poster-mabchem-ppt file

Bioconjugation Chemistries for ADC Preparation Rongliang Lou, Dev Sharma, Daniel Wang, Ping Ge

mAbChem Lab LLC 23 Business Park Drive, Branford, CT 06405

World ADC Summit, San Diego, Oct 26-29th, 2014

Introduction Antibody drug conjugates (ADCs) represent novel structure-modified monoclonal antibodies designed to

deliver cytotoxic drugs selectively to antigen expressing tumor cells. Attaching a toxin or payload to an

antibody can be accomplished through a variety of approaches, conventionally via lysine residues utilizing

amide bonds (as in Kadcyla) or cysteine residues utilizing thiother bonds (as in Adcetris). More recently a

number of site directed approaches have been employed, however, the clinical evaluation of these remain

to be seen.

ADCs are typically characterized by the following techniques:

(i) UV spectrophotometry for protein concentration & Drug Antibody Ratio (DAR)

(ii) Hydrophobic interaction chromatography (HIC) for DAR, amount of unconjugated antibody (UmAb%)

and distribution of various loaded species (0, 2, 4, 6, 8)

(iii) Size exclusion chromatography (SEC) for amount of higher molecular mass species (Agg%)

(iv) Reversed phase chromatography (RP-HPLC) for DAR, amount of residual linker payload and related

species

(v) Mass spectrometry for DAR, residual linker payload and related species and generally more in depth

characterization (peptide mapping, sequence variations, etc)

Different conjugation chemistries pose different challenges from a development perspective in terms of

targeting desired product profile. In this poster, we would like to share our experience in different

bioconjugation chemistries (lysine & cysteine mediated) using IgG1, IgG4 mAbs and NHS ester containing

linker payload (NHS-LP) or non-cleavable maleimide containing LP (mc-LP). We would also share some

initial results using site directed conjugation involving mutant cysteine residues.

Lysine Mediated Conjugation A conventional strategy for preparation of Lysine mediated ADC is based on classical organic chemistry:

amide bond formation from the lysine amino residues of antibody and the activated ester, such as N-

hydroxysuccinimide ester, which is prepared from the corresponding acid of cytotoxic drug/payload (Fig1).

Fig1. Chemistry Strategy Applied for Lysine Mediated Conjugation

After Design Of Experiment (DOE) screening of various reaction parameters, a mixture of lysine mediated

ADC was made from coupling of IgG1 and NHS-LP following the optimum conditions:

4.5 mM NHS-LP in organic solvent

LP/mAb-1 input: 4

Reaction concentration: 5-25 mg/mL;

Reaction Buffer Conc (Ionic strength): 0.1-0.5 M

HIC and SEC chromatograms are shown in Fig 2.

min0 2.5 5 7.5 10 12.5 15 17.5

mAU

0

10

20

30

40

DAD1 E, Sig=280,16 Ref=360,100 (042914-SEC\042914SEC 2014-05-01 02-26-14\043014-2.D)

Area: 55.71 8.579

Area: 1358

.83 9.619

ADC

Aggregate

(3.9%)

Fig 2. HIC and SEC Chromatograms for Lysine Mediated ADC (Crude)

• ADC products with an average 4.3 of DAR determined using UV absorption

• 2.5% of Umab% measured by hydrophobic interaction chromatography (HIC)

• 3.9% of Agg% measured by size exclusive chromatography (SEC)

min2 4 6 8 10 12 14

mAU

-15

-10

-5

0

5

10

15

20

DAD1 E, Sig=280,16 Ref=360,100 (042914-HIC\042914HIC 2014-04-30 17-00-56\043014-2.D)

Area: 53.70

63

5.274

Area: 2074

.22 6.525

Unconjugated mAb

(2.5%)

ADC mixture

with average DAR of 4.3

In a typical preparation of cysteine mediated ADC, the interchain disulfide bonds are partially reduced

with a reducing agent such as tris(carboxyethyl) phosphine (TCEP) and then the resulting free thiols are

conjugated to a maleimide-containing linker-payload (Fig 3).

