Insulin production process -
coli inclusion bodies is complex and involves multiple steps, and the specifics of the processes established by different companies are proprietary. The approaches adopted in each step will have a big impact on the next step in terms of product purity and yield.
coli inclusion bodies will be summarized and discussed. The heterologous proteins are expressed in the form of insoluble cytoplasmic inclusion bodies and they are not being excreted into the culture media.
Therefore, the inclusion bodies have to be recovered from the bacterial cells by either a mechanical or lysozyme-based method Astolfi Filho et al. Mechanical methods to rupture the cell envelope involve using sonication, grinding the cell suspension in a colloidal mill such as a Dyno-Mill, or by passing the cell paste through a Manton-Gaulin press or French press.
In a lysozyme-based method, the lysozyme is added to digest the cell envelope. Thereafter, the inclusion bodies, which are denser than the other cellular components, can be easily isolated from the whole cell lysate by techniques such as membrane microfiltration or centrifugation.
Generally, mechanical methods can effectively disrupt cells, but may compromise the protein quality in inclusion bodies more than lysozyme-based methods Singhvi et al. Neither centrifugation nor membrane filtration has any effect in the extraction and washing of inclusion bodies Tikhonov et al.
Inclusion bodies recovered from bacterial cell lysates can be heavily contaminated with intact whole cells, host proteins, RNA, peptidoglycan cell wall, and membrane fragments Schoner et al.
After lysis of the E. coli cells, the inclusion bodies have to be washed sequentially to remove contaminants which often have strong ionic and hydrophobic interactions with the inclusion body proteins Astolfi Filho et al.
Inclusion body washing is critical in recombinant insulin purification, without which numerous impurities will persist and may interfere with the following steps, such as sulfitolysis, renaturation, and enzymatic digestion Min et al.
This could lead to a reduction in purification yield. Recovery of washed inclusion bodies is performed by multiple rounds of centrifugation combined with re-suspension and washing of pellets with detergents and denaturants. After the wash, the supernatant is discarded to leave behind the inclusion body pellet.
Alternatively, instead of using centrifugation, isolation of inclusion bodies from bacteria can also be achieved via membrane filtration Yuan et al. A list of additives used in washing the proinsulin fusion protein-containing inclusion bodies are summarized in Table 2.
The two most common wash buffer additives are urea and Triton X The E. coli cell wall is constituted of phospholipid, protein, peptidoglycan, and lipopolysaccharide Clark Selective extraction with detergents, low concentrations of urea, lysozyme, and EDTA facilitates the removal of these bacterial cell wall components.
Typically, the pH of the wash buffers ranged from pH 7. Wash optimization is required to evaluate the efficacy of additives in removing impurities.
It is also important to be mindful of ensuring the wash buffer does not inadvertently solubilize the desired recombinant protein, which may negatively impact its recovery.
Other time and cost-saving optimization include number of centrifugation rounds, speed of centrifugation, and wash temperature. Inclusion bodies contain protein in a stable non-native conformation. The protein aggregates may be amorphous, with partial or complete denaturation Astolfi Filho et al.
Inclusion bodies are relatively insoluble in aqueous buffers and this has introduced substantial challenges in purification. Therefore, the washed inclusion bodies have to be solubilized with solubilization buffer solution to recover the recombinant protein. Inclusion bodies are conventionally solubilized using high concentration of denaturants, such as guanidine hydrochloride GdnHCl and urea, which results in a complete disruption of protein structure Singh et al.
For proteins that contain numerous cysteine residues, dithiothreitol DTT or β-mercaptoethanol BME may be added to reduce incorrect disulfide-bond formation Harrison et al. Triton X and sodium dodecyl sulfate can be used to extract proteins from inclusion bodies Astolfi Filho et al.
Oxidative sulfitolysis involves the addition of —SO 3 groups to the —SH of cysteine residues in proinsulin polypeptides to form protein- S -sulfonate in the presence of a high concentration of denaturing reagent e.
As proinsulin expressed in E. coli is not folded in the correct conformation, the sulfitolysis step is crucial to maintain its unfolded form Petrides et al. This will prevent the formation of potentially incorrect disulfide bonds in solubilization and during initial purification steps preceding protein renaturation Harrison et al.
Furthermore, oxidative sulfitolysis can aid in improving the subsequent refolding yield Min et al. Some workflows would start with inclusion body solubilization first, followed by oxidative sulfitolysis Min et al. The conditions for solubilization of proinsulin fusion protein-containing inclusion bodies without concurrent oxidative sulfitolysis , ranked in order of ascending duration of solubilization, are presented in Table 3.
The 8 M urea is the most common solubilization agent. Reducing agents e. Sulfitolysis of denatured proinsulin is carried out with sodium sulfite and sodium tetrathionate at a molar ratio of at least Table 4.
In oxidative sulfitolysis, sodium sulfite assumes the role as a reducing agent. To achieve maximal reaction effectiveness, it is critical to use only freshly prepared tetrathionate Tikhonov et al.
