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Bioprocess

Downstream Processing

The downstream processing of our product involves the following:

 

  1. Ultrafiltration

  2. Preparation of membrane filters

  3. High-Performance Liquid Chromatography

  4. Ion exchange chromatography

  5. Hydrophobic interaction chromatography

  6. Size exclusion

  7. Bioactivity assays

1. Ultrafiltration

Membranes are selective barriers allowing transport of certain components based on pore size by applying pressure. Different membranes processes can be used for isolating compounds of various molecular weight ranges. The main classifications are microfiltration, nanofiltration and ultrafiltration.

Membrane

Figure 39. Different types of membrane sizes (GEA Process Engineering, 2016)

a. Nano filtration

 

Nano filtration membranes have the smallest pore sizes of 0.1- 10nm which results in molecular weight cut off points ranging from 0.3-1 kDa. This small pore size only permits the passage of low molecular weight molecules while retaining larger components >1kDa such as sugars, amino acids, proteins, polysaccharides, colloids and particulates and is also capable of removing all bacteria, viruses and humic materials (Minnesota Rural Water Association, 2005). Due to the small pore size this process operates under high pressure levels typically between 600 – 1000 kPa.

 

 

b. Ultrafiltration

 

Ultrafiltration membranes have pore sizes of 1-100nm and a molecular weight cut off point range of between 1-200 kDa. Like microfiltration, due to the membranes small pore size, high pressure ranges are required for this membrane process, typically between 200 - 700 kPa. Ultrafiltration membranes are capable of retaining all microorganisms, humic materials and larger molecules such as proteins, polysaccharides and other particulates while amino acids, salts & water molecules are small enough to pass through the membrane pores.

 

 

c. Microfiltration

 

Microfiltration membranes have the largest pore size of between 0.1 – 10um and an approximate molecular weight cut off point of 200kDA (Synder Filtration, n.d.). Their larger pore size means this process operates under lower pressure levels than the likes of nano & ultrafiltration membranes and only tend to retain the likes of particulates and colloids while simultaneously allowing the passage of most compounds, microorganisms and viruses.

Specific to the field of bioactive peptides, an ultrafiltration membrane will be applied to isolate the desired bioactive peptides with ACE inhibitory properties from the meat protein hydrolysates (Bazinet & Firdaous, 2009). Aforementioned, this membrane system will be attached to the bioreactor so protein hydrolysates flow directly from the reaction vessel into the membrane. Typically, bioactive peptides are short in length and have a low molecular weight of 10kDA and below (Lafarga & Hayes, 2014). Taking this into account an ultrafiltration membrane was most applicable to isolate such peptides as its pore sizes cover this range while simultaneously it would retain any unhydrolyzed proteins & enzymes and remove all microorganisms which may be present within the meat waste (Fig. 39 (b)). The pores of a microfiltration membrane would be too big to retain the desired peptides while nanofiltration membrane would only result in the isolation of some bioactive peptides with very low molecular weights while removing the rest meaning this membrane process wouldn’t assure complete recovery of all bioactive peptides present within the substrate thus resulting in a lower peptide yield.

 

A series of ultrafiltration membranes with different molecular cut off points of 10kDa, 5kDa and 1kDa will be employed to finely isolate the desired range of peptides with discrete molecular weights (fig.40). Utilizing a multichannel ultrafiltration membrane rather than a single ultrafiltration membrane with only one pore size will assure a higher extraction yield as various peptides of different sizes can be recovered all at once.

