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Tuesday, July 30, 2013

World's Largest Chromatographic Plant for API Production

Novasep's New plant on its Mourenx, France site to produce a large volume commercial API.

Novasep, a leading provider of purification based manufacturing solutions for life science molecules, announces today an investment of EUR 30 million to build in Europe what will be the world's largest chromatography plant used for the production of a large volume commercial API(active pharmaceutical ingredient).

The plant will be built on Novasep's existing Mourenx site in France and will be operational and validated within another few months. Both the development of an advanced purification process and the plant expansion within the challenging timescale are results of the sharp increase in the projected demand for a large volume, highly purified API.

chromatography plant


Novasep has pioneered continuous chromatography for pharmaceutical manufacturing for more than 20 years. Its SMB and Varicol® continuous chromatography technologies are currently used for commercial scale production of several chiral APIs. The new plant, designed by a Novasep in-house engineering team, will include Varicol® systems with 1,200 mm diameter columns operated at up to 70 bars, the largest ever built in the pharmaceutical industry. The plant will enable the production of a highly purified API from a complex mixture. The systems will also integrate sophisticated solvent recovery systems for the recycling of 99.9% of solvents, resulting in both a very cost-effective and environmentally friendly process.

"Demand for Novasep's advanced, purification-based manufacturing capabilities in the life science industries continues to increase as drugs in development and reaching the market become more complex and specific. This necessitates our third, and largest global plant expansion in 2012," said Patrick Glaser, President and CEO of Novasep. "This successful project, the world's largest chromatography plant in the pharmaceutical industry, demonstrates the validity of the Novasep strategy which is based on the combination of synthesis, biosynthesis and purification. We thank our institutional partners for their active support and our shareholders for their strong involvement to enable this transforming project."

Beyond Novasep's commitment, the EUR 30 million investment has been made possible through financial and/or logistic support by the following partners: the association of local authorities of Lacq, the General Council of Pyrénées Atlantiques, the Regional Council of Aquitaine, the Economic Development Bureau, DIRECCTE Aquitaine, the Prefecture of Pyrénées Atlantiques, Total Group, and OSEO, France's innovation partner. The plant will leverage the SOBEGI platform's utility supply.

Sources

Novasep Website
Bioprocess News Channels

Friday, July 26, 2013

Do We Have Enough Sustainable Energy Sources??

Reports claims, within three decades global energy demand will increase by 56%. 

Due to over-consumption, soon there will be oil shortage. how do we cope with the ever growing global energy demands.

Sticking to the sustainable energy strategy and making active contributions to the sustainable energy developments can make big difference in the global energy demands. 

Sustainable energy is the sustainable provision of energy that meets the needs of the present without compromising the ability of future generations to meet their needs. Technologies that promote sustainable energy include renewable energy sources,such as hydroelectricity, solar energy,wind energy, wave power, geothermal energy and also technologies designed to improve energy efficiency.

HYDROPOWER


hydropower


  1. Hydropower or water power is power derived from the energy of falling water and running water, which may be harnessed for useful purposes mainly for electricity.
  2. Hydropower has been used for irrigation and the operation of various mechanical devices,such as watermills,sawmills,textile mills,domestic lifts,power houses and paint making.
  3. Hydro power is a renewable energy source.
  4. Hydropower types: 
Conventional hydroelectric, referring to hydroelectric dams.

Run-of-the-river hydroelectricity,which captures the kinetic energy in rivers or streams, without the use of dams.

Small hydro projects are 10 megawatts or less and often have no artificial reservoirs.

Micro hydro projects provide a few kilowatts to a few hundred kilowatts to isolated homes,villages, or small industries.

Pumped-storage hydroelectricity stores water pumped during periods of low demand to be released for generation when demand is high.

WIND POWER

Wind power is the conversion of wind energy into a useful form of energy,such as using wind turbines to make electrical power,windmills for mechanical power,wind pumps for water pumping or drainage,or sails to propel ships.
  1. Wind power is a renewable energy source.
  2. Types of wind power: 
Wind turbine– a turbine that converts wind energy into mechanical energy.