If not, the formation of side products could affect the yields and subsequent folding step. pH and temperature are factors which could affect the solubilization and sulfitolysis rate of reaction. The solubilization and sulfitolysis reaction are typically carried out under alkaline conditions above pH 8 Tables 3 , 4.
Min et al. The pH adjustment to Raising both the pH from pH 9. The sulfitolysis reaction can be completed in 40 min at 37 °C, as compared to 2—3 h at room temperature Tikhonov et al.
To achieve maximum yield, it is important to monitor the temperature and duration of the sulfitolysis reaction to ensure that the reaction goes to completion.
However, going beyond what is required could be undesirable for the following fusion protein isolation and folding steps as the harsh conditions could lead to irreversible changes in protein structure Tikhonov et al. Analysis of fraction aliquots on a Mono-Q column can be performed to monitor the progression of sulfitolysis reaction Astolfi et al.
A straightforward way to stop the reaction is by diluting the sample with water Castellanos-Serra et al. Buffer exchange and desalting can be carried out to remove the sulfitolysis salts see " Buffer exchange " section. coli protein translation, the recombinant protein must be translated as a fusion protein in which the N-terminal extension provides the initiator N -formylmethionine fMet Laursen et al.
fMet is a modified methionine used as the first amino acid in most bacterial proteins. Since fMet is recognized by the human immune system as a foreign body, it is important to remove it so as to avoid unwanted immunogenic reaction. To remove the signal sequence, the methionine linker of proinsulin can be cleaved off with cyanogen bromide CNBr before purification.
The disadvantages of this method are low cleavage specificity, prolonged evaporation of CNBr, high toxicity and volatility of CNBr, and possible chemical modifications of the released products Mackin and Choquette An alternative method uses protein proteases to cleave the fusion proteins, though one has to be cautious to ensure that the protease cleaves at the correct site to remove N-terminal fused peptide.
Some of the reagents used in protein extraction, sample preparation, and purification may have adverse effects on protein function and stability.
This could interfere with subsequent downstream processes. Therefore, it is necessary to remove or reduce these contaminants using one or more protein clean-up methods. The aim is to make the extracted or purified protein samples compatible with subsequent downstream applications.
Here, we discuss the usage of buffer exchange techniques i. Dialysis is the traditional method for desalting or buffer exchange, using osmotic pressure to drive solutes across a membrane Merck Millipore The main disadvantages of dialysis are extended time taken to complete the exchange and the requirement of a large surface membrane area for exchange.
It is however suitable for sensitive proteins which precipitate easily. In laboratory scale, the solution containing solubilized proinsulin-containing inclusion bodies can be dialyzed to eliminate urea Nilsson et al. Diafiltration achieves desalting or buffer exchange through the use of centrifugal force or other external pressure to drive small microsolutes through a porous membrane Merck Millipore The membrane does not allow macrosolutes bigger than the pore size to pass through.
The main advantage of diafiltration lies in its ability to concentrate protein samples. In the industry, diafiltration is applied after major reaction steps to remove interfering reagents.
For example, diafiltration is applied after inclusion body solubilization and sulfitolysis reaction to remove the high concentration of urea and sulfitolysis reagents Petrides et al. Besides that, the renatured samples can be concentrated and buffer exchanged by diafiltration into a suitable buffer for enzymatic conversion to occur Astolfi et al.
The third method for desalting and buffer exchange is to use size exclusion chromatography SEC , also known as gel filtration chromatography.
SEC separates molecules according to their relative sizes Pharmacia Biotech Using a group separation technique, the small molecules, such as salts, can be separated from the larger peptides. SEC is a faster alternative to dialysis, and it requires a low dilution factor.
It is useful for the removal of salts and other small impurities from molecules with molecular mass above Da Cytiva The use of Sephadex G for buffer exchange has been reported after sulfitolysis reaction Cowley and Mackin and before proinsulin renaturation Astolfi et al.
There was also a report using Sephadex G after lysis, but the gel filtration media became heavily contaminated with non-chromatographable material that they were discarded after every experiment Mackin It is also possible to use Sephadex G after the RP-HPLC polishing step to exchange the sample buffer and to remove residual acetonitrile Zieliński et al.
Bio-Gel P2 was used to remove formic acid and remaining cyanogen bromide after fusion protein cleavage Mackin and Choquette The entire purification process is based on a combination of different modes of chromatography which exploits differences in size, molecular charge, and hydrophobicity.
After recovering fusion precursor peptide from inclusion body, the following capture step usually entails affinity chromatography AC or ion-exchange chromatography IEX. It is important to ensure that the intermediate product is of high purity prior to enzymatic conversion step.
IEX is also the chromatography of choice following protein renaturation and enzymatic conversion step. For insulin polishing step, it is common to use reversed-phase chromatography RP and size exclusion chromatography SEC. Some of the purification strategies have incorporated folding and cleavage step early in the scheme, thus completely eliminating the need to purify insulin precursor.
