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Mouse retrovirus present in cell lines used for production of biologicals

natasa778

Senior Member
Messages
1,774
Characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals
J. Shepherd et al, Biologicals, Volume 31, Issue 4, December 2003, Pages 251-260

Abstract
Rodent cells are used widely to manufacture recombinant proteins for pharmaceutical use in humans and animals. However, all rodent cell lines express endogenous retroviruses that require appropriate testing regimes for identification and characterisation. In this communication we report the results of transmission electron microscopy, reverse transcriptase assay and infectious virus assays for retrovirus in 185 manufacturer cell banks of mouse, rat or hamster origin. The results indicated considerable variability of retroviral expression levels by transmission electron microscopy and reverse transcriptase assay, but nevertheless characteristic features of each cell type were observed. Infectious retrovirus was detected in mouse myeloma and hybridoma cell lines, but not in cell lines of hamster or rat origin….

Rodent cell lines have for many years been used as substrates for production of biological therapeutics such as monoclonal antibodies, recombinant proteins, vaccines and gene therapy virus vectors. It has long been recognised that such cell lines contain retrovirus elements that may be expressed as particles detectable by electron microscopy. Such particles may be infectious, as in the case of Murine leukaemia virus (MLV), or defective and non-infectious, as in the case of the Chinese hamster ovary (CHO) cell retrovirus.]. Despite the lack of evidence for an association between murine retrovirus and disease in man, the potential contamination of therapeutics with agents associated with oncogenicity and immunosuppression in therapeutic products is a cause of regulatory concern. Detection and characterisation of retrovirus in manufacturer‘s master and end of production cell banks is recommended by regulatory agencies using techniques such as electron microscopy, reverse transcriptase assay and appropriate infectivity or co-cultivation assays. In addition, determination of retrovirus particle load and experimental demonstration of appropriate removal or inactivation of retrovirus during purification is required for each product [ref: Committee for Proprietary Medicinal Products. Notes for guidance on quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (CPMP/ICH/295/95), European Commission, Brussels (1997) – see below
…The study indicated characteristic features of retroviral expression in each cell type tested. All RT-positive cell lines demonstrated preference for manganese-dependent RT, characteristic of the Gammaretroviridae.



NOTE FOR GUIDANCE ON QUALITY OF BIOTECHNOLOGICAL PRODUCTS: VIRAL SAFETY
EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN (CPMP/ICH/295/95)
http://www.ema.europa.eu/pdfs/human/ich/029595en.pdf

3.2.1 Tests for Retroviruses
For the MCB and for cells cultured up to or beyond the limit of in vitro cell age used for
production, tests for retroviruses, including infectivity assays in sensitive cell cultures and
electron microscopy (EM) studies, should be carried out. If infectivity is not detected and no retrovirus or retrovirus-like particles have been observed by EM, reverse transcriptase (RT) or other appropriate assays should be performed to detect retroviruses which may be noninfectious. Induction studies have not been found to be useful.

.....
Case B: Where only a rodent retrovirus (or a retrovirus-like particle which is believed to be non-pathogenic, such as rodent A- and R-type particles) is present, process evaluation using a`specific "model" virus, such as a murine leukemia virus, should be performed. Purified bulk`should be tested using suitable methods having high specificity and sensitivity for the`detection of the virus in question. For marketing authorisation, data from at least 3 lots of`purified bulk at pilot-plant scale or commercial scale should be provided. Cell lines such as`CHO, C127, BHK and murine hybridoma cell lines have frequently been used as substrates`for drug production with no reported safety problems related to viral contamination of the`products. For these cell lines in which the endogenous particles have been extensively`characterised and clearance has been demonstrated (note limitations below), it is not usually necessary to assay for the`presence of the non-infectious particles in purified bulk. Studies with non-specific "model"`viruses, as in Case A, are appropriate.
… Cell lines derived from rodents usually contain endogenous retrovirus particles or retrovirus-like particles, which may be infectious (C-type particles) or non-infectious (cytoplasmic A- and R-type particles). The capacity of the manufacturing process to remove and/or inactivate rodent retroviruses from products obtained from such cells should be determined. This may be accomplished by using a murine leukemia virus, a specific "model'' virus in the case of cells of murine origin

6.4 Limitations of Viral Clearance Studies
Viral clearance studies are useful for contributing to the assurance that an acceptable level of safety in the final product is achieved but do not by themselves establish safety. number of factors in the design and execution of viral clearance studies may lead to an incorrect estimate of the ability of the process to remove virus infectivity These factors include the following:

1. Virus preparations used in clearance studies for a production process are likely to be
produced in tissue culture. The behaviour of a tissue culture virus in a production step
may be different from that of the native virus; for example, if native and cultured
viruses differ in purity or degree of aggregation.
2. Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid
initial phase is followed by a slower phase. It is possible that virus escaping a first
inactivation step may be more resistant to subsequent steps. For example, if the
resistant fraction takes the form of virus aggregates, infectivity may be resistant to a
range of different chemical treatments and to heating.
3. The ability of the overall process to remove infectivity is expressed as the sum of the
logarithm of the reductions at each step. The summation of the reduction factors of
multiple steps, particularly of steps with little reduction (e.g., below 1 log10), may
overestimate the true potential for virus elimination. Furthermore, reduction values
achieved by repetition of identical or near identical procedures should not be included
unless justified.
4. The expression of reduction factors as logarithmic reductions in titer implies that,
while residual virus infectivity may be greatly reduced, it will never be reduced to
zero. For example, a reduction in the infectivity of a preparation containing 8 log10
infectious units per ml by a factor of 8 log10 leaves zero log10 per ml or one infectious
unit per ml, taking into consideration the limit of detection of the assay.
5. Pilot-plant scale processing may differ from commercial-scale processing despite care
taken to design the scaled-down process.
6. Addition of individual virus reduction factors resulting from similar inactivation
mechanisms along the manufacturing process may overestimate overall viral clearance.



Considering that XMRV has proven to be very difficult to detect and requires repeated and various approaches for successful detection, does the above sound reassuring??



Another later document (2004) from European Medicines Agency’s CONCEPT PAPER ON THE DEVELOPMENT OF A GUIDELINE ON VIRAL SAFETY EVALUATION OF BIOTECHNOLOGICAL PRODUCTS TO BE USED IN CLINICAL TRIALS

states the following:

… Validation studies are complex.and expensive and a clear position on this is important. The extent to which manufacturers are able to refer to in-house experience concerning virus safety evaluation. For such cases, the virus safety of a given cell culture system and/or the capacity of established manufacturing process steps to inactivate/remove potential virus contaminants may have been demonstrated with several previously developed products. Such a database may serve as
supportive data to justify a reduced virus safety evaluation program for new products that enter development.
 

flybro

Senior Member
Messages
706
Location
pluto
is this saying that lab supplies are contaminated,

it looks like it saying bits of retro-vrirus are in vaccines.

I'm quite ditzy today so any help would be great.
 

MEKoan

Senior Member
Messages
2,630
Vaccines!

Autism and XMRV.

Whoa!

ETA I should say that I had to get the bit of info using the "find" function because I can't really read. So, take anything I say with a grain or more. Still...
 
Messages
34
This is dynamite - this is actually a documentation that they found MuLV in cell lines used to produce vaccines in 2003.

Cort, regardsless of XMRV being the cause of ME/CFS or not, this is a pretty serious worldwide matter given the structure of the large enterprises producing vaccines.

We need your excellent journalism on this issue - are you able to do an interview with J. Shepherd et al?

What is the implication of finding a MuLV-based retrovirus in man given the study above?

What is the implication of finding it in 3,7 % of the population?

Is it the most likely way of contamination into people?

And, just a further speculation - is this the issue that Hillary Johnson is working when she is taking time off?

- Funkster
 

justinreilly

Senior Member
Messages
2,498
Location
NYC (& RI)
So, endogenous mouse retroviruses are exposed to many species of pathogens which infect humans (to manufacture different vaccines). Viruses like to incorporate new DNA or RNA when so they can infect new hosts. Then these are injected into hundreds of millions of humans. Then a larger percentage (as in GWS and autism for example) than for nonvaccinated people come down with chronic neurological and immunilogical diseases. Hmmm.

Thanks natasia.
 

natasa778

Senior Member
Messages
1,774
What about other things they mention are made via animal cell lines: "monoclonal antibodies, recombinant proteins, and gene therapy virus vectors" - are any of those be used in large-scale trials nowdays? any other way they would be entering human population?
 

sproggle

Jan
Messages
235
Location
Teesside, England UK
Vaccines have to be a MAJOR suspect in the transference of XMRV to humans (in my humble opinion that is :D).