Figure 40. Multichannel ultrafiltration model​ (Triqua International, n.d)​

 An ultrafiltration membrane system will not only allow for the continuous isolation & production of bioactive peptides, additionally it allow for the immobilization of enzymes as it will entrap the enzymes as well as unhydrolyzed proteins and any other components exceeding a molecular size of 10kDA. Employing an ultrafiltration membrane in our bioprocess will result in a tangential flow type filtration (cross – flow filtration) which is one of two possible types of filtration modes which exist 1) Tangential flow, 2) Direct flow. A tangential flow is where the feed stream is passed through the membrane, parallel to the membrane face allowing the permeate (product) to pass through the membrane pores while simultaneously the retentate which in our case would be the likes of the enzymes & unhydrolyzed protein molecules will be recycled back into the feed reservoir in the reaction vessel (fig. 41). This mode of filtration is more desirable because it results in a higher flow rate & is more efficient because the flow of the feed stream across the membrane assists the removal of aggregating molecules from the membrane face which would usually cause clogging and so this process in its self is self-cleaning while also reducing the incidence of clogging & membrane fouling – two common problems associated with using membranes for filtration purposes (Schwartz, 2012). For our bioprocess a tangenial flow will additionally allow for the recycling and reuse of enzymes & unhydrolyzed proteins thus meaning improved peptide productivity and enzyme yield.

Figure 41. Cross flow filtration  (SpectrumLabs, 2015)

Table 10. Summary of different flow patterns

Membrane Construction Material

 

The ultrafiltration membranes will be composed of polyvinyldiene fluoride as opposed to the more popular cellulose acetate due to its greater strength and stability and it is a highly unreactive thermoplastic. Cellulose acetate is susceptible to degradation by acids and alkaline solutions and partial hydrolysis by enzymes and so this material was avoided because our bioprocess involves a high salt solution, enzymes and citric acid all of which could influence the membrane material.

 

The membranes will be tubular in shape as they are more suited for difficult feed streams which will contain suspended solids or fat/ oil which ours most likely will due to the nature of the substrate. The larger size of tubular modules make them more desirable for our process and feed stream as it means membrane fouling or clogging is minimized (Synder Inc, 2016). Tubular membranes simply comprise of inner tubes followed by and external tube which acts as a shell which the permeate is filtered out into (fig. 42). Tubular membranes are well known for their strong sturdy nature which allows them to be subjected to high pressures and robust cleaning processes without little to know damage occurring and they also have a long life span. A hollow fiber membrane was another option being considered as it is commonly used in membrane based bioprocesses however the team figured this would particularly be more prone to blocking with the feed stream if small solids or fat particles were present from the meat due to its dense compact structure. These membranes are also more prone to chemical oxidation and damage from stresses like high pressure or harsh cleaning methods and from a financial perspective, tubular membranes are the more cost effective choice as well for small scale productions in comparison to hollow fiber membranes making them the worthy choice of material.

Figure 42. Tubular membrane model (Synder Inc, 2016)​

Table 11. Summary of membrane materials

2. Preparation of filters

Figure 43. Flow chart of stages involved in preparation of filters

Filters intended for use for filtering products in the pharmaceutical industry are required to be validated by destructive & non-destructive integrity testing by the FDA to meet the requirements of their good manufacturing regulations when manufacturing sterile drug and biological products. Validation of sterile grade filters is necessary because the filter itself can be the cause of contamination of products so testing helps verify the sterile condition of the filter, to assure its integral, that it fulfils its intended purpose and it also confirms that there are no leakages in the system, security of seals, correct grade filter and the presence of a cartridge.

 

As outlined in Annex I - Manufacture of Sterile Medicinal Products of the EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use “The integrity of the sterilised filter should be verified before use and should be confirmed immediately after use by an appropriate method such as a bubble point, diffusive flow or pressure hold test. The time taken to filter a known volume of bulk solution and the pressure difference to be used across the filter should be determined during validation and any significant differences from this during routine manufacturing should be noted and investigated. Results of these checks should be included in the batch record” (European Commission, 2008). With this is mind sterile filter testing of our ultrafiltration membranes will take place pre-sterilization, post sterilization and post use. There are two types of testing’s which will be performed, destructive and non-destructive.

 

 

Destructive

 

Destructive testing includes biological/ bacterial challenge testing. Prior to this test, the bioburden of the product solution will be assessed via viability testing. An inoculum of the product solution to be filtered will be taken and diluted in ringers. Then at regular intervals, an aliquot will be taken & plated on plate count agar and the number of colony forming units will be recorded and converted into log values to determine the bioburden level of the bioburden will be recorded. This test can help the team determine the best parameters for the final sterilization step and will also help the team determine the effectiveness of the sterilization process based on the bacterial counts.

