10 replies to this topic

[MouseMab]

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

[klinmed]

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

[MouseMab]

Thanks for the info.  Yes, we do pristane prime the mice - 1 week before injection of the cells (I believe that this has been the practice for many years in our lab - I myself am fairly new to the hybridoma field).  Interestingly, we actually do have the product from Integra that you mentioned, but are still in the process of optimizing the system.  So in the meantime, we wanted to produce a small quantity of ascitic fluid so that we can continue with our downstream processes.  Have you used the Integra CELLline before?  What do you think of the product?

klinmed, on Nov 13 2009, 10:06 PM, said:

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

[klinmed]

MouseMab, on Nov 14 2009, 07:50 AM, said:

Thanks for the info.  Yes, we do pristane prime the mice - 1 week before injection of the cells (I believe that this has been the practice for many years in our lab - I myself am fairly new to the hybridoma field).  Interestingly, we actually do have the product from Integra that you mentioned, but are still in the process of optimizing the system.  So in the meantime, we wanted to produce a small quantity of ascitic fluid so that we can continue with our downstream processes.  Have you used the Integra CELLline before?  What do you think of the product?

klinmed, on Nov 13 2009, 10:06 PM, said:

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

I really like the Integra flasks.  Have used them for a number of years.  In fact, I have 3 of the CL-1000 ones running at the moment.

The cell compartment contains 15 ml DMEM /15% FCS whilst the nutrient one 1000ml DMEM/1% FCS.  The DMEM is the high glucose variety and contains Na pyruvate and 2-mercaptoethanol.

At the moment I harvest ever 5 days and get ca 14 ml supernatant (1 - 1.5 mg Mab/ml) from each flask.

[MouseMab]

Thanks for the info about the CELLline flasks.  What method do you use for quantifying the the MAbs in the media?  How do you purify your MAbs from the supernatant?

klinmed, on Nov 14 2009, 11:21 AM, said:

MouseMab, on Nov 14 2009, 07:50 AM, said:

Thanks for the info.  Yes, we do pristane prime the mice - 1 week before injection of the cells (I believe that this has been the practice for many years in our lab - I myself am fairly new to the hybridoma field).  Interestingly, we actually do have the product from Integra that you mentioned, but are still in the process of optimizing the system.  So in the meantime, we wanted to produce a small quantity of ascitic fluid so that we can continue with our downstream processes.  Have you used the Integra CELLline before?  What do you think of the product?

klinmed, on Nov 13 2009, 10:06 PM, said:

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

I really like the Integra flasks.  Have used them for a number of years.  In fact, I have 3 of the CL-1000 ones running at the moment.

The cell compartment contains 15 ml DMEM /15% FCS whilst the nutrient one 1000ml DMEM/1% FCS.  The DMEM is the high glucose variety and contains Na pyruvate and 2-mercaptoethanol.

At the moment I harvest ever 5 days and get ca 14 ml supernatant (1 - 1.5 mg Mab/ml) from each flask.

[klinmed]

MouseMab, on Nov 16 2009, 12:24 PM, said:

Thanks for the info about the CELLline flasks.  What method do you use for quantifying the the MAbs in the media?  How do you purify your MAbs from the supernatant?

klinmed, on Nov 14 2009, 11:21 AM, said:

MouseMab, on Nov 14 2009, 07:50 AM, said:

Thanks for the info.  Yes, we do pristane prime the mice - 1 week before injection of the cells (I believe that this has been the practice for many years in our lab - I myself am fairly new to the hybridoma field).  Interestingly, we actually do have the product from Integra that you mentioned, but are still in the process of optimizing the system.  So in the meantime, we wanted to produce a small quantity of ascitic fluid so that we can continue with our downstream processes.  Have you used the Integra CELLline before?  What do you think of the product?

klinmed, on Nov 13 2009, 10:06 PM, said:

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

I really like the Integra flasks.  Have used them for a number of years.  In fact, I have 3 of the CL-1000 ones running at the moment.

The cell compartment contains 15 ml DMEM /15% FCS whilst the nutrient one 1000ml DMEM/1% FCS.  The DMEM is the high glucose variety and contains Na pyruvate and 2-mercaptoethanol.

At the moment I harvest ever 5 days and get ca 14 ml supernatant (1 - 1.5 mg Mab/ml) from each flask.

We use protein-A chromatography to isolate IgG from culture supernatants.
The concentration of the purified immunoglobulin is determined by absorbance at 280 nm using a 1 % extinction coefficient of 13.4. The yield from protein-A CMT is used to approximate the Mab concentration in the Integra supernatants.

[MouseMab]

Do you find the presence of bovine IgG in the media a problem when you are purifying your monoclonals?  We have the CL350 which is a smaller system to the one that you are using - have you used this model before?

klinmed, on Nov 16 2009, 08:51 PM, said:

MouseMab, on Nov 16 2009, 12:24 PM, said:

Thanks for the info about the CELLline flasks.  What method do you use for quantifying the the MAbs in the media?  How do you purify your MAbs from the supernatant?

klinmed, on Nov 14 2009, 11:21 AM, said:

MouseMab, on Nov 14 2009, 07:50 AM, said:

Thanks for the info.  Yes, we do pristane prime the mice - 1 week before injection of the cells (I believe that this has been the practice for many years in our lab - I myself am fairly new to the hybridoma field).  Interestingly, we actually do have the product from Integra that you mentioned, but are still in the process of optimizing the system.  So in the meantime, we wanted to produce a small quantity of ascitic fluid so that we can continue with our downstream processes.  Have you used the Integra CELLline before?  What do you think of the product?

klinmed, on Nov 13 2009, 10:06 PM, said:

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

I really like the Integra flasks.  Have used them for a number of years.  In fact, I have 3 of the CL-1000 ones running at the moment.

