CA1147679A

CA1147679A - Process for the surface culture of nucleated cells and the production of cell-culture-dependent substances

Info

Publication number: CA1147679A
Application number: CA000354880A
Authority: CA
Inventors: Walter Merk
Original assignee: Individual
Current assignee: Boehringer Ingelheim Pharma GmbH and Co KG
Priority date: 1979-06-28
Filing date: 1980-06-26
Publication date: 1983-06-07
Also published as: IL60419A; NO801932L; FI71948B; IE49845B1; EP0021257A1; DE3065415D1; NZ194169A; NO162914C; AU5967380A; FI71948C; AU536599B2; DK272680A; DK152678B; NO162914B; EP0021257B1; DK152678C; IL60419A0; IE801332L; FI802040A; ES492846A0

Abstract

ABSTRACT OF THE DISCLOSURE

This invention relates to a process for the surface culture of eukaryotic cells and in particular to such a process in which the cells are cultured on a solid carrier so dimensioned and disposed that when the culture medium 18 drained off, less than 8% of the culture medium (based on the bulk volume of the carrier minus the displacement volume) is retained by adhesion to the carrier.
Preferred carriers include coils,spirals and saddle-shaped carriers. The process enables higher yields of cell culture dependent substances e.g. fibroblast interferon to be prepared by reducing adverse interfacial effects between the carriers.

Description

.
The present invention relates to processes for the surface culture of eukaryotic cel~s and to substances thereby obtained.
It is known that non-transformed nucleated cells ~eukaryotic cells) can only be successfully cultured by the surface culture method. Thus eukaryotic cells held in suspension do not grow. If however the eukaryotic cells are allowed to settle on a suitable surface, they can multiply up to contact inhibition.
In its simplest form the surface culture method may.be effected in a vessel having a flat bottom, it being necessary to ensure that the bottom of the vessel is as exactly horizontal as possible. Such a vessel is preferably made of glass or of a suitable plastics material, the size of the container being limited, in the final analysis, only by handling requirements. In order to obtain as lar~e a surface area as possible containers also exist in which several flat faces lie horizontally one above the other, the faces being connected to one another by appropriate openings. In accordance with the same principle a tray stack has also been described which has 10 flat culture faces lying one above the other, each face having a surface area of 600 cm2. This latter container may also be connected in parallel with other containers to form blocks of 4.
Where it is desired to use other cell culture techniques the entire inner face may be used Eor ~ cell culture by rotation of the container, for example using so-called roller flasks. Such roller flasks may be fitted with discs or other bodies so that the available surface area can be enlarged whilst ensuring not only the supply of nutrients to the cells via the medium but also the necessary provision ~5 of oxygen.
These known processes suffer from a number of disadvantages including the fact that they can ___~_ ., ,.

; ~'?,~

' no longer be handled or can only be handled with difficulty as a result of the size of the container used. Furthermore it is difficult to effect the necessary hose connections/ requirecl for effecting changes in the culture medium as a result of the movement of the container.
Appliances comprising a container having several cell culture faces and a pump have also been described in the literature. The necessary supply of nutrient and oxygen to the cells is ensured by the circulation of the medium as a result oE the pump. Thus, for exa~ple, an appliance having a volume of 2 litres has been described in which the flat culture faces --are replaced by small glass cylinders and the pH
value of the circulating medium is regulated during operation by gassing. The procedure is the same as in the known organ perfusion technique.
In the above-mentioned surface culture process in which small glass cylinders having a length of about 6 mm, an outside diameter of about 4 mm and an inside diameter of about 2 mm are used in a container wherein simultaneous circulation of the medium is effected during operation by pumping, a covering of all culture faces is not possible as a result of the physical effects, such as wetting, capillary action and other interfacial effects, which arise especially due to the formation of air bubbles.
For the same reasons, it is not possible to adequately wash the culture faces covered with cells.
Thus there is no complete interchange of washings with original medium since part of the original medium is persistently retained. The physical forces associated with glass cylinders or balls hitherto used in surface propagation, e.g. in fibroblast interferon production, prevent the necessary complete removal~ by washing, of both the induction medium and foreign protein.
Moreover, the pumps which are conventionally used in interferon production give rise to shearing forces , .

