See also Mass Spectrometry in Diagnostic Techniques

Companies: Refeyn (mass photometry)

Definitions

Amide: includes a derivative form of carboxylic acid in which the hydroxyl group has been replaced by an amine or ammonia. Due to the existence of strong electronegative atoms, oxygen adn nitrogen, next to carbon, dipole moment is produced and the molcule with amide group presents polarity or hydrophilicity.  

Analytics for Antibody Drug Conjugates (ADCs): 

Analytics for Drug to Antibody Ration (DAR):

A variety of techniques have been used to emasure the DAR or payload that can be delviered to a tumor cell using an ADC. The simplest techniqued relies on a UV/VIS spectroscopic analysis of the ADC. Depending on teh chemisty used to attach the drug to the antibdoy, alternative methods for determining the DAR such as HIC can be used. Reduction of itner-chain disulfides to produce free sulfydryl groups allows conjugation at specific residues using maleimide-containing linekrs and genereates conjugates with a mixture of 0, 2, 4 and 6-8 drugs per antibody. As a result of the significantly reduced heterogeneity of this linkage chemistry relative to lysine-linked conjugates, the mixture has proven to be amenable to anaysis to HIC. HIC is performed under non-denaturing conditions at neutral pH with a gradient from high salt to low sat and often incldues a low concentraiton of an organic modifier in the low salt mobile phages to improve the elution of the mAb laoded with hydrophobic drugs. As a result, even though some of the itner-chain disulfides are disrupted due to the conjugation reaction, the combination of covalent binding and strong non-covalent forces between the chains are sufficient to keep the mAb intact during analysis. Thus, each peak observed correspodns to an intact mAb species with an increasing nubmer of bound drugs. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Size Variant Analysis of Conjugates:

The presence of HMW variants (i.e., aggregates) in an ADC has the potentail to elicit the production of the anti-therapeutic antibody (ATA) response. Many of the drugs that are conjugated to antibodies to produce ADCs are relatively hydrophobic and can increase the likelihood of aggregate formation during manufacturing and storage. Most published methdos of ADCs use the same size-exclusion chromatography analysis that are used for teh parent monoclonal antibodies. SEC is perforemd under non-denaturing conditions. CD-SDS has emrged as a preferred technique that offers clear advantages over SDS-PAGE in terms of speed, reroducibiity, resolution, robustness and ease of automation for non-conjugated antibodies. One of the earliest applications to ADCs was the analysis of a BR96-DOX conjugated formed by linkage of drug to inter-cahin suolfhydryls. The addition of SDS to the sampel dissocaites antibody chains taht are not covalently linked by intact disulfides due to partial reeduction and reaciton with linekr-drug, and provdies information about the sites of conjugation along with a comparison to traditional SDS-PAGE. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Charge-Based Separations:

Analysis of charge variants such as those resulting from deamidation or formation of N-temrinal pyroglutamates may be an important quality attribute of a mAb, expecially if the changes affect binding or biologcial activity. Depending on the characteristics of the drug, the linker and the conjguation site, methods that can be applied to the aprent mAb may not be applicable to the ADC or may give significantly different informaiton. Attachmetn of an uncharged linker and drug thorugh lysine residues decreases the net positive charge by one for each bound drug-liner. In this case, spearation based on charge, such as ion exchange chromatography IEC) or iso-electric focusing (IEF) resutls in profiels taht characterize the drug leoad, rather than proving information about the udnerslying mAb. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Analysis of Unconjugated Drug:

Another improtant quality attribute shared by all aDCs is the quantity of free (unconjguated) drug, which poses concerns for differential toxicity and potentail safety issues. ELISA, capillary electrophoresis (CD) and high performance liquid chromatography (HPLC) are methods which ahve been sued to determine free drug levels in various ADCs. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

See also purification of bi-specific antibodies under particular antibodies purified

In general, IgG type bispecific antibodies are composed of two types of H chains (an H chain for antigen A and an H chain for antigen B and two types of L chains a L chain for antigen A and a L chain for antigen B). When such IgG type bispecific antibodies are expressed, 10 types of combinations are possible as combination of H2L2 since two types of H chains and two types of L chains are expressed. Among these, there is one type of combination that has the desired binding specificity for antigeb A on one arm and antigen B on the other arm. Consequently, in order to aquire the desired bispecific antibody, it is necessary to purify one type of antibody of interest from among ten types of antibodies, which is extremely low in efficiency and difficult. (Kuramochi (US 2014/0370020)

