmaceuticals.

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Biopharmaceutical Manufacturing Introduction

  1. 1. APR_Wheelwright 6/3/08 2:51 PM Page 1 MANUFACTURING Biopharmaceutical Manufacturing: An Introduction Scott M. Wheelwright Strategic Manufacturing Worldwide, Inc. T hough the detailed processes for production of biopharmaceu- The next step is to insert this DNA (our gene) into the cells ticals may seem intricate and complex, the basic ideas behind (this step is also referred to as cell transfection). At this point we protein manufacturing are easy to understand and are within must decide what type of cell to use: microbial (such as E. coli) or the capability of everyone working in the field. mammalian (such as Chinese Hamster Ovary or CHO) cells. There The set of activities that are performed to produce finished is a standard set of criteria to help us decide, based on our product vials of drug product can be divided into two groups: those that are behavior and what we need it to do. For example, full size mono- done once and those that are repeated each time a batch of drug is clonal antibodies are always produced in mammalian cells while made. Those that are done once take us from a research idea to a many other proteins are more cost effectively produced in microbial Working Cell Bank (WCB); those that are repeated for each batch cells. start with a vial of cells from the WCB and end up with a batch of The most common way to get our gene into the cell is a process vials ready for sale or for the clinic. These steps are summarized in known as electroporation, in which a sample of cells is suspended in Table 1. a solution of salt and water and our DNA is mixed in, then an elec- tric field is applied. When this happens, the cell wall becomes slight- Table 1. From Research to WCB ly permeable, and the DNA slides in. Once inside the cell, the DNA is capable of replicating as the cells replicate. That is, as the cells divide and produce daughter cells, these daughter cells will contain STEPS PERFORMED ONCE STEPS PERFORMED EACH BATCH copies of our gene and the daughter cells will be capable of making Synthesize DNA Expand WCB cells our protein. Insert DNA into cells Produce protein by fermentation or cell culture Now comes a very important step, namely, the selection of the Select and clone h ighest expressing cell lin e Recover protein from cells highest producing cell. When we completed the electroporation step Develop production process Purify protein we ended up with a library: many (maybe a million) different cells, Generate Master Cell Bank (MCB) Formulate bulk drug substance (DS) some with our gene correctly inserted and many without. During the Generate Working Cell Bank (WCB) Formulate and fill drug product (DP) selection process we want to separate each cell from its siblings. Then we will grow multiple copies of genetically identical progeny List of steps prior to batch production and steps for batch production. from each of those original cells. Because these progeny are genet- The goal of research is to identify the protein that is to be man- ically identical they are called clones, and this step is sometimes ufactured. Once we have identified that protein, we need to develop referred to as cloning. (A note of caution: the word cloning is used a cell that will produce it. Three main steps take us there: copying several different ways in this industry, and this is only one of those the gene, inserting it into the cell, and selecting the highest produc- ways.) ing clone. Now we assign several scientists and research associates to We start with the protein selected by research for which the spend several months identifying from all of these many clones the research group provides a computer document that lists the DNA one that has the highest production rate of our desired protein. Why sequence. We send this off to the sequencing laboratory (typically an is this so important? Because the cost of production is highly outside vendor who specializes in this esoteric activity) and receive dependent on the amount of our protein produced by the cells we will back a vial containing snippets of DNA, which is a bunch of copies grow. If we can double the expression, we can cut the cost of our of our gene of interest. This is the genetic material that will tell the final product almost in half. If we increase expression by ten-fold, cell to make our protein. our costs become one-tenth what they were. 124 American Pharmaceutical Review
  2. 2. APR_Wheelwright 6/3/08 2:51 PM Page 2 MANUFACTURING Once we have selected the highest expressing clone, we grow up a flask and divide it into about 200 separate vials. This is our Table 2. Master Cell Bank (MCB). From the MCB we take one vial, grow up a flask of cells, and divide these into about 300 separate vials. This WCB Vial is our Working Cell Bank (WCB), which generally lasts for about two ↓ Fermentation years. Then we will take another vial from the MCB and make a new Cell Expansion ↓ WCB. We perform a battery of tests on the cell banks to confirm they Fermentation are pure and meet other criteria. These cell banks are carefully stored ↓ in multiple locations. We use cell banks so that every batch we make Centrifugation or filtration ↓ will start with identical cells. Cell disruption by homogenization Recovery ↓ Before we can run our manufacturing batch, which starts with Solubilization and refolding the WCB, we also need to develop a production process, which we ↓ will perform initially at pilot scale. This process may undergo sever- Concentration by ultrafiltration ↓ al iterations and process changes as our clinical program moves from Anion exchange