1 00:00:19,005 --> 00:00:25,005 Today, we rely heavily on biotechnology for many of the necessities of life: 2 00:00:26,009 --> 00:00:33,011 For the food we eat, the clothes we wear, fuels for transportation, earth-friendly plastics, 3 00:00:34,018 --> 00:00:40,019 pharmaceuticals to treat our illnesses, and dietary supplements to help us stay healthy. 4 00:00:40,028 --> 00:00:47,028 Many of these products are produced using three core biotechnology processes: Fermentation, 5 00:00:47,092 --> 00:00:54,092 Recovery and Purification. Fermentation is basically cell farming. 6 00:00:55,001 --> 00:01:00,072 We program cells to produce a product, we nurture them as they grow and reproduce, 7 00:01:00,072 --> 00:01:04,129 and then we harvest them! In recovery we separate our product from the 8 00:01:05,029 --> 00:01:10,058 cells where they were housed... And then in purification we go a step further 9 00:01:10,319 --> 00:01:15,780 by removing everything else that's contaminating our product... 10 00:01:15,078 --> 00:01:19,847 leaving us with a very pure, concentrated solution. 11 00:01:20,549 --> 00:01:26,240 In this program, we're going to look at a typical purification process used in the manufacture 12 00:01:26,024 --> 00:01:33,024 of GFP - Green Fluorescent Protein. GFP is broadly used as a biological marker 13 00:01:33,959 --> 00:01:39,963 because it's very well tolerated by most cells and doesn't interfere with normal cellular 14 00:01:39,999 --> 00:01:44,081 function. We'll examine the technologies, equipment 15 00:01:44,819 --> 00:01:49,810 and materials used... how to prepare for the process... 16 00:01:49,081 --> 00:01:56,081 and how the GFP purification process is managed, step-by-step. 17 00:01:59,969 --> 00:02:06,969 There are two main operations used within purification: Chromatography and Filtration. 18 00:02:07,659 --> 00:02:12,230 Before we can appreciate exactly what these process steps are accomplishing, we need to 19 00:02:12,023 --> 00:02:19,023 take a closer look at what our Green Fluorescent Protein has been through so far. 20 00:02:19,569 --> 00:02:24,600 Back in Recovery, our harvested cells from Fermentation were homogenized. 21 00:02:24,879 --> 00:02:31,530 Our target protein was inside each cell, so to get at it, the cells had to be ruptured. 22 00:02:31,053 --> 00:02:37,055 Homogenization freed a flood of cellular components: including membrane debris, cytoplasm, DNA, 23 00:02:37,073 --> 00:02:44,073 and proteins - including our target protein, GFP -- and they were all mixed into the buffering 24 00:02:45,959 --> 00:02:51,971 solution used to suspend the cells. Most of the solids were then removed through 25 00:02:52,079 --> 00:02:57,610 centrifugation (or "by centrifuge"), but the liquid that remained -- the clarified 26 00:02:57,061 --> 00:03:01,530 lysate, was still rich in biological products, including 27 00:03:02,079 --> 00:03:09,079 dissolved chemicals, proteins and other impurities. Amazingly, through Purification, we can target 28 00:03:09,319 --> 00:03:16,319 a single protein within this biological soup. In our GFP purification process, we'll be 29 00:03:21,078 --> 00:03:27,907 using multiple types of Column Chromatography. The "column" is a cylinder filled with glass, 30 00:03:28,609 --> 00:03:34,645 ceramic or polymeric beads which are engineered to interact with or bind with molecules based 31 00:03:34,969 --> 00:03:40,730 on one or more physical properties. Chromatography relies on differences. Each 32 00:03:40,073 --> 00:03:46,582 molecule has a unique set of physical characteristics; such as size, charge, or extent of interaction 33 00:03:47,239 --> 00:03:52,336 with water. Chromatography uses these differences to separate the target protein from other 34 00:03:53,209 --> 00:03:59,209 proteins and chemicals. Sometimes size is used to differentiate. Some 35 00:03:59,209 --> 00:04:05,210 beads have small holes in them and can temporarily trap or at least slow down smaller molecules 36 00:04:05,219 --> 00:04:10,290 as they travel through the column of resin beads, while molecules too large to enter 37 00:04:10,029 --> 00:04:16,448 the pores move around the beads and exit the column first. This type of chromatography 38 00:04:16,709 --> 00:04:22,920 is called size-exclusion. In the case of charge, opposites attract, 39 00:04:22,092 --> 00:04:27,156 so a negatively charged chromatography bead will attract -- and bind to -- positively 40 00:04:28,056 --> 00:04:34,089 charged components in the process stream. Likewise, a positively charged bead will bind 41 00:04:34,089 --> 00:04:39,128 negatively charged components in the process stream. This charge-based chromatography is 42 00:04:40,028 --> 00:04:47,028 called ion-exchange chromatography. And then there's water. Molecules that readily 43 00:04:47,032 --> 00:04:53,033 interact with and dissolve in water are called hydrophilic (water-loving), while those that 44 00:04:53,042 --> 00:04:59,117 don't are called hydrophobic (water-hating). Proteins contain regions that are hydrophobic 45 00:05:00,017 --> 00:05:05,023 and regions that are hydrophilic. Because water tends to form a shield around the hydrophobic 46 00:05:05,077 --> 00:05:11,078 patches within the proteins, they are not exposed to interact with the resin beads. 47 00:05:11,078 --> 00:05:16,152 By adding salt to the protein solution, we remove the water shield, exposing these hydrophobic 48 00:05:17,052 --> 00:05:24,052 patches on the protein and resin so they can interact. This is how HIC or Hydrophobic-Interaction 49 00:05:26,048 --> 00:05:33,048 Chromatography works. In our process, which uses ion-exchange and 50 00:05:35,098 --> 00:05:39,133 hydrophobic-interaction chromatography, the chromatography equipment is housed on a skid 51 00:05:40,033 --> 00:05:44,117 to make it compact and mobile. The main part of the apparatus is a glass 52 00:05:45,017 --> 00:05:48,091 column filled with resin beads, but we also have... 53 00:05:48,091 --> 00:05:51,128 ...pumps to move the clarified lysate through the process... 54 00:05:52,028 --> 00:05:58,034 ...a supply hose and port to feed the column... ...a pre-filter to remove any remaining particulates 55 00:05:58,088 --> 00:06:02,176 -- usually solid cell debris that has not previously been removed - before the clarified 56 00:06:03,076 --> 00:06:08,112 lysate enters the column... ...an exit port for the processed solution... 57 00:06:09,012 --> 00:06:15,060 ...and auto-switching valves for directing processed solution to either waste or collection. 58 00:06:15,006 --> 00:06:19,077 To help monitor the chromatography equipment - and the solutions flowing through the unit 59 00:06:20,031 --> 00:06:26,031 -- during the process, a number of sensors are located along the product flow path. 60 00:06:26,031 --> 00:06:29,102 There's an electrical-conductivity sensor at the column inlet... 61 00:06:30,002 --> 00:06:36,073 ...a pressure sensor just before the pre-filter to help identify a filter clog... 62 00:06:36,073 --> 00:06:39,151 ...a flow meter to measure the rate of solution movement through the column... 63 00:06:40,051 --> 00:06:45,125 ...and an air sensor to ensure that no air has entered the flow path. 64 00:06:46,025 --> 00:06:52,041 As a solution leaves the column, it passes... ...a UV sensor that reads optical density... 65 00:06:52,041 --> 00:06:57,047 ...a second conductivity sensor... ...and a pH sensor that measures how acidic 66 00:06:57,047 --> 00:07:03,062 or basic the solution is. The conductivity sensors let us know when 67 00:07:03,062 --> 00:07:08,143 a new buffered solution has filled the column. When the conductivity reading on the exit 68 00:07:09,043 --> 00:07:13,082 of the column matches the reading from the sensor at the inlet of the column, then we 69 00:07:13,082 --> 00:07:17,119 know the new solution has completely displaced the old one. 70 00:07:18,019 --> 00:07:24,038 The Ultraviolet (UV) sensor monitors the concentration of protein in the product by observing the 71 00:07:24,038 --> 00:07:29,116 optical density of the passing solution. This sensor works hand-in-hand with the valves 72 00:07:30,016 --> 00:07:34,072 on the exit of the column. Through the controller program, we can set 73 00:07:34,072 --> 00:07:39,097 a protein concentration threshold. When the optical density of the solution leaving 74 00:07:39,097 --> 00:07:46,097 the column is below the threshold, the valve directs the flow to waste. 75 00:07:47,062 --> 00:07:52,097 When the optical density of the solution leaving the column is at or above the threshold, this 76 00:07:52,097 --> 00:07:55,146 means that solution contains our purified product... 77 00:07:56,046 --> 00:08:03,046 and the solution is directed to a collection vessel. 