and these forced tend to destroy a substantial part of the interferon which is produced during culturing.
A further technique termed the microcarrier technique, comprises the use of suitable balls made for example of dextran, with a diameter of about 0.15 to 0.~5 mm suspended in a culture medium and held in suspension by stirring. Cells can be cultured on the surface of these balls using suit-able techniques ~see Biotechnology and Bioengineering, volume XXI, 433-~2 ~1979)). The available surface area given by the medium/volume relation is especially large in the case of this technique. Thus a surface area of 3,000 cm2 is available with 0.5 g of dextran carriers in 100 ml of medium. However, the balls on which the cells grow are very sensitive to mechanical influence and the medium has to be removed by allowing the carriers to settle after which the supernatant liquid is drawn off. ~gain, no co~plete washing can consequently be effected.
The present invention is based on the discovery that the ~bove-mentioned disadvantages in the surface culture method, especially in the production of cell culture producible substances such as interferon, viruses, enzymes and antibodies e.g. immunoglobulins, may be overcome, at least in part by effecting the cell culture according to the surface culture method on carriers dimensioned to reduce adverse interfacial effects, such as wet-ting and capillary action, e.g. dimensioned to possess no substantial adverse interfacial effects thus permitting complete covering and sufficient washing of the culture face.
Thus according to one feature of the present invention there is provided an improvement in a process for the surface culture of nucleated cells in aqueous meclium and for the production of cell culture-dependent substances from the cells thus obtained which comprises using as culture face moving bodies comprising saddle-shaped or cylindrical filling bodies made of metal which after the release of the aqueous culture medium hold back less than 8 percent of the medium relative to free volume due to their form and where the ratio of surface of the filling bodies used to free volume _ ~ _ . "`~

7~
is greater than about 5:1~
The culture medium is generally any convenient aqueous medium.
Minimum essential medium, e.g. basal medium, Dulbeco's* medium with Hank's or Earle salts, Glasgow* medium, medium 199*, Liebowitz medium L15*, Weymonth's MB 752/1* medium and McCoy's 5A* medium may all for example be used in the process of the present invention.
The bulk volume of the carrier minus the clisplacement volume is al-so referred to herein as the "free volume", which term is known in the liter-ature.
Thus the carrier is for example dimensioned to retain by adhesion less than 8% of the culture medium based on the free volume of the carrier after draining off of said medium.
The carriers for use with the present invention convenientLy include fixed carriers such as for example, the plates or screens e.g bubble plates used in fractional distillation apparatus where it is also desired to provide the maximum surface area for phase contact and flowable carriers such as sad-dle-shaped, spherical or cylindrical carriers which may, if desired be made from materials such as for example glass; ceramic materials, e.g. clay or por-celain; plastics, e.g. Duranite*, Azidur* or Teflon*; metals, e.g. titanium, V2A or V~A steel; or sintered products, e.g. corundum.
The term flowable carriers as used throughout the present specific-ation means especially rings such as Pall* rings, Raschig* rings, Prym* rings, porcelain rings, Perfo* rings, expanded metal rings and Intos* rings; coils and spirals such as glass spirals, Wilson* spirals, spring coils, gLass coils, wire spirals, spools and rolls; saddle-shaped carrier bodies such as bead saddles, Super* saddles and Novalox* saddles, Interlox* and Interpack* carri-ers, beads, balls, startype carriers, twin carriers and solid carriers.
Especially preferred flowable carriers for cell culture include spirals, coils and saddle-shaped carriers. Coils and wire spirals for example in expanded form, e.g. made of V2A or V~A steel, in which the individual turns do not touch one another, and saddles, are particularly preferred since these forms present a large surface area to the circulating medium whilst giving *Trade Mark rise to only a small flow resistance and, during washing, have only a small retention of the medium. Especially preferred wire spirals are those which have a total length of 4 to 8 mm, preferably 5 to 7 mm, a total width of 4 to 8 mm, preferably 5 to 7 mm and a wire diameter of 0.4 to 0.8 mm, preferably 0O5 to 0.7 mm, the gap between the individual turns being 0.1 to 0.5 mm, pre-ferably 0.2 to 0.4 mm.
The following tests may, for example, be employed to determine whether a given carrier is suitable for use ln the process of the present in-vention and the following carriers were tested, by way of example, for their suitability:
A = Glass rods (diameter: 10 mm, length: 30 mm).
B = Teflon rings (outside diameter: 10.5 mm, inside diameter: 5.5 mm, length:
10.2 mm).
C = Duranite cylinders (outside diameter: 10.5 mm, inside diameter: 6.5 mm, length: 11.5 mm).
D = V4A spirals (outside diameter: 6 mm, wire diameter: 0.6 mm, length: 6mm).
E = Glass balls ~diameter: 10 mm).
F = Duranite cylinders (outside diameter: 8 mm, inside diameter: 5 mm, length: 9.5 mm).
G = Glass rods (diame~er: 6 mm, length: 10 mm).
H = Saddles (diameter: 6 mm, length: 6 mm).
I = V4A wire gauze cylinders (diameter: 3 mm, length: 5 mm) and J = Glass balls (diameter: 3.5 to 5 mm) in comparison with K = Glass cylinders (outside diameter: 4 mm, inside diameter: 2 mm, length:
6 mm).
~lethod:
1. The apparatus used for conducting the above-mentioned -tests com-prises a glass tube (inside diameter: 3 to 5 cm3 tapered downwardly to a 7~

