The Large Scale Cultivation of Microbes - Fermenation Dylan May.05.,2014

Transcription

1 Customer Presentation The Large Scale Cultivation of Microbes - Fermenation Dylan May.05.,2014 turning science into solutions Page 1

2 Contents Principles of growth conditions Basic Fermentor Design Fermentor Operation Page 2

3 Principles of growth conditions

4 Principles BIOSTAT of growth A Plusconditions

5 Principles of growth conditions Shaker incubator Setting Parameter: 1. Temperature (37 ) 2. Stir speed (200~300rpm) 3. Aeration 4. Moisture

6 Principles of growth conditions What is a Bioreactor? An apparatus for growing organisms (yeast, bacteria, or animal cells) under controlled conditions.(t, DO, ph.tec.) Bioreactors supply a homogeneous (same throughout) environment by constantly stirring the contents.

7 Principles Introduction of growth conditions 1. 生物技術藉微生物的大量培養 (100kl~500kl) 來生產微生物細胞本身 ( 生質量 biomass) 或有用的代謝產物及蛋白質 也就叫工業醱酵 (Fermentation) 2. 最早利用微生物大量培養是應用在釀酒與丙酮 - 丁醇 (acetone-butanol) 的生產, 是一種無氧的醱酵過程 (anaerobe) 目前工業醱酵已多數在好氣的 (aerobe) 條件下進行, 氧氣的供應成為醱酵效率決定最重要的因子 3. 現代醱酵槽之設計事實上均將重點放在如何適當地供應氧氣

8 Principles of growth conditions

9 Principles Introduction of growth conditions 1. 生物技術藉微生物的大量培養 (100kl~500kl) 來生產微生物細胞本身 ( 生質量 biomass) 或有用的代謝產物及蛋白質 也就叫工業醱酵 (Fermentation) 2. 最早利用微生物大量培養是應用在釀酒與丙酮 - 丁醇 (acetone-butanol) 的生產, 是一種無氧的醱酵過程 (anaerobe) 目前工業醱酵已多數在好氣的 (aerobe) 條件下進行, 氧氣的供應成為醱酵效率決定最重要的因子 3. 現代醱酵槽之設計事實上均將重點放在如何適當地供應氧氣

10 Principles of growth conditions 氧氣供應的問題 (The Problem of Oxygen Supply) 1. 充裕的氧氣供應, 才能讓微生物快速地生長培養 但氧氣必須溶解在生長培基中, 才能透過與細胞膜結合的電子傳遞系統 (electron transport system) 產生還原性嘧啶核苷酸之氧化作用 ( 圖

11 Principles of growth conditions 氧氣供應的問題 (The Problem of Oxygen Supply) 2. 氧的溶解度隨溫度與物質濃度增加而減少, 增加氧分壓可提高溶氧 在許多醱酵中, 如絲狀 (filamentous) 微生物的真菌或放射線菌 (actinomycetes), 培養基往往變得非常黏稠, 因此溶氧的傳送控制因子由集形液 (bulk liquid) 至細胞而非由氣相至液相

12 Principles of growth conditions 氧氣供應的問題 (The Problem of Oxygen Supply) 3. 小規模醱酵可經由三角瓶振盪培養而成 於振盪過程中三角瓶的氧氣很容易從氣象傳達至液相 但培養基的量不能超過三角瓶容量之 10~20% ( 如此培養之細胞乾物重約 1~2g/l, 因氧的需用率已超過氧自氣相傳至液相的速率 )

13 Principles of growth conditions 氧氣供應的問題 (The Problem of Oxygen Supply) 4. 在大量或高密度培養時, 基於對氧氣需求必須以通氣 (aeration) 方式進行, 就是將空氣打入培養基中 5. 氧至氣泡 (air bubbles) 傳送至液相中的效率主要取決兩個因素 : (1) 氣泡表面積與氣泡體積之比 : 氣泡越小表面積與體積之比就越大, 氧的傳送也越大 (2) 氣泡在液相中的滯留時間 : 氣泡滯留於液相越久, 氧自氣泡擴散至液相的量也越大 (3) 縮小氣泡大小的方法, 乃利用具有許多小孔的空氣分散器 (sparger) 將空氣打入液相中 (4) 另一個方法是利用強力的攪拌器 (stirrer) 攪動培養基 就攪拌而言有兩個目的 : 一是氣泡之滯留於液相時間因氣泡精由彎曲之途徑而增加, 其次在攪拌過程時氧氣更容易由集形液進入細胞表面