 

 

Biological challenge testing

The aim of the bacterial challenge testing is to test the filter membranes ability to retain bacteria to assure it meets the criteria of a sterilizing filter. The membrane filters employed in our bioprocess have molecular weight cut off points of 10, 5 and 1kDa meaning the pore sizes are below 0.2um and so the membranes should be capable of removing bacteria. This test will be performed in accordance with the ASTM F838 method using Brevundimonas diminuta at a minimum concentration of 10^7 CFU/cm2  in a sample of our product solution and the process parameters specific to our bioprocess including temperature, pH, pressure and time (Bioprocesses Resources LLC, 2015). In this way, the bacterial challenge test will help us determine the filters compatibility with the product, its capability in retaining bacteria and assure it can withstand the process parameters and is not degraded by such factors. Testing will be performed in triplicate and will take place before and after sterilization. Since it is a destructive test it means the filter cannot be used afterward for filtration of the product.

 

 

Non- destructive

 

Non-destructive testing then is essentially the integrity testing for filters which includes a bubble point test, pressure hold test and diffusion test.

 

Bubble point testing

This test will help us to determine the pore size in the membranes to ensure they are the correct size for efficient isolation of the desired bioactive peptides. This testing is based on the principle that for a given pore size with constant wetting, the pressure which is required to force air bubbles through the largest pores is inversely proportional to the size of the filter membrane pores (Lenntech, n.d). So the larger the pore, the smaller the bubble point pressure and vice versa. The membrane will be wetted and placed in a type of housing as in figure 34. A source of compressed air pressure will be attached to the upstream end while tubing will run downstream from the housing into a bath of water from which the gas is driven out from and into the water. The pressure will continue to be increased until a rapid flow of bubbles is observed at the outlet in the water, the pressure value at this point will be taken as the bubble point. If the bubble point value appears to be below the manufacturers specification then this could be an indication of the wrong pore size or a non-integral membrane or seal (Merck Millipore, 2017).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Diffusion test

The diffusion test is used to measure a membrane filter's diffusive flow integrity and is based on Fick’s Law of Diffusion whereby the gas diffusional flow rate is proportional to the differential pressure and total surface area of the filter. For this test, the filters will be wetted in water and placed in housing from which a source of gas will be attached to the upstream side. Tubing will be attached at the other end and submerged in a bath of water as in figure 45. so the flow of gas can be observed. The gas pressure will be gradually increased to the recommended pressure as per manufacturers specifications which is usually 80% of the bubble point pressure. The gas flow at the other end will be measured for one minute and compared to the specified value in the manufacturers specifications. If the diffusional flow rate value is below this then this is could be a sign of the wrong poor size or a non-integral membrane/seal (Merck Millipore, 2017).

 

 

Pressure hold test

The pressure hold test is a variant of the diffusion test. This test employs a highly accurate gauge to monitor the pressure change due to gas diffusion through the membrane without breaching the downstream side of the filter. This method will be conducted in a similar manner to the diffusion test, gas pressure will be applied and increased to the pressure recommended by the manufacturer but the gas flow is not measured at the downstream end instead once this pressure has been reached, the valve will be shut off and the decrease in pressure is observed from the gauge as a function of time. If the decay in pressure is within the specified range then the loss of pressure is simply due to the diffusion through the wet membrane. However, if the decrease in pressure appears to exceed the maximal value then this is an indicator the rapid loss of pressure is due to a leak in the system which needs to be addressed (Bioprocesses Resources LLC, 2015).