Since in bispecific antibodies the two H chains as well as the 2 L chains are different and can randomly associate, expression of these four chains leads to the formation of 10 different antibody variants. Correct H chain association resulting in a heterodimeric Fc can be enforced wuing KiH technology by introducing a builky tryptophan (Trp) resiude in one Fc fragment and forming a corresponding cavity on the other Fc fragment that can accommodate the Trip residues. More recently, multip alternative approaches to enable correct H chain association have been described such as relying on charge interactions. Although kiH technology was developed in the late 1990s, enabling correct L chain assocaition remained a major problem, and the only approach to achieve this at the time relied on the sue of common L chains for both specificities. However, the use of a common light chain required the de novo identificaiton of the corresponding antibody pairs, which can be challenging and/or time consuming depending on the desired target, and restrict the availabity and diversity of antibodies that can be sued; those methods allowing the generation fo bispecific antibodies from pre-existing antibody pairs were highly desired. (Keiine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Common Heavy chain with two light chains:

lamda/kappa bodies (common heavy change, one Kappa and one lambda): 

Fischer (US2014/0179547) discloses the generation of bispecific antibodies where two antibodies having different specificties sharing the same variable heavy chain domain but different variable light chain domains are isolated. The variable heavy chain domain is fused to the constant region of a heavy chain, one light chain variable domain is fused to a Kappa constant domain and the other variable light chain domain is fused to a Lambda constant domain. Preferably, the light chain variable domain fused to the Kappa constant domain is of the Kappa type and the light chain variable domain fused to the Lambda constant domain is of the Lambda type. However, it is also possible to generate hybrid light chains so that two variable light chain domains of the same type can be used to generate bispecific antibodies. The three chains are co-expressed in mamalian cells leading to the assembly and secretion in the supernatant of a mixture of 3 antibodies; two monospecific antibodies and one bispecific antibody carrying two different light chains. The antibody mixture is purified using standard chromatography techniques used for antibody purification. See antibody purification

Introduction:

A problem with KiH technology remains correct light chain associrion. While a common light chain can be used, a common light chain requires the de novo identification of the corresponding antibody pairs, which can be challenging and/or time-consuming depending on the desired target and restricts the avaiability and diversity of antibodies that can be used. CrossMab technology enables this correct light-chain association in bispecific antibodies. By incorporating the original heavy chain VH-CH1 domain in the Fab of the second specificity of the bispecific antibody as the novel “lgiht chain” and the original light chain VL-CL domain for the novel “heavy chain” by fusing them to the hing region of the Fc fragment, correct lgiht-chain association can be enfored. The Crossmab technology has evolved in the past decade into one of the most mature, versatile and broadly applied technologies in the field for teh generation of various bispecific antibody formats. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)). 

Roche developed the CrossMab approach as a possibility to enforce correct light chain pairing in bispecific hterodimeric IgG antibodies, when combining it with the KH technology. In this format, one arm of the intended bispecific antibody is left untouched. In the second arm, the whole Fab region, or the VH-VL domains or the CH1-CL domains are exchanged by domain crossover between the H and L chain. As a consequence, the newly formed “crossed” L chain does not associated with the (normal, i.e., not-crossed) H chain Fab reigon of the other arm of the bipsecific antibody any longer. Thus, the correct L chain aossication can be enfored by this minimal change in domain arrangement. (Schlothauer, WO 2016/071377)

Two problems must be solved to produce the desired bispecific antibody exclusively: effective induction of heterodimerization of the two heavy chains and discrimination between the two light- chain/heavy-chain interactions. The former can be overcome by introducing large amino acid side chains into the CH3 domain of one heavy chain that fit into an appropriately designed cavity in the CH3 domain of the other heavy chain [the “knobs into holes” (KiH) The latter problem is more difficult to address, because a total of four possible pairings of heavy and light chains remain, with only one of which represents the desired compound. To solve this, the heavy chain 1 and light chain 1 are unmodified. On the opposite side, a new “heavy” chain 2 consisting of an Fc part and the Fab of the original light chain. As the new “light” chain 2 the heavy chain domains VH and CH1 are used. Because the sequence of the modified heavy chain now is crossing over between light- and heavy-chain domains, the term “crossover” for this kind of domain interchange and the term “CrossMab” for antibodies based on this technology. Hetero- dimerization of the two heavy chains is achieved by using the KiH method. As a consequence of this domain rearrangement, associations between unrelated partners can no longer occur. The new “light” chain 2 on the crossover side consists of heavy-chain domains only; thus it cannot assemble with the remaining original heavy chain 1. On the other hand, the original light chain 1 on the unmodified side cannot interact with the new “heavy” chain 2 on the crossover side, because both partners contain the same light- chain heterodimerization interfaces, which do not interact. A bispecific antibody (Fig. 1C) with correct light-/ heavy-chain pairing in both Fabs and almost no deviation from the original IgG is thus obtained. (Klein, “Immunoglobulin domain crossover as a generic approach for the prouciton of bispecific IgG antibodies”. PNAS, July 5, 2011, 108(27). 