14 Principles of growth conditions Bubble surface area Residence time

15 Principles of growth conditions 氧氣供應的問題 (The Problem of Oxygen Supply) (4) 另一個方法是利用強力的攪拌器 (stirrer) 攪動培養基 就攪拌而言有兩個目的 : 一是氣泡之滯留於液相時間因氣泡精由彎曲之途徑而增加, 其次在攪拌過程時氧氣更容易由集形液進入細胞表面 (5) 攪拌器的設計 (impeller) 對攪拌具有重要的影響, 尤其是 拉西頓式 (Rushton type) 較傳統的螺旋槳 (propeller) 能在縮小氣泡大小更有效率 攪拌器上的扇葉 (blade) 之數目與大小隊與氧氣傳送有相當大的影響

16 Principles of growth conditions Marine impeller 6-blade disk impeller (Rushton type impeller)

17 Contents Principles of growth conditions Basic Fermentor Design Fermentor Operation Page 9

18 Basic Fermentor Design

19 Vessel Vessel : Single-wall Double-wall (jacketed) glass vessels Page 48

20 Vessel 材質為高級不銹鋼能抗腐蝕, 依攪拌結構又分頂部 (Top drive) 與底部攪拌 (Bottom drive)

21 Stirrer control Stirrer shaft with single mechanical seal The stirrer shaft with single mechanical seal provides industrial biotech-proven and contamination-free operation. Direct coupling Two motor couplings are available: - Direct coupling - Magnetic coupling for even more process safety Magnetic coupling

22 Impeller & Baffles Impeller & Baffles for microbial applications 6-blade disk (Rushton) Impeller & Baffles The 6-blade disk impeller produces a flow which moves radially away from the shaft and has a high energy dissipation density. This impeller breaks up air bubbles effectively and thereby improves oxygen transport in the liquid phase. At the same time, baffles can be inserted into the vessel, which avoids the swirl that might occur at high stirring speeds and can affect mixing times.

23 Impellers Impellers for cell culture applications 3-blade segment impeller Paddle impeller 3-blade segment impeller & paddle impeller 3-blade segment impellers and paddle impellers are specially designed for use with cell cultures sensitive to shear stress and especially for cell cultures immobilized in suspension or on microcarriers. The impeller creates an axial direction of flow and thereby achieves homogenous and thorough mixing of the culture medium while minimizing shear forces. The angles of the 3-blade impellers can be adjusted to either create a downward or upward movement along the stirrer shaft.

24 Aeration spargers Aeration Spargers Microbial applications Ring Sparger for 19 mm top plate port Best suited for use with Rushton impeller Ring sparger µ-sparger Cell culture applications Micro Sparger for 19 mm top plate port Sintered stainless steel frit 20 µm

25 Aeration systems Overview Aeration Systems Aeration method Application Shear stresses OTR Material Up-scale Remarks Ring Sparger Microbial Cell culture Stainless steel + + Micro Sparger Cell culture Stainless steel + + Flotation (MC), foaming Bubble-free Cell culture Silicone membrane - - Limited by tubing length (scalable only up to 30L) ++ very high, + high, - low, -- very low; MC = Microcarrier, OTR: Oxygen Transfer Rate

26 Exhaust Cooler Exhaust Cooler The exhaust cooler is used to prevent evaporation and therefore liquid loss in the culture vessel. Moreover it prevents liquid from entering and eventually clogging the exhaust filter. It is available for UniVessel glass. For small autoclaves an adapter is available to reduce the total height of the culture vessel during the autoclaving procedure.

27 Headplate ports 19 mm 12 mm 6 mm :

28 Culture Vessels UniVessel Port and Top plate sealing's - O-Ring sealing Direct or Magnetic motor coupling All product wetted parts are labeled with material no. and lot no. Mirror polished top plate 6 mm port 12 mm port 19 mm port Direct ph & DO probe ports - no installation adaptor required Metal quick couplings - Jacket in (self closing) - Jacket out - Exhaust cooler in (self closing) - Exhaust cooler out - Cooling finger in (self closing) - Cooling finger out Vertical lifting handles Non-losable top plate screws 14mm top plate thickness Single mechanical seal Strong stirrer shaft Removable bottle holder Stainless steel stand Single-wall or jacketed glass vessel

29 Culture Vessels Sensors & Connections DO Sensor Hamilton (standard) or Mettler (optional) Clark or Optical electrode ph Sensor Hamilton (standard) or Mettler (optional) Foam & Level sensor Conductive probes Temperature sensor Pt100 sensor with sleeve Page 51