78 00:08:06,068 --> 00:08:12,109 TFF - Tangential Flow Filtration - is at its heart, a simple process step. 79 00:08:13,009 --> 00:08:18,094 We're going to pump a fluid through and across a special type of filter known as an ultrafiltration 80 00:08:18,094 --> 00:08:21,168 membrane. The size of the pores in the filter material 81 00:08:22,068 --> 00:08:25,145 determines what passes through and what's held back. 82 00:08:26,045 --> 00:08:31,047 As with almost any filtering process, we can choose what we want to keep. 83 00:08:31,047 --> 00:08:36,053 The solution that passes through the membrane is referred to as the permeate. 84 00:08:37,007 --> 00:08:41,045 Because the pores of the ultrafiltration membrane are small enough to keep the product from 85 00:08:41,045 --> 00:08:46,093 passing through, the permeate contains no product and is sent to waste. 86 00:08:46,093 --> 00:08:51,118 The portion of the feed stream that does not permeate the membrane is called the retentate. 87 00:08:52,018 --> 00:08:57,076 It contains the retained product and is the stream we are most interested in. 88 00:08:57,076 --> 00:09:03,082 What makes TFF different is a core technology that enables it to be faster, more efficient, 89 00:09:04,036 --> 00:09:10,080 more flexible and even self-cleaning! In conventional -- or terminal - filtration, 90 00:09:10,008 --> 00:09:16,011 a fluid is pumped directly into a filter. The particles within the stream that can't 91 00:09:16,083 --> 00:09:21,089 fit through the pores of the filter build up at the filter surface, eventually clogging 92 00:09:22,043 --> 00:09:25,124 it. In Tangential Flow Filtration, the stream 93 00:09:26,024 --> 00:09:32,037 moves across the filter -- that is, tangential to the filter - instead of directly at it. 94 00:09:32,037 --> 00:09:37,074 The cross-flow current actually picks material back out of the filter media or membrane and 95 00:09:37,074 --> 00:09:41,105 into the stream. This retained material -- called retentate 96 00:09:42,005 --> 00:09:47,023 -- is recirculated to the supply tank and will continue to loop through the filter for 97 00:09:47,023 --> 00:09:52,117 as long as the process runs. We'll be using TFF for two different tasks 98 00:09:53,017 --> 00:09:58,084 within the purification process: Concentration and Diafiltration. 99 00:09:58,084 --> 00:10:05,023 We are processing purified GFP from a chromatography step. The fluid from the chromatography step 100 00:10:05,779 --> 00:10:11,830 is purified Green fluorescent protein dissolved in a buffering solution 101 00:10:11,083 --> 00:10:17,117 In Diafiltration, we add new buffer to the GFP retentate, while displacing the old buffer; 102 00:10:18,017 --> 00:10:25,017 effectively exchanging buffer solutions! NOTE: the GFP is retained by the membrane. 103 00:10:26,068 --> 00:10:32,071 If we don't add a new buffer, then we're concentrating our solution. 104 00:10:32,098 --> 00:10:36,156 Concentration is simply the removal of water and buffer components from the feed solution 105 00:10:37,056 --> 00:10:44,056 that results in a more concentrated solution of Green Fluorescent Protein. 106 00:10:47,045 --> 00:10:52,071 From operation to operation or product to product, the purification process can look 107 00:10:52,071 --> 00:10:56,107 quite different. It could be as simple as a single Chromatography 108 00:10:57,007 --> 00:11:03,044 step, one cycle of TFF to concentrate the product, and final filtration... 109 00:11:03,044 --> 00:11:09,085 Or it could involve several different types of Chromatography, Diafiltration between Chromatography 110 00:11:09,085 --> 00:11:15,118 steps, and a final conventional filtration. Our Green Fluorescent Protein process is pretty 111 00:11:16,018 --> 00:11:20,029 typical of the process for biopharmaceutical products: 112 00:11:20,029 --> 00:11:26,086 The clarified lysate will be pre-filtered... Go through Anion Exchange Chromatography... 113 00:11:26,086 --> 00:11:31,152 and the GFP collected and pooled. Next, Ammonium sulfate will be added to make 114 00:11:32,052 --> 00:11:37,098 the solution high-salt... Followed by Hydrophobic Interaction Chromatography- 115 00:11:37,098 --> 00:11:43,140 HIC. The GFP is then collected and pooled again... 