Applications:

–Autoimmune diseases:

Bispecific CrommMab based antibodies have been generated with the goal of treating autoimmune diseases. Fischer showed that combined inhibition of TNFalpha and IL-17 was more effective in inhibting the development of inlammation and bone and carilage destruction in arthritic mice compared to the respective monotherapies. For this purpose bispecific TNFalpha/IL-17 1+1 and 2+2 CroossMab (HC1-CL) antibodies were rpepared that showed superior efficacy in blocking cytokine ahd chemokine resosnes in vitro. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

–Cancer therapy and opthalmology:

For many years, anti-angiogenesis approaches blocking the vascular endothelial growth factor-A (FEGF-A) have been a major area of targeted cancer therapy. One of the first IgG based antibodies and the first bispecific CrossMab to enter clinical trails, in 2012, was the hterodimeric 1+1 VEGF/Ang-2 CrossMab (HC1-CL) vanuucizumab (RG7221) tarteting the pro-angioneic ligands VEGF-A and angiopoietin-2 (Ang-2), which are invovled in tumor angioenesis. VEGF and Ang-2 ahve also been shown to play an important role in ocular angionesis in diseases liek wet age related macular degerenation a. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Another major field in targeted cancer therpay has been and continues to be apoptosis induction through death receptor (DR) signlaing. As conventional DR5 anitobdies have not been successful in clinical trails, approaches for tumor targeted DR5 agonism have been persued. Expression of the fibroblast activaiton prtoein (FAP) on tumor fibroblasts is found in the majority of solid tumors, making fAAP an attractive antigen for tumor targeting. Based on this rationale, FAP targeted bispecific antibodies and fusion proteins have been created using CrossMab technology that rely on FAP binding with one moiety to induce, with their second moeity, hyper-clustering of TNF receptor superfamily members like DR5 for apoptosis induction. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

With the advent of cancer immunotherpay and checkpoint inhibitor antibodies during the past decase, the development of bispecific antibodies for immunotherapy has attracted substantial attention in industry and academia, wehreas the interest in anti-angioenic and pro-apoptotic therapies has declined. In this context, bispecific monovalent dual checkpoint inhibitory PD-1 antibodies co-targeting the checkpoint inhibitory receptors TIM-2 or LAG-3 ahve been designed based on a bispecific 1+1 CrossMab (VH-VL+/-) format, allowing avidity mediated selectivity gain and thus enhanced selectivity for PD-1+ nad PD-1+TIM=3+/LAG-3+ double positive T cells. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Many of bispecific antibodies currently being developed are bispecific T-cell engarers. One of the first IgG based and Roche’s firt, T cell bispecific antibody (TCB) to enter clinical trails was  the heterodimeric and invalent CEA/CD3E 2+1 TCB bisatamab. It is a hterodimeric CEA/CD3e bispecific antibodyin the 1+1 CrossMab (CH1-C2) format to whcih a single additional Fab targeting CEZ is fused to the N-terminus of the nokb containg H chain. (Kleine “Ten years in the making: applicaition of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

The msot advanted 2+1 T cell bispecific antibody is glofitamab which, in constrat to cibisatamab, is based on a 2+1 CrossMab (VH-VL) formal with charge interactions using vriable regions dervied from obinutuzumab. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Central nervous system diseases:

Th treatment of CNS diseases with mAbs is hampered by the low penetration of antibodies thorugh the blood brain barrier. To overcome this limitation, Nieoether generated transferrin receptor targeted bispecific antibodies that allowed delivery of these antibodies through the blood brain barrrier and showed improved brain exposure and prevented plaque formation. Using this approach, BS-GANT was generated based on the amyloid-beta antibody gantenerumab as a trivalent C terminally fused amyloid-beta-TfR 2+ 1 bispecific antibodies in a 2+1 CrossMaby (VH-VL=/-) format with charges. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

–Viral Infections:

The appliction of CrossMab technology ahs become popular for the geenraiton of bispecific and multispecific antibodies targeting various viruses. Curing the past years, multiple highly potent bispecific antibodies targeting HIV-1 have been generated using CrossMab technoloyg for the prevention and treamtent of HIV-1. (Kleine “Ten years in the making: applicaiton of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins” 2021, MABS, 13(1)).

