ProteoCool Pills#25: Choice of the right material is essential to perform DNA and/or protein UV Spectrophotometric quantifications

 As already reported in the ProteoCool Pills#13, and ProteoCool Pills #24 several different methods are currently available to perform quantification of purified DNA fragments and plasmid as well as recombinant proteins and antibodies.

One simple method common to both, DNA and protein quantification is the spectrophotometric determination in the UV range (260nm for DNA and RNA and 280nm for proteins and monoclonal antibodies)

Spectrophotometric quantification has several advantages:

 - Cheap (do not require any specific reagent);

-  Fast (do not require sample pre-incubations);

- Non-destructive (the sample could be recovered);

The main drawback that limits in the past the use of the UV quantification was the fact that standard glass and standard plastic absorbs strongly in UV region and the quartz cuvettes were necessary to perform protein (280nm) and DNA (260nm) quantification.

Quarts cuvettes work well in both UV and visible regions (right from 190 nm) but are expensive, fragile and time consuming, because not disposable and therefore it need to be carefully washed between the different samples.

Of course the same limitation is applicable also to the multiplate reader, because the standard plastic bottom plates cannot be used for measurements in <300-320 nm due to the plastic absorbance.

Solutions:

1) Use a microvolume UV-Vis spectrophotometers (eg Nanodrop) that do not require any specific support (plate or cuvette):

  Pros:

 Low sample volume (2 µl)

 Fast

      Simple

       Cons:

          Less sensitive than cuvettes because the optical path is 1mm instead 10mM of the cuvettes

  Lambert-beer law à  Abs=ebc where b is the optical path

For the same sample e= constant à 1/10 of optical path à 1/10 of Abs at the same c (concentration)

 Need to be carefully cleaned (protein buffers are rich of salt and the surface properties of the pedestals can be compromised and the samples drop Flattens out and the read are not reproducible

       2) Use Plastic UV-Cuvette or  UVclear multiplates:

In the recent years special plastic compounds with low absorbance at wavelength >220nm were developed:

In this post I would like just to provide some example of comparision of background Abs260 and Abs280 signal obtained with standard and UV-transparent plastic matherial:

Semimicro BRAND 1.5ml PMMA cuvettes (Link)

BRAND UV-Cuvette SEMIMICRO

In conclusion, expecially for DNA determination, standard plastic cuvette and multiwell plates cannot be used. The UV transparent plastics represent a nice, cheaper alternative to quartz cuvette. If it is true that those support are little more expensive than the corresponding made by standard plastic matherial, it is also true that in the most of the cases, after carefull washing with milliQ water and ethanol20%  can be re-used many times. 

 

ProteoCool Pills #23: Selection of the right post-coloumn filter is essential to detect protein/antibody aggregates in static light scattering

Static light scattering (SLS) is a technique to measure absolute molecular weight using the relationship between the intensity of light scattered by a molecule and its molecular weight and size. 

Some SLS technologies exist: multiangle light scattering (MALS), right-angle light scattering (RALS), low-angle light scattering (LALS) and RALS/LALS hybrid systems 

MALS or RALS/LALS when coupled with other detectors (eg UV-vis, RI, densitometry, fluorimeter) in an advanced GPC-SEC system can be applied to investigate solution properties, stability testing and process development for different kind of  conjugated and unconjugateed biologics

Light scattering detectors (low-angle LALS, right-angle RALS, or multi-angle MALS) are very sensitive to the presence of particulates, when those are used for molecular weight detection in chromatography. Unfortuantelly even the best  columns can shed some particulates from their packing material. Although undetected by most conventional detectors, such as UV and RI, these particles scatter significant amounts of light, produce noise that affect the light scattering signals and baselines. To mitigate this, the MALS, LALS/RALS sistems include an in-line coloumn filter that can significantly improve the quality and thus the accuracy of both the data and results.

In this post i would like to share with you some data acquired loading different mabs in a Cytiva Supedex200 increase 10/30 coloumn in the OMNISEC instrument equiped wih the LALS/RALS detector in the presence/absence of post coloumn filter. 

First of all we compared the baseline signals that can be obtained 

- Without filter;

- With a 0,2um nylon filter;

- With 0,2um cellulose filter; 

The baseline signals suggest that nylon filter is the best in terms of signal/noise ratio 

Is it really the best choiche for analisys of protein/antibody preparations? 