The cell compartment contains 15 ml DMEM /15% FCS whilst the nutrient one 1000ml DMEM/1% FCS.  The DMEM is the high glucose variety and contains Na pyruvate and 2-mercaptoethanol.

At the moment I harvest ever 5 days and get ca 14 ml supernatant (1 - 1.5 mg Mab/ml) from each flask.

We use protein-A chromatography to isolate IgG from culture supernatants.
The concentration of the purified immunoglobulin is determined by absorbance at 280 nm using a 1 % extinction coefficient of 13.4. The yield from protein-A CMT is used to approximate the Mab concentration in the Integra supernatants.

[klinmed]

MouseMab, on Nov 18 2009, 03:16 PM, said:

Do you find the presence of bovine IgG in the media a problem when you are purifying your monoclonals?  We have the CL350 which is a smaller system to the one that you are using - have you used this model before?

klinmed, on Nov 16 2009, 08:51 PM, said:

MouseMab, on Nov 16 2009, 12:24 PM, said:

Thanks for the info about the CELLline flasks.  What method do you use for quantifying the the MAbs in the media?  How do you purify your MAbs from the supernatant?

klinmed, on Nov 14 2009, 11:21 AM, said:

MouseMab, on Nov 14 2009, 07:50 AM, said:

Thanks for the info.  Yes, we do pristane prime the mice - 1 week before injection of the cells (I believe that this has been the practice for many years in our lab - I myself am fairly new to the hybridoma field).  Interestingly, we actually do have the product from Integra that you mentioned, but are still in the process of optimizing the system.  So in the meantime, we wanted to produce a small quantity of ascitic fluid so that we can continue with our downstream processes.  Have you used the Integra CELLline before?  What do you think of the product?

klinmed, on Nov 13 2009, 10:06 PM, said:

MouseMab, on Nov 13 2009, 09:44 AM, said:

Has anyone ever had trouble producing ascitic fluid in mice?  We have injected numerous monoclonal hybridoma cultures, but in most instances ascitic fluid is never produced.  The cells are healthy prior to injection and are secreting the specific antibody as determined by ELISA.  Any suggestions?

These days we are not allowed to make Mabs as ascites (by law in my country).  In the old days, we never had many problems. Occasionally a particular hybridoma would make a solid tumour rather than ascites and very occasionally a particular cell line would kill the animals. I assume that you prime the mice using pristane a few weeks before injecting the hybridomas?  Sometimes you need to find the optimal time between pristane priming and cell injection.
An alternative way (and much better for the mice!) is to make Mabs invitro using two-compartment dialysis flasks (check out the Integra web site).

Hope this helps

I really like the Integra flasks.  Have used them for a number of years.  In fact, I have 3 of the CL-1000 ones running at the moment.

The cell compartment contains 15 ml DMEM /15% FCS whilst the nutrient one 1000ml DMEM/1% FCS.  The DMEM is the high glucose variety and contains Na pyruvate and 2-mercaptoethanol.

At the moment I harvest ever 5 days and get ca 14 ml supernatant (1 - 1.5 mg Mab/ml) from each flask.

We use protein-A chromatography to isolate IgG from culture supernatants.
The concentration of the purified immunoglobulin is determined by absorbance at 280 nm using a 1 % extinction coefficient of 13.4. The yield from protein-A CMT is used to approximate the Mab concentration in the Integra supernatants.

The CL350 flasks work as well as the CL1000 ones. The concentration of Mab is usually similar > 1 mg/ml but the harvest volume is of course lower.
The Mab will be contaminated with a little bovine IgG.  If your Mab is a IgG1 it will elute from protein-A at a comparatively high pH and bovine IgG content will be minimal.  In this regard, it should be remembered that Mab produced as ascites contains significant concentration of polyclonal "normal" mouse IgG.
Have never has a problem caused by the low levels of bovine IgG in downstream applications.

[MouseMab]

With regard to the collection of harvests from the flasks - do you wait until you have a sufficient volume of harvest and then pool them and subsequently purify OR do you purify as you collect?  If you wait and subsequently purify then how do you store them in the meantime?

Do you try and maintain the viability of the culture at a certain level?  What would be the lowest that you'd allow the viability to get to?

Sorry that I have so many questions - I am struggling with the purification and trying to identify what the problems might be.

[klinmed]

MouseMab, on Apr 28 2010, 12:16 PM, said:

With regard to the collection of harvests from the flasks - do you wait until you have a sufficient volume of harvest and then pool them and subsequently purify OR do you purify as you collect?  If you wait and subsequently purify then how do you store them in the meantime?

Do you try and maintain the viability of the culture at a certain level?  What would be the lowest that you'd allow the viability to get to?

Sorry that I have so many questions - I am struggling with the purification and trying to identify what the problems might be.

I harvest the flasks every 4 - 5 days and store the supernatants at -20 oC until purification. Usually wait until I have ca 70 ml and then purify on a 30 ml rprotein-A column (usually get 50 - 120 mg of Mab).  When I have looked at viability at harvest I have always ended up totally worried (usually ca 50 %). However, the volume of the cell pellet at each refeeding seems a better index of how things are going. If you get a pellet about 10 % of the sup harvest volume (at each refeeding) then things are going well.
The Integra flasks seem to me fairly "idiot proof" as long as you can keep them sterile. Usually get at least ca 200 - 450 mg/CL1000 flask before disaster (contamination) strikes.

Hope this helps.

[MouseMab]

Hi,

Thanks for the info - I really appreciate it.  Our antibodies appear to be IgM, have you had experience with purification of IgM MAbs?