chromatography phase 1 to phase 3. This is the end of those steps we perform just ↓ Purification once. Hydrophobic interaction chromatography ↓ Cation exch ange chromatography ↓ From WCB to Drug Substance and Drug Drug substance formulation ↓ DS Product Lyophilization ↓ Once we have our working cell bank we can now manufacture DP Drug product packaging drug substance, which is the bulk purified protein that is the active ingredient. We will go through a series of steps that are repeated Typical flow chart for manufacture of a biopharmaceutical from E. coli every time we manufacture a new batch. Our goal is to repeat these Fermentation and Cell Culture steps exactly, without changes, in order to ensure the drug substance made from each batch is identical in its performance. During fermentation we grow the cells, which produce our pro- The manufacturing process is divided into three parts, each of tein. (In the United States, we usually use the word fermentation to which contains multiple unit operations. (A unit operation is a refer to microbial growth processes and we use the phrase cell culture process step using a particular type of equipment. For example, fil- to refer to mammalian cell growth processes.) The fermentation or tration is a unit operation; centrifugation is a unit operation, and so cell culture process starts with a vial of cells (our WCB vial) and forth.) The three parts of our process are fermentation, recovery and expands the number of cells by culturing them in increasingly larger purification. Concerned about Safety? The First Raman Analyzer Certified to ATEX Standards Applications • Pharmaceutical / Biotech • PAC & PAT • Specialty Chemical • Reaction Monitoring • Refining & Distillations • Olefins & Polymers RAMANRXN3: From the Leader in Process Raman WWW.KOSI.COM 371 Parkland Plaza Ann Arbor, MI. 48103 phone (734) 665-8083 / fax (734)665-8199 125 American Pharmaceutical Review
  3. 3. APR_Wheelwright 6/3/08 2:51 PM Page 3 MANUFACTURING volumes until we reach our full scale production fermenter or biore- gation with filtration to remove the last few small particles that actor. These vessels may be 15,000 L or larger. The series of biore- escape the centrifuge. Filters come in two main varieties: depth fil- actors of increasing volumes is referred to as the bioreactor train. ters, which consist of fiber beds that entrap particles, and membrane The fermentation process is optimized for many parameters, filters, which have a defined pore size that restricts passage of mate- including the composition of the growth media containing carbon rials larger than the pores. and nitrogen sources; the rate of addition of the growth media to the Microfiltration, that is, the separation of particles from solu- bioreactor; and the oxygen transfer rate, which is controlled by the tion, may be performed with either depth filters or membrane filters. agitation and aeration rates, and is coupled with control of the rate Ultrafiltration, which is the separation of proteins from other con- at which growth media is added. Many experiments will be con- stituents of the solution (such as salt or smaller proteins), is per- ducted during the development of the process, but once we have formed only with membrane filters. Ultrafiltration is often used to implemented the process in manufacturing, each batch will be concentrate our protein by removing excess water. Diafiltration is a repeated with the same operating parameters and in the same way. unit operation that uses ultrafiltration to replace one set of salts in The fermentation process is the step where we generate our the solution with another. protein. After fermentation stops we do not make any more of our If our protein is inside the cell, then we must disrupt the cell protein; on the contrary, from here on we will lose much of our to open it up. This is done with an homogenizer that splits the cell product due to inefficiencies in the process. So the goal of fermen- apart and degrades the cell wall into small pieces. The homogeniz- tation is to get as much of the product as we can from each batch. er also disrupts the cellular DNA, which otherwise may be very vis- Similarly, the goal of each of the following steps will be minimize cous and impede the recovery and purification operations. the loss at each step. In the case of E. coli inclusion bodies, we must solubilize the precipitated protein. This is done by adding various chemical Recovery agents that disrupt the interactions between the protein molecules (so-called chaotropes, which induce chaos), such as urea or guana- Following fermentation we enter the recovery stage. The dine, often at concentrations as high as 8 M. Proteins solubilized recovery unit operations are designed to separate our protein from this way must be refolded from this disrupted condition into their cells and other solid materials so that we have a particle-free homo- native state, in which the different parts of the protein wrap around geneous solution. After recovery we move to the purification stage, each other just as they do in nature. This is done by adding a reduc- which is where we separate our protein from the other proteins that ing agent such as mercaptoethanol and then diluting the protein as contaminate the solution. much as 40 fold while allowing it to gently mix and reassemble The first step in recovery is always a solid-liquid separation. itself into a native (and therefore active) configuration. Refolding Our protein is either in the liquid surrounding the cells or it is inside steps often have poor yields and may represent a substantial loss of the cells (or sometimes both). In the case of mammalian cell cul- our protein. ture, our