Figure 44. Schematic of bubble point test  (Merck Millipore, 2017)

Figure 45. Schematic of diffusion/ pressure hold test  (Merck Millipore, 2017)

Sterilization

Once all testing has been performed and results are satisfactory the filters will then be sterilized in line in a filter housing. Since integrity testing takes place pre-sterilization, the membrane filters will be dried with pressurized air in order to ensure subsequent steam flow during sterilization  (Lutkewitte, 2017). The valves of the filtering system are then open to bleed the air and any condensation from the filter and closed again. Steam will be injected into the filter housing and once the steam reaches the appropriate temperature the sterilization cycle begins. For our process, the sterilization process will last a duration of 30mins at 121C and the pressure is constantly monitored by the gauges to ensure it doesn’t exceed 350 millibars to ensure the filters integrity is maintained. After steaming is complete, the bleed valves are opened again slowly to release condensation & air. Compressed air will then be applied again to dry the filter. Figure 47 is a proposed model for the configuration of our filtration system except instead of one, there will be three membrane filters in-line connected to each other with piping so they can be sterilized as one rather than individually.

Figure 46. Phases in the sterilization cycle  (Lutkewitte, 2017)

Validation of Sterilization

To validate the sterilization process, temperature sensors will be placed at the condensation outlets of the system to verify the desired sterilization temperature was maintained through the process (fig.47). If an incident happens where the temperature recorded from the outlets is below 121C then this is an indication there was a fault and the sterilization cycle will be repeated. After the sterilization cycle is complete, integrity testing will then be performed again before beginning the bioprocess to ensure the membrane filters integrity has been maintained.

Figure 47. Filtration system model  (Merck Millipore, 2017)

3. High Performance Liquid Chromatography

The analytical method used to separate the target peptides will be high performance liquid chromatography (HPLC). HPLC is a rapid method used for decades in pharmaceutical, chemical, agricultural and food processing industries amongst a plethora of others (Ali et al., 2009). In chromatography there are two main types of analysis: analytical and preparative analysis. For the purpose of this project, HPLC will be applied in the sense of a preparatory step and collection of the analyte. To understand the optimum rate of separation, first we must understand the compounds we are trying to generate. Represented in the schematic below are the characteristics of each of our compounds that we will be separating them by.

 

 

MNPPK

  • Soluble in water

  • pH 9.8

  • Net charge of 1

RMLGQTPTK

  • Soluble in water

  • pH 11.3

  • Net charge of 2

PAGNPGADGQPG

AKGANGAP

  • Soluble in water

  • pH 6.98

  • Net charge of 2

ITTNP

  • Not very water soluble

  • pH 3

  • Net charge of 0

Column choice

Monolithic columns have been gaining more traction in the area of separation of small peptides and proteins. While they have been used for decades in separation analytics, it is only recently that columns made of solely monolithic silica as an effective method to separate biomolecules according to their size (Ali et al, 2009). These monolithic columns have a mixed porous structure consisting of slightly larger macropores (2um) and smaller mesopores (13nm). The larger macropores allows for higher flow rates while the mesopores increase the surface area, inversley increasing the separation efficiency. This column will play a vital role in the size exclusion separation. Size exclusion chromatography and hydrophobic interaction chromatography and was extensively applied in the study by Pownall et al, (2010) in which they analysed the functional and structural properties of  bioactives extracted from vegetables.

 

In these monolithic columns two stationary phases dominate the market, the first being methacrylate columns and the second being monolithic silica gel. Both of these must be prepared for the column, yet since preparation or polymerization is essential, more control is given to the user with regard to differing pore sizes and ratio of the skeleton(Ali et al, 2009). As depicted by Freitag (2014) monolithic columns are restricted for use as they have a maximum volume capacity of 20ml, yet for this project this volume capacity is ideal.