funnel, fitted with a tube having an inside diameter of about 0.5 cm as an outlet and provided with a stop-cock. A marking is made at the transition from the funnel to the 0.5 cm tube. With the stop-cock closed the glass tube is then filled with water up to this mark and the volume determined by weigh-ing. The tube is subsequently filled with ~ater to this mark again and a weighed quantity of water (e.g. 200 ml) added. The upper level is marked on the tube. The water is then drained off down to the lower mark. The quantity of water which remains adhering to the glass wall is simultaneously determined by weighing the drained quantity of water and subtracting this quantity from the fixed nominal volume ~e.g. 200 ml) referred to above. The entire opera-tion is repeated se-veral times and the average value is fixed as the value when empty.
Before each test the test tube must be completely dry (e.g. by wash-ing with acetone and subsequent drying by blowing with air). The carrier bo-dies to be tested are similarly dried in a drying chamber. ~or testing, the glass tube is filled up to the upper mark with the carriers. The weight of the carriers introduced and the quantity are determined. Subsequently~ with the stop-cock closed the glass tube is filled up to the upper mark with a weighed quantity of water. The volume of water added is determined by weigh-ing the quantity of water remaining and subtracting from the previously deter-mined total. The free volume (i.e. the bulk volume of the carriers minus the displacement volume of the carriers) may then be calculated using thP above determined measurements. The stop-cock is subsequently opened to allow the water to drain out to the lower mark, the water being weighed on a balance after collection in a tared vessel. By subtracting the drained quantity from the "free volume", the "remaining quantity" of water left on the carrier bo-dies may be calculated. The weight of the tube when emptied of the carriers is also taken into consideration. The surface area of the carrier bodies is either calculated or taken from the manufacturer's specification.

The experimentally determined data is calculated for the following values: free volume per litre in cm3, ratio of the surface area of the car-riers to the free volume and the volume of water remaining on the carriers after draining as a percentage of the free volume.

2. Using the same glass tube ~dry) the same quantity of carriers is employed, but the tube is topped up to the upper mark with dyed water (e.g 1%
neutraL red stock solution = 100%). The upper opening is then closed in an air-tight manner with a hose which is filled with water and clampe~ in posi-tion by a hose pump. After the stop-cock has been opened water is added via the hose pump in a measured quantity of 100 ml per minute and the washing effect per minute is determined by measuring the colour intensity in a photo-meter. The measured value is related to the initial value and calculated to give the washing effect as a percentage of residual dye after washing x times ~1 with a free volume quanti-ty. 5 ~inutes after the start of washing the intake of water is stopped, the column is shaken briefly and the value after 30 se-conds of pumping is measured. The value is given in the form of residual dye as a percentage after washing x times with a free volume quantity and after subsequent shaking. ~lthough water is used in this test the same results are obtained when using Hank's solution without calcium and magnesium ions at pH8, instead of water.
The following table contains the results of the above-mentioned tests:

a) a a)~~1~3)~ ... . ~ ....
I h O
~ rd 3 X 3 ~1 >
,~ a~
O ~ ';P O O O ~ ~
O . ~ O . O O . .
p~ C::~ O O o o o o ~-1 ~1 3 ~--~ r-l O + O ~ O O ~1 ~: ~ ~ O O ~ O. O O
a d' O O O O O O
~) ~IJ ~ tJ~
O
~r ~ In ~ ~1 ~ ~ o r~
a~ x ~ ~ ~ ~ o ~1 )-I O O O O
~0 a ~
tn ~ al ~ o~ co ~ ~ ~ ~ I` ~ o ~ a~
trJ ~ ~I VJ 3 . . . ~ .. . . . . ~1 ~: 3 ~ J r~ 10 ~ O 0~1 0 ~1 ~ . O
r~
rl 4 ~1 S ~ ~1 1 O ~ O
rl ~ ~1 ~
o aJ ~ ) .
S~ Ll E3 t~7 ~ ~ o ~ Q~
a) ~IJ oY~ :~ . . . ., . . , , . . ~
1~ 1--1 r l r-l ~ ~~~r ~~D~r r~CO ~1 ~ ~ O
Ir; 1~1 rl C- rl ~_1 a) o ~ ...................... a~
r~ ~ ~1 o a~ L~ O ~ U~ a U h U~ ~ r~ 1 O~ D ~ Ul ~r ~ OD d~ r o ~ .
,, ~ u ,, a) In r~ ~ o r` c~ ~ Lr) ~ ~ ~ ~
a) r~ l ~1 t~ ~ ~1 0 Ll a ~ ~0 0~4~ ~
+
a~ ~

I ~ rl r-l 3 I~ Il') ~ ~D ~ t- ~ ~`1 0 ~) ~ ~a ~ o a~ oo ~ ~ ~ oo s~l ~r 1` f'l a~ ~ ~ ~ u~ D ~ W a~ ~
~ ~ ~ (a ~ U ~
U~

a) u~
,~ a~
~-rl ~ m c~ Q ~ ~ H 1~ ~C
Ll ~ O
C) ~ E~

. ~P____ ~ ~`'~'' ' ' '''' ' .
. ! . , ~ _ .
,~
.

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On the basis of the above testsr carriers A-J possess the following favourable characteristics:-1. after release of the medium the carriers retain less than 8% of the medium relative to the free volume (medium retained due to adhesion~, and 2. have a maximum residual dye percentage of 0.15 after washing four times with a free volume quantity or a maximum residual dye percentage of 0.5% after washing four times with a free volume quantity and shaking.
Such carriers enable the suspended eukaryotic cells, e.g. fibroblast cells or epithelial cells, to adhere to them within a short timer for example 1 to 3 hours. Furthermore, the nutrients of the circulating medi~m are also absorbed, so that rapid growth of the cells is ensured. It is especially advantageous, however, that the closely grown cultures are brought into contact with inducers, such as for example Poly I:C cycloheximide, Mitomycin C, viruses or other known inducers described in the literature, within the desired time and that the necessary removal of the inducers by simple washing subsequently presents no difficulties. Washing can be effected continuously or intermittently, for example by replacing the circu-lating medium with a washing medium or by optionally draining off the medium several times and subsequent replacement by a washing medium; in the latter case, the washing medium is preferably introduced from the bottom of the culture vessel.
The carriers for use in the process of the present invention may be cleaned without difficulty and subsequently used again. Thus for example the carriers for use in the process of the present invention are such that cells adhere sufficiently strongly to their surface to ensure undisturbed cell growth,whilst not adhering so firmly that the cells can no longer be detached from the carrier upon subsequent cleanlng .