The renaturation of insulin precursor immediately following their recovery from inclusion body also eliminates the need to work with a high concentration of urea. Some of the more unconventional techniques in insulin downstream processing have been explored.
The use of simulated moving bed SMB , in place of traditional batch chromatography mode, has been reported for the purification of insulin Wang et al. The SMB mode uses solid phase much more efficiently and require much less column volume for the same throughput.
Furthermore, it can produce products at a purity similar to or higher than that of batch chromatography, and at substantially higher yields. There have been investigations on the use of protein-folding liquid chromatography PFLC to achieve simultaneous purification and renaturation of proinsulin Bai et al.
The SFC is more ecologically friendly as the major mobile phase, carbon dioxide, can be efficiently recycled. AC is a separation technique based on specific binding interaction between an immobilized ligand and its binding partner.
Depending on the specificity of the interaction, the degree of purification can be quite high. Some of the reported usage of AC in proinsulin purification is presented in Table 6. For AC to work, a suitable fusion tag has to be fused to the proinsulin molecule. Table 7 provides a list of fusion tags adopted in various insulin purification schemes and their advantages.
Polyhistidine tag is the most commonly adopted fusion tag in insulin production. For affinity capture of His-tagged proinsulin, it is common to use nickel column with imidazole elution.
Sample loading in the presence of a low concentration of imidazole and a moderate concentration of salt significantly reduced nonspecific binding of contaminating proteins Mackin As high concentrations of urea are compatible with immobilized metal ion affinity chromatography IMAC , it is useful to purify denatured proinsulin from inclusion bodies.
A well-designed fusion tag is desirable for both upstream and downstream processes. This is especially useful given that the proinsulin is a short-length peptide and is prone to degradation Min et al. Other than that, an appropriately engineered fusion tag can achieve an improvement in proinsulin peptide expression levels Min et al.
The introduction of hydrophilic amino acids to fusion tag enhanced the solubility of the molecule and resulted in an improvement in renaturation yield downstream Min et al. Gene fusions may be tricky to handle in downstream processing, owing to the fact that the fusion tail has to be cleaved off and removed.
To avoid using the toxic CNBr to cleave fusion proteins, it is possible to engineer lysine and arginine linkers that serve as trypsin cleavage site to join the fusion protein and proinsulin together. In this case, a simultaneous removal of both C-peptide and N-fused sequence can be achieved in just a single enzymatic conversion step Min et al.
If the fusion tag has a molecular weight and hydrophobic properties similar to the insulin molecule, after enzymatic conversion, the two molecules will be poorly resolved during analyses such as on RP-HPLC and SDS-PAGE.
Finally, the cost of resin also constitutes an important factor for consideration. The AC resin is generally more expensive than IEX resin. It may be cost-effective to switch to IEX as the capture step if the latter has equal or better purification performance.
Nonetheless, the fusion tag can still be preserved for the aforementioned benefits other than for affinity binding. IEX involves the separation of ionizable molecules based on their total charge.
The significant negative charge of S -sulfonated fusion protein can bind strongly to AEX. However, negatively charged non-protein impurities such as nucleic acids can also compete for binding.
Therefore, in some instances, it is advantageous to use CEX. Commonly, IEX is adopted as the capture step of precursor insulin or as the first step after enzymatic cleavage. Its high binding capacity and the possibility of including multiple wash steps enable the removal of a large number of contaminants which may complicate subsequent purification.
These contaminants comprised of host cell impurities protein, DNA, endotoxin , proteolytic enzymes, and product impurities produced during enzymatic digestion excised fragments and miscleavages.
IEX is characterized by a charged surface on stationary phase, and the use of buffer, salt, and pH control on mobile phase composition.
After the impure protein sample was loaded onto an IEX column, the column is washed to remove undesired proteins and other impurities. Using either a salt or pH gradient, the protein of interest is then eluted.
In salt gradient elution, the salt ions compete with bound proteins for the charged functional groups on the resin. Proteins with many charged groups will elute at high salt concentrations and thus have greater retention times.
On the other hand, proteins with few charged groups will elute at earlier retention times. In pH gradient elution, the number and type of ionizable amino acid side chain groups will determine the charge on the protein.
Protein elution occurs at the point when the pH gradient meets their pI as the protein no longer have a net charge that allows binding to the resin.
An increasing pH gradient is used to elute protein from a cation exchange resin, whereas a decreasing pH gradient is used to elute proteins from an anion exchanger.
It is important to take into consideration the pI of the insulin or insulin analogue during IEX purification as it will influence the choice of resin. Insulin analogue may have a pI that is different from the native insulin.
For example, the pI of native insulin is 5. A weak cation exchanger may be applicable for insulin analogues with a pI greater than the pI of native insulin, whereas a strong cation exchanger is used for insulin analogues with a pI similar to that of native insulin Coleman et al. A strong cation exchanger is ionized across a wide range of pH levels, whereas a weak cation exchanger is ionized within a narrower pH range and they start to lose their ionization below pH 6.
