mAbChem poster-2015 ADC meeting

Process Development of ADCs in mAbChem
Rongliang Lou, Dev Sharma, Daniel Wang, Ping Ge
23 Business Park Drive, Branford, CT 06405
lou_r@mabchemlab.com www.mabchemlab.com
World ADC Summit, San Diego, Oct 19-22th, 2015

Abstract
Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies
(selectivity) and cytotoxic small molecules (potency) for targeted delivery to cancer cells
thereby enhancing their antitumor activity and minimizing off target toxicity. Preparation of
ADCs with desired profile is a key step in ADC development. Attaching a toxin or
payload to an antibody can be accomplished through a variety of conjugation approaches,
conventionally via lysine residues utilizing amide bonds (as in Kadcyla) or cysteine residues
utilizing thiother bonds (as in Adcetris), or more recently a number of site directed protocols
utilizing a variety of reliable chemistries.
The concept of ADCs appears simple, however, the development of efficacious, safe and
reproducible product presents a considerable challenge. In this poster, we would like to share
our experience in the process development of ADCs via conventional cysteine-mediated
conjugation. Several parameters involved in the bioconjugation including reaction time,
scale, pH, stoichiometry, etc have been investigated. Conjugates with different drug loading
were also separated as individual species from the crude mixture by using hydrophobic
interaction chromatography (HIC).

Introduction
ADC Drugs on the market
 Kadcyla: conventional lysine conjugated ADC, used for treatment of HER2 positive
metastatic breast cancer
 Adcetris: conventional cysteine conjugated ADC, used for treatment of Hodgkin’s
lymphoma or systemic anaplastic large cell lymphoma
Methods used to prepare ADCs
 Lysine mediated conjugation: conjugation of the drugs to lysine residues scattered
throughout the entire mAb structure, leading to a multitude of species, many of which
may display undesirable physico-chemical and biological characteristics
 Cysteine mediated conjugation: involving the use of endogenous interchain cysteine
residues for conjugation with maleimide-containing linker-payloads
 Site specific conjugation: using engineered mAbs designed for conjugation at specific
cysteine residues or other functional groups
 Enzymatic based conjugations: conjugation to specific tags/residues on mAbs utilizing
enzyme

0
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0 1 2 3 4 5 6 7 8 9
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0 1 2 3 4 5 6 7 8 9
0
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0 1 2 3 4 5 6 7 8 9
Multiple ADC Architectures
Lysine mediated
conjugation
Cysteine mediated
Interchain disulfide
conjugation
Site specific
conjugation
%
Drug loading
% %
Drug loading Drug loading

Advantages of Cysteine Mediated Conjugation
 Less number of conjugated species with simpler HIC than lysine mediated conjugates;
i.e., more selective
 Wild type antibody can be used directly for conjugation
 The robustness of required chemical steps: disulfide bond reduction and conjugation
with maleimide also makes this a very convenient approach for generating ADCs from
microgram scale for discovery research through kilogram scale necessary to support the
marketing of an approved agent
 Maleimides react with thiols very quickly at neutral pH and with high selectivity over
amines, enabling well-defined conjugation
 Maleimide linker payload are stable for months to years as stock solutions in appropriate
solvent and their rate of hydrolysis in aqueous solutions is negligible

Two step reaction scheme
 The interchain disulfide bonds are partially reduced with a reducing agent such as tris(carboxyethyl)
phosphine (TCEP), Dithiothreitol (DTT), 3-(diphenylphosphino) propionic acid etc.
 The resulting free thiols are conjugated to a maleimide-containing linker-payload (MC-LP)
Procedure of Cysteine Mediated Conjugation
Challenges in cysteine mediated conjugation
 Many factors, including stoichiometry, pH, buffer concentration, temperature, mAb concentration,
co-solvent and its percentage affect cysteine mediated conjugation
 In some cases, there is difficulty to identify the conjugation products due to positional isomers for the
conjugates with the same drug loading
 Different mAb and linker payload show quite different behavior in conjugation
 Hydrophobic linker-payloads may result in hydrophobic ADC that leads to rapid clearance in vivo
and aggregation issues