Windmill– a machine which converts the energy of wind into rotational energy by means of vanes called sails or blades.

wind mill


Windpump– a windmill used for pumping water, either as a source of fresh water from wells, or for draining low-lying areas of land.

Sail– any type of surface intended to move a vessel, vehicle or rotor by being placed in a wind – in essence a propulsion wing.

MARINE ENERGY

marine energy

  1. Marine Energy or Ocean energy refers to the energy carried by ocean waves,tides,salinity, and ocean temperature differences.
  2. The movement of water in the world’s oceans creates a vast store of kinetic energy,or energy in motion.This energy can be harnessed to generate electricity to power homes, transport and industries.
  3. Marinepower can be renewable or non-renewable energy source.
  4. 4.Forms of ocean energy:
RENEWABLE:

Marine current power- The energy obtained from ocean currents.

Osmotic power-The energy from salinity gradients.

Ocean thermal energy The power from temperature differences at varying depths.

Tidal power -The energy from moving masses of water — a popular form of hydroelectric power generation.Tidal power generation comprises three main forms, namely:tidal stream power,tidal barrage power, and dynamic tidal power.

Wave power-The power from surface waves.

NON-RENEWABLE:

Petroleum and natural gas beneath the ocean floor are also sometimes considered a form of ocean energy.

SOLAR POWER


solar power


  1. Solar power is the conversion of sunlight into electricity, either directly using photovoltaics(PV),or indirectly using concentrated solar power(CSP).Its Renewable source of energy.
  2. Concentrated solar power systems use lenses or mirrors and tracking systems to focus a large area of sunlight into a small beam.Photovoltaics convert light into electric current using the photoelectric effect.

Types of Solar energy: 

Solar water heater– Heating water with solar energy.


Solar Electricity-Using the sun's heat to produce electricity.

Photovoltaic Systems– Producing electricity directly from sunlight.

Passive Solar Heating and Daylighting-Using solar energy to heat and light buildings.

Solar Process Space Heating and Cooling-Industrial and commercial uses of the sun's heat.


Solar Impulse - First Solar Powered Flight 

The world’s first solar airplane able to fly day and night powered solely by the sun has flown San Francisco (CA) – Phoenix (AZ) – Dallas (TX) – St. Louis (MO) – Cincinnati (OH) – Washington D.C. (District of Columbia) – New York City over a period of two months.


solar impulse




BIOENERGY

Bioenergy is renewable energy made available from materials derived from biological sources.

biofuel

Types of Bioenergy

Biopower is electricity generated from combustion of biomass,either alone or in combination with coal, natural gas or other fuel (termed co-firing).

Biofuel is liquid,gas and solid fuels produced from two types of biomass materials – plant sugars and starches (e.g., grains), and lignocellulosic materials (e.g., leaves, stems and stalks).Liquid and gas biofuels are produced through fermentation,gasification,pyrolysis,torrefaction,and transesterification conversion technologies.

*This is a guest post from one of the blog reader.

References:
Wikipedia
Internet Sources
Image Source - Google Images

Tuesday, July 23, 2013

Single Use Bioreactor - Types, Advantages & Disadvantanges

A single-use bioreactor (S.U.B) or disposable bioreactor is a bioreactor with a disposable bag instead of a culture vessel made from stainless steel or glass, a single-use bioreactor is equipped with a disposable bag. The disposable bag is usually made of a three-layer plastic foil.

Many mammalian cell based biotech producers are moving to single use bioreactor modules leveraging the advantages of single use bioreactors.