In addition, the insulin molecule is unstable at both the extremes of pH. At a high pH, there is an increased risk of deamidation and aggregation Helmerhorst and Stokes On the other hand, a pH that is overly acidic also poses the risk for deamidation Brange and Langkjœr Hence, a suitable pH range for purification of insulin with CEX is typically set at around pH 3 to 4.
In a salt gradient elution, the pH can be used to refine the eluted peak resolution. An optimal pH should provide maximal binding of insulin peptide and minimal binding of contaminants.
Acetic acid is a preferred buffer in CEX as it has been shown to reduce the risk of insulin fibrillation Whittingham et al. For example, the addition of ethanol may increase the solubility of insulin as the molecule is relatively hydrophobic GE application note AB ; Mollerup and Frederiksen The presence of ethanol may improve chromatographic performance, such as by decreasing elution volume and increasing yield Heldin et al.
The wash buffers should be optimized with respect to pH and salt content to ensure maximal removal of impurities. Some impurities may be very challenging to remove, especially if they carry the same charge as the target peptide.
It has been found that in the presence of di- or polyvalent metal ions in binding buffer, the insulin peptides are capable of self-association or having structural change Mollerup and Frederiksen This results in an improvement in the control of peak shapes and leads to a good resolution of the protein of interest from closely related impurities.
Also, a steep insulin peak tail makes it possible to obtain a very concentrated pool of purified insulin. This is advantageous in industrial production scale, in which the load can be increased while maintaining the same capacity to remove impurities.
RP involves separation of molecules based on the hydrophobic interactions between ligands attached to the stationary phase and solute molecules in the mobile phase. There is a difference in mechanism by which polypeptides interact with the RP surface, as compared to the small molecules Carr In the separation of small molecules, there is continuous partitioning of the molecules between the mobile phase and the hydrophobic stationary phase.
On the other hand, polypeptides are too big to partition into the stationary phase. RP is usually placed after the enzymatic conversion of proinsulin to insulin.
Following enzymatic digestion, the purification of insulin or insulin analogues usually requires two to three orthogonal chromatographic purification steps Table 5. Purification with RP complements IEX and SEC by providing selectivity based on differences in hydrophobicity.
RP is typically placed late in the overall purification scheme e. This will be beneficial in prolonging the effective lifetime of RP stationary phase as it is prone to fouling as a result of irreversible binding or low solubility of contaminants Kroeff et al.
This is critical as the silica-based packings have pH limitations which prevent the use of pH extremes for chromatography clean-up and regeneration.
Such product impurities include C-peptide, N-terminal signal sequences, dipeptides, aggregated insulin, misfolds, miscleaves, deamidated insulin, and any residual proinsulin Watson et al.
Often, minor modifications on the insulin molecule result in the generation of A21 desamido insulin hydrolysis of A21 asparagine to aspartic acid , B30 des-threonine insulin deletion of B30 threonine , or derivatization of amines by formylation or carbamoylation Petrides et al.
The most significant ones among these contaminants are A21 desamido insulin and B30 des-threonine insulin Balcerek et al.
As des-threonine insulin carries the same net charge as insulin, IEX is not ideal in resolving the two molecules. However, there have been many reports which have demonstrated the capability of RP to resolve such close-related insulin variants and fragments generated after trypsin digestion Table 9.
C18 is usually preferred for small hydrophilic peptides in the range of 2— Da, whereas C4 is most suitable for the separation of proteins, and large or hydrophobic peptides Carr The size of insulin at about Da after digestion and its relative hydrophobicity makes C8 column, which is an intermediate between C4 and C18, ideal for insulin separation.
It is best to try several different hydrophobic phases to ascertain which has the best selectivity for that specific mixture of peptides generated from protein digestion.
Also, subtle changes in RP adsorbent surfaces may result in differences in RP selectivity Carr Due to the highly hydrophobic nature of RP adsorbents, an organic solvent as the mobile phase has to be used in peptide elution.
Its long history of use, high volatility and thus easy removal after purification, low viscosity and thus low back pressure, transparency to low wavelength UV light, and ability to provide good chromatographic selectivity and peptide solubility make it preferable over many other solvents such as ethanol, isopropanol, and acetone Carr ; Kroeff et al.
There are also reports on the usage of isopropanol for insulin purification Dickhardt et al. Isopropanol, which is less polar than acetonitrile, may enhance recovery or elution of hydrophobic insulin molecule. The RP mobile phases used in insulin purification are generally adjusted to a low pH.
At a low pH, protonation of the carboxylic acid groups on the side chains of glutamic and aspartic acids, and on the carboxy terminal group will occur and this will make the protein more hydrophobic Carr This will increase the retention of peptides.
