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8.4 SENATE ENQUIRY BATCH 3


INFORMATION PROVIDED ON BATCH 3

The information provided by the Department on Batch 3 in the recent HPH Newsletter is incorrect.



REFERENCE:14


Mr. Elton Humphrey
The Secretary
Senate Community Affairs References Committee
Suite S1 59
Parliament House
CANBERRA ACT 2600

BY FACSIMILE - (06) 277 5829

URGENT

Dear Sir,


Re: INQUIRY INTO CJD SETTLEMENT - MISLEADING
INFORMATION ABOUT BATCH 003 FROM THE DEPARTMENT
AND CSL

We refer to the above Inquiry and to the recent announcement from the Commonwealth Department of Health and Family Services that hPG Batch 003-2 appears to be implicated in the most recent case of CJD which has developed in the Australian recipient community.

One of our clients has provided us with a copy of the Department's HPH Newsletter of September, 1997 wherein the testing of Batch 003 is described. The information provided by the Department on this batch is most disturbing and it would appear that recipients have been misled.

Our perusal of the relevant records indicates that the information contained in this Newsletter is far from factual. As we have had enquiries from a recipient who received injections from Batch 3, we believe it is imperative that the Department's response be corrected for the record.

For the Committee's immediate consideration and convenience we provide a summary of the extraction, firactionation and testing of Batch 003 below.

FSH Batch 003 and 004

Extraction of this batch commenced on 28 September, 1967. it can be presumed that the glands used in this batch were collected at various times prior to this date indicating that the incubation period of the infective agent is probably in excess of thirty years. The date on which the treatment was administered to the recipient who has recently developed CJD should not therefore be used as the time against which the incubation period is measured.

A Report prepared by A. Telford (CSL) dated 2 November, 1967 (contained in CSL File 67/406) details the production of the batch. A copy of this report, and all other reports referred to below will be forwarded to the Committee by express post with the original of this facsimile.

Mr. Telford noted that difficulties were experienced during the sterilisation of the batch. The batch was split into two parts, FSH Batch 003 and FSH Batch 004. In relation to FSH Batch 003, Mr. Telford noted that it was found to be unsterile in two out of six vials and that it was to be retreated. Mr. Telford warned that it should not be released until uncertainty relating to its potency had been resolved.

In a further report dated 30 November, 1967, Mr. Telford notes that prior to the 1,335 vials being retreated, one in three vials of the Batch which became FSH Batch 003-2 were found to be pyrogenic, contaminated with a gram negative bacillus, probably a Pseudomonas. It was redissolved, refiltered and redispensed into 1,054 vials of FSH Batch 003-2.

Significantly, Mr. Telford makes no mention of any of these vials being tested for sterility. We have been unable to locate any documents within the documents discovered by the Commonwealth and CSL which suggest that ampoules from the reprocessed FSH Batch 003-2 were in fact tested for sterility or that these tests were satisfactory.

Moreover it is clear from a Report of Mr. Mathews (CSL, Chief of Quality Control) dated 28 June, 1968 that FSH Batch 003 and 004 were contaminated with pyrogens. The date of this Report is significant. The Pyrogen tests were conducted in June of 1968. However ampoules from this batch had been distributed to numerous obstetrician/gynaecologists for use in their patients before the results of the tests were obtained. Patients were therefore administered injections of pyrogenic hPG prior to the results of the pyrogen tests being obtained by CSL. This can hardly be considered to be 'best practice' at the time. There is no doubt that the batches were contaminated with pyrogens and any claim by CSL, or the Department to the contrary is incorrect

We therefore fail to see how the Department can claim that:

"During preparation, a routine test found that batch 003 contained bacteria. It was therefore refiltered to remove the bacteria and was tested again. This test showed that the batch was sterile i.e. it contained no bacteria. The batch was then distributed as batch 003-2"

[emphasis supplied]

Obviously there are a number of alternatives to explain the discrepancy in the information provided to recipients by the Department and CSL and the information which is actually contained in CSL's flies:

The reprocessed FSH Batch 003-2 was not tested for sterility as the second Report of Mr. Telford suggests. As a result it must be assumed that it remained contaminated and unsterile. One wonders how CSL would have been able to extract the pituitary powder from the vials to reprocess it in any event. There were in excess of 1.000 vials and we understand that the powder would stick to the glass of the vials; or

The reprocessed FSH Batch 003-2 was retested and found to be sterile, as claimed by the Department, and presumably CSL, in the HPH Newsletter of September, 1997. If this is the case, we wonder whether the Committee could seek copies of the documentation upon which the Department and CSL rely in making this claim. Moreover, it would appear that if such documentation does in fact exist, the Commonwealth and/or CSL have failed to discover it to this firm, and presumably, to Rennicks Briggs during informal discovery in the litigation.