Quality Attributes for Conjugate Evaluation
Drug Antibody Ratio (DAR): One of the most important quality attributes of an ADC is the average
number of drugs that are conjugated because this determines the amount of “payload” that can be
delivered to the tumor cell and can directly affect both safety and efficacy. Most investigators in the
ADC field have considered a drug loading range of 2–4 drugs/antibody as giving the optimal balance
between potency, pharmacokinetics and tolerability
Distribution of conjugates with different drug loading: This is an important ADC attribute
because different forms may have different pharmacokinetic and toxicological properties, the conjugates
with appropriate drug loading exert the optimum therapeutic effect
Unconjugated antibody (UmAb%): The competitive binding of naked mAb to antigen will block
the binding of ADC, and as a result, may cause the loss of therapeutic potency
Aggregation (Agg%): Aggregate levels in drug substance and final drug product are a key factor
when assessing quality attributes of the molecule, since aggregation might impact biological activity of
the biopharmaceutical; aggregated mAb will lose partially or totally its therapeutic properties or even
cause immunogenic reactions thus potentially further endangering patients' health
Residual drug: the quantity of free drug/degradants, poses concerns for differential toxicity and
potential safety issues. Residual amounts of drug or drug-related impurities may remain in the final
product as a result of incomplete removal by purification steps down-stream of the conjugation reaction

Goals
 Develop reliable processes for ADC preparation of different scales
 Investigate the kinetics of conjugation
 Study the effect of stoichiometry of reducing reagent on DAR
 Separation of conjugate species using HIC
 Identification of these conjugate species
Materials
 mAb-1 (10 mg/mL)
 MC-LP (10 mM in DMSO)
 VC-LP (10 mM in DMSO)
 TCEP (10 mM in water)
Methods for characterization
 UV spectrophotometry for protein concentration & Drug Antibody Ratio (DAR)
 Hydrophobic interaction chromatography (HIC) for DAR, amount of unconjugated antibody
(UmAb%) and distribution of various loaded species (0, 2, 4, 6, 8)
 Size exclusion chromatography (SEC) for amount of higher molecular mass species (Agg%)
 Reversed phase chromatography (RP-HPLC) for DAR, amount of residual linker payload and related
species
 Mass spectrometry for DAR, residual linker payload and related species and generally more in depth
characterization (peptide mapping, sequence variations, etc)
Goals, Materials, General Conditions and
Methods for Characterization
General conditions (previously explored)
 pH: 6-8
 Buffer: histidine, PBS
 Temperature: 25-37°C
 Co-solvent and percentage: 5-15% DMSO

min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
120
*DAD1 E, Sig=280,16 Ref=360,100 (072815-HIC072815HIC 2015-07-29 22-23-23072815-1 1H.D) - DAD1 E, Sig=280,16 Ref=360,1
min3 4 5 6 7 8 9
mAU
0
2.5
5
7.5
10
12.5
15
17.5
20
*DAD1E,Sig=280,16Ref=360,100(063015-HIC063015HIC2015-07-0115-08-05MAB-1.D)-DAD1E,Sig=280,16Ref=360,100(06
min3 4 5 6 7 8 9
mAU
0
25
50
75
100
125
150
175
min3 4 5 6 7 8 9
mAU
0
50
100
150
200
min3 4 5 6 7 8 9
mAU
0
50
100
150
200
250
*DAD1 E, Sig=280,16 Ref=360,100 (072915-HIC072915-1HIC 2015-07-30 13-36-09072815-1 20H.D) - DAD1 E, Sig=280,16 Ref=36
min3 4 5 6 7 8 9
mAU
0
50
100
150
200
250
*DAD1 E, Sig=280,16 Ref=360,100 (072915-HIC072915HIC 2015-07-30 09-12-13072815-1 10H.D) - DAD1 E, Sig=280,16 Ref=360,
mAb-1
1 h
Effect of Reaction Time on the Conjugation Step
 Conditions: mAb-1 and TCEP (3.2 eq), 2 h at RT; MC-LP (9.6 eq), 6.5% DMSO, RT
 In the two step procedure for cysteine mediated conjugation, reduction is usually considered to be the
key step which finally controls the drug loading. As reported in literature, conjugation of the cysteine
thiols and the maleimide group is very facile, a fully completed reaction can be reached within 1h at 0°C
 In our case, the conjugation of mAb-1 and MC-LP requires 10 h
Completed conjugation observed at 10 h
2 h
5 h
10 h
20 h