Types of single-Use Bioreactor:
  • Single-use bioreactors use stirrers like conventional bioreactors, but with stirrers that are integrated into the plastic bag.The closed bag and the stirrer are pre-sterilized. In use the bag is mounted in the bioreactor and the stirrer is connected to a driver mechanically or magnetically. 
Single Use Bioreactor


  • Single-use bioreactors are agitated by a rocking motion. This type of bioreactor does not need any mechanical agitators inside the single-use bag.
single use bioreactor
Single Use Bioreactor - Agitated Type



Advantages:
  1. Eliminates validation issues because the cleaning process is avoided. 
  2. Shortens downtime and turnaround time because no cleaning is required. 
  3. Lowers risk of cross-contamination with use of new bags for each run. 
  4. Offers multiple advantages based on the elimination of the cleaning process. 
  5. Decreased operating costs and capital investment: savings on start-up capital costs as well as utilities, space, and labor requirements. 
  6. Cost savings from reduced cleaning needs: less space and stainless steel equipment needed; decreased cleaning validation and decreased use of WFI and cleaning solutions; cleaning and sterilization of bioreactors or process tanks can be ‘outsourced’ to disposable bag suppliers. 
  7. Elimination of the design elements of traditional stainless steel vessels that are dictated by CIP and SIP requirements: validation is reduced because systems are less complex. 
  8. Ease of installation. 
  9. Ease of moving when empty. 
Disadvantages:
  1. Limitation in liquid transfer. 
  2. Scalability is an issue: larger-scale bioreactor bags are needed than are available. 
  3. Expensive to use:repetitive purchases required. 
  4. Performance is not completely proven: new technology. 
  5. Slight increase in variable costs per run 
  6. Disposable bag systems difficult to justify for dedicated products or greater than 10,000L bioreactor scale processes. 
  7. Most of the larger scale facilities already have the investment in the ground for the tanks and the required cleaning validations to support multiple product use. 
  8. Leachables and inability to store hot liquids. 
  9. Potential for puncture. 
  10. Difficulty in moving when full. 
  11. Pressure and temperature sensitivity. 
  12. Disposal costs. 
Progress for Single Use Bioreactor
  • Wide acceptance of bioprocess bags 
  • Single use bioreactors are scalable and performance is comparable to Stainless Steel bioreactors. 
  • 1000L Bioreactor scales can open up opportunitiesin commercial applications. 

List of Commercially available Single Use Bioreactor Systems
  • XDR - Xcellerex 
  • Mobius - Millipore 
  • CelliGen BLU - eppendorf 
  • Hyclone S.U.B - Thermo Scientific 
  • Single Use Bioreactor - Applikon 

Helicase Dependent Amplification...DNA Amplification Without Thermocycler

Helicase Dependent Amplification (HDA) is an invitro nucleic acid amplificatio method similar to Polymerase Chain Reaction (PCR).



HDA works at constant temperature, where as PCR requires a thermal cycler for the amplification.

Nucleic acids can be amplified by various technologies and the product can then be used in research or in diagnostics. The Polymerase Chain Reaction (PCR) dominates the nucleic acid amplification market. However, the requirement for thermocycling in PCR limits its application in certain areas, such as field-testing and point-of-care diagnostics.

Biohelix Corporation has developed Kits based on Helicase Dependent Amplification Technology.

Helicase Dependent Amplification is  an isothermal DNA amplification system that uses a helicase enzyme to unwind double stranded DNA (dsDNA), referred to as Helicase-Dependent Amplification. In HDA reactions, duplex DNA is separated into single strands by a helicase. Compared with the other techniques, this amplification method is closer to nature's method of performing DNA replication and it has the following advantages:


1. Low cost for instrumentation because use of helicase to separate DNA eliminates need for thermocycler.

2. Easy-to-use for assay development (using two primers).

3. Versatile platform: can amplify both DNA and RNA (with a reverse transcriptase) and is compatible with multiple detection technologies.

4. High sensitivity and specificity.

5. HDA is a rapid method of amplifying nucleic acid at isothermal temperature that doesn't require thermal cycler.

Disadvantages

Mass diagniosis from large no. of samples cannot be achieved.

Cost of HDA reagents are costly compared to PCR reagents.

Progress and Developments of HDA Technology

A team of researchers have successfully implemented the HDA technology for amplyfing the target DNA in combination with an integrated disposable device for DNA extraction. Full Publication can be found in the below link.