_____ `,~ i d~i '7~

Thus whilst it is essential that the carrier body for cell culture is dimensioned and disposed to retain by adhesion less than 8~ of the culture medium, based on the free volume of the carrier body, after draining off of the said medium, it i5 preferred that the carrier body should also be such that less than 0.15~ of the said medium is retained adhered thereto after four washings of the c:arrier, each washing being effected using a "free volume" o a washing medium~ Each washing may for example by effected by adding a free volume of the washing medium to the carriers and then allowing the washing medium to drain away. The washlng medium may for example be minimum essential medium without serum.
The process of the present invention is advan-tageously effected using a pump for circulatlng the culture medium, the pump being adapted to avoid the introduction of shear forces into the medium. Thus for example a peristaltic pump or a hose pump, bellows or siphon pump, diaphragm pump or air-lift pump may be employed for circulating the culture medium.
Especially preferred carrier bodies for cell culture, include those in which the ratio of the surface area of the carrier to the free volume is 20:1 to 2:1, preferably 15:1 to 5:1.
The process according to the invention has proved especially advantageous in the production of fibroblast interferon. For this purpose, a reaction vessel, preferably a pressure-stable reaction vessel, for example a 25-litre double~jacket reaction vessel of Duran glass, provided conventionally with an inlet and outlet, pH electrodes and permeator, is filled about 3/4 full with carefully washed carriers, preferably V~A wire spirals of expanded form (length: 6 mm, width: 6 mm, wire diameter: 0.6 mm, pitch: 0.3 mm) and is preferably sterilised with superheated steam.
The ratio of the surface area of the carriers to the free volume is conveniently about 11:1. Subsequently, .~
.....

cells preferably obtained by trypsination of surface cell cultures are suspended at a cell density of approximately 20,000 cells/cm2 in minimum essential medium to which or example 10~ foetal calf serum and preferably an antibiotic such as kanamycin, e.g.
in a concentration of 200 ug/ml are added.
The cell culture suspension introduced conveniently remains at about 37C for 2 to 4 hours without pump circulation; subsequently, the medium is circulated preferably with a peristaltic pump at about 30 revolutions per minute and the pH is preferably adjusted to from 7.3 to 7.5. 24 to 48 hours after introduction of the cell culture suspension the medium is conveniently removed at regular intervals and fresh growth medium of the same composition is added; altogether, preferably 80 litres of medium are added and removed over the following 3 to 4 days. Further processing depends on the substance which i5 to be obtained Erom the cell culture. With, for example, interferon the ~0 total medium is preferably removed 8 to 10 days after preparation of the cell culture and induction is conveniently subsequently effected by known processes, eOg. with Poly I:C, Mitomycin C, viruses or any other convenient substance. The entire induction medium is thereafter preferably removed and the cells washed several times with minimum essential medium. Subsequently, additional medium is added, to which a stabiliser, e.g. serum albumin, suitable for interferon has been added. This liquid is again circulated by pu~ping as specified above. After a further 12 to 24 hours the medium is harvested and the crude interferon obtained is concentrated and purified by a process known per se.
According to the invention, a peristaltic pump is preferably used which has a rate of revolution of 25 to 60 revolutions per minute and conveniently a delivery rate of 50 to 400~ of the total volume per hour. The process according to the invention _ 7~
can therefore be effected with considerably large volumes than hiterto known processes and is limited only by the requirement for a sufficient supply of nutrient and oxygen as well as by the need to keep -~he pH within an appro-priate range for the cells.
The process accorcling to the invention enables cell-culture-dependent substances, such as for example interferon, formed during the production phase to be removed from circulation, batchwise or continuously, and replaced by new medium. The substance, e.g. interferon9 thus removed can then be purified or concentrated on a purification and/or concentration column.
Moreover, the process of the present invention enables the addi-tion of a so-called stimulator substance or a mixture of such substances to be made at any desired stage in the process J in order to increase the yield of a cell-culture-dependent substance, and the removal thereof present no difficulties as may be seen from our European Patent A2-0.005.476 filed 30th April 1979 and published on November 28, 1979.
The following Example illustrates the present invention:
~xample A 20-litre double-jacket reaction vessel of Duran glass is filled with 17 litres of carefully washed carriers for cell culture [V4A wire spirals, expanded formJ length 6 mm, width 6 mm, wire diameter 0.6 mm, pitch 0.3 mm ~ratio of surface area of filling bodies to free volume is about 11:1)~.
The inlet and outlet of the vessel are provided with silicone rubber hoses, having an inside diameter of 8 to 10 mm, and equipped with p~l electrodes con-nected to a p~l meter and a permeator and connected to the circuit. The apparatus is sterilised with superheated steam. Cells obtained by trypsina-tion of surface cell cultures are suspended in 17 litres of minimum essential medium, with 10% foetal calf serum and 200 ug/ml of kanamycin, 7~i~7~