Of equal significance is the fact that Mr. Telford in his Report of 2 November, 1967 emphatically warned that ampoules from FSH Batch 003 "should not be issued without prior assays as it is of very uncertain potency. Nevertheless, ampoules were distributed to clinicians for use in patients without any warning of the uncertainty.


This batch was divided into two parts - hGH Batch 003-2 and hGH Batch 003-3 as a result of the loss of some of the material during freeze drying. The batches were found to be sterile. However the batches were Pyrogenic as the Report of Mr. Mathews of 17 June, 1968 indicates. He noted that there were "marked pyrogenic reactions" and that he understood that Batch 003-3 "was found to be contaminated". A further Report from Mr. Mathews of 19 June, 1968 notes that some of the mice used in the pyrogen tests suffered from "slight convulsions", "partial paralysis" and "collapsed momentarily" after injections.

Again, the results of the Pyrogen tests were obtained by CSL, after ampoules of the batch had been distributed to clinicians for use in patients. As a result recipients also received injections from a pyrogenic batch of hGH. This can not be regarded to be 'best practice' at the time.

In addition, Mr. Telford in his Report on Batch 003 recommended that hGH Batch 003 be accompanied with a warning as to the uncertainty of the potency of the batch. It would seem that from the documents discovered by the Commonwealth and CSL no warnings were hGH Batch 003
in fact given to practitioners when ampoules of the batch were provided to them for use in their patients.

An Experimental Batch

The above batches were experimental batches. CSL were still attempting to "fine tune" their extraction and fractionation method. It was the first batch using a large number of glands. Ampoules from this batch (as with the earlier and some later batches) were distributed to clinicians for a clinical trial of the product. These batches were described by CSL as being 'experimental'. The purpose of the administration of the hGH and hPH was to ascertain the in vivo potency of the products - its use in patients was also 'experimental'.

Documents confirming the status of this batch can be provided to the Committee if required.

In short hGH Batch 003 and FSH Batch 003 and 004 should never have been distributed for use in humans.

We are very concerned that the Department and CSL continues to mislead recipients and the Australian public in relation to the problems it experienced during the extraction, fractionation and distribution of the hGH and hPG.

Should you have any further enquiries, please do not hesitate to contact the writer.
We look forward to your earliest reply.

Yours faithfully,
MACEDONE CHRISTIE W11LIS
SOLARI PARTNERS


KAREN REWEEKS

CC:

Senator Harradine (06) 277 3739, Senator Foreshaw (06) 277 3809, Senator Lees (06) 277 3996, Senator Lightfoot (06) 277 5825, Senator Bishop (06) 277 3123 & Senator Neil (06) 277 3092





Senate Community Affairs References Committee

Dr. P. Schiff,

HUMAN PITUITARY HORMONES BATCH 003

Human Growth Hormone Batch 003 has been dispensed. and freeze dried, and the sterility test found to be satisfactory. A total of 2450 vials at approximately 4.0 I.U./vial are available for issue to interested parties. This represents a yield of 10.3 I.U./gland from 960 glands, c.f. 7.9 I.U./gland for batch 002.

The HGH was dispensed in two sub-batches (003-2 and 003-3) and an assay was done on one of these prior to dispensing. The assay result was 1.9 I.U./ml. (95% F.L. - 1.4 - 2 . 6 I.U./ml.), and 2.0 ml./vial nominally dispensed, but in fact there was probably an approximate 10% overfill, as fewer vials were obtained than calculated from the bulk volume. Thus the actual activity per vial is about 4 I.U., but may be somewhat higher than this, and may be different for the two halves of the batch. A further assay will therefore be done on each sub-batch but the results will possibly not be available until January 1968. The present assay figure of 4.0 I.U./vial should be sufficiently accurate to allow issue of the batch. if accompanied by a warning of the possible inaccuracy of the figure.

