Theory of freeze-drying
1、Freeze-dry (brief named Freeze-dry) is a stable process of material dried which will put the material contained with water to freeze to solid first, then make among them of the water sublimate from solid to air condition, removing water to preserve materials
2、Products at solution stage are frozen through sublimation and desorption ,then the solvent reduce to a certain extent,thereby preventing the formation of micro-organisms or chemical reaction between the solute and solvent that products can be preserved for a long time and maintained its nature.
3、Vacuum freeze-drying method is a liquid → solid → gaseous process. In freeze-drying process, "liquid bridge" between the solution particles has been frozen into "solid bridge". The relative position of particles has been fixed and does not exist surface tension on gas-liquid interface between the two particles . With the continuous distillation of solvent, "solid bridge" continuously decrease, but the the relative position between two particles will not longer change until the "solid bridge" completely disappear.

The advantages of freeze-dry(compared to the usual dry method such as drying, dry cooking, spray-dried and vacuum drying)
1、It drys at low temperature in order to make protein degeneration, micro-organisms loss biological activity.
2、The material in the volatile components and nutrients heated degeneration of and aromatic ingredients loss a little because of the low-temperature drying.
3、It is almost impossible for microorganisms and the role to grow so that the material can maintain the original character in the low-temperature drying process.
4、The volume and shape essentially does't change and has a strong water uptake after drying.
5、Because it has little oxygen under the vacuum drying,the vulnerable materials have been protected.
6、It can remove 95-99.5 percent of water , and the retention period lasts longer.

The use of freeze-drying technology
1、The bio-products, medicine aspects: such as the anti-virus medicine, anti-toxin medicine, examination products for patients and vaccine conservancy.
2、The microbial and algae aspects: such as various bacteria, yeast,enzyme, protozoon, micro-algal etc for the long-term conservancy.
3、The bio-products specimen, bio-organism aspect: such as manufacturing various plants specimen, the dry conservancy used for the skin, skeleton, aorta, the heart valve transplantation of different or the same race animal
4、The manufacture used for the optical microscope, electronic scan and watch through the small group of the microscope.
5、The drying aspect for food: such as the coffee, tea-leaf, meat, fish, egg, seaweed, fruit, vegetable, dressing, bean curd, instant food etc.
6、The top-class nourishment and herb medicine aspect: such as the queen bee syrup, honey, pollen, and chinese herb medicine products etc.
7、The making aspect of the micro powder: such as making to micro powder, such as Al2 O3,ZrO2,TiO2,Ba2 Cu3 O7~ 8, and the Ba2 Ti9 O20 etc.
8、Other aspects: If the catalyst in the chemical engineering, the can raise 5-20 times of the catalyst efficiency after freeze-dry; Keep the plant, leaf, soil freeze-dry, study soil, fertilizer, weather in order to the plant to grow the function by influence and factor; The damp wood system cultural object, flood bad book manuscript etc. use the freeze-dry method to dry, which can keep original shape etc. with maximum limit.
Water and the nature of solution

The material has three states of solid,liquid,gas which is related to temperature and pressure.As showed on the drawing ,it is the state drawing for water balance.There are there curves on the drawing ,OA:ice to water,OB:water to steam,OC:ice to steam,which presents that the relationship between the pressure and temperature when the two states exist .The three curves separately are the melting curve ,boiling curve and sublimation curve ,boiling curve and sublimation curve.The three curves make the drawing into solid area,liquid area and gas area. The arrow in the drawing shows the process that the ice melts into water ,water becomes steam,and that icce sublimates into steam. K point on the top of the OB curves,the temperatures around 374℃,which is called critical point .If the steam temperature is higher than the critical point of 374℃, the steam is difficult to become water no matter how big pressure adds. The joint O of the three curves is that the three states can exist at the same time,which called the triple point ,the
temperature is 0.01℃,the pressure is 610Pa .Under triple point ,the liquid state doesn't exist.If the pressure on the ice surface can maintain lower than 610Pa and heat the ice ,the ice will directly become ice without getting throgh liquid state .The process is called sublimation .

Solution in the freeze-dry process
Freeze-dry solution are normally configured to contain 4 to 15 percent solid substances of the dilute solution.
The composition of water in solution
1、Most water exists in free water of solution in the form of water molecules.
2、A little of water is adsorped in the lattice of solid material or combined with polarity genes of hydrogen bonds.
3、Most of the water fixed in cells and organisms is free water which can freeze and sublime ,and also contain some binding water which can not freeze and remove . The freeze-dry aim is to remove the free water in material and a part of the binding water which is absorbed in solid material lattice in the gap at low temperatures and vacuum environment.