4. Ion exchange chromatography

Based on the charge of each peptide, they will have varying elution times due to their differing charges and will be the first of three steps of chromatographic separation of the target peptides. For optimum elution using the monolithic column, it will be packed with an anionic resin as all of the bioactive peptides are either positively or neutrally charged. Taking advantage of these electrostatic interactions is imperative in achieving a high quality end product. As the solution following filtration will already be of a high salt content, the mobile phase to begin with will be a basic ammonia buffer of a pH of 11.5, calculated using the Henderson - Hasselbach equation solution in order to support the gradient elution of these target molecules. After 5 minutes of elution times, the mobile phase will be switched to a 1M ammonia solution of 9.25pH value. This will follow the same protocol until the last mobile phase which is acidic in nature is swapped out. As Freitag (2014) has highlighted, for the buffers to work optimally they they must be within 0.5pH units away from the iso-electric point of the target molecule. The schematic above details the iso-electric point of each peptide, which all differ greatly, meaning that using four differing mobile phases will elute each peptide accordingly.

 

The video below, created by GE life sciences provides a detailed description as to what is happening during ion exchange chromatography.

Video 1. Ion exchange chromatography

5. Hydrophobic interaction chromatography

Taking advantage of the hydrophobic or hydrophilic interactions of the peptide is the main principle of hydrophobic interaction chromatography. The chromatographic taking place for this technique is reversed phase high performance liquid chromatography (RP-HPLC) and is widely applied in protein purification in biotechnological processes. This is effectively done through the choice of stationery phase and changing the concentrations of the mobile phase or elution gradients. As outlined above, three of the four target peptides are water soluble, or hydrophilic and only one is hydrophobic. Taking advantage of this fact, the monolithic column will be prepared with polymerized methacrylate stationary phase due to its high polarity and effectiveness in the chosen column. The mobile phase used will be polar, deionised water as the first three peptides targeted for elution are polar, then the column will be rinsed with methane to ensure the final elution of the only peptide that is hydrophobic. A video below again, GE life sciences demonstrate the intricate processes that are occurring within hydrophobic interaction chromatography.

Video 2. Hydrophobic interaction chromatography

6. Size exclusion CHromatography

Size exclusion chromatography, in my opinion, is just a more justifiably correct term for a method of filtration. In its most basic principle, chromatography is a separation technique - as is filtration, although of course, chromatographic techniques are a more complex method of separation. In addition to this, chromatographic techniques in industry are expensive. Upon the embarkment of this project, the aim was to gain a high quality product, which we believed  using every possible chromatography technique would help us achieve this.

 

The first stage of primary processing involves very specific filtration methods and even in our secondary processing involving two chromatographic techniques used in conjunction with the monolithic column. In the column there is an underlying method of size exlcusion occurring as the target peptides travel in and out the mesopores in the column in addition to the method of target separation such as ion exchange or hydrophobicity separation.

 

Originally, the idea of a size exclusion chromatographic method was planned to be included inthe secondary stage of downstream processing. Yet, when comparing our peptides to synthetically created peptides already existing on the market, the cost of our process is a major disadvantage. Therefore, the size exclusion chromatography step was omitted as it was deemed that the HIC and IEC methods were sufficient.

 

7. Bioactive assays

The last stage of our bioprocess is conducting bioactive assays on the bioactive peptides we have produced to determine how effective they will be for their intended use. Since the bioactive peptides are intended for oral consumption it is imperative that they remain active during gastrointestinal digestion & absorption so it can be assured they reach the renin-angiotensin system for activity against the ACE enzyme. Although peptides obtained from meat sources are typically known to be able to withstand such harsh conditions of the gastrointestinal tract and resist digestion by such enzymes as they are true inhibitor type peptides, it is still a requirement to perform such tests to verify this for the peptides extracted during our bioprocess each time. Originally the team didn’t consider performing any testing on the produced bioactive peptides to assess their activity and stability, I think we just presumed they would work because literature says so. However from a recent critical review I done on the article “Bioactive peptide production by hydrolysis of porcine blood proteins in a continuous enzymatic membrane reactor” by Jen-Ting Wel & Been-Huang Chiang and comparing similar literature I was made aware the importance of conductive bioavailability and bioactive assays as many bioactive peptides which have proven to be active in vitro actually have shown to have little to no ACE inhibitory effects in vivo. From reviewing literature during the critical review process the following assays were decided upon for anlysisng our bioactive peptides:

 

Gastrointestinal digestion assay

The stability of bioactive peptides against gastrointestinal enzymes will be investigated by exposing them to the enzymes pepsin, trypsin and pancreatin which are representative of the enzymes they would encounter during digestion in the stomach and small intestine. For this the peptide fraction will be solubilized water and 0.05% test protease will be added. They will then be left to digest for 24hrs at body temperature (37C). Following this time period the solution will be heated to 90C to inactivate the proteases and the solution will be centrifuged. The IC50 value of the supernatant will then be determined to assess whether the peptides resisted hydrolyzation by the test enzymes or not.