To assess it, a 2% BSA standard and 2 different purified monoclonal antibodies (igG1 human) were loaded in the superdex200 10/30 increase coloumn and analized with the OMNISEC instrumentation  

In all the 3 cases, the Nylon filter guarantee the best signal/noise ratio at LALS but it seems to mask the presence of a significant fraction of high molecular weight proteins.

This was most evident for the mabs, since the dimeric-trimeric and MW aggregates were not detected, even if at UV and RI, using the nylon filter.

ThIs differences may lead to overtimating the quality of a certain mab or protein preparation and it can lead to false positive results in case of stability studies that  would like investigate the mab/protein aggregation propensity under stress (as acid pH, 37°C, freeze/thaw)

For this kind of studies cellulose filter seem to be the best compromise between quality of LALS signal and recovery of the protein polimers. 

As a general comment: Often the diffence is in the details! It is important be able to critically review the Positive results to distinguish the real positives than false positives! 

Suggested links: 

https://www.materials-talks.com/how-to-change-the-light-scattering-post-column-filter-membrane/

ProteoCool Pills#22: Tips for cleaning the OMNISEC RALS/LALS flow cell if get dirty after the passage of biological samples

 

                                                                           OMNISEC 

is an advanced GPC/SEC system combining a pump, degasser, autosampler for mobile phase delivery and sample injection module with an integrated multi detector incorporating refractive index, UV/Vis, light scattering and viscosity detectors.

I have used the OMNISEC system to analyze the aggregation state of several biological samples (eg. recombinant antibody/protein preparations) in standard Phospate, MES or Tris buffers (pH range 5.5-8).

I found amazing the performances of the OMNISEC RESOLVE module, that thanks to the presence of a temperature controlled Autosampler (4^oC – 60^oC), allow to load in reproducible way an high number of samples and guarantee a very good throughput.

One of the main drawbacks (which i'm not sure if is it is common or not to other light scattering systems) that i found is the high tendency of the LALS detector to get dirty after the passage of some biological samples.

In this post i would like to share with you my experience about the cleaning procedure to use when you  see a strong increase of the LALS baseline signal after the passage of biological samples.

For example in the following video yoo can see the baseline LALS signal that was detected some months ago after the run of about 20 mab samples in a SEC coloumn (all the mabs were expressed from ExpiCHO cells, purified with MAbselect resin and buffer exchanged in PBS by desalting) :

If the RALS signal is not much higher than the optimal (aobut 80mV), the LALS signal was very high (optimal range is 200-300mV) and a cleaning procedure was required.

Since we do not observe any improvement (data not shown) from the passage of any routine cleaning solutions (methanol 10%, acetonitrile 10%, sodium azide 0,02%) suggested on pag106 OMNISEC SYSYEM Basic Guide manual provided with the instrument, we then decided to proceed with Deep cleaning (pag.105) using a 5% HNO3 solution:

        BUT UNFORTUNATELLY WE OBSERVE ONLY A WEAK REDUCTION IN THE LALS BASELINES SIGNAL

we than tested SDS (1% solution) which may be able to resuspend and remove protein aggregates/precipitate:

BUT UNFORTUNATELLY  ALSO IN THIS CASE WE DO NOT OBSERVE A REDUCTION IN THE LALS BASELINES SIGNAL

Finally we tested NAOH 0,1M solution, which is routinelly used for the cleaning of  several chromatographic resins  used for biologic purification  (e,g proteinA, proteinG, sepharose)

NAOH 0,1M works very well 

and restore the LALS BASELINES SIGNAL in the optimal range

 Thanks a lot to 

 Mirco Toccafondi 

ProteoCool Pills#20: Micro PEG solubility screening, a simple Tool for Biologics Design and Formulation Development.

Adequate protein solubility is an important prerequisite for development, manufacture, and administration of biotherapeutic drug candidates, especially for high-concentration protein formulations. 

For example, in monoclonal antibody discovery, early identification of monoclonal antibody candidates whose development, as high concentration (≥100 mg/mL) drug products, could prove challenging, due to self-interaction that may induce high viscosity, can help define strategies for candidate engineering and selection.

If in theory, Rheology measurements are an effective means for characterizing therapeutic protein/antibody solutions, practically, the conventional measurements are hindered by the limited amount of material, especially during early development, when it is necessary to screen and compare several different molecules over a wide range of conditions (e.g different pH, additives, concentration)

Therefore alternative techniques able to provide hints about aggregation propensity and solubility using a smaller sample volume are essential to compare and select the best candidates in the early development and reduce the risk of move forward an candidate with high developability risk. 