An integrated disposable device for DNA extraction and helicase dependent amplification.

http://www.ncbi.nlm.nih.gov/pubmed/20066496

Monday, July 22, 2013

Tangential Flow Filtration: Membrane Types and Process Parameters

Tangential Flow Filtration [TFF] is used for clarifying, concentrating and purifying proteins.

Filtration is a pressure driven separation process that uses a membrane to separate components in liquid solution or suspension based on size and charge difference.

Types of Membrane Filtration




1. Normal Flow Filtration - Dead End Filtration
2. Tangential Flow Filtration - Cross Flow Filtration

Tangential Flow Filtration:

Tangential Flow Filtration is divided into categories based on the size of the components being separated.

Generally membrane pore size is given as micron value, particles sized above this value will be retained.

Types of TFF

1. Micro Filtration 
2. Ultra Filtration

Ultra Filtration is catagorized into two 
  • Virus Filtration
  • High Performance Tangential Flow Filtration
Process Goals
  1. Final Product Concentration
  2. Feed Volume Reduction
  3. Extent of Buffer Exchange
  4. Contaminant Removal Specification
Primary Protein Processing Goals
  1. High Product Yield
  2. High Product Quality
  3. High Product Purity
  4. Controlled Bioburden
Membranes
  • Polyethersulfone (PES)
  • Regenerated Cellulose
Types of Membranes
  1. Flat Plate
  2. Spiral Wound
  3. Hollow Fiber
Key Parameters to Optimize
  1. Cross Flow Rate
  2. Transmembrane Pressure
  3. Filtrate Control
  4. Membrane Area
  5. Diafiltration Design
Factors affecting Product Yield

These are the four contribution of product loss during Tangential FLow Filtration
  1. Retention Loss
  2. Adsorption Loss
  3. Solubility Loss
  4. Unrecoverable hold up volume loss
Factors affecting Product Quality

Quality of product is compromised due to aggregation or denaturation caused by
  1. Micro cavitation
  2. Air-Liquid Interface
  3. High Protein Concentration
  4. Temperature Effects

Friday, July 19, 2013

An Insight on Ion Exchange Chromatography

Ion Exchange Chromatography is based on the electrostatic interaction of charged surfaces.



Ion Exchange Chromatography is of Two types:
  • Cation Exchange
  • Anion Exchange


    Ion Exchange Chromatography Principle

Factors affecting Ion Exchange Chromatography
  1. pH of mobile phase
  2. Ionic Strength
  3. Mobile Phase Modifiers
  4. Temperature
Operating pH of Ion Exchange Chromatography (IEX) is mostly 2-9.

Buffers Used in Ion Exchange Chromatography

Use Cationic Buffers in Anion Exchanger : Eg: Alkyl Amines, Tris, Amino Ethyl Alcohol

Use Anionic Buffers in Cation Exchanger: Eg: Phosphate, Acetate, Citrate, Barbiturate

Ionic Strength:

Care should be taken while choosing ionic strength since the use of >1M salt concentration can induce hydrophobic interaction.

Mobile Phase Modifiers
  • EDTA – For Chelation
  • Chaotropic Salts – Eg: Urea for stabilization
  • PEG – For Enhanced Selectivity
  • DTT – To Prevent Oxidation
Stationary Phase

Selectivity is Based on the following parameters
  1. Ligand Density
  2. Spacer Arm
  3. Backbone of the resin material
Particle size generally used is in the range of 20 – 200um.

Ion Exchange Functional group

Stationary phase in Ion Exchange chromatographic matrix is functionalized with Basic (Anion Exchange) or Acidic (Cation Exchange) groups.

Strong or Weak Ion Exchanger

Strong Ion Exchangers retain their charge over a wide range of pH
Weak Ion Exchanger retain their charge only in a narrow range of pH.

Strong Ion Exhangers will have very low or very high pKa.

Example for Strong Exchanger – Sulfopropyl, Quarternary Ammonium

Binding Capacity

Total no of charges per unit stationary phase volume or the mass of protein adsorbed per unit volume.