 

ACE inhibitory activity

The ACE inhibitory activity of the produced bioactive peptides will be measured based on their activity when administered in spontaneously hypertensive rats in accordance with the method of  Jamhari, et al. (2007). For this assay, the bioactive peptides will be dissolved in distilled water (10mg/ml) and a volume of 10mg/kg of body weight will be orally administered to the hypertensive rats with a sonde.  The systolic blood pressure of the rats will then be measured at 0, 3, 6, 9 and 24hrs after administration with a sphygmomanometer to determine the antihypertensive effects of the peptides.

 

Limitations of bioprocess & Product

Process limitations

 

Mechanical issues

Firstly mechanical issues may arise with the processing equipment such as leakages broken pumps or valves, damaged seals which would essentially impact our process as it cannot be continued if these problems arise. To address this limitation the process will be continuously monitored and prior to each bioprocessing cycle the valves, pumps and all other components will be checked to ensure they are in sound conditions and integrity tests are implemented to be performed before and after sterilization and post use to ensure no leakages are present in the system.

 

Electrical issues

Electrical issues is another limitation of the process since all the monitoring equipment is run on electricity. A power cut or loss of power for some other reason could essentially cause the control systems such as probes and gauges to fail and as a result, the process won't be adequately monitored and it can't be assured the conditions are being maintained at optimum. To eliminate this potential limitation a backup generator will be on site which will automatically kick in if a powercut arises. Electrical equipment may also fail due to faulty cables or physical damage. In the event of this happening duplicates of all probes and measuring devices will be in stock to replace any damaged ones. Prior to each bioprocess run cables of all electrical will be inspected for any visible damage.

 

 

Contamination

Contamination is a huge limitation of the bioprocess particularly since our bioprocess is continuous, contamination could prove detrimental for the Nutriplus company as it means large volumes of product will be lost. Due to the perishable nature of our meat substrate contamination is a major risk factor as it is prone to bacterial contamination. Apart from this contamination may also arise from other sources such as the employees, equipment itself and the surrounding environment. To reduce the risk of contamination to an absolute minimum several steps are undertaken which are as follows:

 

1. Any meat waste which appears visibly contaminated or gone off is discarded and not used for processing.

 

2. The homogenized meat substrate is filtered upon filling through a series of membranes with pore sizes of 0.01um and below to retain any microorganisms which may be present which assures sterility of the produced product.

 

3. Any suppliers used for the supply of materials and equipment will be assured to have the appropriate documentation and correct certification.

 

4. All staff will receive full health & safety at work training and will receive documented procedures as well as full training in performing the stages of the bioprocess. Only upon completion of all training will employees be allowed to partake in performing the bioprocess.

 

5. Adequate personal protective equipment including goggles, boots, gloves, facemask, head cover and a full body suit will be provided as in figure 48 for any staff members who will come into contact with or are working with the process and it will be assured they are wearing it at all times.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6. All equipment including filters and membranes will undergo CIP and SIP cycles prior to processing to assure it is sterile by ensuring its free from contamination and any residue or debris from the previous run.

 

7. Air filters will be fitted within the processing area where the process is taken place to filter out contaminants which may be present in the air that could potentially contaminate any stage of the process.