Introduction of difference in the charges to Heavy chains:

If two heavy chains and two light chains are expressed, there are 10 possible heavy chain and 10 possible light chain combinations. Accordingly, introducing difference in the charges of two heavy chains has been used to facilitation purification of BiAb. (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012).

Knobs-into-holes strategy:

The homodimerization of the two heavy chains in an IgG is mediated by the interaction between the CH3 domains alone. Heavy chains were first engineered for heterodimerization in the 1990s using a “knowbs-into-holes” strategy. Starting fromm the a “knob” mutation (T366W) that disfavors CH3 homodimerization, compensation “hole” mutations (T366S, L368A, and Y407V) were identified by phage display providing efficient pairing witht the “knob” while disfavoring homodimerization. (Spies, Molecular Immunology 67 (2015) 95-106). 

Bascially, the concept relies on modifications of the interface between the two CH3 domains of the two H cahins of an antibody where most interactions occur. A bulky residue is introduced inot the CH# domain of one antibody H chain and acts similarly to a key (“knob”). In the other H chain, a “hole” is formed that is able to accommodate this builky residue, mimicking a lock. The resulting heterodimeric Fc region can be further stabilized by the introduction of artificial disulfide bridges. Notably, all KiH mutations are buried within the CH3 domains and not “visible” to the immune system. Correct H chain assocaition with hterodimierizaiton yield above 97% can be acheived by introducing xix mutaitons: S354C, T366A in the “knob” H chain and Y349C, T366S, L368A and Y407V in the “hole” H chain. (Schlothauer, WO 2016/071377). 

In the knobs in holes technology, one or more small amino acid side chains from the interfact of the first antibody molcule are replaced with larger side chains (e.g., tyrosine or tryptophan) (knobs or protuberances). The proteuberance may exist in the original interface or may be introduced synthetically. Compensatory “cavities” (holes) of identical or similar size to the large side chains are created on the on the interact of the second antibody molecule by replacing large amino acid side chainswith smaller ones. (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-proudcts such as homodimers. (Giese, US 16/221,369 published as US 2019/0256556)

Schlothauer (WO 2016/071377) disclsoes variant Fc regions that specifically bind to SpA and that do or do not bind to human FcRn. Thesse variant Fc region contain specific amino acid mutations in the CH2 domain whereas the CH3 domain is not changed with respect to protoein A binding. The mutations when used in the hole-chain of a heterodimeric Fc regioulfide brdige(s) and wherein the CH3 domain of the first and second polypeptide both bind or both do not bind to protein A and allow for the purification of the heterodimeric Fc region, i.e., the separation of the heterodimeric Fc region from the homodimeric Fc region by-produce (hole-chain-hold-chain dimer). Thus in one embodiment, the heterodimeric polypetide includes a first polypeptide which includes in N to C terminal direction at least a pottion of the hinge region, which includes one or more cysteine residues, a CH2 domain, a CH3 domain and a second polypetide that includes in the N to C terminal direction at least a protion of the hinge resgion which includes one or mroe cysteine residues, a CH1-domain and a CH3 domain, wherein the first polypeptide includes the mutations Y349C, T366S, L368A and Y407V (hole chain) and the second polypetide includes the mutations S354C and T366W (knob-chain) and wherein the first polypetpide (hole-chain) icludes the mtuations I253A or I253G and L314A or L314G or L314D, wherein the first and second polypetpidds are connected by dis

Introduction of + charged amino acids into H chain CH3 and – charged amino acids into L chain CH3: 

Zhang (WO 2017/034770) discloses modifying antibody CH3 domain to promote heterodimerization between a first CH3 polypeptide and a second CH3 harboring polypeptide. In particular, at least two positively charged amino acids such as arginine, lysine or histidine are introduced into the first polypeptide by amino acid substitution to increase the positive charge while at least 2 negatively charged amino acids such as aspartic acid or glutamic acid are introduced into the seocnd polypeptide to increase negative charge of the polypeptide chain. Those different modifications at the itnerfact of two complementary CH3 omains presumably provides much stronger electrostatic attractions between those two chain to favor heterodimer formation rather than homodimer formation. In addition, cysteine molecules may be intoduced togehter wtih the charged amino acids to the appropriate position of both interfaces of the two complementary CH3 domains to allow inter-chain disulfide bond formation and further strenghten the heterodimer formation. In cerain embodiments, the molecule may be a bispecific, trispecific or qudrospecific antibody. 