Freeze-drying process is divided into the following steps
Pre-freeze
Pre-freeze is to cure the free water in the solution , endow products with the same shape before and after drying , prevent products from foaming , concentrated, and systolic ,solute movement and other irreversible changement.
Solution should be below freezing point in the freezing process which makes it product nuclei,then free water will clystallize in the form of pure crystal, at the same time the crystal give off heat that makes temperature rise to freezing point. With the growth of crystals and increasing of solution concentration , the solution will be frozen when the concentrations reach eutectic concentration and the temperature drop to below the eutectic point.
The quicker the cooling rate is , the lower the supercooling temperature is , that can form more nuclei.Crystals will be frozen if they are too late to grow ,and form more grain which is also fine. Slow cooling rate makes grains few , grains coarse.
Freeze-dried products sublimation must be frozen to a certain temperature, which should be located below melting point around 10 to 20 ℃ .If not pre-freeze and take out the vacuum directly, the liquid will be taken out when the pressures drop to a certain extent.It is called evaporation and the steam is called unsaturated steam. If the product is not frozen and taken out vacuum, gas will escape from the liquid quickly and result in "boiling".
If products are frozen in "boiling",some may escape out of the bottles and cause the drug loss or manufactures surface unsmooth. This shows that the melting temperature is the safest temperature to make products dry normally which can only lower than that, not be higher than the melting temperature.

Sublimation drying (the frist dry)
Sublimation drying is to place frozen products to heat in the vacuum-sealed container, its ice crystal will sublimate into steam to make the products dehydrate drying. Drying is beginning gradually from the outward appearance to inside, the ice crystal remains in the bottom after sublimation.The space becomes sublimation steam to overflow piping. Drying layer and the frozen-part boundary had boundary interface (one thin layer actually) which is called sublimation interface. Sublimation interface about the velocity of the 1 mm/h in bio-products drying process to push inside. When all ice eutectics remove and sublimate complete drying, which can remove 90% water at this time.
The ice in the products sublime through sublimation interface. The heat supplied by the heating equipment (through the shelf) and the heat from the shelf spread to the sublimation interface through the following several ways : solid conduction, radiation and gas convection.

Products sublimation at the following temperature restrictions:
1、The temperature of the frozen products should be lower than the melting temperature.
2、The temperature of the frozen products must be below its collapse temperature or the maximum faculative temperature (not charred or variable).
3、The highest shelf temperature

Analysis of dry(The second stage drying)
The first drying stage is to remove water in the form of ice-crystal removal, so the temperature of the freeze-drying layer and the pressure of sublimation interface should be controlled under eutectic points of products (or under the collapse temperature), which can make the ice-crystal melt. But for adsorbed water, it absorbs high energy, if don't provide enough energy for them, they are impossible to adsorb from the solution. Therefore, the temperature of this stage products should be enough high, as long as not exceed to allowable highest temperature, not be destroied by firing product which won’t result in the product overheating to change the quality. At the meantime, for making the steam that solution absorbed out have enough motivation to divert product, have to make shape internal products become bigger steam difference, therefore internal space in this stage have high vacuum degree. After second drying stage, the remaining water in the product depends on the products category and requirements during 0.5%~4%.

Several key concepts in freeze-dry process
Eutectic temperature
Hybrid solutions compose with some material .At the beginning，some components of crystal precipite in the freezing process makes the remaining concentration solution change.When the temperature reaches a certain or region, its liquid and solid-state formed by the identical components, then the solution is known as eutectic solution. This temperature or the temperature range is known as the eutectic points of the solution or eutectic range , also known as fully curing temperature which is the maximum temperature in the process of cooling products from liquid to solid . Eutectic temperature is the highest temperature that pre-freeze should meet in the course of freeze-drying .Most pre-freeze process should be less than eutectic temperature of 10-20 ℃.
Common solution temperature
When the mixed solution with solid-state in warming melting process reaches a certain temperature, the temperature that solid melts liquid is known as melting point or start to melt temperature.It is the minimum temperature in the process of heating that products begin solid-state to liquid . The freeze-drying material temperature must be below the common melting point.
The determination ways of the eutectic point have the determination of resistance, differental thermal analysis , low-temperature microscope directly observed, the number of formula.Because of the ion's drift-rate with the temperature droping and gradually reducing in the course of solution frozen , the resistance will increase and the current can flow as long as the liquid exists. Once it is frozen all, the ion with electric can not move and then the resistance will suddenly increase.We measure the eutectic point of solution in accordance with this principle .
Collapse temperature
When the ice in freeze-dried material disappears, the space occupied by the ice becomeing a hole,so the freeze-dried layer shows porous honeycomb cavernous structure. The structure relates with temperature.When the solid matrix of the honeycomb structure has a high temperature, its rigidity will low . When the temperature reaches a certain threshold and the solid matrix is too insufficient to maintain the honeycomb structure, the solid matrix hole in the wall will collapse,the channel will be closed which steam spred.The critical temperature is known as freeze-dried materials temperature or collapse temperature.
Glass transits temperature
There are two different states - crystalline and amorphous when the temperature decreases, the liquid changes into a solid. Atoms, molecules or ions are no rules in amorphous solid material.Because people have been accustomed to call melt materialthe which is not occurrence of inorganic substances in the cooling process as glass, so later other amorphous states are called a glassy state .
As drugs have stringent requirements in freeze-dry which is hoped to below glass transition temperature. But the glass transition temperature is not referring to full glass here, because full glass is that entire samples form glass.It is almost impossible to implement full glass which demands high cooling rate.The glass transition temperature in freeze-dry process is the largest glass transition temperature from frozen concentrated liquid .
With the precipitation of ice crystals in the process of freezing , the remaining solutions of concentration will increase gradually. when it reaches a certain level, the remaining water will not crystallize. Then the solutions reach the largest state to freeze and enrich,and the corresponding temperature is known as the largest glass transition temperature from frozen concentrated liquid .