Also, an acidic mobile phase typically elutes all the insulin derivative impurities after the insulin peak and this will simplify the collection step Petrides In contrast, a buffer with mildly alkaline pH causes the elution of derivatives to be on either side of the parent insulin peak, thus sacrificing on the yield recovery and purity of the insulin pool.
In addition, the mildly acidic conditions will reduce the formation of monodesamido B-3 insulin generated at pH 7 or slightly above Brange ; Kossiakoff As the typical pH range for RP on a silica-based packing is pH 2—8 Verma , the mildly acidic mobile phase will be within the pH tolerance of stationary phase.
It is important to select an appropriate acidic buffer as mobile phase in insulin purification. Insulin can form fibrillation elongated insoluble aggregates at a high salt concentration Chatani et al. Thus, the mobile phase should be capable of maintaining a stable pH without the addition of salts.
Acetic acid-containing mobile phase is a good option, because acetic acid is a weak acid that buffers in the region of pH 3 without the need for other salts Kroeff et al.
Also, insulin can be readily crystallized from the acetic acid buffer by adding zinc chloride. A proprietary ion-pairing chromatography method on RP was reportedly able to improve the recovery of pure insulin collected as the des-threonine insulin impurity is displaced away from the insulin peak Kromasil This is due to a few reasons.
First, the sensitivity of polypeptide retention to subtle changes in the organic modifier concentration makes isocratic elution challenging, due to the fact that any small changes in modifier concentration can significantly affect protein retention Carr To separate impurities from the insulin peak in isocratic elution, the concentration of the organic modifier must be maintained very precisely in every batch.
Second, a gradient elution of polypeptide yields much sharper peaks than in an isocratic elution Carr Finally, a gradient elution can minimize the run time as compared to using isocratic elution in RP Healthcare GE HIC, similar to RP, separates molecules based on their hydrophobicity.
However, unlike RP, HIC adsorbents are not as hydrophobic. Thus, organic solvents are not a prerequisite for successful elution.
HIC is useful for purifying sensitive proteins as their biological activities are preserved under less denaturing chromatographic conditions.
It is also possible to adopt an elution gradient of a calcium chelating compound e. Using the salting-out effect, the retention of peptide can be modulated by adjusting the salt concentration Johansson et al.
It is however important to select a suitable salt for proinsulin purification on HIC, as the type of salt chosen for binding and elution can have an effect on product yield and purity Bai et al.
Some of the example applications of HIC in proinsulin purification are presented in Table As a gentler purification technique as compared to RP, it may be suitable to use HIC following proinsulin renaturation, so that the peptide conformation is maintained Petrides et al.
The use of HIC for a simultaneous purification and refolding of proinsulin has also been reported Bai et al. This may make RP preferable over HIC as RP is known to be the highest resolution chromatography technique available Healthcare GE To ensure insulin adsorption on a HIC column, the feed should have the same high salt concentration as the mobile phase during elution.
Therefore, another disadvantage of HIC is that diluted large insulin feed volume had to be used as the insulin solubility was reduced due to the high salt content in the feed. It has been reported that column load for HIC with butyl adsorbent was 0. SEC, also known as gel filtration, is the mildest of all the chromatography techniques.
SEC separates molecules by differences in size as they pass through the resin. There is no binding of the molecules to the chromatography resin, unlike chromatography techniques such as AC and IEX. It has also been adopted in protein-folding liquid chromatography PFLC for simultaneous purification and renaturation of proinsulin Yuan et al.
During elution with refolding buffer, the folded proinsulin was separated from the misfolded and aggregated forms. MMC utilizes more than one form of interaction between the stationary phase and analytes to achieve their separation Zhang and Liu The multiple modes of interaction include affinity, ion exchange, size exclusion, hydroxyapatite, and hydrophobic interactions.
The mixed-mode ion exchangers give a different selectivity as compared to the conventional ion exchangers.
It also enables the possibility of proteins binding at a high salt condition, which eliminates the need for desalting or dilution prior to carrying out MMC. This method can also be used to concentrate the protein for further purification steps. Purification and concentration of the solubilized protein from inclusion bodies can be achieved by pH precipitation.
As a protein has the lowest solubility at its isoelectric point pI , it will precipitate out of the solution at its pI. Following that, centrifugation can be performed to recover the protein of interest. For example, solubilized fusion protein can be precipitated by lowering the pH to 5. After sulfitolysis reaction, the S -sulfonated preproinsulin can be precipitated by adjusting the pH to 4.
The pH precipitation step can also be carried out after proinsulin refolding Hwang et al. The pH of the refolded proinsulin solution can be lowered to pH 4. The supernatant containing correctly folded proinsulin is collected by centrifugation.
It is important to determine the exact pH required to precipitate the impurities, while maintaining the correctly refolded proinsulin in solution.
In the presence of zinc ions, insulin is capable of self-association to form hexamers, di-hexamers, or bigger complexes of insulin peptide Mollerup and Frederiksen The hexamer consists of three dimers closely associated through interacting with two zinc ions at its core positioned on the threefold axis.
