HIC is a well studied major polishing step in the purificaiton process of IgG based products and known for its capability to remove aggregated from of antibody. (Chen, J. Chromatography A, 1177 (2008) 272-281

Modes of operation:

Bind & elute mode: 

When used in bind-elution mode, there are two main limitation to HIC. HIC resins have relatively lower binding capacity and lower step yield compared to the other chromatography steps used in mAb purificaiton. Second, sufficient binding of mAb proteins to HIC resins is usually achieved with increasing salt concentrations in the binding buffers and the elution product may thus contain a fair amount of salt. However, optimization of resin pore size can significantly improve the binding capacity of standard HIC resins to increase HIC unit process efficiency. (Chen, J. Chromatography A, 1177 (2008) 272-281).

There are two main limitations when using bind elution mode. In general, HIC resins have relatively lower binding capacity and lower step yield compared to other chromatography steps used in mAb purificaiton. Secondly, sufficient binding of mAb proteins to HIC resins is usually acheived with increasing salt concentrations in the binding buffers, and the elution product pool from HI step purificaiton may still contain fair amount of salt, which often complicates sample manipulations and process flow transitions during large scale manufacture. In this respect, efforts have been made to optimize resin pore size to facilitate mass transport of mAb molecuels into ligand binding sites to increase binding capacity and hydrophobic charge induction (HCIC) mied mode chromatography design to allow mAb molecules to bind tot he resin at elatively low salt conditions. (Chen J Chromatography A, 1177 (2008) 272-281). 

Hunter (US2016/0083453) discloses a method from removing multimers from a sample of polyclonal antibodies which includes HIC operated under bind and elute conditions, where monomers are multimers are both bound to the column and then monoerms are selectively eluted with a change in salt concentration and/or pH. The loading buffer is substantially the same as the equilibration buffer and the resin may be washed in a buffer that is substantially the same as the loading buffer. For elution, the buffer may be a lower ionic strengt phosphate buffer. In some embodiments, the buffer comprises a step wise or linear gradient of decreasing salt. 

–Sequential HIC

(Mccue (US 2011/0129468) discloses a method of purifying an Ig (constant part) fusion protein using HIC in bind and elute mode and then contacting the eluate with a second HIC in bind and elute mode.

Flow through mode:

For flow through, process related impurities such as HCPs and aggregates will bind to the HIC media while product flows through the column Herigstad ((US13831181, published as US 9,249,182)

Ghose (US 9,994,609) discloses purification of antibodies by HIC under no-salt conditions with a pH of about 5-7 in flow through mode. 

Herigstad (US 14/077574, published as US 8,946,395) discloses purification of an antibody using HIC that combines flow through and bind-elute tehniques (also known as weak-partitioning mode). A porition of the antibody binds to the HIC and a substantial protion of a contaminant also binds. The flow through of the antibody is collected and by washing the HIC meida another fraction of the antibody is collected. 

Pan (US2008/0167450) teaches that while HIC conducted in bind and elution fashion is an efficient mode to remove dimers and aggregates, this mode has realtively low yield and separation resolution for other product related isomers and has to contend with high salt concentration in the eltuion pools. Thus, HIC in bind and elution mode is becoming less popular in antiboyd production. Instead, HIC chromatotraphy in a flow through mode is gaining interest as a way to remov a large percentage of aggregates.

(Nti-Gyabaah, US 14/355014, published as US 2014/0288278) discloses hydrophobic interaction chromatography (HIC) for the purification of monoclonal antibodies from heterogeneous aggregates. n. In one embodiment, the HIC is conducted in flowthrough mode with operating codnitions of pH 7, conductivity of 110 mS/cm, protein loading of at least 425 gm of protein per liter of resin. 

Shukla (US7,427,659; see also US 2005/0136521) discloses a method for separating a target protein such as an antibody using HIC with branched hdyrocarbon functional groups in an aqueous salt solution and collecting the unbound flow through fraction containing the target protein.

–No salt conditions:

HIC has two primary challenges: In general, binding capacity has been limited, especially in comparison to IEX. To circumvent this issue, HIC is sometimes used in flowthrough mode in which the product of interest flows through wile the more hydrophobic impurities remain bound to the column. This strategy is popular as a polishing step in antibody processes since aggregates are usually more highy retained on HIC. Second, the use of high concentraitons of salts is highly undersirable in any manufacturing prcoess because it can cause correosion of stainless stell tanks. Efforts to operate HIC under reduced or no-salt conditions have those been atempted. (Ghose, “purification of monocloanl antibodies by hydrophobic interaction chromatography under no-salt conditions”, Monoclonal antibodies, 5(5), 2013, pp. 795-800). 

Ghose, “purification of monocloanl antibodies by hydrophobic interaction chromatography under no-salt conditions”, Monoclonal antibodies, 5(5), 2013, pp. 795-800) discloses operating HIC in flwothrough mode with no kosmotropic salt in the mobile phase. A very hydrophobic resin was selected and the pH of the mobile phase was modulated to acheive the required selectively. Under the pH 6 and below, antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product flowed through while impurities such as aggreagates and HCPs bound to the column. 

Rinderknecht (WO/1996/033208) discloses purifying antibody using HIC at low sat concentration. The antibody is eluted from the column in the fraction which does not bind to it.

Ghose (US14/775,774, US 2016/0024145; see also US 16/005642, published as US2019/0144495) discloses a method of purifying an antibody using HIC in flow through mode where the solution does not contain a salt and has a pH of about 5-7.