Pore Size of Ion Exchange resin can range from 10 – 1500A.

Dynamic Binding Capacity can be found out by doing breakthrough Curve.

Thursday, July 18, 2013

A Brief note on Chromatographic Process Scale Up

To get the same results as in lab scale these following parameters need to be kept constant:

Chromatography process scale up


  • Column Length
  • Linear Flow Rate
  • Ratio Between Gradient and Column Length
Constant Column Length

scale up of chromatography column


Desired Capacity should be proportional to the cross sectional area of the column, because same proportion of the column should be occupied by the samples on both the scales

Gradient

gradient elution

Gradient volume is decided when the diameter of the column is set. Gradient elution in process scale will take the same time as in laboratory scale.

Sometimes a lower flow rate will compensate the decrease in resolution due to ineffective solvent distribution.

Things to look for in a Hydrophobic Interaction Chroamtography

Things to look for in a Hydrophobic Interaction chroamtography are
  • Purity
  • Speed
  • Resolution
Sample viscosity can interfere in resolution so sample should be diluted prior to loading if the sample is viscous.

In HIC media is described based on
  1. Ligand
  2. Ligand Density
  3. Available Capacity: It is the actual amount of protein that can bind to the media. If flow rate is included in the defined conditions then it is called as Dynamic Binding Capacity.
HIC matrix dynamic binding capacity
Dynamic Binding Capacities of Different Matrices

Dynamic Binding Capacity is based on these factors:
  • Salt Concentration
  • Flow Rate
  • Temparature
  • pH to a lesser extent

Most commonly used salts in HIC are

Ammonium Sulfate, Sodium Sulfate, Sodium Chloride, Potassium Chloride, and Ammonium acetate.

Characteristics of good HIC Matrix

  • High Binding Capacity - Larger Area will results in high capacity binding
  • Physical Stability
  • Chemical Stability
  • Matrix should be Inert

Check the stability of protein at different salt concentrations.

Proteins shouldn't precipitate at high salt concentration, if protein precipitates there will no or very low yield.

Elution from the HIC column is in the increasing order of hydrophobicity.

Ligands Used Hydrophobic Interaction Chromatography meida has Alkyl or Aryl Groups.
Phenyl 650


  • Alkyl - Butyl, Octyl, Ether, Isopropyl - Eg: Butyl S
  • Aryl - Phenyl - Eg: Phenyl 650 - 
Alkyl shows pure hydrophobic character while Aryl shows mixed behaviour.

Binding Condition: 1M - 2M Ammonium Sulfate or 3M NaCl most frequently used.


For tightly bound proteins use harsh conditions like washing with 0.5M - 1.0M NaOH, 70% Ethanol or 30% IPA followed by water washes or salt free buffer washes.

References

Encyclopedia of Bioprocess Technology: FERMENTATION,BIOCATALYSIS, & BIOSEPARATION

Technical Resources on Chromatographic Media From GE, TOSOH, Shimadzu

Monday, July 15, 2013

Protein Purification : Practice Problems

I am sharing some the questions on protein purification on request by some of the readers , you can answer these questions by commenting. Full Question & Answer booklet (PDF) will be sent to those who are interested for Free.


questions on protein purification


Protein Purification: General Biochemistry Questions
  • An enzyme (MW 24 kDa, pI 5.5) is contaminated with two other proteins, one with a similar molecular mass and a pI of 7.0 while the other has a molecular mass of 100 kDa and a pI of 5.4. Suggest a procedure to purify the contaminated enzyme. 
  • A mixture of lysine, glycine, alanine, isoleucine and glutamic acid are separated by ionic exchange chromatography. What is the order of elution of these amino acids if you use gradient buffer system from pH 10 to pH 2: 
  • a) with a cation exchange resin?
    b) with an anion exchange resin? 
Which column would give the best separation? 
  • Which is the best UV wavelength that can be used to detect Aromatic amino acids?
a) 220nm
b) 214nm
c) 280nm
d) 205nm