Conventional Cysteine Mediated Conjugation

Fig 3. Chemistry Strategy Applied for Cysteine Mediated Conjugation

Several parameters were evaluated in the conjugation of IgG1 (or IgG4) mAb with non-cleavable linker

payload (mc-LP), these include:

• Stoichiometry of TCEP

• Stoichiometry of LP

• Reaction pH

• Buffer Conc (Ionic Strength)

• Antibody Concentration

• Temperature

All results are summarized in Fig. 4, Table 1~7 and Chart 1~7.

min2 4 6 8 10 12 14

mAU

0

10

20

30

40

50

DAD1 E, Sig=280,16 Ref=360,100 (060914-HIC\060914-MABHIC 2014-06-09 18-47-51\MAB-1-RE.D)

5.994

min2 4 6 8 10 12 14

mAU

-15

-10

-5

0

5

10


5.887 6.571

7.427

min2 4 6 8 10 12 14

mAU

-15

-10

-5

0

5


5.924

6.604 7.452 7.811

min2 4 6 8 10 12 14

mAU

-18

-16

-14

-12

-10

-8

-6

-4

-2

0


6.608 7.463 7.816 8.782

9.599

min2 4 6 8 10 12 14

mAU

-18

-16

-14

-12

-10

-8

-6

-4

-2

0


7.500 8.827 9.653min2 4 6 8 10 12 14

mAU

-17.5

-15

-12.5

-10

-7.5

-5

-2.5

0

2.5


9.688

min2 4 6 8 10 12 14

mAU

-17.5

-15

-12.5

-10

-7.5

-5

-2.5

0

2.5


9.754

LP Loading = 0 2 4 6 8

mAb-1

TCEP 1 eq; LP 3 eq

TCEP 2 eq; LP 6 eq

TCEP 3 eq; LP 9 eq

TCEP 4 eq; LP 12 eq

TCEP 5 eq; LP 15 eq

TCEP 6 eq; LP 18 eq

Fig. 4: Effects of TCEP stoichiometry on loading distribution for

conjugation of IgG1 mAb and mc-LP

TCEP

equiv DAR

HIC

(UmAb%)

SEC

(Agg%)

1 1.77 34.2 0.52

2 3.78 7.0 0.64

3 5.31 1.5 0.68

4 6.82 0 0.75

5 7.83 0 0.77

6 8.00 0 0.78

Table 1. Stoichiometry study results for the


• Starting materials: 25 mg/mL IgG1 in 50 mM histidine; 20 mM mc-LP in DMSO, 10 mM TCEP in H2O

• Other condition: reduction, 37°C, 2h; conjugation, 37°C, 1h

• DAR and UmAb% were determined based on the HIC chromatogram as shown in Fig 4 and 5

• Agg% was measured by using SEC chromatogram (not shown)

-5

0

5

10

15

20

25

30

35

40

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8

UmAb%

DAR Agg%

TCEP Equivalent

Chart 1. Effects of TCEP Stoichiometry on DAR , UmAb% and Agg%

DAR

Agg%

UmAb%

Table 2. Stoichiometry study results for the conjugation

of IgG4 mAb and mc-LP

• Starting materials: 25 mg/mL IgG4 in 50 mM histidine; 20 mM mc-LP in DMSO, 10 mM TCEP in H2O

• Other condition: reduction, 37°C, 2h; conjugation, 37°C, 1h

• DAR and UmAb% were determined based on the HIC chromatogram

• Agg% was measured by using SEC chromatogram

TCEP

equiv DAR

HIC

(UmAb%)

SEC

(Agg%)

1 1.24 57.63 0.76

2 2.47 33.77 0.8

3 3.43 20.37 0.64

4 4.07 13.49 0.65

5 4.55 10.01 0.63

6 4.96 6.46 0.61 0

10

20

30

40

50

60

70

0

1

2

3

4

5

6

0 2 4 6 8

UmAb%

DAR Agg%

TCEP Equivalent

Chart 2. Effects of TCEP Stoichiometry on DAR , UmAb% and Agg%

DAR

Agg%

UmAb%

• IgG1 mAb is more easily reduced by TCEP than IgG4 mAb

• With enough TCEP, the disulfide bonds in IgG1 can be completely reduced

• Using same equivalent of TCEP, higher DAR and lower UmAb% obtained with IgG1 than IgG4