Dynamic light Scattering (DLS) is probably the most used technique for this purpose since it allows to:

1) Characterize the sample intermolecular interactions (attractive or repulsive?) comparing how the diffusion coefficient (Dt) is affected by concentration since: 

In an ideal dilute solution, the diffusion coefficient (Dt) measured by DLS is not dependent on solute concentration. As concentration increases, the solution becomes less ideal. 

           Dt=D0(1+kD*C)

Attractive interactions (kD < 0) cause an apparent decrease in Dt and an apparent increase in Rh, while repulsive interactions (kD > 0) cause an apparent increase in Dt and an apparent decrease in Rh 

Therefore, decrease of Dt in function of the concentration, indicating the presence of repulsive intermolecular interactions while increase of Dt in function of the concentration, indicate presence of attractive interactions (sample more prone to aggregation)

2) Perform viscosity assessment by Microrheology: Using polystyrene beads with known values of R allows for the determination of the viscosity of the protein solution that the beads are suspended in. The size of the beads is larger than that of the protein molecules, and thus the DLS signals can easily be separated.

Bilayer interferometry (BLI) was also recently proposed as an alternative to DLS to assess protein self-interaction. (Sun et.al mabs 2013;  Domnowsky et. al International Journal of Pharmaceutics 2020) 

All those methods are fast and require a small amount of material but they require specific and expensive instrumentations those are not present in all the laboratories.

In this post, I would like to introduce you a simple method that could be done in every laboratory (since it requires the presence of a centrifuge for plates and nanodrop UV spectrophotometer or similar) based on PEG precipitation, a previously established method for determining the relative apparent solubility of adapted for screening in small scale and which is reported to correlate with the Kd values obtained by DLS (Scannell et.al, Pharm Res 2021) 

This method, adapted in 96 well plate allow to compare monoclonal antibody (mAb) candidates also if only limited quantities (eg. 1 mg) are available. 

Protocol 

(for A280nm reading with Nanodrop or similar)

(adapted from Toprani et.al J Pharm Sci. 2016) 

Day1 (afternoon)

1) 25ul of monoclonal antibody at 1mg/ml mixed with 25ul of PEG10K solutions at different concentrations (from 32% to 8%) in a 96well V-bottom plate

                           Example of a plate assembled to test 6 different mabs in duplicate: 

2) The plate was covered by aluminum foil and incubated O/N at 4°C

Day2

3) The plate was centrifuged 1h at 3200g at 4°C; 

4)10ul of surnatant were carefully transferred in a 96 well PCR plate (using a multichannel pipette). V bottom plates are preferable since the form of the well reduce the probability to resuspend the precipitate during the surnatant pick-up;

5) Amount of mab present in the surnatant was quantified by measuring the A(280nm) by NanoDrop Spectrophotometer;

6) Relative soluble fraction is calculated and plotted as function of PEG concentration;

Example of results

Example 1

 Comparision of  PEG solubility for 3 different mabs  (human igG1-CLk) in PBS buffer

Example 2

Comparision of PEG solubility for 2 different mabs in 2 different buffers (different pH)

The main limit of this protocoll is represented by the throughput, since sample reading by nanodrop allow to scale down the protocol and reduce a lot the protein amount but it is not very fast. 

6 mabs in duplicate --> 96 well --> more than 1h at nanodrop

For high number of samples, you can run a modified verision of the protocol, based on A280 deterination using 96well half volume UV clear plates (Greiner cod. 675801) in a multiplate reader. 

Since, in multiwell reader, the A280nm value change in fuction of both, concentration and optical path (that is function of the sample volume)  in this case to obtain a good sensitivitty the reaction volume (step1) were doubled  (50ul of mab 1mg/l + 50ul of PEG solution) and after centrifugation 70ul of the surnatant were tranfered to the  UV-clear 96 plate for the A280 determination with the multiplate reader. 