Modification of H chain CH1 and L chain constant region;

Kuramochi (US 2014/0370020) discloses regulation of the association of the H and L chain such that in one eobmdiment the amino acids repel electrically or in a separate embodiment the amino acids do not repeal electrially. By making amino acid residues at given locations in the H constant region (CH1) and L chain constant region of a desired bomination that do not mutually repel electrically, a desired combination of H and L chain can be foremd by suing the attractive force of the electric charges. Amino acids that do not mutually repeal electrially include amino acids in which one of the amino acids is a positively charged amino acid and the other is a negatively charged amino acid. 

Modifying AAs of first and/or second polypeptides to create differences in isoelectric point:

Igawa (US 2009/0263392) discloses modifing both or either one of a nucleic acid encoding the amino acid residue of the first polypetpide and a nucleic acid encoding the amino acid residues of the second polypeptide, usch that the difference between the isoelectirc point of the rift and second polypeptide will be incdreased. The modification results in peaks of the homomultimer for the first polypeptide and second polypeptide and heteromultimer of the first polypeptide and the second polypeptides that can be seaprated. 

Common Light chain with two Heavy Chains:

If two heavy chains and two light chains are epxressed, there are 10 possible H and 10 possible L chain combination. But if a common light chain is used for two H chains, combinations become only three. (“Proprietary Innovative Antibody Engineering Technologies in Chugai Pharmaceutical” 12/18/2012)

A common light chain strategy was applied to assemble IgG like bispecific antibodies which can be combined with the knobs-into-holes approach. The mechanism of a common light chain is based on the fact that antibodies discvoered form phase display screening against diverse antigens often share the same VL domain, reflecting the very limited size of the L chain repertoire in the phage library. One of the advantages of the common L chain format is that it allows the use of methods that simplify the antibody engineering and the purificaiton process in industrial production. (Wang, “Design and Production of Bispecific Antibodies” Antibodies, 2019)

See also simulated moving bed chromatography (this is a form of continuous chromatography) under moving bed. 

Definitions:

Binding capacity and mass transfer: The binding capacity strongly depends on the affinity ligand, its density on the surface and its accessibility. Due to the improvements in the modificaiton technologies, high binding capacities >30 g/L can be reached at residence times of >3 min independently form teh support matrix chemistry. On the other hand, mass transfer properites are quite variable, cuasing radical differences in the application. Film mass transfer and pore diffusion are two main parameters, which influence the target molecule diffusivity towards the adsorption sites. Film mass transfer is mainly influenced by the particle size and shape. The smaller the particle and the interparticle volume the great the mass transfer and the flow resistance. Thus, for the analytical scale applicaitons smaller particle size is used to assure a fast mass transfer and high efficiency. But due to the pressure resistance issues, the industrial scale operations are performed at <3 bar operation pressures. Bigger particules (60-120 um) are usually choses. Due to these issues, the pore diffusion is one of the most influential properties defining the mass transfer. It has been shown that some materials exhibit side pores (50-500 nm) to enable a fast mass transfer. But these materials show low binding capacities (about 20 g/L) due to the low surface area. To enhance binding capacity, higher surface area is required, susually acheived through smaller pore size. Materials exhibiting aobut 100 nm pore sizes have shown 40 g/L binding acpaicites, even more about 70 nm pore size exhibiting materials ahve shown 56 g/L binding capacities. Due to the smaller pore size, the diffusion of the target molecule is slower, requriing certain target molecule residence time to acheive binding capacities abbove 40 g/L. The usual range of used residence times is in the range 3-6 minutes to assure that the target molecules diffuses towards the binding sites. (Skudas, US 20130280788)

Breakthrough curve: a plot of product concetnraiton at the outlet of a column as the column is being laoded with fluid at its inlet (this can be plotted v. time, mass loaded or volumn loaded). (Gjoka (US 2017/0016864)

Breakthrough experiment: an experiment where a column is overlaoded with product in order to ensure product breakthrough at the outlet so that a plot of the breakthrough curve can be obtained (column is typically loaded to 100% of its dynamic binding capacity). (Gjoka (US 2017/0016864)

Cycle time: amount of time requried for one column to complet an entire set (load, wash, elution, reneration and equilibration) of chromatogrpahy unit operations. (Gjoka (US 2017/0016864)

Load residence time: the resident time condtiion at which feed material (containing product) is loaded onto the column. 