The three key elements of freeze-dried
1、Prescription
It is the most crucial step to confirm the prescription.
The composition of solution relations with freeing and sublimation steps to implement .
Prescriptions include active component, excipients and process water.
2、The importance of the rmodynamic properties
The extent of the cold,the degree of crystallization,collapse temperature or eutectic???? temperature,the changement in the metastable state of the gap material ,the solution of the crystallization heat,the melting temperature of the gap material.
3、A good lyophilizer
Meeting the GMP requirements;A high performance; The energy ajusting itself; A strong recurrence .

Making a freeze-dried curve
Freeze-dry curve is that products temperature and pressure change with time in the course of freeze-dry . The shape of freeze-dry curve relates with products performance, the number of installed, the type of packaging containers, equipment conditions, and any other factors. Making a freeze-dry curve mainly determines the following parameters:
A、Pre-freeze rate
Pre-freeze rate has a direct impact on the size of grain products, the survival rate of viable and sublimation rate. Slow pre-freeze rate makes grain big, products appearance rough and viable difficult to injury, sublimation rate fast. Fast pre-freeze rate makes grain small, products appearance delicate, sublimation rate slow.
B、Pre-freez temperature
Products temperature should be below its common melting point at 5-10 ℃.
C、Pre-freeze time
The pre-freeze time required depends on the specific conditions , the general principle is that all parts of products are completely frozen . It will maintain at this temperature for 1-2 h in order to sublimate after products temperature drops to the scheduled and lowest temperature .
D、 Condenser cooling time and temperature.
The high or low temperature of the condenser should be based on the distillation temperature of products. Products distillation are at low temperature, which requires condenser at low temperature correspondly .The best rate of distillation is around the half of saturated steam pressure of distillation temperatures of the products . Low pressure not only can not speed up the sublimation, contrary to that it will lead products heating poor and decrease the rate of sublimation.
E、Sublimation rate and drying time
Sublimation rate mainly depends on the heating capacity of the shelves and the catching water capability of the condensers.Heating rate can increase as long as the pressure within the drying oven maintains under the allowable maximum pressure At the first dry, they can roughly calculate an hour of 1 mm of the thickness of the dried products .We can judge it according to the following phenomena:
1、The interface of freeze-drying layer arriving at the bottom of bottle and disappearing

2、Product temperature rises to nearly conducting oil temperature.

3、The pressure within the drying oven down to the pressure of the condenser, both close and remain unchanged.
4、Closing the partition valve, the rising rate of pressure inside the box is close to the leakage rate of stem .
The first drying ends after the above-mentioned phenomena occers and further extends for 0.5-1 h.