The aptitude of insulin to form crystals readily can be utilized in the purification scheme to isolate insulin from impurities that do not co-crystallize with it Mollerup et al. Zinc crystallization is a convenient and efficient method to concentrate and hold the insulin temporarily prior to further processing Kroeff et al.
The hexameric form also protects insulin from physical and chemical degradation during storage Mollerup and Frederiksen In proinsulin purification, the addition of 0. Talk with your provider about the insulin regimen that is most suitable for you.
Self assessment quizzes are available for topics covered in this website. To find out how much you have learned about Insulin Therapy , take our self assessment quiz when you have completed this section. The quiz is multiple choice. Please choose the single best answer to each question.
At the end of the quiz, your score will display. All rights reserved. University of California, San Francisco About UCSF Search UCSF UCSF Medical Center. Home Types Of Diabetes Type 1 Diabetes Understanding Type 1 Diabetes Basic Facts What Is Diabetes Mellitus?
What Are The Symptoms Of Diabetes? Diagnosing Diabetes Treatment Goals What is Type 1 Diabetes? What Causes Autoimmune Diabetes? Who Is At Risk? Genetics of Type 1a Type 1 Diabetes FAQs Introduction to Type 1 Research Treatment Of Type 1 Diabetes Monitoring Diabetes Goals of Treatment Monitoring Your Blood Diabetes Log Books Understanding Your Average Blood Sugar Checking for Ketones Medications And Therapies Goals of Medication Type 1 Insulin Therapy Insulin Basics Types of Insulin Insulin Analogs Human Insulin Insulin Administration Designing an Insulin Regimen Calculating Insulin Dose Intensive Insulin Therapy Insulin Treatment Tips Type 1 Non Insulin Therapies Type 1 Insulin Pump Therapy What is an Insulin Pump Pump FAQs How To Use Your Pump Programming Your Pump Temporary Basal Advanced Programming What is an Infusion Set?
Diagnosing Diabetes Treatment Goals What is Type 2 Diabetes? Home » Types Of Diabetes » Type 2 Diabetes » Treatment Of Type 2 Diabetes » Medications And Therapies » Type 2 Insulin Rx » Insulin Basics.
View Article PubMed Google Scholar Yuan J. Biomedical Chromatography. View Article PubMed Google Scholar Kim C. Journal of Microbiology and Biotechnology. View Article PubMed Google Scholar Min C. Journal of Biotechnology. View Article PubMed Google Scholar Mbanya J. View Article PubMed Google Scholar Nilsson J.
View Article PubMed Google Scholar Ladisch M. Biotechnology Progress. View Article PubMed Google Scholar Newsome C. Theses, Dissertations and Capstones. Slattery D. Diabetic Medicine. View Article PubMed Google Scholar Niloy K. Theses and Dissertations ETD. Paper View Article Google Scholar Gutka H.
Lyophilized Biologics and Vaccines. View Article Google Scholar Chausmer A. Journal of the American College of Nutrition.
View Article PubMed Google Scholar Frost G. Google Patents Google Scholar Dave S. Advanced Biomedical Research. View Article PubMed Google Scholar Begg A. Google Scholar Dingermann T. Biotechnology Journal. View Article PubMed Google Scholar Piri H.
International Journal of Endocrinology and Metabolism. View Article PubMed Google Scholar Wild S. Diabetes Care. View Article PubMed Google Scholar Walsh G. Applied Microbiology and Biotechnology. View Article PubMed Google Scholar Akbarian M.
View Article PubMed Google Scholar Cowley D. FEBS Letters. View Article PubMed Google Scholar Zhao M. View Article Google Scholar Redwan E. Scientific Reports. View Article PubMed Google Scholar Jafari S. coli expression system. Research in Pharmaceutical Sciences. PubMed Google Scholar Jenkins N.
View Article PubMed Google Scholar Bill R. Nature Biotechnology. View Article PubMed Google Scholar Baeshen M. coli: current scenario and future perspectives. View Article PubMed Google Scholar Carrió M. View Article PubMed Google Scholar Wildt S. View Article PubMed Google Scholar Baghban R.
Molecular Biotechnology. View Article PubMed Google Scholar Gerngross T. View Article PubMed Google Scholar Kildegaard J. View Article PubMed Google Scholar Landgraf W. View Article PubMed Google Scholar Borowicz P.
Journal of Biomolecular NMR. View Article PubMed Google Scholar Safder I. Biometrical Letters. Polez S. View Article PubMed Google Scholar Cereghino J. FEMS Microbiology Reviews. View Article PubMed Google Scholar Tschopp J. View Article Google Scholar Schillberg S.
Frontiers in Plant Science. View Article PubMed Google Scholar Khan A. View Article PubMed Google Scholar Exposito-Alonso M. View Article PubMed Google Scholar Tzen J. International Scholarly Research Notices.