 

 

Product limitations

The possible limitation with regards to our product is in relation to its stability during digestion. The peptides must reach the site of action intact in order to be able to have antihypertensive effects. Although peptides in meat sources are known to be true inhibitor types which remain stabilized upon exposure to digestive enzymes, these studies are limited to specific peptides and so this cannot be assured for all recovered antihypertensive peptides from or bioprocess. To combat this problem if it were to occur, if the product proves to have antihypertensive activity we plan to nano encapsulate the bioactive peptides in liposomes which can be recovered from the meat itself. The will be entrapped in the aqueous core of liposomes and kept intact and protected by the lipid bilayer. Encapsulating the bioactive peptides will enable protections against degradation, mask any flavor, allow for timely release of the peptides and targeted drug delivery.

 

 

 

Think you know our bioprocess? Why not test your learning and partake in our quizzes!

Figure 48. Personal protective equipment

References

Bazinet, L., & Firdaous, L. (2009). Membrane Processes and Devices for Separation of Bioactive Peptides. Recent Patents on Biotechnology, 31, 61-72. Retrieved from https://www.researchgate.net/publication/23796822_Membrane_Processes_and_Devices_for_Separation_of_Bioactive_Peptides

 

Bioprocesses Resources LLC. (2015). Filter Integrity Testing. Retrieved November 30, 2017, from Bioprocess resources: http://www.bioprocessresources.com/servicelist/information/filterintegritytesting

 

Cappia, J.-M. (2004). Principles of Steam-InPlace. Retrieved December 10, 2017, from PharmaTech: http://alfresco.ubm-us.net/alfresco_images/pharma/2014/08/22/042e6702-31d4-40fb-955a-42f8d8134ca9/article-128401.pdf

 

Carvalho, L., de Almeida, G., de Oliveira, J., Skorupa Parachin, N., & Souza Carmo, T. (2017). Production Processes for Monoclonal Antibodies. In Fermentation Processes (pp. 182-198). InTech. Retrieved from https://www.researchgate.net/publication/313542678_Production_Processes_for_Monoclonal_Antibodies

 

European Commission. (2008, November). EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use. Retrieved November 30, 2017, from European Commission: https://ec.europa.eu/health/sites/health/files/files/eudralex/vol-4/2008_11_25_gmp-an1_en.pdf

 

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GEA Process Engineering. (2016). Membrane Filtration Systems. Retrieved November 14, 2017, from GEA Engineering for a better world: http://www.gea.com/en/binaries/membrane-filtration-ultrafiltration-nanofiltration-microfiltration-reverse-osmosis-gea_tcm11-34841.pdf

 

Jagani, H., Hebbar, K., Gang, S., Raj, P., Chandrashekhar H., R., & Rao, J. (2010). An Overview of Fermenter and the Design Considerations to Enhance Its Productivity. Pharmacologyonline, 1, 261-301.

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Jamhari, K., Mori, T., Kawahara, S., Miake, K., Kodama, Y., Sugiyama, M., . . . Muguruma, M. (2007). Angiotensin-I Converting Enzyme Inhibitory Peptide Derived from Porcine Skeletal Muscle Myosin and Its Antihypertensive Activity in Spontaneously Hypertensive Rats. Journal of Food Science, 72(9), 702-706.

 

Lafarga, T., & Hayes, M. (2014). Bioactive peptides from meat muscle and by-products: generation, functionality and application as functional ingredients. Meat Science, 98, 227–239.

 

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Synder Filtration. (n.d.). Membrane Processes. Retrieved November 14, 2017, from Synder Filtration: http://synderfiltration.com/learning-center/articles/membranes/membrane-processes/

 

Synder Inc. (2016). Tubular membranes. Retrieved November 25, 2017, from Synder Filtration: http://synderfiltration.com/learning-center/articles/module-configurations-process/tubular-membranes/

 

Triqua International. (n.d). MemTriq Membrane bioreactor MBR. Retrieved October 20, 2017, from Triqua International bv: https://www.triqua.eu/site/mem-triq-mbr

 

US Food & Drug Administration. (2004). Guidance for Industry Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice. Retrieved November 30, 2017, from US Food & Drug Administration: https://www.fda.gov/downloads/Drugs/Guidances/ucm070342.pdf

 

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