• In order to reach same DAR or UmAb%, more TCEP is needed for conjugation with IgG4 than IgG1

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8

DAR

TCEP Equivalent

Chart 3. Effects of TCEP Stoichiometry on DAR Comparison of IgG1 and IgG4

IgG1

IgG4

-10

0

10

20

30

40

50

60

70

0 2 4 6 8

UmAb%

TCEP Equivalent

Chart 4. Effects of TCEP Stoichiometry on UmAb% Comparison of IgG1 and IgG4

IgG1

IgG4

pH DAR HIC (UmAb%) SEC (Agg%)

4 2.24 27.09 0.75

5 2.49 20.37 0.95

6 3.37 8.11 0.89

7 3.88 4.48 0.77

8 1.55 43.47 0.93

Table 3. pH study results for the conjugation

IgG1 mAb and mc-LP

• Antibody: 25 mg/mL IgG1 in 50 mM histidine with different pH

• 10 mM TCEP in H2O, 2.2 eq., reduction, 4°C, 2h

• 20 mM MC-LP in DMSO, 6.6 eq., conjugation, 25°C, 1h

• The optimum pH for conjugation: 6~7

0

5

10

15

20

25

30

35

40

45

50

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

3 4 5 6 7 8 9

UmAb%

DAR Agg%

pH

Chart 5. Effects of pH on DAR , UmAb% and Agg%

DAR

Agg%

UmAb%

• Antibody: IgG1 with different concentration in 20 mM histidine (10, 25, 50, 75 and 100 mM)


• 20 mM MC-LP in DMSO, 6.6 eq., conjugation, 25°C, 1h

• Conjugation is more completely at higher antibody concentration (lower UmAb%)

Table 4. Antibody concentration study results

for the conjugation IgG1 mAb and mc-LP

mAb Conc.

(mg/mL) DAR

HIC

(UmAb%)

SEC

(Agg%)

10 4.69 1.60 0.70

25 4.70 1.05 0.71

50 4.70 1.07 0.84

75 4.72 0.94 0.87

100 4.73 0.99 0.89 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

4.685

4.69

4.695

4.7

4.705

4.71

4.715

4.72

4.725

4.73

4.735

0 20 40 60 80 100 120

UmAb% Agg%

DAR

mAb Conc. (mg/mL)

Chart 6. Effects of mAb Conc. on DAR , UmAb% and Agg%

DAR

UmAb%

Agg%

• Antibody: 25 mg/mL IgG1 in histidine (10, 25, 50, 75 and 100 mM)


• 20 mM mc-LP in DMSO, 6.6 eq., conjugation, 25°C, 1h

• Higher ion strength gave the better conjugation result

Table 5. Ion Strength study results for the


Buffer Conc.

(mM) DAR HIC (UmAb%) SEC (Agg%)

0 4.10 3.09 0.73

10 4.26 2.40 0.68

20 4.28 2.28 0.67

50 4.33 2.25 0.67

75 4.39 1.88 0.66

100 4.42 1.52 0.67 0

0.5

1

1.5

2

2.5

3

3.5

4.05

4.1

4.15

4.2

4.25

4.3

4.35

4.4

4.45

0 50 100 150

UmAb% Agg%

DAR

Buffer Conc. (mM)

Chart 7. Effects of Buffer Conc. on DAR , UmAb% and Agg%

DAR

UmAb%

Buffer DAR HIC

(UmAb%)

SEC

(Agg%)

Succinate 3.7 8.4 0.76

PBS 3.74 9.05 0.83

Histidine 4.05 4.1 0.73

HEPES 4.07 4.91 0.77

Tris 3.96 6.36 0.73

• Antibody: 25 mg/mL IgG1 mAb in 50 mM

different buffer

• 10 mM TCEP in H2O, 2.2 eq., 37°C, 2h

• 20 mM mc-LP in DMSO, 6.6 eq., 25°C, 1h

• Histidine and HEPES are two best buffers

among all 5 tested

Table 6. Buffer study results for the


Temp. DAR HIC

(UmAb%) SEC (Agg%)