It was originally intended that the above assay should be done on the finished product. The complications arose because (a) the Delvac drier was not available at the time originally arranged, so that the batch had to be divided into two, and (b) when the first portion was dried In the Leybold drier, the drying was unsatisfactory and the sub-batch had to be redissolved, sterilized and dispensed, resulting in an overall loss of material of 5% of the total batch.

Follicle Stimulating Hormone

Trouble was experienced in filtering this product through the sterilizing Millipore membrane. Consequently, one half of the batch (004) was further treated with DEAE-Cellulose according to the method of Roos and Gemzell. Biochem. and Biophys. Acta 93 217 (1964), i.e. it was absorbed from a .02M potassium phosphate solution pH 7.0 onto Whatmen DEAE-Cellulose and eluted with .06M phosphate. The solution was then sterilized and dispensed at 1/2 gland per vial. The result of drying this material in the Leybold is not yet known.

To the other half of batch (003) was added 5% lactose and dispensed at 1/3 gland per vial. This was dried in the Delvac and an assay of this dried material showed it to be of much higher potency than anticipated on the basin of the previous batch, but it was found to be non-sterile in 2 out of 6 vials. Consequently, it will have to be retreated. Assays on these two batches will probably be done, one in late December and the other in January 1968. This material should not be issued without prior assay as it is of very uncertain potency.

It is also desirable that assays for luteinizing hormone be done on these two batches. Miss D. Fell is looking into the possibility of establishing an LH assay at C.S.L., on the basis of published methods, but it may be worthwhile making inquiries of those concerned with this product, as to what is the recommended method of assay and/or whether the LH assays on this batch could be done by somebody else. Also as our FSH assay does not give the same result as that of Lamond in Brisbane (using hypophysectomized mice) it might be preferable to have FSH assays done on the two batches by him as well as was done for batch 001.
(ALAN P. TELFORD)
Dept. 512.

67/406

BATCH 3. CSL

PITUITARY HORMONE ASSAYS

An ad hoc meeting was held on 21/11/67 at C.S.L. to discuss G.H and F.S.H. assays.

Present : Drs. Brown, Martin and Schiff, Miss Fell, Mr. Telford.

Processing of Batch 003 has now been completed and has yielded a total of 2450 vials of G.H. at an average potency of 4.2 I.U./vial.

Some 1900 ampoules of F.S.H. are also available. This represents two sub-batches, both of which will have to be assayed separately. Difficulties have been encountered in a preliminary assay using C.S.L. rats. Dr. Martin suggested that we try another rat strain. The Sprague-Dawley strain was usually recommended for the Steelman-Pohley assay. Miss Fell agreed to obtain this strain and repeat the assay.

The.mouse augmentation assay can be used as a check on F.S.H. activity, although it is not as meaningful. Dr. Martin will carry out this assay on Batch oo3.

Assays for L.H. activitiy were also discussed. Dr. H. P. Taft is using the Parlow assay (ascorbic acid depletion in hypophysectomized rats) with success. Miss Fell will contact Miss Eda Ekkel in Dr. Traft's laboratory for details of this assay. Dr. Burger at Prince Henry's Hospital can measure L.H. by radio-immunoassay. It was agreed that samples should be sent to Dr. Burger so that the R.I.A. and biological assay results could be compared.

Dr. Schiff will notify Dr. Lazarus that G.H. is available for immediate distribution. He will also ask Dr. R. Cox for any available information concerning the in vivo potency of H.S.H. Batch 002 in women.





(Peter Schiff)
21.11.PREPARATION OF HUMAN PITUITARY HORMONES BATCH 003

The extraction of the third batch of human pituitary hormones was commenced on 28th September, 1967. From 960 glands (180 acetone-dried and 780 frozen), 2,450 vials of approximately 4.4 I.U./vial of HGH have been obtained, and 1,950 vials of F.S.H. containing the equivalent of 0.5 glands/vial. This represents a yield of 11.2 I.U./gland of HGH c.f. 7.9 I.U./gland for batch 002.