6 mabs in duplicate --> 96 well --> 1-2 minutes

Example 3

Comparision of PEG solubility for a wild tipe mab vs some mutant in PBS 

A(280nm) measure with Biotek- cytation5 multiplate reader

Microplate reader vs nanodrop:

Pros:

 Throughput (A plate could be acquired in few minutes)

Cons: 

Double amout of material required: (0,4 mg vs 0,2mg of mab each coloum)

Cost of the plates (about 8 euro/plates

Materials: 

- PEG 10K (Alfa Aesar cod. B21955)

- 96 well V-bottom plates (Costar cod. 3897)

- 96 UV-clear half volume (Greiner cod. 675801 cost ~ 8 euro/plate)

Other references: 

https://www.americanlaboratory.com/media/20/Document/DLS-Microrheology.pdf

ProteoCool Pills#17: Alternative cheaper trasfection agents for recombinant protein/antibody expression with Expi293

 As already reported in the ProteoCool n° 29, Expi293 are a powerfull cell line those, thanks to their ability to growth up to very high cell density and be transfected with high efficiency are able to secrete in the surnatant high yields of recombinant proteins.

The main factor that currently limit the application of this system is certanilly the cost, since the volumetric cost (euro/liter of culture) is very high and comparable with the cost of a 13C/15N labelled sample in E.coli.

Cost of 30ml culture of Expi293 (updated to July 2022)

Components:

a) Expi293 expression medium (cod. A1435101 Thermo)  cost: 331euro (quotation 2022) --> 1 liter 

b) Expifectamine trasfection kit (cod. A14524 Thermo) cost:  1100 euro  (quotation 2022) --> For 1 liter

c) Cup vented Corning shaking flaks 125ml cod. 734-1885 (VWR quotation 2022) --> 8,8 euro/flask

d) DNA preparation  (EZNA mini kit II cod. D6945-02) 1 -> euro/1 sample – Thermo midi kit cod. K210004 cost 4euro/1sample)

Therefore the total cost of a 30ml tranfection 

 9,9(a)+ 33(b)+8,8(c)+1-4(d) = 50-55euro/flask  --> Around  1.800 euro/liter

and the tranfection reagent contribute to more than 50% of the costs (33 out of 55 euro)

Since the Expi293 become progressivelly more popular, several company develope alternative  tranfection agents that are suggested to be suitable for Expi 293.

In this post i briefly sgow you the results that i obtained by comparing;

- The standard Expifectamine 293 Transfection kit ;

- Endofectin Expi293 transfection kit;

- FectroPro transfection kit 

2 different expression trials in 2ml format (6 well plates)

Trial 1: 2 recombinant His-tagger protein; 

Trial2; 2 recombinant monoclonal antibodies)

 performed to compare the  producivity of the different trasferction agents;

In all cases the only reagents that are different are those part of the transfection kits

, while culture media (Expi293 culture media) and antibiotics, are the same 

alteady listed in ProteoCool n°29

Summary:

Those preliminary data suggest that both Endofectin Expi293 and Fectopro

 show performances close to the Expifectamin293  Thermo reagent with Expi293 cells

Of course the trails was performed with a limited number of protens and few replicates and more data need to be performed to confirm it, but it seems that the differences in protein/antibody productivity, if there are differences, are much less important than the difference in terms of costs.

The same comparision performed with the Expi-CHO kit was not successfull (data not shown) 

probably because the Endofectamine293 and Fectopro Kit do not contain any medium Feed

 which seems to be essential to allow the ExpiCHO cells to growth until high cell densities

  (trasfection with ExpiCHO is performed at 6*10^6 cells/ml vs 3*10^6 cells/ml used for the Expi293) and retain good viability during the entire trasfection(8 days).

ProteoCool Pills#16: Loading small sample volumes on AKTA instruments

AKTA instruments (as AKTA-FPLC, AKTA-PRIME, AKTA-Purifiers, AKTA-Go, AKTA-pure) represent in the last 30 years the point of reference for protein/antibody purification in both research, development and pharmaceutical production departments since they are robust, easy to use and provided with a software (the Unicorn)  which is a reall uses friendly software.

On the contrary, their application for protein/antibody analytics characterization is mainly limited to some specific columns and approaches (eg SEC chromatography with the Superdex 10/30 or 5/15 coloumns) in those laboratories that do not have budget dedicate to acquire an analitical HPLC. 

 In those case the loading and injection of a small sample volume in reproducible way it a prerequisite.

How we can inject small volumes in an FPLC or other AKTA instruments? 

For volumes between 200-300µl to 1ml,  the standard injection port could be used with an 1ml insulin plastic syringe (with out needle)

For volumes <250µl ,  the standard injection port need to be replaced with a injection filling port designed for small volumes and use an more precise Hamilton syringe (250µl or 100µl volume) for sample injection.  