116 00:11:44,004 --> 00:11:50,036 The solution then goes through a TFF ultrafiltration step -- which includes concentration of the 117 00:11:50,072 --> 00:11:57,072 GFP followed by Diafiltration to exchange buffer solutions and remove salt. 118 00:11:57,839 --> 00:12:04,839 And the process finishes up with Final Filtration into bulk bottles or bags 119 00:12:08,068 --> 00:12:13,068 It's time to gather everything we'll need for the Purification process and ensure that 120 00:12:13,068 --> 00:12:19,113 the area and equipment are ready to go. The Chromatography skid and TFF system are 121 00:12:20,013 --> 00:12:25,040 checked for proper operation... process hoses are attached and examined for 122 00:12:25,004 --> 00:12:28,883 leaks... We verify that we are using the correct column 123 00:12:29,279 --> 00:12:36,070 resin and that the resin is properly packed. the column is filled with a storage buffer... 124 00:12:36,007 --> 00:12:41,041 and the product path is checked for trapped air and purged if necessary. 125 00:12:41,041 --> 00:12:45,124 Our raw materials include the clarified lysate from the Recovery process, 126 00:12:46,024 --> 00:12:50,096 various buffer solutions that are tailored to specific process steps, 127 00:12:50,096 --> 00:12:54,183 and Ammonium Sulfate that we add to one of the buffers to make it high-salt. 128 00:12:55,083 --> 00:13:00,112 The Purification process is managed through the use of a Batch Record. There is a separate 129 00:13:01,012 --> 00:13:06,056 Batch Record for each processing operation. These documents lead the operator through 130 00:13:06,056 --> 00:13:11,097 the process, step-by-step... with each step requiring a sign-off and separate 131 00:13:11,097 --> 00:13:16,181 verification by a second operator. The Batch Record also includes spaces for 132 00:13:17,081 --> 00:13:23,130 documenting times, activities, operation steps and instrument readings. 133 00:13:24,003 --> 00:13:31,003 Before the process can begin, the Purification area must be cleaned, disinfected and organized. 134 00:13:31,046 --> 00:13:35,063 Any unnecessary equipment or materials are removed... 135 00:13:35,063 --> 00:13:40,080 All equipment must be cleaned, sanitized, and set up as required by Standard Operating 136 00:13:40,008 --> 00:13:44,487 Procedures... All required materials and documentation must 137 00:13:45,279 --> 00:13:52,279 be gathered and prepared...before the process may begin. 138 00:13:54,062 --> 00:13:59,127 The Purification process begins as the transfer tank of clarified lysate from the Recovery 139 00:14:00,027 --> 00:14:05,656 process is connected to the inlet pump on the Chromatography skid. 140 00:14:05,899 --> 00:14:11,290 The first Chromatography step in our Green Fluorescent Protein purification process is 141 00:14:11,029 --> 00:14:15,038 Anion-Exchange. At this point in the process, the pH of the 142 00:14:16,019 --> 00:14:23,019 clarified lysate is about 8.0, which means that the protein is negatively charged. Because 143 00:14:23,899 --> 00:14:30,899 it is negatively charged, GFP will bind to the positively charged anion exchange resin. 144 00:14:31,279 --> 00:14:37,360 The pump draws the lysate from the vessel... past the first conductivity sensor and pressure 145 00:14:37,036 --> 00:14:42,077 sensor... and through the 0.45 micron pre-filter. The 146 00:14:42,077 --> 00:14:47,586 pre-filter removes any residual cell debris or other particulates that may have contaminated 147 00:14:48,279 --> 00:14:53,360 the solution. If the pre-filter begins to clog, the pressure 148 00:14:53,036 --> 00:14:57,087 sensor at the inlet side of the filter will register a rising pressure... 149 00:14:57,087 --> 00:15:00,164 and the controller will signal the need for a filter change. 150 00:15:01,064 --> 00:15:06,093 After pre-filtering and before the column, the lysate passes through... 151 00:15:06,093 --> 00:15:09,189 a flow meter... and an air sensor. 152 00:15:10,089 --> 00:15:16,152 Then, as the lysate passes over the resin beads, the negatively charged protein binds 153 00:15:17,052 --> 00:15:23,055 to the positively-charged beads. The solution leaving the column passes a UV 154 00:15:23,055 --> 00:15:29,141 optical density sensor, a conductivity sensor and a pH sensor. 155 00:15:30,041 --> 00:15:36,041 The optical density sensor's low readings confirm that the GFP is not in the solution, 156 00:15:36,041 --> 00:15:39,130 so the outlet valve sends the solution to waste. 157 00:15:40,003 --> 00:15:44,085 When all the lysate has entered the column -- or when the capacity of the beads to bind 158 00:15:45,012 --> 00:15:48,036 the protein has been reached, it's time for Elution. 