Operating binding capacity: amount of product that is loaded divided by the column volume. (Gjoka (US 2017/0016864)

Productivity: grams of product processed per liter of sorbent per hour (operating binding capacity/cycle time) (Gjoka (US 2017/0016864)

Residence time: the amount of time it takes for a non-interacitng particle in the mobile phase to pass through the volumn of stationary phase (sorbent volumn/flow rate). (Gjoka (US 2017/0016864)

Static binding capacity: amount of product htat stationary phase is capable of binding under the condition of no flow. (Gjoka (US 2017/0016864)

Introduction: 

In continous chromatography, several identical columns are connected in an arrangement that allows columns to be operated in series and/or in parallel. Comared to a single column or batch chromatogrpahy, wherein a single chromatogrpahy cycle is based on several consecutive steps, such as loading, wash, elution and regeneraiton, in continous chromatogrpahy based on multiple identical columns all these steps occur simultaneously but on different columnes each. Continous chromatogrpay operation results in a better utlization of chromatography resin, reduced proessing time and reuded buffer requirements, all of which beenfits process economy. Rose (WO 2017/140081).

In continous chromatography, several identical columns are connected in an arrangement that allows columns to be operated in eseries and/or in parallel, depending on the method requirements. Thus, all columns can be run in principle simultaneously, but slightly shifted in method steps. The rpcoedure can be reepated. so taht each column is laoded, eluted and regenerated several times in the process. Compared to conventional chromatogrpahy, wherein a single chromatogrpahy cycle is based on several consecutive steps, such as laoding, wash, elution and regeneration, in continous chromatography based on multiple identical columns all these steps occur simultaneously but on different columns each. Contuous chromatogrpahy oepration results in a better utilization of chromatogrpahy resin, reduced processing time and reduced buffer requirements, all of which benefits process economy. One example of continous chromataphy is simulated moving bed (SMB) chromatography. (Skudas, US 20130280788)

Multiple-column chromatogrpahy (MC) involves loading two or more columns connected in series, where feed sample is passed form the iinlet of a first column through the outlet and into the inlet of a second column, through the outlet of the second column and so on, depending on how many columns are connected. This allows the first column to be over-loaded and the target product passing form the first column (that would otherwise be lost to waste) is captured by a subsequent column. In MCC, one column can be loaded while another stage in the cycle can be carried out on another column. When a firt column is over loaded adn the target is passed to a second column, this can be referred to as a “second pass” as the over loaded target product is passing into the second column. Depending on the number of columns, there can be a third pass as the over-laoded target is passing into a third column and so on. (Gjoka (US 2017/0016864). 

Sequential multicolumn chromatography (SMCC): 

SMCC uses full automationa and flexible, asynchronous scheduling of multiple columns (up to six) in enabling processes to run at linerar flow rates over 1,000 cm/h and effectively make use of almost 100% of resin capacity. The end result of approaching the maxiumum loading capacity while increasing process flow rates is 1.5-40 fold improvement in productivity.  Furthermore, several chromatographic and tangential-flow filtration (TFF) steps can be integrated to make continous downstream processing possible. SMCC uses the same type of stationary phase and the same separation techniques as are used in any given batch chromatography prcoess. With SMCC, a single batch column is divided into four smaller columns. Three of those four columns are available for laoding in linear sequence while the fourth column is eluted, regenerated, and equilibrated. This happens out of sequence with continual laoding until each column is ready to return to the linear sequence in rotation. (Holzer, “multicolumn chromatography” Bio-Process Intl 2008, 6, 74-82).