View Article Google Scholar Rooijen G. van, Moloney M. View Article PubMed Google Scholar Damerum A. Postharvest Biology and Technology. View Article PubMed Google Scholar Sun H.
View Article PubMed Google Scholar Boyhan D. Plant Biotechnology Journal. View Article PubMed Google Scholar Burberry D. Plant Biotechnol J. View Article Google Scholar Baby B.
Critical Reviews in Food Science and Nutrition. View Article PubMed Google Scholar Dzhanfezova T. Food Bioscience. View Article Google Scholar Tavizi A.
Molecular Biology Reports. View Article PubMed Google Scholar Dong O. Plant Physiology. View Article Google Scholar Schroeder I.
Nature Protocols. View Article PubMed Google Scholar León-Quinto T. View Article PubMed Google Scholar Jiang W. Cell Research. View Article PubMed Google Scholar Shi Y.
Drug Discovery. View Article PubMed Google Scholar Shahjalal H. Abdal, Lim K. View Article PubMed Google Scholar Bar-Nur O. Cell Stem Cell. View Article PubMed Google Scholar Pellegrini S.
Acta Diabetologica. View Article PubMed Google Scholar Huzes G. Methods in Molecular Biology. View Article PubMed Google Scholar Chen Y.
View Article PubMed Google Scholar Mundra V. Molecular Pharmaceutics. View Article PubMed Google Scholar Bhartiya D. The Indian Journal of Medical Research. View Article PubMed Google Scholar Frenette P. Annual Review of Immunology. View Article PubMed Google Scholar Bernardo M. Cancer Research.
View Article PubMed Google Scholar Negi N. Stem Cells Dayton, Ohio. View Article PubMed Google Scholar Düber S. View Article PubMed Google Scholar Hashemian S. Journal of diabetes research. View Article Google Scholar Nemati M. Ranjbar, Ebrahimi B.
Stem cells international. View Article Google Scholar Lilly M. American Journal of Stem Cells. PubMed Google Scholar Solis M. Moreno, Correa R.
View Article PubMed Google Scholar Ratajczak M. Polskie Archiwum Medycyny Wewnetrznej. View Article PubMed Google Scholar Prabakar K. Cell Transplantation.
View Article PubMed Google Scholar Huang C. View Article PubMed Google Scholar Rosano G. Frontiers in Microbiology. View Article PubMed Google Scholar Tekarslan-Sahin S. View Article Google Scholar Karbalaei M.
Journal of Cellular Physiology. View Article PubMed Google Scholar Wang J. View Article PubMed Google Scholar Ghag S. Biotechnology and Bioprocess Engineering.
PubMed Google Scholar Tremblay R. Biotechnology Advances. View Article PubMed Google Scholar Su H. View Article PubMed Google Scholar McNulty M.
Food and Bioproducts Processing. View Article Google Scholar Castellanos-Morales V. at different nitrogen levels. Journal of the Science of Food and Agriculture. View Article PubMed Google Scholar Kollu L. International Journal of Current Research and Review.
View Article Google Scholar Afify S. Cancers Basel. View Article PubMed Google Scholar Beghini D. International Journal of Molecular Sciences.
View Article PubMed Google Scholar Lee S. Osteoporosis and Sarcopenia. View Article PubMed Google Scholar Oggu G. Stem Cell Reviews and Reports. View Article PubMed Google Scholar P. Gentile, S. Garcovich, Systematic review: adipose-derived mesenchymal stem cells, platelet-rich plasma and biomaterials as new regenerative strategies in chronic skin wounds and soft tissue defects.
View Article Google Scholar Y. Qi, J. Ma, S. Li, W. Liu, Applicability of adipose-derived mesenchymal stem cells in treatment of patients with type 2 diabetes.
Stem Cell Research and Therapy. View Article PubMed Google Scholar Y. Su, T. Zhang, T. Hang, J. Gao, Current advances and challenges of mesenchymal stem cells-based drug delivery system and their improvements. International Journal of Pharmaceutics. View Article PubMed Google Scholar C.
Eguizabal, B. Aran, S. Chuva de Sousa Lopes, M. Geens, B. Heindryckx, S. Panula, Two decades of embryonic stem cells: a historical overview.
Human Reproduction Open. Shufaro, B. Reubinoff, Therapeutic applications of embryonic stem cells. View Article PubMed Google Scholar F. Yu, R. Wei, J. Yang, J. Liu, K. Yang, H. Wang, FoxO1 inhibition promotes differentiation of human embryonic stem cells into insulin producing cells.
Experimental cell research. View Article PubMed Google Scholar A. Rezania, J. Bruin, J. Xu, K. Narayan, J. Fox, J. Stem Cells. View Article PubMed Google Scholar S.
Kadam, S. Muthyala, P. Nair, R. Bhonde, Human placenta-derived mesenchymal stem cells and islet-like cell clusters generated from these cells as a novel source for stem cell therapy in diabetes.