EXPERIMENTAL

Extraction

The human pituitary glands (180 stored under acetone and 780 frozen) were mixed with an approximately equal weight of dry ice and ground in a cooled meat grinder. The brown powder was stirred with 4 8 L (5mL/gland) of Tris buffer (0.01M Tris, 0.1M KCl to pH 8.4) for 20 hours at 4o C, separated by centrifugation for 2 hours at 2,000 r.p.m. in an IEC-PR2 centrifuge the residue extracted for 2 hours with a further 0.96 L Tris buffer, and centrifuged again. Some care was required in separating the supernatent frorn the second spin, as a portion of the residue was very lightly sedimented. The pooled opaque red supernatent was extracted with 2 X 400 mL iso-octane, and the de-fatted aqueous solution dialysed overnight VS. 60 L distilled water and then concentrated by dialysis VS. 30%. carbowax solution. Dialysis tubing of 1 7/8" flat width was found to be most satisfactory, allowing 2-fold concentration over a period of 8 hours; 3" tubing only allowed 25% reduction in volume during 12 hours.

At this stage, the batch was divided into two unequal portions for, Sephadex fractionation. It was impossible to divide the batch equally because insufficient concentration had been achieved at the scheduled time for starting the Sephadex treatment. The first portion (A) represented 40% of the total batch, in a volume of 900 mL., estimated to contain approximately 1.8% of solute. The other 60% (portion B).was concentrated to 900 mL for fractionation and estimated to contain approximately 2.6% solute.

Fractionation
A glass column, 5" x 6", was filled with. Sephadex G75 (2.5 kg. dry weight, 27.7 L wet volume) in 0.15M ammonium bicarbonate/I% butanol. 80% of the Sephadex was that which had been used for the previous batches and had, therefore, been kept for 20 months at room temperature in the wet state. 20% of fresh material was mixed with it. To pour the column, a long glass rod was placed inside the column, the top portion protruding through a large funnel. The slurry of Sephadex in water was poured into the funnel and down the stirring rod, to avoid the possible formation of air bubbles. A total of approximately 20 L. water was run through the column while topping up with Sephadex to within 4 inches of the top. At this stage, the Sephadex was equilibrated with 0.15 M ammonium bicarbonated/l% butanol buffer, by running 18 L of buffer through the column. The last portion was filled with a very thick slurry of Sephadex in buffer, the conical hollow filled by applying suction to the outlet tube of the cap while lowering
bicarbonate buffer prio to use. The evenness of Packing; was checked by passing through a band of blue Dextran 2000 during the washing.

To each portion of concentrated extract, ammonium bicarbonate was added to 0.15 M and butanol to 1%, the solution filtered through a bed of 40 g. Sephadex G25 in bicarbonate buffer and run onto the bottom of the column at 0/5 mL/min. It was necessary to use a fresh lot of G25 for each treatment, due to clogging. This was followed, onto tne column by 100 mL. 1% sucrose in bicarbonate buffer, to sharpen the trailing edge of the protein band. The elution was carried out with bicarbonate buffer initially at 9 mL/min for portion A. Initially the maximum rate attainable with a head of seven feet of water was 12 mL/min, but this decreased to 5.5 for the latter half of portion A and for all portion B.

It was originally intended to wash the column through with 1% sodium hydroxide solution between batches, but time prevented this and it was only possible to wash it with 6mL bicarborbonate buffer. The effluent from the top of the column was collected using a time operated fraction collector, in fractions to 50 - 140 mL, the optIcal density at 280 mu of each fraction determined, and these pooled in lots of about 350mL.

The optical density U.S. effluent volume of the various fractions and the pooling of the fractions is shown on the graph. The fractions suspected of containing F.S.H. were assayed roughly biologically. The results of the assay indicated that most of the activity was either in the fractions which as shown on the graph, were subsequently pooled - ie, fraction 63-92 A and 24 - 39 B. The appearance of the fractions vary considerably between the two halves of the batch, A being very similar to 002, but B was much more cloudy in the early stages. It is apparent from the graph that B had 1.75 times the absorbance of A, although the supposed concentration of solution to the column was only 1.5 times that of A. Despite these discrepancies between the portions, the elution volumes for FSH and HGH were found to be almost identical. The HGH pool was decided on the basis of the O.D. graph, the appropriate peak being decided by comparison with previous batches. The KD's in this case are FSH: 18, HGH: 50 c.f. batches. 17, 50, 13, 46 for the batches 002 and 001 respectively.

Some of the HGH fractions were concentrated VS. carbowax enable dispensing of a 2.0 I.U./mL solution at a 2.0mL/vial, but this was unnecessary, as up to 3.0 mL could be satisfactorily dried in the 14 cc vials, and the concentration of the pool without concentration would have been about 1.5 I.U./mL corresponding to 2.2 I.U./vial.