Until some years ago (as I already wrote to answer to a question in Research gate in 2019) https://www.researchgate.net/post/Loading-small-sample-volumes-on-FPLC), Citiva bioscience (previously GE bioscience) supplied for small sample injection a metal injection filling port (CITIVA cod. 19768701) supplied with a plastic syringe stand suitable for injection of small volumes using Hamilton syringes

Recently those Injection fill port was discontinued and replaced by a plastic filling post (Fill Port, INV-907 cod.18112766)

Also if this filling port, is not supplied with any syringe stand, it could be used directly with an hamilton syringe, as you can see from the following imagine:

Of course to inject so small volumes you need to select also the appropriate:

•          Injection loop (with a small volume)
•         Syringes (with BLUNT tips:

Suggested:

50-100ul sample:  Hamilton 1710N volume 100 μL, needle size 22s ga, needle L 51 mm; Sigma-Aldrich cod. 28634-U cost ~100€;

250-100ul samples: Hamilton® 1725N, volume 250 μL, needle size 22 ga (blunt tip), needle L 51 mm; Sigma-Aldrich cod. 28636-U cost ~80€;

 

 

ProteoCool Pills#15: An alternative way to fix the sticky pad to the Eppendorf S41i CO2 shaker

In the last 20 years, thanks to the adaptation in suspension of several cell lines and to the development of more efficient transfection agents (eg lipofectamines) and culture media, the scientist were able to obtain amazing progresses in the performance of mammalian expression systems, 

As I already reported in the ProteoCool n° 29 and in the tips of the week#8 , modern cell lines, as  ExpiCHO and Expi293, are able to produce high levels (up to 300-500mg/l) of recombinant antigens and/or antibodies in the culture surnatants also in shaking flask culture. 

The availability of an orbital shaker able to maintain 5-8% of CO₂ atmosphere is essential during cell propagation and maintenance.

Differently to bacteria, the cell lines do not require high shaking rates (generally 120-125rpm) but are more prone to contamination, therefore since the cleanliness is essential, the use of sticky pads, those can be easily removed and sainted with ethanol or isopropanol solutions is preferable respect than the conventional flask clamps.

To my knowledge Eppendorf S41i (previously New Brunswick) and  the Infors multitron  are the mostly used for mammalian cell cultures.

In this presentation I would like to share with you,  my experience with the Eppendorf S41i

In our lab we have 2 Eppendorf S41i shakers:

- 1st setted at 37°C- 8% CO₂ and 120rpm for propagation of both Expi293 and ExpiCHO and for transfection of Expi293 cells;

-  2nd setted at 32°C- 5%CO₂ and 120rpm for the transfection of the ExpiCHO cells; 

The first shaker was acquired in 2016-2017 while the second in the 2020. 

Our older S41i shaker carry a platforms characterized by a smooth surface and the sticky pads attach well  directly to its surface

                   while our newest  S41i shaker carry a platforms characterized by a rough surface and the                         sticky pads don’t stick well to its surface.

                   Eppendorf sell a Stickpad ADAPTOR KIT which contains 2 metal smooth plates

                                but this kit seems to be designed for a different shaker format since

               the sizes of those plates are not appropriate to cover the entire area of the shaking platform.

Since the Eppendorf S41i is smallest than other shaking incubator, e.g. the multitron, 

 giving up such a large part of the plate surface represent an important limitation that may 

induce the scientist to choose a different shaker for their laboratories. 

In this post, I would like to show how we were able to cover with the sticky pad the entire shaking platform of both our shakers

           You can use the fixing screws supplied with the conventional flask clamps or wtih the Stickpad ADAPTOR KIT  to drilling and fixing the sticky pads directly on the black  shaker platform.

Standard Washers with 6mm in diamteter

 those can acquired in the most hardware stores /e,g  Leroy Merlin, Obi, Bricoman, Brico ) to ensure the sticky pad integrity and extend the life time of this solution.

N.B I'm happy to share with you that, as responce to this post, Eppendorf specify  that all the platforms actually produced are chatacterized by a smooth surface and the sticky pads are able to attach directly to it. 

