ProteoCool Pills#21: Chromas a free tool to check your Sanger sequencing results

In the last 20 years several different software packages able to support the scientist in their molecular cloning activities  (e.g primer desing, sequence assembly, transaltiion, vector desing  desing to manipulate DNA and protein sequences  as CloneManager, SnapGene, VectorNTI (discontinued by Thermo from 2019)

Those software are fully integrated set of tools for e cloning simulation, graphic map drawing, primer design and analysis. They are charaterized by a huge number of functions and they require the payment of a licence. 

Are those advanced softwares striclty necessary for a molecular biologist that would like just to check the result of a gene cloning into a mammalian or bacterial expression vector ?

In my opinion NO! 

Those softwares could be certanilly useful (for example to map the primer annealing region, to desing the vector map) but not essential expecially if you have to produce a limited number of clones in parallel.

For example, in this post i would like to present youm  Chromas, which  is a free simple, easy-to-use sequence viewer and editor (able to open chromatogram files(.ab1) produced from automated Sanger sequencers) that could be used to check your sequencing results.

7A. Allign the Chormas sequence with the “Theoretical” DNA template 

using ClustalW or ClustalOmega

or 

7B. Translate the sequence in AA format 

 (Generally I’m using the Translate Tool available at the Expasy web server) 

and allign the translated sequence with the “theoretical AA” template

 using ClulstaW or ClustalOmega

If the sequence show some point mutation or shift, do not  discard the clone but check carefully the chromatogram to see if the mutation/shift is real or is it due to a non-correct Chromas assignment that may happen in case of:

-  overlapping peaks:

- peak with low intensity;

- a sequence regions with multiple residues of the same species;

  

Other similar softwares:

Finch TV

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#18: MabSelect@ a cheaper alternative to proteinA sepharose FF resin for small scale gravity flow purification of recombinant mab

Affinity chromatography which is based on the interaction the Fc region of the mAb molecule with  specifi bacterial proteins as proteinA, proteinG or protein L immobilized on the resin is generally used for the isolation of antibodies from culture surnantants of the cell lines (eg CHO) used for their recombinant production.

The binding specificity and strength of protein A, protein G and protein L is not equally strong for all immunoglobulins and, in the case of IgG, not equal for all isotypes (Link1, Link2)

A generally stronger binding to the Fc region is observed by protein G, however higher binding strength however does not automatically result in better results since also the presence of impurities may influence the binding capacity.

In both, protein A and protein G affinity chromatography, the elution is carried out using a low pH buffer (eg glicine pH 2,7 for proteinG, and citrate pH3,0 for protein A). Generally in protein G chromatography, a stronger eluent is required to elute the  captured antibody from the coloumn.

Thus, protein A chromatography is preferred over protein G since lower levels of impurities are generally obtained and it currently represent the gold standard in mAb pruficiation. 

In general an efficient protein A resin should have: 

- High dynamic binding capacity (able to bind large amounts of mAbs in a short time) which allow high flow-rate without losing mAbs in the flow-through

-  High stability of the resin under regeneration with sodium hydroxide. The number of cycles you can run with the same resin has a huge economic impact;

Often, in the preliminary phases of the pre-clinical research, to identify the best mabs, scientists have to a large panel of different mabs in small amount (from ug to mgs). In absence of robotic platforms dedicated to mab purification, the gravity flow purification may represent a simple and powerful alternative.   

Gravity flow purification require a resin with high porosity, rigidity and low backpressure to avoid resin clogging and guarantee reasonable flow-rate and purification timelines.

The Citivya rProteinA Fast flow resins  (90uM of particle diameter), which represent the gold standard for the gravity flow purification of recombinant monoclonal antibodies is very expensive  (more than 80euro/ml). 

If it is true that this resin could be cleaned and re-generated several time and that thanks to its high binding capability (>35mg/ml)  generally small volumes (100-500ul/sample) of resin are enough to purify mgs of mab samples requited in the preliminary mab screening phase i however its cost may have an high impact expecially in academic laboratories and the identification of an alternative resin with similar performances but low cost is preferable.

In this post i would like to share with you some enocuraging results that i have recently obtained with gravity flow purification by replacing the rprotA FF with the cheaper Mabselect resin which is reported to have 

- similar binding capability  (30mg/ml of Mabselect vs 35mg/ml of rproA FF)

and

- similar particle size  (85um of Mabselect vs 90um of rproA FF)

but it is at least 5 time cheaper (25ml of mabselect cost = 5ml of rprotA resin)

Mab Select resin tested in the 2 following  formats

format 1 (small)  --> 75ul of resin in a Poliprep coloum (Biorad cod. #731-550)

format2 (medium) --> 750ul of resin in a PD-10 empty coloumn  (GE cod. 17-0851-01)

showed flow rate very close to the FF (as you can see in the following videos: protA FF on the left, MAbselect on the right) 

Video 1: Equilibration with buffer

                                                 Video 2: Expi-CHO surnatant loading

and similar results in terms of binding capacity and final mab purity level

In the following picture, 2 examples of purification performed with the MabSelect resin

In both cases the following buffers were used: 

- Tris 2M pH=8 (1ml for 20ml culture) to correct the surnatant pH before coloumn loading;

- Equilibration and washing buffer; Tris 25mM, NaCl 25mM pH 7,2  buffer;

- Elution buffer: 300ul di Citrate 100mM NaCl 60mM pH=3;

- Tris 1M pH=9 (30ul for small size; 300ul for medium size) to neutralize the acid pH after elution

Of course there are many other interesting proteinA resins cheaper than the rProtA FF ND produded from suppliers different from Cytiva as  Tosoh. Biorad,  Thermo that could be also tested.

Personally, i selected the Mabselect since was the one with the particle size more close to the FF and i suspect that this detail can be essential to guarantee a good flow rare  in gravity flow purification. 

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#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.