Sterilization and Dispensing

The HGH fractions were dialysed VS. distilled and pooled as indicated above. The optical density at 250 mu was adjusted to 1.04 by dilution to 6.18 litres with water. This solution was filtered through a 14 cm 5 micron Millipore membrane and then through a 0.45 micron membrane, the filtration proceeding quite satisfactorily. The sterile solution was dispensed as 2.0 mL. aliquots into 14 cc vials (VI/14), one half of it (003-2, 1396 vials) kept frozen for 1 week, and the other half (003-1, 1299 vials) dried in the Leybold freeze-drier.

This drying resulted in the loss of some of the material out of many of the vials, presumably due to incomplete freezing during drying. Consequently, batch 003-1 had to be re-dissolved in 0.07 M ammonia solution, resteriIzed and re-dispensed to give 1,075 vials(003-3).

Batches OO3-2 and OO3-3 were both dried in the Delvac freeze-drier, sealed, and found to be sterile. Batch 003-2 was assayed at 4.4 I.U./vial (3.7 - 5.3 I.U./vial, 95% F.L.). Batch 003-3 was assayed prior to dispensing at 3.9 I.U./mL. As only approximately 90% of the theoretical number of vials was obtained, there was probably an 11% overfill, so that the dispensed assay should be ca. 4.4 I.U./vial for this batch also. Thus, the yield of HGH is 1,075 +1,396 = 2,471 vial's at 4.4 I.U./vial = 10,870 I.U. from 960 glands = 11.3 I.U./gland.

The F.S.H. fractions were dialysed VS. distilled water, pooled as indicated above, and diluted to 5.4 litres with water (6 mL /gland) and 270 g. lactose B.P. added (to 5% concentration). This was passed through 5 micron millipore membranes - due to clogging of the filter, three of these were required. Attempted filtration through two 0.45 micro- membranes in parallel, resulted in only 50% passing the filter during 20 hours. The portion which had passed through the filter (2.7 L) was dispensed as1,335 x 2.0 mL aliquots and dried in the Delvac. Approximately 1 in 3 of these vials were found to be contaminated with a gram negative bacillus, probably a Pseudomonas which is known to pass 0.45 micron-membranes, but not 0.22 microns. The batch was, therefore, re-dissolved, filtered through a 0.22 micron membrane and redIspensed, yielding 1,054 vials equivalents to 0.46 glands/vial and containing approximately 125 mg lactose per vial, as batch 003-2.

The other portion (batch 004) was treated with DEAE-cellulose to make it more easily filtered and to improve it's purity. The 2.7 litres of F.S.H. solution was concentrated to 0.92 litres by dialysis VS. carbowax and the lactose removed by dialysis VS. distilled water. To the concentrated solution, was added potassium phosphate buffer pH 7.0 to a concentration of 0.02 M phosphate. This was mixed with 150 g DEAE-cellulose (Whatman DE-50 Floc.) previously washed with 0.02 M phosphate buffer, for 1 hour at 4oC and filtered on an 18 cm buchner funnel during several hours . The DEAE-C was washed on the
funnel with 1 litre of 0.02 M phosphate and then slurried with 1 litre of 0.06M phosphate pH 7.0. The mixture was filtered and washed through with 1 litre of 0.06 M phosphate.

Rough assays of these filtrates showed the largest portion .... expected in the first wash with 0.06 M phosphate, but there was still a significant quantity in the second wash, but practically none in the initial filtrate. The whole of the 0.06 M phosphate wash was pooled and filtered through a 14 cm 0.45 micron MillIpore filter to sterilize it. This proceeded satisfactorily - i.e., the DEAE-C treatment removed the material causing the clogging of the filter and also removed approximately 50% of the colour, changing the red solution to deep straw coloured. The product was dispensed as 2.2 mL aliquots into 897 vials and freeze-dried in the Delvac. Thus, the yield of F.S.H. is 1,054 + 897 = 1,951 vials corresponding to approximately 0.5 glands/vial. An assay was done on the non-sterile batch 003-1, but the assay was invalid because the potency was higher than the assumption based on the result of the previous batch. No accurate assays have been determined.

The yields etc. of the batch are summarized below


H.G.H
F.S.H.

