HIC (Hydrophobic Interaction Chromatography) is used to purify Abs (Antibodies) by conventional procedure. This presentation gives a brief about non-conventional mode of HIC process operation for optimization of conditions like ligand nature, mobile phase pH, column loading, product selectivity and more to avoid harsh nature of salts like Ammonium sulfate.

1. Purification of monoclonal
antibodies by hydrophobic
interaction
chromatography under no-
salt conditions
( Paper by Sanchayita Ghose , Yinying Tao , Lynn Conley and Douglas Cecchini )
Summary of above paper
SOURABH SANTOSH GURAV

2. Summary
• Polishing step in mab antibody purification processes
• Limitation of use of high concentrations of kosmotropic salts
• Unconventional way of operating HIC
i. no kosmotropic salt
ii. very hydrophobic resin
iii. Modulation of pH of the mobile phase (pH<6)
iv. Selectivity such that aggregates & HCP bound to the column

3. Conventional HIC and Challenges
• Hydrophobic (aliphatic or aromatic) ligands
• Aggregate ,hcp,leached protein A and endogenous viruses removal
• Kosmotropic salts, e.G., Ammonium sulfate, sodium citrate,
potassium phosphate
• Elution by decreasing salt concentration or by using organic mobile
phase modifiers.
• Challenges –
a) optimize the pore size and ligand density
b) binding capacity compared to IEC
c) Salt present in elution pool (Expensive)
d) Waste water concerns/disposing cost

4. Optimisation of HIC (NO SALT)
• In the FT mode, only a more hydrophobic resin than the control resin has the potential of achieving the same
separation under reduced salt conditions. A lesser hydrophobic resin would require even higher salt
concentration to provide the same selectivity.
• To compare the hydrophobicity of various resins on an even basis, linear retention of lysozyme in a
decreasing salt (ammonium sulfate) gradient was determined on commonly used commercial HIC resins.

5. • Mabs B and D were practically
unretained and hence eluted at pH 6.0,
the starting point of the gradient
• Optimum pH should give the best
compromise between recovery and
HMW clearance.
• Higher pH -lower yeild
• Lower pH -HMW species flow along
with monomer
• Optimum pH needed by each molecule
was influenced by both its pi and
surface hydrophobicity
• Average loading of ~100 g/L
B

6. • Plots step yield and HMW
level of the FT pool as a
function of column loading on
the hexyl resin.
• Both yield and hmw levels
increased as a function of
column loading.
• The optimum column loading
is selected based on best
compromise between yield and
desired hmw level.
• 100 mg/ml resin loading

7. Advantages of NO-SALT
unconventional HIC
• Method eliminates the need for the additio ammonium sulfate or
other kosmotropic salts to the mobile phase
• Overcomes tank volume limitation
• Reduce the size of the costly viral filter
• Helped reduce disposal costs of ammonium sulfate
• No corrosion of steel tanks & less expense
• More compatible with environmental considerations

8. Materials and Methods
Materials Provider / Company Remark
• Monoclonal ab (A-D) Biogen idec Produced internally in a CHO
cell line
• Protein lysozyme Sigma
• Phenyl sepharose HS, capto
phenyl HS, butyl sepharose
4FF and octyl sepharose
4FF
GE healthcare Agarose-based resins
• Phenyl toyopearl 650M,
butyl toyopearl 650M, and
hexyl toyopearl 650C
Tosoh bioscience
• Chemicals and salts JT baker

9. Equipments
Equipments Company/Provider Remark
• AKTA Explorer
chromatographic systems
GE Healthcare
• Lambda 25 UV/VIS
spectrophotometer
Perkin Elmer
• pulse injection (0.1 mL of protein at ~5 mg/ml
concentration) using a 0.66 cm
D × 10 cm L column
• online Monitor pH/C-900
unit
part of the AKTA system
• analytical Size Exclusion
Chromatography (TSK gel
G3000 SWXL column)
To measure HMW levels
• ELISA-based immunoassay Meso Scale Discovery platform. To determine HCP levels in
preparative experiments

10. REFERENCE
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851231/
• FOR ANY QUESTION FEEL FREE TO MAIL sourabhgurav23@gmail.com