Rev Diabet Stud. View Article PubMed Google Scholar X. Feng, J. Liu, Y. Xu, J. Zhu, W. Chen, B. View Article PubMed Google Scholar L. Accomasso, C. Gallina, V. Turinetto, C. Giachino, Stem cell tracking with nanoparticles for regenerative medicine purposes: an overview.
Stem Cells International. View Article PubMed Google Scholar T. Nagamura-Inoue, H. He, Umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical utility.
World journal of stem cells. View Article PubMed Google Scholar C-G. Fan, Q-j. Zhang, J-r. Zhou, Therapeutic potentials of mesenchymal stem cells derived from human umbilical cord.
Omole, A. Fakoya, Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications.
View Article PubMed Google Scholar M. Hossain, Dayem A. Han, Saha S. Choi, Recent advances in disease modeling and drug discovery for diabetes mellitus using induced pluripotent stem cells.
Int J Mol Sci. Agrawal, G. Narayan, R. Gogoi, R. Thummer, Recent Advances in the Generation of β-Cells from Induced Pluripotent Stem Cells as a Potential Cure for Diabetes Mellitus. Advances in Experimental Medicine and Biology.
View Article PubMed Google Scholar. Content Top. PDF XML. Article Details. DOI : More Formats ACM ACS APA ABNT Chicago Harvard IEEE MLA Turabian Vancouver. Jessica Alyas, Ayesha Rafiq, Horia Amir, Safir Ullah Khan, Tahira Sultana, Amir Ali, Asma Hameed, Ilyas Ahmad, Abeer Kazmi, Tehmina Sajid, Ayaz Ahmad, Search Panel.
Jessica Alyas. Downloads Download data is not yet available. Prokaryotic Expression System. Escherichia coli. Versatility and cost-effectiveness for large scale production.
US FDA in Plasmid loss, high probability of translation errors which can cause adverse immunological responses in human. Colony PCR strategies for similarity verification, strain modifications, codon optimizations and addition of chaperones.
Yeast Expression System. Saccharomyces cerevisiae. Eukaryotic model systemwhich enables the quality production and proper folding. Plasmid degradation and Hyper-glycosylation on large scale.
Pichia pastoris. High cell density of Methylotrophic yeast with methanol-inducible alcohol oxidase-1 AOX-1 promoter, do not hyper glycosylate insulin. High mannose structures and glycosylation heterogeneity can cause batch-to-batch variation.
Engineered strains for higher secretion levels, AOX1-based methanol-free protein manufacture, modified N-glycosylation machinery. Transgenic Plants. Arabidopsis thaliana. Large amount of seed generation in 6 weeks, easy purification of oil bodies, no human pathogen, shockable.
Approval required. Protein stability issues Inappropriate containment strategies. Finding tissue-specific promoters to avoid toxicity. High number of leaves yield contains proinsulin, high purity protein with minimal PTMs requirement, less transgene containment.
Chloroplast glycoproteins interference. Transformed chloroplast, CTB-insulin fusion. Long term stability in leaves, more leaf protein yield then tobacco plant. Lack of regulatory approval due to food-chain contamination, Improper glycosylation.
Transformed chloroplast. Oral consumption, using hairy roots as bioreactor. lack of regulatory approval, unwanted allergic reactions.
No gene escape. Stem cells. Embryonic stem cells ESC. Can differentiate into any cell type.
Processs Natural anti-cancer supplements of ;roduction has saved the pfocess of millions of people with Natural anti-cancer supplements worldwide, but little is known about nIsulin first Natural anti-cancer supplements of insulin synthesis. Researchers Nutrition periodization for overall wellness the University of Insuulin have uncovered part of this mystery. Examining messenger RNAs involved in the production of insulin in fruit flies, they found that a chemical tag on the mRNA is crucial to translating the insulin mRNA into the protein insulin. The alteration of this chemical tag can affect how much insulin is produced. The study, conducted by researchers Daniel Wilinski and Monica Dusis published in the journal Nature Structural and Molecular Biology. Natural anti-cancer supplements ultimate goal of Womens fitness supplements therapy is to mimic productkon insulin levels. Unfortunately, current insulin replacement procss can peoduction approximate normal insulin levels. Insulin therapy for Productino 2 lroduction ranges from one injection a day to multiple injections and using an insulin pump continuous subcutaneous insulin infusion — CSII. The more frequent the insulin injections, the better the approximation of natural or normal insulin levels. Discuss with your medical provider the insulin regimen that is best for you. Natural insulin i. insulin released from your pancreas keeps your blood sugar in a very narrow range.
Bescheidener sein es muss
Nach meiner Meinung lassen Sie den Fehler zu. Geben Sie wir werden besprechen.
Ich werde besser einfach stillschweigen
Ich denke, dass Sie den Fehler zulassen. Ich biete es an, zu besprechen. Schreiben Sie mir in PM.
Auch als es zu verstehen