But given the amount of resistance we've had over whether people with ME/CFS are sick at all &the fact government agencies, politicians &most doctors &scientists all seem completely incapable of admitting they could ever possibly be mistaken or wrong about anything... Plus the fact AIDS is widely thought to have been transmitted by infected polio vaccine but of course that ideas considered sooo toxic it will never be allowed to be widely accepted until at least all involved in it are well dead &burried..... I have to say What are the chances of getting anywhere near a consensus of public or scientific opinion that it is even a lightly suspect ????? Any scientist even suggesting such a link is likely to be bombarded with accusations of poor practice &attempts to widely discredit them...

Think we will have to settle, once again, with having our own opinion on what's what &the rest of the world at best not having a blinking clue &more likely thinking we're all paranoid, out for revenge etc.... hmph! :worried:

Sorry if that was a bit negative lol! :ashamed:

My own opinion is XMRV will turn out to be causative for ME. But the battle to prove it causes ANY disease process in humans &to gain acceptance of this hasn't even really started...

I also believe vaccines are, if not THE then at least A, prime suspect. But blaming vaccines is pretty much the same as blaming the few select people who create/control vaccines &anyone even remotely involved is bound to fight to the death to resist such an accusation.

Anyway, I don't know what my point was gona be lol! :tongue:

Except I would definitely love to hear a scientific approach to likelyhood of transmission via vaccines etc... It is certainly very interesting &also of course a major public health concern...

Jan xx
 

natasa778

Senior Member
Messages
1,774
Jan, I agree completely it is a battle not worth fighting as would be lost before it even starts. Even if someone came with and absolute undeniable scientific 'proof' it would never be published or made public in any way.

But it would be good to "know" as you say, to understand the scientific mechanisms and possibilities, that is why I posted.
 

sproggle

Jan
Messages
235
Location
Teesside, England UK
Jan, I agree completely it is a battle not worth fighting as would be lost before it even starts. Even if someone came with and absolute undeniable scientific 'proof' it would never be published or made public in any way.

But it would be good to "know" as you say, to understand the scientific mechanisms and possibilities, that is why I posted.

I hear ya!!
I just wanna clarify -I'm extremely glad you did post it! :thumbsup:

This is exactly the kind of thing we need to read &know more about. The more discussion regarding XMRV transmission on here the better as far as I'm concerned!

The powers that be not wanting to know, unfortunately, will not make it go away. We all need to know the risks. I'd like to think that even if it was initially transmitted to humans through vaccines (&/or other products), that modern techniques would have improved to the extent that it would no longer be possible (needless to say, that will be the line we're fed regardless :rolleyes:)

Then again, I'd also like to think we will soon be vindicated in the media &this injustice will come to an end... I wouldn't bet in either!

Jan xx
 

lostinthedesert

Killer, Clown, Priestess
Messages
115
Medicines

Many medicines/peptides are made in modified rodent cell lines. Much of the human growth hormone on the market is produced by modified mouse cells, though some companies use modified bacterial cells. I'm not sure about EPO though it is likely to vary depensing on the source. Anything made with recombinent technology is suspect. If they state they use mammalian cells they almost always mean rodent. This means that a lot of the treatments many of us use have this sort of danger. s
 

CBS

Senior Member
Messages
1,522
George,

I'd be interested n your thoughts on John Coffin's talk at the CROI conference, specifically from minute 10 to approx. minute 15 (http://app2.capitalreach.com/esp120...&s=20431&&espmt=2&mp3file=12369&m4bfile=12369).

He talks at length about how easily human cells "efficiently and almost invariably" pick up xenotropic MLV's if they pass through mice, how casually labs handle most MLV's (not XMRV - at least not today) and how in the beginning MLV's were not recognized as being endogenous mouse viruses but rather thought to be a new human retrovirus (a "rumor" virus).
 

Gemini

Senior Member
Messages
1,176
Location
East Coast USA
George,

You said "Plus, documentation of this virus goes back to 1934 at least so...."

Funny you should mention 1934. Byron Hyde's book "The Clinical and Scientific Basis of ME/CFS" lists the 1934 outbreak at Los Angeles County Hospital as the first documented worldwide outbreak in a list covering the timeframe 1934-1990, including Incline Village in 1984.

Gemini
 

natasa778

Senior Member
Messages
1,774
Funny you should mention 1934. Byron Hyde's book "The Clinical and Scientific Basis of ME/CFS" lists the 1934 outbreak at Los Angeles County Hospital as the first documented worldwide outbreak in a list covering the timeframe 1934-1990, including Incline Village in 1984.

First documented cases of autism go back to children born in early 30s...
 
G

George

Guest
First inbreed strains of mice used in Laboratories goes back to 1922. (eye brow wiggle)