ProteoCool Pills #32: HemA, a powerfull selection marker for antibiotic free plasmid mantainance for recombinant protein expression in E.coli

Antibiotic resistance genes  (e.g Ampicillin, Kanamycin) are the most commonly used markers for plamisd selection in DNA production and recombinant protein expression processes. 

Adding an antibiotic resistance gene to the plasmid solves 2 problems at once:  It allows a scientist to easily select the plasmid-containing bacteria when the cells are grown on selective media and at the same time provides those bacteria with a pressure to keep your plasmid.

For the reason the most of commercial vectors suitable for recombinant protein expression in E.coli  as pET,  pQE, pMal, pCold, pGEx carry Ampicillin, Kanamicin selection marker. . 

Even if this strategy work very well in R&D setting, on the contrary it has several drawbacks for biological manufacturing. 

The spreading of antibiotics in the environment and consequent emergence of multi-resistant pathogenic bacterial strains has become a general promise to even further increasome categories of biological products such as DNA vaccines where potential issue of allergic reesponses to some classes of antibiotics is evoked and the necessity to document the trace amount of antibiotics. 

Apart from the use of the antibiotic itself, there is another emerging limitation in the use the antibiotic resistance gene used as a selection marker due to the potential risk of horizontal transfer of antibiotic resistance gene to environmental microbes

Behind the regulatory issues, the use of antibiotic resistance gene marker imposes a significant metabolic burden on the cells and may. also impact the process yield. 

At least but not last,antibiotics themselves are expemsive and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. 

In this context novel strategies to efficiently replace antibiotic-based selection are required. 

To date, several systems have been developed based on different principles, each presenting advantages and drawbacks. 

The most common way to achieve selection in the absence of antibiotics is the complementation of an essential gene making use of an expression vector in a strain with a defect or inhibited expression of the same essential gene.

Several examples are reported in literature where the plasmid selection is achieved through the complementation of amino acid auxotrophy (e,g Proline;  Glycine)  and more recently of QAPRTase, an enzyme implied in de novo nicotinamide adenine dinucleotide biosynthesis. 

Many different E.coli auxothopic strains were already generated but to ensure the proper selective pressure, the auxotrophy complementation systems reported so far require the use of chemically defined media since the standard complex media contain variable amounts of aminoacids as well as other catabolites that covercome the need of biosynthetic pathway and as a consequence the loss of selective pressure for the complementation plasmid maintenance.

About 10 years ago I was involved in a project aimed to developed a novel antibiotic free plasmid selection approach for protein selection in E.coli. 

Some experiences gained in previous work activities such as: 

1) The positive effect that the supplementation  δ-ALA (δ-aminolevulinic acid) has on recombinant Human cyt c expression in E.coli (CERM)

2) The use of 5-aminolevulinic acid (ALA) as  a prodrug to stimulate  intracellular Heme biosynthesis to produce the natural photosensitizer (PS) Protoporphyrin IX (PpIX) in antimicrobial photodinamic therapy (Molteni Therapeutics)

lead me tot think that the complementation of hemA deficient bacteria by a vector carrying a functional hemA gene, as a selection marker, which confers to the transformed bacteria the ability to grow and maintain the vector in any medium that does not support growth of hemA deficient bacteria of a mutation in heme biosynthetic pathway and at the same time the complementation of empty cells with 5-ALA allow to easly propagate and prepare the empty competent cells.

After several months of work we produced an interesting data package supporting our hipotesis and a patent application was filled and submitted (WO2015165841 - AN ANTIBIOTIC-FREE METHOD FOR SELECTWO2015165841 - AN ANTIBIOTIC-FREE METHOD FOR SELECTION OF TRANSFORMED BACTERIA)

Even if the patent was not accepted since the examing authority do not recognize its inventive steps since the HemA was already show to work as selectable marker in Aspergillus Oryzae, i still think that it is work very well in E.coli since we was able to show that the Delta HemA E.coli  show negligible growth in both chemical defined and complex media but the growth it readly restored when there are complemented with 5-ala or plasmid carryng (see Fig2a,2b and Fig3a,3b of the WO2015165841 patent application)   the HemA gene and that the expression was mantained after a several different passages.