003-2
003-3
003
004
No. of glands
Wt. of glands
No. of vials
Activity/vial
Weight/vial
Potency
Total activity
Activity/gram
Activity/gland
960
ca. 480 g
1396
4.4 I.U.
3.7 mg
960
ca. 480 g
1075

960
ca. 480 g
1054

960
ca. 480 g
897


DISCUSSION

Batch Size

The comments on this aspect made in the report on batch 002 (File 66/4, folios 26 - 30; are still aplicable, but more relevant data is now available. In the above purification using the same column twice for one batch, it was found impossible to pool the corresponding fractions from the two runs because of the considerable variation in ultraviolet absorbance graphs. Thus, qualitative assays for F.S.H. had to be done for each run. The total time taken for each run was longer than anticipated so that there was insufficient time to wash the column between runs. More time could, of course, be allowed, but this would result in a longer time in solution for the F.S.H. It would, therefore, be much more convenient if three columns were used.

The qKd values for this batch were very similar to those for 002, so that allowing for the presence of 20% of new Sephadex, the deterioration was not great during the intervening period. It would, therefore, appear that the life-time of a column could be two years or one column should be replaced every 3 months.

The shape of the graph of ultraviolet absorbance VS volume was somewhat different for both portions of the batch. Portion A resembles batch 002 and portion B resembles batch 001, the former pair giving much better resolution of peaks. The apparent reason for the differences is the quality of solute run onto the column, the following being the load for each batch:-

001 800mL x 2.4% 19.4 g/21.0 L 0.95 g/L'
002 550mL x 2.5% - 13.6 g/21.0 L 0.65 g/L
003- A 900mL x 1.8% 16.2 g/27.7 L 0.58 g/L
003- B 900mL x 2.6% 23.4 g/27.7 L 0.84 g/L

Thus, it appears that the maximum load to be used for good resolution is approximately 0.65g per litre of column volume. For a column of 27.7 litres, this corresponds to 18.0g in a volume of 720 - 900 mL (2.0 - 2.5%). This mass of material should be obtained from approximately 450 glands, as originally estimated. The above batch, unfortunately, was unevenly divided.

Flow Rate

In the present batch, difficulty was experienced in obtaining a sufficiently high flow rate. It is estimated that a flow rate of ca. 10 mL/min. through a 6" diameter coIumn (3.0 mL/cm/hr) would be suitable for obtaining good resolution. But the maximum attainable with the seven feet of water pressure was 5.5 mL/min. except at the beginning of the run. To increase this to 10 mL/min, approximately 14 feet would be required, and for three columns in series, a pressure of 42 feet of water would be required. As this is most inconvenient to obtain by gravitiy, a pump must be used to supply the pressure. This pump should give a constant rate of flow to enable the use of a time operated fraction collector for obtaining constant volume fractions. The flow rate should be unaffected by changes in the resistance to flow of the column (back pressure) and should also have a maximun rate of flow of approximately 1L/hr (for washing the column) at 50psi output pressure. It should also have a safety device in case the back pressure exceeds this value. The pump should preferably be placed between the first and second columns, to minimize the pressure required at the outlet. The pressure drop over the second and third columns combined, at 10mL/min should be approximately 14 psi.

A considerable amount of work is involved in manually determining the absorption at 280mu of each fraction, approximately 300 separate readings being required for the above fractionation. This would be considerably simplified by the use of a continuous recording monitor. No such recorders are available within C.S.L., although a suitable recorder (LK3 Uvicord Model II) could be purchased from Watson Victor for $2235. But a monitor for 254mu is available and it is suggested that during the running of the next batch of pituitary hormones, a recording at 254 mu be obtained and compared with the graph at 280 mu, to see if it could be used in future.

Sterility of Product

Problems of early sterility have been encountered with these products on two occasions; HGH batch 001 and FSH batch 003; no special precautions are taken at present to avoid contamination of the product during processing. The sterility of the final product relies entirely on the efficient removal of bacteria by Millipore filtration (in the case of batch 001, an ultrafine sintered glass filter was used) . To ensure this efficiency it is necessary, as mentioned earlier to use a 0.22 micron membrane. Because of the high cost and inconvenience of reprocessing the batch as was done above, it is suggested that a sterility test should be done on the bulk solution prior to dispensing. This was previously avoided because some loss of potency of the FSH can be expected on standing in solution for a.week. But considering the relatively high risk of contamination so far experienced, (2 out of 9 batches dispensed), it seems that the small potency loss can be justified. It is, of course, hoped that use of finer membranes will eliminate the problem, and if this proves to be the case, the bulk sterility testing prior to dispensing could again be eliminated.