min4 6 8 10 12
mAU
0
10
20
30
40
*DAD1 E, Sig=280,16 Ref=360,100 (080615-HIC080615HIC2015-08-06 18-51-43080515-1.D) - DAD1 E, Sig=280,16 Ref=360,100
min4 6 8 10 12
mAU
0
20
40
60
80
100
*DAD1 E, Sig=280,16 Ref=360,100 (080615-HIC080615HIC2015-08-06 18-51-43080615-3.D) - DAD1 E, Sig=280,16 Ref=360,100
mAb-1+ VC-LP
easy to identify the conjugates
with different drug loading
mAb-1+ MC-LP
More complicated
conjugates
Standard HIC Chromatogram of Cysteine Mediated
Conjugation
 Conjugation of mAb-1 and VC-LP gave an ADC mixtures in which conjugates with
different drug loading is easily differentiated in HIC chromatogram
 The conjugates from mAb-1 and MC-LP show a complicated chromatogram, which are
difficult to identify for their drug loading
0 drug
2 drug
4 drug
6 drug
8 drug
0 drug
2 drug
4 drug difficult to identify the drug
loading of conjugates

Positional Isomers from Cysteine Mediated
Conjugation
 Positional isomers from cysteine mediated conjugation cause additional complexity of
conjugate mixture

min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
120
*DAD1 E, Sig=280,16 Ref=360,100 (073015-HIC073015HIC 2015-07-31 14-57-50072815-1.D) - DAD1 E, Sig=280,16 Ref=360,100
Crude conjugate
min3 4 5 6 7 8 9
mAU
0
1
2
3
4
5
6
*DAD1 E, Sig=280,16 Ref=360,100 (073015-HIC073015HIC 2015-07-31 19-44-46072815-1 2-2.D) - DAD1 E, Sig=280,16 Ref=360,
min3 4 5 6 7 8 9
mAU
0
2
4
6
8
10
*DAD1 E, Sig=280,16 Ref=360,100 (073015-HIC073015HIC 2015-07-31 14-57-50072815-1 P2.D) - DAD1 E, Sig=280,16 Ref=360,1
min3 4 5 6 7 8 9
mAU
0
2.5
5
7.5
10
12.5
15
17.5
20
min3 4 5 6 7 8 9
mAU
0
1
2
3
4
min3 4 5 6 7 8 9
mAU
0
0.25
0.5
0.75
1
1.25
1.5
1.75
min3 4 5 6 7 8 9
mAU
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
Separation of Conjugate Isomers by Preparative
Chromatography (HIC)
 Several conjugate isomers were separated by preparative HIC
 Their drug loading was measured by UV absorbance and all peaks in crude mixtures were identified
based on drug loading
 Those conjugates with higher drug loading (6 and 8) were not retrieved from column due to their higher
hydrophobicity
Loading = 0 2 4 6 8
Fraction 1
Fraction 2
Fraction 3
Fraction 4
Fraction 5
Fraction 6