At the second drying stage,the products temperature can increase below the allowable maximum temperature that make combined water and the wate absorbed in dry layer get enough energy to analysis from elements .Second drying time is 0.35-0.5 times than the first drying time .
Optimization the freeze-dry curve continually:
A good freeze-dry technology should have personalized, with stable product quality, short time and low energy consumption features. In freeze-dry process study, We must apply Chemistry, molecular point of view to observe the problem,apply mechanical, electronic,and computer methods to control problem.
Several frozen and distillation methods：
Products frozen method (freezing fast / slow freezing at low temperature)
Slow freezing at low temperature (1 ℃ / points) forms thick crystal that can improve the freeze-dried efficiency, but the general quality of products at slow freezing stage extremely impact on the survival rate particular with the activity of enzymes or viable live virus.
The requestion of synthetic drugs at fast freezing or slow freezing stage is not strict, but both ways can be used
The first distillation : The common melting of general application is around -10 ~ -20 ℃,which structure is single, viscosity and concentrations are not high, installed in 10 mm or 15 mm thickness . It can heat and sublimate as long as the products temperature reduce to below the common melting at-10 ℃ ~ -21 ℃ .And then we should control the speed of warming up which can guarantee success.
Pre-repeated freeze distillation method: Some products with low melting point can not be completely frozen even at very low temperatures . In view ,it seems to have been frozen, but frozen block often be soften in the distillation process which cause bubbles and form thick mesh-like structure on the surface ,which impact on the dry distillation.For example ,honey, royal jelly and others which have complex and viscous structure often difficult to freeze, appear the above situation in sublimation. Solution is to use pre-repeated freeze distillation method. Application of this method can increase the efficiency of freeze-dried, but also improve the quality of products, shorten the cycle of freeze-dry.
Division frozen distillation method: Platters load products which have thickness and high concentration in the freeze-dry. The bottom of the water vapor can not escape so that all the frozen blocks deform and demesh from the end in the sublimation.Solution is to divise the installed plate which can improve the efficiency of heating conduction.

Failure of freeze-dried products:
1、Products have eruption phenomenon when vacuum is pumped : This is because products have not been frozen and pumped vacuum so that pre-freeze temperatures have not be below eutectic temperature, or have been below the eutectic temperature, but the time is not enough for products to complete. Solution is to reduce pre-freeze temperatures and extend pre-freeze time.
2、Products have shrinkage and bubbling phenomenon: This is due to that products have partial melting in the process of drying and distillation liquid evapores to vapour, resulting in volume decreased or dry products dissolved into the liquid, causing the volume reduced.The serious melting will cause a bubbling phenomenon, because heating is too high or partial bad vacuum make product temperature oven than the total crystal, or collapse of temperature. Solution is to reduce the temperature and raise the vacuum in freeze-dried box, and control products temperature which make it less 5 ℃ than crystal points or collapse temperature.
3、No fixed shape: this is due to not come into skeleton because of few dry matter, low product concentration, and even dried products sublimate into steam which show to container outside. Solution is to increase the concentration of products or add excipients.
4、Products unfreeze-dried:When we finish freeze-dried in the presence of ice, Frozen parts begin to melt into liquid . A little of liquid is absobed by dried-products,and form into a "vacancy". Dried products all dissolve into the liquid with a large quantity which form concentrated liquid. T If the containers touch the bottom of a cool feeling, and even the product looks good, the residual water content will not be qualified. Solution is to increase energy supply, raise the board temperature or use vacuum regulation.If products may also be lack of time, solution is to extend the desorption or distillation dry of the time.
5、Uneven products color: Products have crystalline pattern, which are due to the slow frozen rate . Solution is to improve the frozen rate, not stay at 0 ℃ which makes products form smaller crystals. Sometimes products can be seen a lap of the darker colors in layer line, which caused by bad vacuum for a short period of time and brief power outage .
6、Top products good and low bad: Products sublimation has not yet ended and then come into the stage desorption in advance, which means to increace plate temperature early that results in products of excessive heat at lower deck melting .The solution is to extend the distillation phase of the time.When we dry products into the lower deck ,distillation resistance will increase because of too thick loading, too large resistance, and partial bad vacuum will also lead to melt . Solution is to low the temperature of the plate and increase the vacuum in freeze-dried box.
7、Upper products bad ,lower good: When the products form gas-tight and glass-like structure on the surface, but do not return to heat . Products begin to heat up aoon after products sublimate, and then some products begin to melt and shrink which makes products surface rupture ,so sublimation at low layer is normal. Solution is to return to heat when products re-freeze.
8、Products water unqualified: The time of desorption stage is not enough , or not use vacuum regulation, or use vacuum regulation but the products reach the permissible maximum temperature and not resume the high vacuum. Solution is to extend the desorption and drying time, use vacuum-conditioning and make products resume the high vacuum after products reach the maximum permitting temperature .
9、Poor solubility of products: Products occurr evaporation in the drying process which will concentrate. For example,products have sandwich lump which melt in the distillation ,evaporate, dry, and then concentrated . The solution is to low the temperature of the plate appropriatly , raise the vacuum freeze-dry box or extend the dry time.
10、Products distortion: There is a good vacuum in the bottle when vacuum press stopper. But it will loss vacuum soon after storage , perhaps stopper is not supporting or aluminum cap pressure is too loose and air leakage result in lossing vacuum.The solution is to replace the closure or adjust elastic pressure with Al cap; It also may be too high water content resulting in lossing vacuum caused by water vapour pressure .Solution is to extend the time of desorption . There is also a problem which doesn't contact with freeze-dry curve ,but impact on the quality of the products, because it leak silicone oil and hydraulic oil in freeze-dried box
Sterile control technology used by freeze-dry equipment
CIP(Clean in place )
SIP(Sterilization in place)
（WIT）
Hydraulic bellows confirmed with aseptic
Leakage Test
Prevention measures to the foreign matters pollution
Gas speed and particles dust test
Charge and discharge into 100 one-way flow (laminar flow) protection
（MFT）
Sterile isolation system
Prevention measures to the foreign matters pollution in the box
Foreign matters pollution caused by external factors
Strict selection on vacuum gas from materials in the freeze-dry box
Strict selection of hydraulic piston oil
Reverse the spread of the vacuum pump oil by dry vacuum pump