159 00:15:48,036 --> 00:15:54,041 Elution is the release of, in this case, Green Fluorescent Protein from the beads by using 160 00:15:54,086 --> 00:16:01,086 a new solution -- in this case a buffer that includes NaCl (sodium chloride) - solution. 161 00:16:01,098 --> 00:16:06,154 As the new buffer is pumped through the beads, at some point the GFP no longer binds to the 162 00:16:07,054 --> 00:16:13,293 beads and is released into the buffer. The resulting product stream is usually referred 163 00:16:13,779 --> 00:16:19,270 to as the eluate (el-you-ate). The UV optical density sensor, which measures 164 00:16:19,027 --> 00:16:24,079 protein concentration, indicates when product begins eluting from the column. 165 00:16:24,079 --> 00:16:28,161 At this point, outlet valves are switched to allow flow of the eluate -- the product 166 00:16:29,061 --> 00:16:34,790 stream -- to a collection vessel. When the UV sensor indicates that all of the 167 00:16:35,339 --> 00:16:42,339 GFP has come off of the chromatography resin, the outlet valves are switched to waste. 168 00:16:43,042 --> 00:16:48,067 When all of the eluate has been collected and pooled, the Anion-Exchange Chromatography 169 00:16:48,067 --> 00:16:55,067 step is finished and it's time for HIC-Hydrophobic-Interaction Chromatography. 170 00:16:57,001 --> 00:17:01,058 Hydrophobic-Interaction Chromatography is based on the principle that hydrophobic chemicals 171 00:17:01,067 --> 00:17:07,346 on the resin surface will bind to hydrophobic patches on the GFP protein. 172 00:17:07,949 --> 00:17:13,047 In order for this to occur, the resin and protein eluate have to be in a high salt environment 173 00:17:13,929 --> 00:17:18,977 to remove the water shielding.  The salt we use is ammonium sulfate. 174 00:17:19,409 --> 00:17:25,485 To remove the attached GFP protein from the HIC column we simply lower the salt concentration 175 00:17:26,169 --> 00:17:32,220 during elution causing the water shielding to reform and the GFP protein detaches from 176 00:17:32,022 --> 00:17:38,721 the resin into the elution stream. The protein-rich eluate is collected and pooled. 177 00:17:38,919 --> 00:17:43,992 The product is now ready for the last major step, TFF. 178 00:17:44,649 --> 00:17:49,684 At this point in the process, Tangential-Flow Filtration will be used to concentrate and 179 00:17:49,999 --> 00:17:56,999 diafilter the GFP product stream. The eluate is rich in Green Fluorescent Protein, 180 00:17:57,249 --> 00:18:01,360 but it's still too dilute... and too high in salt. 181 00:18:01,036 --> 00:18:07,665 As the solution moves through the TFF apparatus, it leaves the supply tank...is pulled through 182 00:18:07,989 --> 00:18:11,360 a pump... past a pressure sensor... 183 00:18:11,036 --> 00:18:16,435 and then across the filter membrane. Everything that passes through the membrane, 184 00:18:16,759 --> 00:18:22,340 including the buffer solution, is known as permeate -- and -- for this process - is sent 185 00:18:22,034 --> 00:18:26,072 to waste. The GFP protein is larger than the pores of 186 00:18:26,072 --> 00:18:31,121 the membrane and therefore is retained. The retained material -- called retentate 187 00:18:32,021 --> 00:18:39,021 -- is recirculated to the supply tank. Recirculation of the feed continues until the desired concentration 188 00:18:39,869 --> 00:18:45,360 of GFP is achieved. Following concentration, and while the protein 189 00:18:45,036 --> 00:18:51,325 solution is recirculating, a new solution -- a storage buffer - is added to the feed. 190 00:18:51,649 --> 00:18:57,440 In effect, the protein is being washed by the flow of a new buffer solution in, and 191 00:18:57,044 --> 00:19:03,883 the old buffer solution out. As this diafiltration step proceeds, the buffer 192 00:19:04,279 --> 00:19:09,375 solution that is being added to the feed replaces the buffer solution that the GFP was originally 193 00:19:10,239 --> 00:19:14,320 in, effectively removing any remaining salt as well. 194 00:19:15,049 --> 00:19:20,610 When this process is complete, the GFP solution is routed through a 0.22 195 00:19:20,061 --> 00:19:24,400 micron final filter... and then collected in appropriate containers 196 00:19:24,949 --> 00:19:31,008 -- usually bottles or bags. The Purification process is complete. The 197 00:19:31,539 --> 00:19:36,615 Green Fluorescent Protein concentrate can now move downstream to final Fill/Finish to 198 00:19:37,299 --> 00:19:44,299 be freeze-dried and packaged.