ProteoCool Pills#27: DSF an useful tool for Monoclonal antibodies selection, optimization and developabiltiy assesment

 

DSF 

(Differential scanning fluorimetry previously named also Thermofluor)

is a very simple tecniques that exploit the ability of a fluorescente probe,  typically either SYPRO Orange or 1-anilino-8-naphthalenesulfonate (ANS) – that is quenched in an aqueous environment but becomes strongly fluorescent when bound to exposed hydrophobic groups of a protein. Therefore, the thermal unfolding of a protein of interest in the presence of such a dye, can be followed spectrophotometrically.

DSF is a 96 well based assay that could be performed on a RT-PCR thermocycler, it is inexpensive,  fast, and require relatively little sample. All these advantages have made this approach attractive for screening applications in drug discovery  and also for protein stability formulation

In the ProteoCool n°24 in suggest as in my opinion the DSF may become an essential assay todetermine lot to lot concistenciy in QC department of company producing recombinant proteins (e.g recombinant vaccine and/or enzymes)

In this post i would like to share with you 2 examples that show as DSF may play an important role also in antibody screening, optimization and developability assesment. 

DSF thanks to its excellent throughput and minimal material consumption (about 50ul of antibody at 0,3 mg/ml for each point) allow to readily compare the thermal stability of:

- different antibodies in the same buffer:

-  different  Fab mutants or Fc mutants in the same buffer;

-  different  antibody formulations; (optimize pH, salt concentration, excipients)

Example 1: 

Comparision of thermal stability of different mabs 

Full length human IgG1-Clk mabs  in PBS may show 1 or 2 separate transitions on the basis of the Fab stability. 

Mabs with lower Fab stability show in general a single transition while mabs with high stability show 2 separate transitions, 1 at about 69°C for the Fc and 1 at higher temperature for the Fab.

Example 2:

Comparision of thermal stability of different Fc mutants

In recent years the enhancement or elimination of the Fc-effector function has led to a growing interest in Fc-engineering, to give antibodies specific mechanisms of action and therapeutic properties.  

Even if several Fc modification, as:

-   LALANA, LALAPG, LALAGR mutations to remove effector function;

-  SDIE, GAALIE mutations as well as afucosilation to enanche the effector function

 were already reported, few data about developability of those solutions are still available and there is a lot work  to do to select an enanched Fc combining the best effector function and good manufacturability, stability and pharmacokinetics.

Those are just 2 examples that show as in short time and using a limited amount od samples, thanks to DSF we can obtain  essential informations to guide our decision in antibody during the screening, desing, formulation phases.

Of course, thermal stability itself do not Guarantee that the selected antibody has good developability profile but  some of the following other tecniques as:

- DLS;

- PEG solubility;

- Self-interaction determined by BLI;

those provide informations about antibody  aggregation propensity 

- Hydrofobic interaction chromatografy (HIC)

those provide informations about antibody hydrofobicity

have to be peformed to complement the DSF data. 

A great thanks  to

 Luca Sorrentino

 

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.