（CIP）
The wood inside the freeze-drt box indirectly contacts with the liquid.There is plates ,metal hoses, nozzles, hydraulic piston, and many other difficulty to clean parts and components.It is possibile to prevent products in the gas .The significance lies in cleaning office automation is to verify records.
Cleaning verification sampling of lyophilizer
1. Checking equipment after cleared
2. Chemical sampling
1) Cotton sampling
2) Washing solvent sampling
3) Final wash water sampling

1. Microbial sampling

?1) Cotton sampling
2) Final wash water sampling

Simulation tests of CIP cleaning
(SIP): (steam sterilization, VHP hydrogen peroxide sterilization, VOP ozone sterilization, EO ethylene oxide sterilization)
We have a strict requirements for producting drugs , including sterile particularly important. Lyophilizer is pharmaceutical machinery which directly contacts with drugs and sterilize. There are mainly two kinds of sterilization methods, namely, physical and chemical sterilization method.
Some old-fashioned lyophilizers use chemical sterilization method, such as the use of alcohol, formaldehyde, lactic acid and ethylene oxide and so on .The new type of lyophilizers also use hydrogen peroxide (VHP) of the sterilization method, but it is also chemical method.
The residues have an impaction on drugs by chemical sterilization method which is not suitable for lyophilizers.
Now modern lyophilizers use high-pressure steam sterilization in physical sterilization method, because this method can completly sterilize without any impaction on drugs residues.
We choose health-class stainless steel tubeins with ASME BPE standards ,joints welded by TIG automatic welder ,check speculum in weld internal when we make CIP, SIP pipelines. external connector all use fast access interface with ISO2852 standard,choose health-class diaphragm valves.The pipelines have not blind period which can be cleaned and sterilized. Each has the number and welding marks in order to trace back.

Gas filter SIP and compete tests
Destructed vacuum by gas filter stsyem with the integrity of filte
Filters must be designed to withstand sterilization
The integrity of filte must be tested after each cycle of freeze-dry.
If possible , we should take SIP and compete tests.

Leakage test (the integrity of the testing methods - bubble point test)
We must do leakage test and verify the process for lyophilizer security . There allowable benchmark is 0.05 mbar L / sec, which is the time for delivery production beween pharmaceutical factories and manufacturers.
The purpose of the bubble point is to confirm the membrane pore size of the filter to be tested.
Bubble point test is a simple and reliable way which test the integrity of the porous membrane filters .
The principle is to use liquid membrane which form on the surface and bear the pressure of gas to test the size of the pore size membrane.Bubble point pressure is different from the membrane of materials and the same materials but different aperture of the membrane. Detection method is to ues proper Liquid moist filter which is determined the pressure difference by gas compression when liquid seepage from filter microporous.According to the pressure ,we determine the quality of filters. When membrane aperture is 0.22 um ,foaming pressure is ≥ 0.35 MPa .Detection test is divided into offline and online testing.

The importance of gas rate
The process what sterilization and completment of the gas filters with two filters,the pressure interdependence of microparticles settling velocity, microparticles freeze-dried particles over the automatic control in the freeze-dry box is verified .
Tamponade works
Aseptic confirmation is recognized as a method of using bellows isolation on surface of hydraulic piston rods , that is, the pistons rod which have retractable stainless steel bellows isolate from intracavity of the dry-box.
Isolaed room technology
Sealed isolation enclosures;Air throgh the HEPA filter:; Chemical cleaning and disinfection within the surface. Human body isolateing the operating environment ; Detection of bacteria; Aseptic filling operations such as handling toxic products.
Dry Vacuum Pump
Oil pumps can completely prevent the pollution from the products.