protein will be in the liquid outside the cells and we will perform a separation to remove the cells, which are disposed of. Purification The usual step following cell separation is a reduction in the volume of the solution so that purification steps have a smaller volume to Following recovery, our protein will be in aqueous solution handle, which reduces the cost. free from particulates. Our next challenge is to separate our protein In the case of microbial fermentation, our protein may remain from the many other proteins (including fragments of our protein) inside the cell, in which case we will recover the cells and break that are also found in the recovery solution. them open. Once again we have a solid-liquid separation. And The primary unit operation in purification is chromatography, again we face the question of whether our protein is in solution or which is a process by which proteins are adsorbed on (adhered to) a remains in the solid phase. Many yeast cell systems retain a sub- solid phase (also referred to as the resin or media) in the chromatog- stantial amount of the protein inside the cell, in the liquid of the cell. raphy column and then selectively removed. The goal of each chro- Disrupting the cells allows recovery of the protein from the liquid matography step is to selectively separate our protein from others in phase. E. coli on the other hand often sequesters the protein into the solution. We can do this either by binding our protein to the solid masses of precipitated protein known as inclusion bodies. resin and eluting it at a different time (either before or after) the con- Thus, in the case of E. coli as an expression system, we may have taminants, or by binding just the contaminants and allowing our several solid-liquid separations in which we save the solid phase. protein to flow through the column. Once we have recovered the inclusion bodies we must solubilize the Chromatography resins are available with many different lig- protein and bring it into the liquid phase. We may also need to ands, or active groups attached to the solid matrix. One set of lig- refold the protein into a native, or active, configuration. ands (ion exchange chromatography) contains an electrically In every case, at the end of recovery we will have an aqueous charged group, either positive or negative, and is capable of binding solution containing our protein, but also containing other proteins ionized forms of our protein, either as anions or cations. Another set that were produced during the fermentation process. of ligands consists of hydrophobic groups that interact with our pro- What are the unit operations we use in recovery? For separat- tein molecule under conditions of high salt (hydrophobic interaction ing solids and liquids we have two main options: centrifugation or chromatography, or HIC) or in the presence of solvents (reversed filtration. Centrifugation separates solids and liquids in a rapidly phase chromatography). Still another set of ligands bind through spinning vessel. With centrifugation we can separate whole cells complex interactions leading to high affinity for our protein (affini- from culture media; separate inclusion bodies from cell wall debris; ty chromatography). A common example of an affinity ligand is and wash cellular debris to improve our recovery of protein. Protein A, which is widely used for purification of monoclonal anti- Filtration relies on a solid sheet that allows our solution to bodies. pass through but retains particulates. We will often follow centrifu- Following completion of the purification steps, our protein is 126 American Pharmaceutical Review
  4. 4. APR_Wheelwright 6/3/08 2:51 PM Page 4 MANUFACTURING held as drug substance, and then tested and released. The drug substance is further formulated by the addition of salts and other excipients after which it is filled into the final container, usually as a liquid or as a lyophilized (freeze-dried) solid. Once labeled and packaged it is known as the drug product and is ready for distribution and sale. Summary This is clearly a simplified description of the process of manufacturing a biopharmaceutical. There are additional unit operations that are used less often but are still important to the industry. There are almost as many different combinations of unit operations making up manufacturing processes as there are different protein products. The reader who desires further detail on the subject is recom- mended to one of the many excellent courses that are taught on the subject and to subscribe to the industry magazines that provide the latest information on developments in the manufacture of biopharmaceu- ticals. Scott M. Wheelwright, Ph.D., is founder and principal of the consulting firm Strategic Manufacturing Worldwide, Inc. Dr. Wheelwright has over twen- ty years experience in bringing novel products to market with work experience that encompasses pharmaceu- tical firms and both large and small biotech companies. Dr. Wheelwright received his Ph.D. in chemical engineering from the University of California at Berkeley and performed post-doc- toral studies at the Max Planck Institute for Biophysics in Frankfurt, Germany. He is the author of a book on protein purification and has published numerous articles on manufacturing and process development. Dr. Wheelwright focuses on technology transfer and outsourcing, particularly in Asia. To correspond with the author, please e-mail: To read more articles on Aseptic, please visit our website (www.americanpharmaceuticalre- and type quot;Asepticquot; in the advanced search box located on the upper right-hand quadrant of the homepage. 127 American Pharmaceutical Review