When the process is being run under more automated conditions - ie., using three columns in series,a metering pump and a UV monitor, it should be possible to practically eliminate the initial contamination, thus facilitating the final sterilization and avoiding the risk of pyrogenicity.

DEAE-cellulose Treatment

The further purification of the FSH by treatment with DEAE-cellulose according to the method of Roos and Gemzell, Biochem & Biophys. Acta 93, 217 (1964), as described above, resulted in a product containing less colour and more easily filtered. But the recovery of activity has not yet been determined. Comparison of the assay results of the two batches, when available, will not give an accurate estimate of the loss of activity on DEAE-cellulose treatment because of the other differences in treatment of the two batches. But providing a reasonableyield has been obtained, the next batch should preferably be given DEAE-C treatment , and the activity of the solution compared pror to and after treatment. A larger quantity of 0.06M phosphate should be used to wash the DEAE-C, as there was an unexpectedly large amount of activity in the second of the above washes.


Construction of Fractionation Column


The column used for Sephadex treatment consisted of a QVF pipe section, (PS6/60) five feet in length and six inches in diameter. The ends were closed by plates of laminated bakelite, cut out in a conical shape, with a small outlet in the centre, and covered with mesh nylon gauze. They worked satisfactorily although some trial and error was necessary to determine the quantity of Sephadex required to just fill the space. When obtaining ends for the other two columns required for the larger batches, the three alternatives are: (a) conical ends as used here, (b) flat ends of similar material which could be slid across the end to close it, (c) a QVF end piece which is approximately hemispherical in shape and could be closed with a rubber stopper. The first of these is of a more ideal shape for smooth flow, although recent information from Pharmacia (the manufacturers of Sephadex) suggests that the shape of the inlet and outlet is not of great importance. The fitting of the ends to the column is illustrated in the accompanying diagram.



(ALAN P. TELFORD)
Dept. 512

30th November, 1967.


Commonwealth Serum Laboratories

INTERNAL COMMUNICATION


From: Chief of Quality Control.

To: Dr. Schiff, Chief of Research,

Subject: Pyrogen Tests on Human Growth Hormone.

Pyrogen tests have been performed on Batches 003-2 and 003-3 of Human Growth Hormone, as shown below:



Batch 003-2
Batch 003-3
Date
Wt of
Rabbit
Dose (Percentage of contents of vial)
Temp.
Response
Wt of
Rabbit
Dose (Percentage of contents of vial)
Temp.
Response

(Kg)
per Rabbit
per Kg
(oC)
(Kg)
per Rabbit
per Kg
(oC)
12/67
1.6
100
63
1.2
2.1
100
68
1.1
12/67




2.0
100*
50*
0.8
12/67




2.0
50
25
1.1
6/68
2.4
48
20
1.65
2.3
46
20
1.0
6/68
2.1
21
10
1.5
1.9
19
10
1.2
6/68
2.6
13
5
1.2
1.8
9
5
0.8
6/68
2.6
7
2.5
0.9
1.9
5
2.5
0.6


* Dose given intramuscularly - all others intravenously.

Apart from the marked pyrogenic reactions, no other reactions were observed during the period of the tests. In the case of the rabbits tested on 14/6/68 they are still alive and well today.

Toxicity tests in mice are in progress.

I understand that Batch 003-3 was found to be contaminated and was reprocessed after 19/12/67 and before 14/6/68, but I have not checked the history of the batch against documents.


(A.G. MATHEWS)
17/6/19

Copies to Mr. Davey, Mr Dennis and Mr Hinton

Commonwealth Serum Laboratories

INTERNAL COMMUNICATION


From: Chief of Quality Control.

To: Dr. Schiff, Chief of Research,

Subject: Pyrogen and Toxicity Tests on Human Growth Hormone.