min3 4 5 6 7 8 9
mAU
0
2.5
5
7.5
10
12.5
15
17.5
20
*DAD1E,Sig=280,16Ref=360,100(063015-HIC063015HIC2015-07-0115-08-05MAB-1.D)-DAD1E,Sig=280,16Ref=360,100(06
min3 4 5 6 7 8 9
mAU
0
25
50
75
100
125
150
175
*DAD1 E, Sig=280,16 Ref=360,100 (072115-2D-HIC072115HIC-2D 2015-07-24 08-55-02072115-2.D) - DAD1 E, Sig=280,16 Ref=36
min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
120
140
min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
min3 4 5 6 7 8 9
mAU
0
20
40
60
80
min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
120
140
TCEP Stoichiometry Study Targeting Optimum DAR
 ADCs with specific DAR can be easily obtained by partial reduction of the native disulfides using
TCEP as reducing reagent. The level of drug loading in the final ADC is controlled at the reduction
step by modulating the molar equivalents of reductant added to the antibody
 TCEP stoichiometry study indicated that in the conjugation of mAb-1 and MC-LP, using about 3.2
eq of TCEP resulted in the ADCs with DAR=4
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7
DAR
TCEP (eq)
Optimal TCEP Stiochiometry
for DAR=4
TCEP (eq) 1 2 3 4 6
DAR 1.52 2.65 3.79 4.65 6.02
mAb-1
TCEP 1eq
TCEP 2eq
TCEP 3eq
TCEP 4eq
TCEP 6eq

min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
min3 4 5 6 7 8 9
mAU
0
20
40
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80
100
min3 4 5 6 7 8 9
mAU
0
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100
min3 4 5 6 7 8 9
mAU
0
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100
min3 4 5 6 7 8 9
mAU
0
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80
100
min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
min3 4 5 6 7 8 9
mAU
0
20
40
60
80
100
0.2 mg, 20 uL
1 mg, 100 uL
Conjugation at Different Scales
 Conditions: mAb-1 (10mg/mL, 20 mM histidine, pH=6) and TCEP (3.2 eq), RT, 2 h;
MC-LP (9.6 eq), 5% DMSO, RT, 15 h
5 g, 500 mL
10 mg, 1 mL
100 mg, 10 mL
1 g, 100 mL
25 g, 2500 mL

Conjugates Profile at Different Scale
Scale Volume DAR Agg% UmAb%
Residual
drug%
0.2 mg 20 uL 4.06 1.79 4.57 <LOQ*
1 mg 100 uL 4.05 2.01 4.38 <LOQ
10 mg 1 mL 4.10 1.86 4.44 <LOQ
100 mg 10 mL 4.04 1.99 4.73 <LOQ
1 g 100 mL 4.02 2.05 4.92 <LOQ
5 g 500 mL 4.04 2.08 4.69 <LOQ
25 g 2500 mL 4.05 1.92 4.75 <LOQ
 Conditions: mAb-1 (10mg/mL, 20 mM histidine, pH=6) and TCEP (3.2 eq), RT, 2 h;
MC-LP (9.6 eq), 5% DMSO, RT, 15 h
 The excess of free drug is removed by buffer exchange using the following three ways:
NAP column (0.1~3 mL); dialysis (casettes, 1~50 mL); ultrafiltration/diafiltration
(50~2000 mL)
 Conjugation of mAb-1 and MC-LP repeated very well at different scale
* Limit of Quantitation

Quality Attributes of ADC at Various Scales
0
1
2
3
4
5
6
1 10 100 1000 5000 25000
DAR %Agg %UmAb
Scale of Reaction (mg)

Conjugation Reactors for Different Scale
300mL insert
Conjugation: 10~150mL Insert used in HPLC
vial
2mL HPLC vial
Conjugation: 100~500mL
8~25mL vial
Conjugation: 0.5~5mL
25~150mL reactor
500~5000mL reactor

Summary
 A practical process was developed in mAbChem for cysteine mediated conjugation of
mAb-1 after thoroughly investigating the parameters involved in the reaction: pH,
stoichiometry, temperature, reaction time, buffer type, mAb concentration, co-solvent and
percentage
 An optimal conjugate was obtained with ideal profile: DAR = 4; Agg% = 2; UmAb%=5,
Residual drug% < 0.1
 The protocol was highly reproducible in our lab to give a consistent result at the scale
range of 0.2 mg~25 g
 Longer conjugation time (10 h) may be needed for the conjugation of some specific
combination of antibody and linker payload, unlike the typical conjugation reported in
literature (0.5~2 h)
 Conjugates with different drug loading were also separated as individual species from the
crude mixture by using hydrophobic interaction chromatography (HIC)
 The drug loading of single positional isomers were successfully determined by UV
measurement

mAbChem poster-2015 ADC meeting

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