 Hybrid mode:

A hybrid mode can involved the use of an HIC media that allows for the product to be immobilized but then removed by successive washes of buffer identical to or substantially similar to the loading buffer such as where the salt concentration is adjusted within about 20% of the concentration of the loading buffer. 

Herigstad ((US13831181, published as US 9,249,182; see aslo 14/978553, published as US 2016/0115193)

Overload Mode:  See HIC conditions generally

Conditions:

A large numbr of factors are known to affect HIC performance such as resin matrix, hydrophobic ligand type and density, resin pore size, antibody concentration, amount of laoding, type and concentration of the salting-out salt, pH and operation temperature. In addition, detergents, alcohols, polyols, amino acids and chaotropic agents can be added to the wash and/or elution to improve resolution and/or recovery. While epxerience chromatographers amy be able to cut through the factors with a limited set of experiments, developing the HIC step for optimal yield and purity can still be time consuming due to the large number of actors involved. (Lu, Current Pharmaceutical Biotechnology, 2009, 10, 427-433). 

Bind and Elute Mode:

–Binding:

—-High salt concentration: 

Geenrally, the protein preparation in a high salt buffer is loaded on the HIC column. The salt in the buffer interats with water molecules to reduce the solvation of the proteins in solution, thereby exposing hydrophobic regions in the protein which are then adsorbed by hydrophobic groups on the matrix. The more hydrophobic the molecule, the less salt is needed to promote binding. Senczuk (US 8,273,707)

Hydrophobic interactions are strongest at high salt concentration but the actual concentraitons can vary over a wide range depending on the nature of the protein of interest, salt type and particular HIC ligand chosen. and thus HIC is conventionally performed folloiwng salt elution step such as one used in connection with ion exchange chromatography. For example the salt concentrations shown to be effective in aggregate reduction are in the range of 80mM-1000mM depending on the salt type and HIC adsorbent. Herigstad ((US13831181)

—-Low salt concentration: 

Rinderknecht (US5,641,870) teaches that in contrast to performing HIC at neutral pH in the presence of high salt concentration to elute the antibody, a low pH HIC at low salt concetnration such as 0-0.25M salt can be used. Preferably, no salt gradient is used to elute the antibody. Rinderknecht teaches that buffers that will control the pH within this range include phosphate, acetate, citrate or ammonium buffers.

Sanchayita (US14/775,774, US 2016/0024145) discloses a method of purifying an antibody using HIC in flow through mode where the solution does not contain a salt and has a pH of about 5-7.

–Wash: 

—-Isocratic conditions

Herigstad ((US13831181, now US 9,249,182; see also 14/978553, published as US 2016/0115193) disclsoes a method for reducing a preparation comprising a protein of interest such as an antibody by contacting the antibody/misture with HIC miedia in the presence of a load buffer and collecting a flow through fraction and then contacting the HIC with a wash buffer that is substantially similar to the load buffer and collecting the wash fraction. In one embodiment, the protein of inters binds to the HIC media at a Kp of greater than 90.  and the antibody comprses adalimumab. In one embodiment the Kd for the binding of adlimumab is at least 0.47 and the Kd for the binding of the at least one impourity is 0.01 and the Qmax is at least 6, In one embodiment, the load and/or wash buffer is in the range of 80 mM-1000 mM. 

–Elution: 

—-Decreasing Salt Gradients:

Usually, a dcreasing salt gradient is used to elute proteins from a HIC. As the ionic strenght decreases, the exposure of the hydrophobilic regions of the protein increases and proteins elute from the column in order of increasing hydrophobicity.Senczuk (US 8,273,707)

—-Low pH and Low salt:

Rinderknecht (US5,641,870) teaches elution of an antibody from HIC with a buffer having a pH of about 2.5-4.5 (see also binding above using low pH).  that in contrast to performing HIC at neutral pH in the presence of high salt concentration to elute the antibody, a low pH HIC at low salt concetnration such as 0-0.25M salt can be used. Preferably, no salt gradient is used to elute the antibody.

Flow through Mode:

Sanchayita (US14/775,774, US 2016/0024145) discloses a method of purifying an antibody using HIC in flow through mode where the solution does not contain a salt and has a pH of about 5-7. 

In conjunction with other Types of Chromatography

Bioprrocess engineers often try to develop processes using chromatographyc steps arranged in order to reduce the number of buffer changes. One well known example is using the high conductivity elution buffer from an ion exchange chromatogrpahy (IEC) step as a basis for formulating a high conductivity adsorption buffer in a following HIC step. The latter typically involves adding even more salt to the IEC elution buffer to form  a suitable HIC adsorption buffer (Van Alstine, WO2011/0352382). 

Herigstad ((US13831181, now US 9,249,182) discloses using HIC in combination with other types of chromatography for the purificaiton of an antibody such as Affinity – HIC, HI-Affinity, IEX-HIC, Mixed Mode -HIC, HIC-filtration

Nti-glyabaah (US2014/0288278) disclsoes methods of purifying an antibody such as adalimumab by binding the antibody to Protein A then to AEX in flow through mode and a polishing step which can be mixed mode resin or HIC. 

Protein A – HIC:

Cunnigham (US 2022/0306727) disclsoes prparing masked antibodies with reduced aggregation which includes Protein A chromatography followed by HIC.