30 Culture Vessels Sensors & Connections Redox sensor Combined ph & Redox sensor Hamilton (standard) or Mettler (optional) Turbidity (cell density) sensor On-line measurement of cell growth and biomass Reduction of sampling frequency and bench analyses Absorption based

31 Culture Vessels Vessel accessories Blind plugs for all ports Septum assembly for 6mm and 19mm port Manual Sampler / Bypass sampler Dip tube, height adjustable Dip tube, with bend Sterile aeration filters Portadapter 19mm -> 12mm Consumable kit Tool Kit for vessel assembly

32 Contents Principles of growth conditions Basic Fermentor Design Fermentor Operation

33 Fermentor Operation Cycle of Fermentor operation Check point Cleaning Sterilization Operation Sterilizat ion Cleaning Operatio n Cleaning Sterilizat ion Operatio n Check point

34 Cleaning Clean Considerations EP Surface finishing: Electropolishing

35 Sterilization 無菌操作 (ASEPTIC OPERATION) 1. 成功的醱酵在於無菌操作, 亦即培養過程沒有任何雜菌汙染發生 2. 除醱酵槽本體外, 其他配管配件與培養基於接種前必須是無菌的 此外醱酵時所供應的空氣也必須除菌, 並且確認醱酵槽沒有任何機械縫隙使得外界微生物得以進入 3. 成功的蒸氣滅菌必須達到醱酵槽各組件, 亦即無任何 死角 要達到無死角的需求必須要有精密的設計, 所有組件必須光滑, 特別是焊接的部分

36 Sterilization 4. 培養基於醱酵槽中, 經由夾套與蛇管通過之熱蒸氣滅菌 或也可用分離方式先以熱交換器 (sterilizer) 滅菌處理後, 再打入已滅菌完成之醱酵槽中 同樣其他添加成分如消泡劑也必須滅菌 5. 醱酵培養使用之空氣是以過濾法滅菌 6. 雖然細菌與真菌可用過濾法去除, 空氣中的噬菌體卻難以過濾法去除 此外大腸桿菌 (E. coli) 之醱酵工廠不應位於廢水處理廠或農場下風處, 因這些地點含有多量的大腸桿菌噬菌體 (coliphages) 7. 攪拌軸 (drive shaft) 進入醱酵槽之接觸點可能是汙染的原因之一 最早的醱酵槽以玻璃棉與酚脂 (phenolic grease) 填封 ( 圖 5.6) 以減少微生物進入 近來雙機械軸封 (double mechanical seals) 已被應用, 其中轉動部位可以蒸氣滅菌及無菌水潤滑之功能

37 Sterilization Sterility Considerations Critical Elements of Steam Sterilization : 1. Time 2. Temperature 3. Moisture 4. Air Removal

38 Sterilization

39 Operation 醱酵槽之操作 (OPERATION OF FERMENTERS) 1. 任何醱酵的開始必先蒸氣滅菌, 然後接種 接種佔培養基之 1~10% 接種量太少會延長 lag period, 醱酵所需時間也會增加 2. 於 10~20L 的小型醱酵槽培養時, 可用三角瓶培養做接種源 超過 20L 則必須以較小的醱酵槽做種槽 如果再大量則必須具備一系列的醱酵槽做擴大化生產之接種源

40 Operation Process Types The traditional forms of cell culture processes are Batch, Fed-batch, Continuous and Perfusion. The type of process selected depends on many factors, including production forecasts, operation costs, available facilities, bioreactor size, media support, robustness of cell line, stability of product, downstream capabilities, etc. CELL RETENTION DEVICE Batch Fed Batch Continuous Chemostat Perfusion Fed-batch and Perfusion are typically used for manufacturing process.

41 Operation ATF filtration/cleaning action - pressurization Please go to our website for the animation

42 Operation ATF filtration/cleaning action - exhaust Please go to our website for the animation

43 Operation A cell culture process involving the continuous replenishment of cell culture media, removing waste products, and harvesting the product, all while retaining the cells within the bioreactor system.

44 Operation The Growth Curve induction feeding

45 生化工程人員使用之名詞定義與重要性 ( 巷仔內的話 - 行話 ) 縱橫比 (Aspect ratio) 醱酵槽高度與直徑之比, 傳統的攪拌槽比值介於 1:1 與 4:1 之間 氧氣傳遞係數 (KLa) 氧氣從氣泡傳送至液相之速率, 以下列式子表示 a: 氣泡表面積 速率 = KLa(C* - CL) C*: 氣泡中的氧濃度 ( 相當於氧的分壓 ) CL: 溶解氧濃度 KL : 比例常數 在醱酵槽內如果氣壓上升,C* 值隨之增加, 氧氣傳送至液相也因此增加