E. coli BL21(DE3) DeltaHemA/pet21-BFP and E. coli BL21(DE3)/pet21-BFP were cultivated without antibiotic selection for many generations by diluting a 12h culture 1:100 in fresh medium for several times. After 1,2,5,10,15 passages the recombinant protein expression was analyzed (1G ≈ 7-8 duplications) and as reported in the Fig5 of the WO2015165841 patent apprication,  the BFP production in HemA mutant strain complemented with BFPpet24-HemA is stable after 15 cultivation cycles without antibiotic while in the wild type strain transformed with standard BFPpet24, it dramatically decreases after 5 cultivation cycles without selection.     

 I would like to Thanks to

Maria Giuliani

that performed the most of the experimental work

and provide an essential contribute in experiment desing 

 

ProteoCool Pills #31: Modern Approaches to modulate the antibody effector function

Full length human IgG antibodies are composed of 2 regions, the N-terminal region of each chain corresponding to the Fab (fragment for antigen binding which contain the variable regions and the CDRs) and the C-terminal region corresponding to the Fc (crystallizable fragment)

Until some years ago the scientist focus mainly their efforts in the optimization of the Fab regions to develop antibodies with highest antigen affinity and lower immunogenicity.

In the last years, especially with the emergence of bispecific antibodies, e.g T-cell engagers there has been a growing interest in modifying the Fc region to modulate (enhance or remove) the antibody effector function.

The Fc region is able to bind with high affinity the Fc gamma receptors (FcγRs), and the neonatal Fc receptor (FcRn) expressed in the surface of the immune system cells (Reference)  by triggering  different effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and complement- dependent cytotoxicity  (CDC) responses (Reference)

Among the four subclasses of human IgG (IgG1, IgG2, IgG3, IgG4), which differ in their constant regions, particularly in their hinges and CH2 domains, IgG1 has the highest FcγR-binding affinity, followed by IgG3, IgG2, and IgG4. As a result, different subclasses modulate in different way the different responses ADCC, ADCP and CDC.  (Reference)

In in the first generation of monoclonal antibodies the Fc activity was modulated by producing of the antibody in different Fc format on the basis of the antigen properties and the MoA (mechanism of action) that they would confer to the antibody: 

Blocking antibodies that do not have to activate the immune response where produced in the IgG4 format

For example Nivolumab which binds to the PD-1 receptor located on the cell membrane and inhibits its interaction with its ligands, PD-L1 and PD-L2. Since PD-1 is expressed on activated T cells, NK cells, regulatory T cells and B cells. Nivolumab has been desinged to block the interaction with the PD-L1 and PD-L2 and therefore releases immune cells from pathological immune suppression, but not activate the immune-response against these immune-cells otherwise we will obtain the unwanted depletion of effector cells inducing  serious adverse events..

On the contrary, mabs targeting antigen expressed in the surface of cancer cells (e.g Cetuximab targeting EGFR) were produced in igG1 form to activate the immune-response against those cells and induce cell clearance. In fact, it has been demonstrated in preclinical models and ex vivo studies that target-bound cetuximab IgG1 isotype mAbs stimulate natural killer (NK) cell–driven cytotoxicity against tumor cells coated in mAbs via the interaction of the constant region and the CD16 receptor on NK cells

More recently  igG1-Fc sequence engineering were largely exploited to further improve the modulation of Fc function:engineering as well as CHO cell line 

Fc Function enhancement:

It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody (N297 of IgG1).

 In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity.

As I reported in the ProteoCool Pills #10, the simple supplementation of the culture media with  2F-Peracetyl-Fucose allow to produce low fucosilated monoclonal antibodies in mg scale to be used for R&D screening, on the other hand, several strategies (as the GlymaxX® and POTELLIGENT^®  have been developed to build stable clones for large scale production based of afucosylated antibodies with improved therapeutic potency.

Since those cell lines may show some drawbacks in terms of growth rate and mab productivity at the same time other scientists focus also in the development of igG1 mutant (eg S239D/1332E  named SDIE and G236A/S239D/A330L/I332E named GASDALIE – US patentUS20230057150A1  those similarly to afucosilated mab show improved affinity for the FcγRs even in produced in the standard CHO cell lines.

In the past I had the opportunity to do some trials comparing the ADCC activity (using the Promega ADCC assay, both F and V versions) with using the igG1 WT afucosilated and igG1 SDIE mutant (produced with standard fucosilation) for the same clone and interestingly  (data not shown) it seems that their effect could be combined at least from the results of this in vitro assay. 