  • What is the net charge (+, 0, -) of the amino acids glycine, serine, aspartic acid, glutamine and arginine at: 
a) pH 2.01
b) pH 3.96 
c) pH 5.68 
d) pH 10.76
  • We load a DEAE-cellulose column adjusted to a pH of 6.5 with the following mixture of proteins: ovalbumin (pI = 4.6), urease (pI = 5.0), and myoglobin (pI = 7.0). The proteins are eluted first with a buffer of weak ionic strength at a pH of 6.5, and then the same buffer containing increasing amounts of sodium chloride is used to elute the proteins. What order are the proteins eluted? 

Sunday, July 14, 2013

Excellent Basic Guide on HPLC

Fundamentals of Liquid Chromatography (HPLC) Guide on HPLC Basics
HPLC Basic

Download Fundamentals of Liquid Chromatography (HPLC) Guide on HPLC Basics

The PDF link attached here is from Agilent Technologies which is available online.

Saturday, July 13, 2013

Steps in Protein Purification


  • Know properties of your Protein
  • Developing a Strategy for Purification
  • Choosing Suitable method for highest yield.
  • Optimize the Protein Purification
steps in protein purification CIPP

Know Your Protein

This is very important step to consider before developing the purification strategy. Knowing your protein means characterizing the protein. Understanding pI value, and other characteristics of protein will aid in the protein purification. Some of the techniques used for protein characterization are:

1. SDS- PAGE
2. Iso Electric Focussing
3. Western Blot
4. Mass Spec, etc

Developing a Strategy for Purification

Once the protein is characterized, it is easy to develop a strategy for purification. Depending on the stability and required purity suitable  method should be choosen. Keep in mind about the scale up during the strategy development. Some of the Common Protein purification mehod includes

  1. Protein Salting out using suitable salts
  2. Chromatographic Techniques like Ion Exchange Chromatography, Size Exclusion chromatography, Hydrophobic interaction Chromatography, Reverse Phase Chromatography, Affinity Chromatography, Etc.
  3. Protein Concentration through Filtration
  4. Lyophilization
Choosing Suitable method for highest yield.
For various reasons suitable method which provides highest yield need to be selected, a few to cite here is the production cost, one should keep in mind about the production cost involved while choosing a method for purification when it comes to scale up all these adds up so one should select a method which will give highest yield. chosen method used should not compromise the protein efficacy, activity, usablity and purity specifically when trying to purify a protein for therapeutic use.

Optimizing Protein Purification

Optimization is important step in any process not only just protein purification. Optimizing a process can have multiple advantages reduced time, improved yield, higher purity, more stability, cost efficient all these are some of the key target points to achieve by optimizing a process. Optimizing targets vary depending on the type of process and the final goal.


Wednesday, July 10, 2013

DEAE Cellulose Column for Protein Purification

DEAE - Diethyl Amino Ethyl Cellulose columns are used for Protein purification. DEAE cellulose is an anionic resin which can be used to bind -vely charged proteins.

Proteins can be eluted by altering either by increasing the salt concentration or by altering the pH.



Structure of DEAE



DEAE is a weak ion exchange resin, which means the pH range at which it can be used is limited, charge density will vary.

The diethylamino group (-CH2-CH2-NH+-CH2-CH3) of DEAE-cellulose carries a positive charge which is responsible for the ion-binding properties of this resin. Effectively, negatively charged
amino acids/proteins will interact with the diethylamino group (via electrostatic interactions), while positively charged amino acids/proteins will be eluted. Since the diethylamino group has a pKa close to 8.5, it will be deprotonated at pH values above 8.5 and will lose all ability to bind negatively charged molecules.

Protonation is the addition of a proton H+ to an atom. In this case protonation results in net positive charge.

Deprotonation is the loss of a proton H+ from an atom, in this case deprotonation results in the loss of postive charge from the DEAE and there by it loses its ability to bind -vely charged proteins when the pH is raised above 8.5.