Further to my report dated 17/6/68, further pyrogen tests have been done, and the results have been incorporated into the composite table below:-



Batch 002
Batch 003-2
Batch 003-3
Dates
Wt of Rabbit (kg)
Dose
Temp
Response (oC)
Wt of Rabbit (kg)
Dose
Temp
Response (oC)
Wt of Rabbit (kg)
Dose
Temp
Response (oC)
6,8/12/67
19/12
"
18,14,14,6/68
`
"
"
18,18/6/68



2.4
2.4
2.6
2.2



20
10
5
2.5



1.2
1.25
1.25
0.65

1.6


2.4
2.1
2.6
2.6
2.3
2.6
63


20
10
50
2.5
2.5
1.25
1.2


1.65
1.5
1.2
0.9
1.0
0.3
2.1
2.0
2.0
2.3
1.9
1.8
1.9
2.2
2.7
48
50*
25
20
10
5
2.5
2.5
1.25
1.1
0.8
1.1
1.0
1.2
0.8
0.6
0.5
0.65

Dose - Expressed as percentage of contents of a vial per kg of rabbit.

* Dose given intramuscularly - all others intravenously.

All of the rabbits tested on 14/6/68 and 18/6/68 are still alive and well.

On 18/6/68 the above batches were injected intravenously into mice, each weighing about 20 g., at the rate of 20, 10, 5, 2.5 and 1.25% of the contents of a vial per mouse. All of the mice are alive and well today, but immediately after the injections the following transient reactions were observed:-

Batch 002 mouse given 2nd largest dose slight convulsions.
Batch 003-2 mouse given largest dose partial paralysis of hind legs for about 5 min Batch 003-3 mouse given largest dose collapsed momentarily

(A.G.MATHEWS)

19/6/1968

Commonwealth Serum Laboratories

INTERNAL COMMUNICATION


From: Chief of Quality Control.

To: Dr. Schiff, Chief of Research,

Subject: Pyrogen and Toxicity Tests on Human Growth Hormone.


Further to my report dated 19/6/68, all of the mice and rabbits referred to therein remained alive and well until the end of the observation period (5 days after injection).

(A.G. MATHEWS)

28.6.1968

Copies to Mr. Davey, Mr. Dennis and Mr. Hinton.


Commonwealth Serum Laboratories

INTERNAL COMMUNICATION


From: Chief of Quality Control.

To: Dr. Schiff, Chief of Research,

Subject: Pyrogen and Toxicity Tests on Follicle Stimulating Growth Hormone.


Pyrogen tests have been performed on Batches 003 and 004 of F.S.H., as shown below. All doses were given intravenously.


Batch 003
Batch 004
Date
Wt of
Rabbit
Dose (Percentage of contents of vial)
Temp.
Response
Wt of
Rabbit
Dose (Percentage of contents of vial)
Temp.
Response


(Kg)
per Rabbit
per Kg
(oC)
(Kg)
per Rabbit
per Kg
(oC)
8/12/67
1.9
100
53
1.3
1.9
100
53
1.3
20/6/68
1.8
36
20
1.05
1.9
38
20
1.85
"
2.0
20
10
1.25
1.7
17
10
2.05
"
1.9
10
5
1.15
2.1
11
5
1.65
"
1.9
5
2.5
0.9
1.9
5
2.5
1.4




On 20/6/68 the above batches were injected intravenously into mice, each weighing about 20 g, at the rate of 20, 10, 5, 2.5 and 1.25% of the contents of a vial per mouse. No adverse reactions were observed.

All rabbits and mice remained alive and well for the 5 days following injection.

(A.G. MATHEWS)

28.6.1968





Mr. A. P. Telford,
Dept. 512.


HUMAN PITUITARY HORMONES - BATCH 003.

Thank you for your minute dated November 2.

The processing does not appear to have proceeded as smoothly, both for G.H. and F.S.H., as was the case with batch 002. Is this due to the larger size of the latent batch, and if so do you think any modifications of the process should be instituted?

G.H. Which sub-batch was assayed to obtain the result of 1.9 I.U./mL? Since the two batches were handled differently they must be regarded as entirely separate, and the only material currently available for issue is that which has been assayed.

F.S.H. Dr. Martin will shortly arrange a meeting of the fractionation sub-committee, at which the question of F.S.H. assays will be discussed. I shall put the question of L.H. assays to that meeting.

For this meeting it would be helpful if I could have the details concerning the fractionation of Batch 003 and also the assay figures available to date. Please let me know on which files these data are recorded.



(Peter Schiff)
CHIEF OF RESEARCH
8/11/67.