B) Fc Function silencing:

The most recent alternative to the use of IgG1, which allow to point mutations in the linker region between hinge and Fc domains were described to reduce or fully abrogate Fcγ binding affinity and downstream effector cell activation

In the following Table are reported some of the most known and used mutations:

From literature the STR (recently developed by MAbSolve seems to be the only mutation that completely abolish all the ADCC, ADCP and CDC activities (Reference) and  it seems that STR do not alter the antibody developability profile.

Even in this case I had the opportunity to do some R&D trials comparing the igG1 human wild type, LALAPG and STR mutants and I was not able to was not able to reveal meaningful differences between LALAPG and STR with the Promega ADCC assay (complete abolishment in both cases – data not shown) but this can be due to the fact that as shown in seems that the advantage of STR vs LALAPG is in the lower affinity of STR for the FcγRI which is probably more involved in trigger the ADCP than ADCC response or that the sensitivity of those kind of assay is not enough to detect so small differences (see Table3)  the authors do not reveal any differences even in ACDP assays

I also performed antibody aggregation propensity detected performing SEC on 10mg/ml samples subjected to different incubations (4°C, 37°C, Freeze/Thaw) and in this case the LALAPG seems to be slightly better (data not show)

However, this post does not would provide any ranking or evaluation of the different mutants but just inform you about the possibility to test them to achieve the wanted MoA for your new monoclonal antibody.

 

 

 

ProteoCool Pills #30:How improve Specific heavy-light chain pairing in the production of bispecific mabs

 

A Bispecific antibodies (BsAb) is a single molecule that comprise two single binding entities that are physically connected, which enables simultaneous binding to two different epitopes  on two different antigens or even in some cases two different epitopes on the same antigens (named biparatopic antibodies). 

The ability of BsAbs to bind to different antigens was largelly exploited in immunotherapy to act as a linker beetween  a cytotoxic cell and a target (a tumour cell) to be killed. 

For example T-cell-redirecting bispecific antibodies  (named T-cell engagers) are specifically designed to bind to tumor-associated antigens, thereby engaging with CD3 on the T cell receptor. This linkage between tumor cells and T cells actively triggers T cell activation and initiates targeted killing of the identified tumor cells. These antibodies have emerged as one of the most promising avenues within tumor immunotherapy.

The main issue associated with asymmetric BsAbs is mispairing of polypeptide chains leading to product-related impurities. In fact BsAbs assemble require co-expression of 4 different chains (2 different heavy chains and 2 different light chains),  each heavy chain can form either homo- or heterodimers, which in turn can associate with the two light chains. From a stochastic view point, this leads to 16 chain arrangements with 10 unique combinations, including only one, that represent only the 10% of the total mixture, that is the desidered BsAb where heavy chain exhibit the correct heterodimer and pairing with distinct light chains and this low probability impairs the manufacturability of bsAbs 

Protein engineering was largelly applied in the last 20 years to solve this issue.

First of all, complementary mutations that favors heavy chain (HC) heterodimerization while disfavouring formation of HC homodimers were reported. The first reported, and most widely used, platform is the knob-into-hole (KiH) which introduces a large bulky tryptophan in one HC and smaller sterically complementary residues in the other HC. The KiH strategy is widely implemented because it is highly effective in suppressing the most of HC homodimers and because its patent has expired 

While the correct heavy chain heterodimerization can be enabled using the knobs-into-holes (KiH) approach, the correct association of generic light chains has remained a problem, in fact even in presence of KiH there are still 4 possible random combinations that limit the fraction of desidered form only to about 25%

To solve this issue the KiH could be coupled with the CrossMAb technology that was described and patented by Roche in 2011 (it is stil protected by IP). which is able to enforce correct light chain association in bispecific heterodimeric IgG antibodies. 

This technology could be applied to any existing antibody pair using domain crossover, without the need for the identification of common light chains, post-translational processing/in vitro chemical assembly or the introduction of a set of mutations enforcing correct light chain association.

In the past I had the opportunity to produce for R&D purpose 2 different bispecific containing both KiH and Crossmab and in both cases the antibodies were produced fron CHO with high yields (transient trasfection) and the an very good rate of HC-LC correct coupling (>90%)    was detected in both cases by mass-spectrometry.

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 #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#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.