4 4.28 2.28 0.67

25 4.23 2.72 0.72

37 4.23 3.19 0.81

Table 7. Temperature study results for

the conjugation IgG1 mAb and mc-LP

• Antibody: 25 mg/mL IgG1 mAb in histidine (10, 25, 50,

75 and 100 mM)

• 10 mM TCEP in H2O, 2.2 eq., different temperature, 2h

• 20 mM mc-LP in DMSO, 6.6 eq., 25°C, 1h

• lower reduction temperature is slightly beneficial to

afford lower UmAb%

By following the standard procedure (Fig 5), a site modified antibody which contains 4-engineered

cysteine residues protected by glutathione/cysteine via disulfide bonds, was treated with excess TCEP to

result in a fully reduced antibody. After buffer exchange to remove excess TCEP and protecting groups,

the resulted product was re-oxidized using excess DHA to re-form interchain S-S bonds while keeping

free thiols at mutant sites. Additional buffer exchange column was applied to get rid of excess DHA. mc-

LP was added to the resulted antibody to give a homogeneous ADC with 4 drugs per antibody.

Site Directed Mutant Cysteine Conjugation

Fig 5. Chemistry Strategy Applied for Site Directed Mutant Cysteine Conjugation

Further exploration in our lab found that the above standard procedure could be even simpler: two steps

of column purification to remove excess TCEP and DHA were unnecessary if the amount of TCEP and

DHA were well controlled. As shown in Fig 6, the ADC products obtained from our modified protocol and

the standard procedure have shown an identical HIC chromatogram.

min4 6 8 10 12

mAU

-5

0

5

10

15

20

DAD1 E, Sig=280,16 Ref=360,100 (090214-HIC\090214HIC 2014-10-04 16-21-16\MAB6.D)

6.2

56

6.9

43

min4 6 8 10 12

mAU

-5

0

5

10


8.6

18

10.

047

min4 6 8 10 12

mAU

-5

0

5

10

15

20


8.6

56

10.

082

Fig 6. HIC Chromatogram for Site Specific Cysteine Mediated ADC (Crude)

Antibody used as starting

material

Homogeneous ADC products 4 drug/mAb

Obtained following literature procedure

Homogeneous ADC products 4 drug/mAb

Obtained via a simpler procedure modified in mAbChem

Discussion

• Both lysine mediated conjugation and conventional cysteine mediated conjugation result in ADCs that

have a heterogeneous mixture of drugs per antibody.

• Site directed mutant cysteine conjugation proved to be a good solution to produce an ADC with a

homogeneous number of drugs per antibody.

• In cysteine mediated conjugation, IgG1 mAb was more easily reduced/conjugated with mc-LP than

IgG4 mAb. Using same equivalents of TCEP and mc-LP, ADC products with higher DAR and lower

UmAb% were obtained in IgG1 than IgG4.

• Cysteine and lysine mediated conjugations are highly sensitive to the pH of conjugation buffer, in the

circumstance with pH range of 6~7 (histidine), best conjugation results were obtained.

• Other factors such as buffer, ionic strength, antibody concentration and temperature have smaller

effects on cysteine mediated conjugation.

• The procedure for site directed mutant cysteine conjugation could be simplified by omitting two

column steps to remove the excess reagents for reduction or re-oxidation.

Conclusion

Several reliable and reproducible protocols using a variety of bioconjugation chemistries to make

ADCs were successfully developed. A facile process for site specific mutant cysteine conjugation was

identified. We believe that this modification will be beneficial to the preparation of homogeneous

ADCs, especially in clinical manufacture. Development of new conjugation technologies, such as

enzyme based conjugation and solid phase conjugation, as well as practical application of these

technologies to the conjugation of other antibodies and linker-payloads are under way.

poster-mabchem-ppt file

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