46 牛頓流體 (Newtonian fluid) 當兩個液體或固體表面以相對之方向接觸時會產生扭力 (shear forces) 牛頓流體是指扭力速率 (rate of shear) 與扭力壓力 (shear stress) 成比例 此比例常數即是流體之黏度 (viscosity) 氧氣吸收率 (Oxygen uptake rate-our) 二氧化碳釋出率 (Carbon dioxide production rate -CER) 呼吸商 (RQ) OUR 即細胞利用溶解氧之速率 CER 即細胞產生二氧化碳之速率 單位容量之功率 (Power per unit volume) 只用於攪拌一特定容積液體所需要的功率 如果在一不同大小之醱酵槽中, 其單位容量之功率相同, 顯示在較大的槽體中, 攪拌葉之端速度 (tip speed) 較大 同時在微生物存在下, 扭力也將增加

47 端速度 (Tip speed) 指攪拌葉邊緣在液體 ( 或空間中 ) 中移動之速度, 端速度 tip speed(m/s) = rpm(2πr) r: 攪拌葉從旋轉中心至其端部之距離 在一定轉速下, 如果攪拌葉的直徑越大, 其端部在液體中移行之速度越快, 在液體中之扭力也越大 醱酵槽越大, 即使是相同 rpm 轉速, 若是養絲狀微生物, 其引起之傷害也越大 空氣流速 (Volumetric air flow rate-vvm) 在一定時間內, 供應至一定量培養基之空氣量 1VVM 是指一分鐘內, 相當於培養液體積的空氣被供應給醱酵液 如果醱酵槽中有 100 公升醱酵液, 在一分鐘內通 100 公升空氣即是 1VVM

48 操作量 (Working volume) 在培養液被通氣攪拌開始前, 醱酵槽所能盛裝之最大培養液量, 在攪拌通氣後容積膨脹 ( 即液面升高 ) 之空間必須預留, 並且包括液面起泡空間之保留

49 Thank You very much for your Attention turning science into solutions Page 64

50 All Scale Fermentors Fermentation Technologies

51 What is Scale-Up? To take a manufacturing process from the laboratory scale to a desired large scale at which it is commercially feasible. 3 Stages Bench Scale ( 2 20 L) Pilot Scale ( L) Plant Scale (500 20,000 L)

52 Scale-Up Parameters Geometry Height to Diameter Ratio is held constant Called aspect ratio

53 Bioreactor Process Steps The set up of the bioreactors and the process steps are performed in a specific order.

54

55 ambr and fill-it support this whole process ambr15 Replaces shake flasks ambr250 Clone screening Early PD More bioreactors For process development Fill-it Fast, consistent For the best cell banks

56 The ambr bioreactor family ambr mL working volume 24 or 48 vessels Cell culture Screening & early PD ambr mL working volume 12 or 24 vessels Cell culture & fermentation Late PD & design space

57 A Profile of Sartorius The Widest Product Portfolio Along Biopharma s Process Chain

58 基本的醱酵槽設計 (BASIC FERMENTER DESIGN) 醱酵槽是由一個具有攪拌器 (agitator) 與空氣出入口之密閉容器所組成, 具備下列幾項配置 : 1. 擋板 (Baffles): 額外的擋板可增加攪拌紊亂度 (turbulence) 來改善氧氣的傳送速率 2. 消泡控制 (Antifoam control): 培養基的攪拌導致起泡 (foaming), 尤其在高密度細胞或較複雜的原料如酵母或豆類萃出物時更易起泡 一般以消泡電極偵測發泡, 在啟動邦浦 (pump) 加入消泡劑 3.PH 控制 : 多數微生物代謝產生酸鹼度變化, 一般利用 PH 電極測定加入酸或鹼來維持固定之酸鹼度 4. 溫度控制 : 當微生物代謝基質時, 會產生醱酵熱 在醱酵過程中產生的熱量必須送出, 可經由夾套 (jacket) 或交換蛇管 (exchang coil) 以冷卻水循環來降低溫度 5. 額外的出入口 (Additional ports): 用於額外的接種 (inoculum) 或追加培養基

59 SCADA Software BioPAT MFCS win/da SCADA Software Besides the core functionality of a real SCADA system BioPAT MFCS/win offers a number of additional modules for adaptation to individual requirements: - Bioprocess optimization with Design of Experiments module (DoE) S88 recipe control Batch control compliant with ANSI ISA CFR, Part 11 compatibility Secure integration into company networks Flexible device connection facility through OPC Expanded data logging and data archiving Logging of alarms and transmission to multimedia devices Page 37