in a concentration of 20,000 cells/cm2 and the suspension is introduced into the reaction vessel. The reaction vessel is heated to 37C via the double jacket and a water-bath thermostat and is maintained at this temperature. The cell culture suspension introduced is left to stand for 2 to 4 hours in the reaction vessel without pump circulation. Subsequently, the silicone hose is inserted into a peristaltic pump and circulation is effected at a rat:e of 30 revolutions per minute with a delivery rate oE 40 to 80 litres per hour. The pH value is adjusted to between 7.3 and 7.5. 24 to 48 hours after addition of the cell suspension the medium is removed at regular intervals and fresh growth medium of the same composition is addedG Altogether, 80 litres of medium are added and removed over the following 3 to 4 days. 8 to 10 days after introduction of the cell culture the entire medium is removed and the cells are washed by the addition of minimum essential medium without serum and the induction medium is subsequently added thereto. The induction medium contains 100 ug/ml of Poly I C and 2.5 ug/ml of cycloheximide. The medium is left to stand for 3 hours in the reaction vessel. 2 ug/ml of actinomycin D are subsequently added. One hour after the addition of the actinomycin D the entire induction medium is removed and the cells are washed at least four times with minimum essential medium without serum. Subsequently, minimum essential medium containing l,000 ug/ml of serum albumin is added and circulated by pumping, as described above. The pH is adjusted to 7.5 to 7.6. l9 to 20 hours later the medium is harvested and is concentrated and purified by known processes.
The yields of interferon in the crude solution are 900 to 2,000 units/cm2 of culture face, referring to the international reference preparation G 023-902-527 of the National Institute of Health, USA.
Using various carrier bodies for cell culture ~ ~ _ 1~
~ . .~
:' 7~

in various amounts fibroblast interferon was prepared in a similar manner with the following results:

Carrier body Starting Interferon reference units/cm2 (as herein-quantityoE culture face beforein litres defined) ~ 0.2 1,147 A 0.2 1,956 A 1.2 593 B 0.3 83 D 2.2 1,580 D 17.0 1,374 D 108.0 1,103 H 0.2 470 _______ ________________________________________ ._____ _ Comparison (K) 0.2 23 0.~ 19 :` .

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In a process for the surface culture of nucleated cells in aqueous medium and for the production of cell culture-dependent substances from the cells thus obtained, the improvement which comprises using as culture face moving bodies comprising saddle-shaped or cylindrical filling bodies made of metal which after the release of the aqueous culture medium hold back less than 8 percent of the medium relative to free volume due to their form and where the ratio of surface of the filling bodies used to free volume is greater than about 5:1.

2. The process according to claim 1, wherein the filling bodies are spirals or wire gauze cylinders.

3. The process according to claim 2, wherein the filling bodies are spirals of expanded form having a total length of from about 4 to 8 mm, a total width of from about 4 to 8 mm, a wire diameter of from about 0.4 to 0.8 mm, and a pitch of from about 0.1 to 0.5 mm, or saddles.

4. The process according to claim 3, wherein the filling bodies are wire spirals having a total length of from about 5 to 7 mm, a total width of from about 5 to 7 mm, a wire diameter of from about 0.5 to 0.7 mm, and a pitch of from about 0.2 to 0.4 mm.

5. The process according to claim 1, wherein the filling bodies are made of V2A or V4A steel.

6. The process according to claim 2, wherein the filling bodies are wire spirals or wire gauze cylinders comprised of V4A steel.

7. The process according to claim 1, wherein the ratio of surface of filling bodies to free volume is from about 20:1 to 5:1.

8. The process according to claim 1, wherein the ratio of surface of filling bodies to free volume is from about 15:1 to 5:1.

9. The process according to claim 1, wherein the improvement comprises using as culture face moving filling bodies comprising spirals, coils, or saddle-shaped bodies made of V2A or V4A steel, which after the release of the culture medium hold back less than 8 percent of the medium relative to free volume due to their form.

10. The process according to claim 1, wherein interferon is the cell culture-dependent substance produced.

11. The process according to claim 10, wherein the interferon formed after the induction of the cells is separated continuously or batchwise.

12. The process according to claim 1, wherein a pump with which no shearing forces occur is used for circulating the medium.

13. The process according to claim 1, wherein the filling bodies are spirals.