 

ProteoCool Pills#14: Hints for cell propagation and recombinant protein expression using Expi-CHO and Expi293 (WT and GNTI-)

For many years the application of the traditional adherent mammalian cell cultures for recombinant protein production was difficult and applicable only in those approaches requiring very low protein amount (ug’s) because cell scale up was very difficult, transfection not very efficient and the final volumetric yields (mg/litre) very low. 

This limitation forces the scientist to use other simple eukaryotes expression systems as Pichia Pastoris or Baculo virus for the production of mammalian protein in mg scale.

However, in the last 20 years some technological improvements as:

 1) The adaptation of several cell lines to suspension cultures;

 2) The development of more efficient transfection agents;

 3) The development of serum free media where the cells are able to growth at high cell density;

Improve drastically the performances of the mammalian expression systems;

As I already mention in previous presentations, theExpi293 and Expi-CHO date, represent 2 of the most performing mammalian transient expression systems. 

If it is true that those systems are quite expensive, on the other hand they are very simple to use and guarantee, high yields also in shake flask. 

For some recombinant protein, Expi293 in shake flask were able to provide me up 300mg/yields. 

Values that until some years ago were reachable only using E. coli expression in high cell density set-up (e.g. feed-batch fermenters)

Expi-CHO are able to provide up to 100-200 mg/l of full length recombinant antibodies. Full length antibodies are big (150KDa), composed from 2 chains and contains several disulfide bonds, their expression in bacterial expression system is very challenging.

However, to reach those results details, as;

-             - The selection of the optimal shake flask format and right culture volume;

-              -  The selection of an antibiotic not affecting cell growth and protein expression;

-                The selection of the optimal signal peptide for protein secretion into the cell surnatant;

can make a huge difference!!

The selection of the right shake flask and culture volume

Expi293F and Expi293 GNT- cells :

In small scale (up to 60ml culture) Expi293 are not so sensitive to the flask format and presence of baffle:

For example, in our experience Expi293 are growing well in both

-        Erlenmeyer flaskscup vented plain (cost ~ 8€/flask for 125ml format)

-        Erlenmeyer flasks cup vented baffled

-        Thomson Optimun growth flaks  (cost ~ 8€/flask for 125ml format and 30€/flask for 1,6l)

Optimal culture volumes: 

125ml flask volume for 30ml Expi293 transfection and 250ml flask volume for 60ml Expi293 transfection

For culture scale up (eg 200-500ml culture),  the Thomson Optimun growth flaks eems to perform better probably  because it guarantee better CO2 and oxygen exchange.

My preferred format is 200-300ml transfection in 1.6liter flask and generally we do not exceed the 500ml single transfection (using a 2.4 litre flask) because in high volumes we more frequent bacterial contamination was observed, therefore in case of large culture volume e.g. 1-2liter, I prefer to split the culture using more flask in parallel.

ExpiCHO:

ExpiCHO show an high tendency to aggregate and are much more susceptible to  the flask format.

Thomson Optimun growth flaks https://htslabs.com/og/ that contain small baffles seem to represent the best compromise and are the format more able to prevent the cell aggregation during the cell maintenance passages.

N.B: Expi-CHO are able to growth up to very high cell density (>10milion/ml) but to avoid cell aggregation is it better do not exceed the 6 milion/ml during the cell propagation passages

Optimal culture volumes: 

   125ml flask for 25ml transfection 

   250ml flask for 50 ml trasfection

    1,8liter flask for 150-300ml tranfection

In all cases we use the max titer protocol à 8day of trasfection at 32°C

The selection of the right antibiotics to prevent cell contamination.

The Expi293 and ExpiCHO Thermofisher manuals do not suggest the addition of any antibiotics to prevent bacterial contaminations. However after that we experienced some culture contaminations, considering the high  cost of the reagents (more than 1000euro/Liter)  we decided to perform some  transfection trials in the presence of  antibiotics to see if they affect or not the cell viability, growth rate and recombinant protein expression

Expi293 are not affected by:

-        Pen/strep (thermo cod. 15070-063) diluted 1:100 (50 unit/ml penicillin and 50ug/ml Streptomycin)  

-        Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml)

Generally, we use for cell propagation and transfection the Pen/Strep 

Expi293 GNT- are not affected by Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml) while reduction in cell growth rate and viability was observed in presence of Pen/Strep;

Expi-CHO are not affected by Pen/strep (thermo cod. 15070-063) diluted 1:100 while reduction in cell growth rate and viability was observed in presence of Gentamicin diluted 200 times.