Particular Ligands Used

(Skudas, US 20130280788) discloses pvoiding at least three separation units having the same matrix, preferably an affinity or ion exchange chromatogrpahy matrix, which are connected so that liquid can flow from one separation unit to the subsequent one and form the last to the frist separation unit, b) feeding the sample on the first separaiton unit so taht while the sample is loaded on this separation unit wherein the sample is at a pH and conductivity enabling the target molecuels to be bound to this separation unit, said separation unit is at least part of the loading time in fluid communication with the next separation unit so taht target molecuels not bound to the frist separation unit can bind to the next separation unit, at the same time at least eluting and reqequilibrating one separaiton unit different formt eh separaiton unit that is loaded and form the one that is in fluid communicaiton with the separation unit that is laoded. That measn that the other process steps like washing, eluting and reequilibrating that are needed in a chromatogrpahic seapration pcoress are perfomred on one or mroe of thsoe separaiton units that are not in fluid communication with the separation unit or units that are being loaded, c) switching the feed to the next separaiton unit. That means when the separation unit that has jsut been laoded is fully loaded, the sample feed to this separation unit is stopped and by simultaenous handling of the vlaves of the system without interruption directed to the separation unit that is next in the circle. The separation unit that is next in the circle is the one which was before at least part of the loading time connected to the outlet of the separation unit that has been laoded. Consequently, this separation unit is already partly loaded with those target molecuels that have not been bound to the frist seapraiton unit. d) feeding the sample on the next separation unit so that while the sample is laoded on said next seapration unit wehrein the sample is at a pH and condcutivity enabling the target molecuels to be bound to said net separation unit, said next separation unit is at least part of the loading time in fluid communication with the separation unit after the next so that target molcuels not bound to said next separation unit can bind to the separation unit after th enext, at the same time at least eluting and reequilbrating one separation unit different form the the separation unit that is laoded and from the one that is in fluid communciation with the separation unit that is laoded. 3) repeating steps c) and d) one ore more times. 

Protein A

Skudas (EP 2656892) discloses continous affinity chromatography such as Protein A to purify a target molecule such as an antibody. At least three separation units have the same chromatography matrix are connected so that liquid can flow from one separation unit to the subsequen one and form the last in the to the first separation unit. The sample is fed onto a first separation unit so that while the sample is loaded and the target molecule is bound to thsi separation, said separation unit is at least fluid communication with the next separation unit so that target molecuels not bound can bind to the next separation unit at the same time that washing, eluting and/or requilibrating one separation unit different from the separation unit that is being lodaed and switching the feed to the next seapration unit.  Skudas discloses using two seprations units A1 and A2 both having the same chromatography matrix such as an affinity chromatoraphy, a cation exchange, AEX or MM matrix and a separation unit B that has a CEX, ZEX or AEX matrix. In a preferred embodiment A1 and A2 have an affinity chramotgraphy matrix and unit B has a CEX matrix. In a preferred emodiment, the sample is continously loaded alternatively to either separation unit A1 or Ab and in another embodiment while loading the sample onto unit A1 the fluid outlet is at least partly in fluid communication with the fluid inlet of unit A2 to enable capture of the starting to leach target molecule from A1 to A2 and while loading the sample onto A2 the fluid outlet of A2 is at least partly in fluid communicaiton with the fluid inlet of A1 to enable the capture of the starting to leach target molecule form A2 to be bound to A1.

Hydrophobic Interaction Media:

Mattila (US 16/459,187, published as US 20200002373) discloses a method for preparing a target polypeptide form a mixture using a chromatography apparatus that includes a plurality of zones/chromatographic columns where the one or more columns include hydrophobic interaction media. Such chromatography apparatuses may include pre-manufactured apparatuses (e.g., Cadence TM BioSMB (Pall Biosciences), BioSCR (novadep), VaricolR (novasep), Octave (SembaR Biosciences) or mroely two or more standard batch chromatography apparatuses used in tandem. In some embodiments, the method includes passing the mixture to a first column, passing an effluent from the first column to a second column, passing mobilde phases to a thrid column wehrein each of the plurality of columns includes an outlet connectable to the other columns and a sum of residence times for teh mixture in the first and second column is substantially the same as the sum of the residence times in the third column. In some ebmodiments, passing mobile phase(s) to a column may include passing a wash buffer and after passing a wash buffer regenerating the column. 

Protein L:

Muller, “Intensification of Fab-fragment Purification, Multicolumn chromatography using prepacked prtoein L columns”, BioProcess international, June 2023, 21(6)) discloses a MCC prcoess using a protein L affinity chromatography (Tosoh Bioscience’s Toyopearl AF r-protein L-650F resin) where the Octave Bio system was equipped with five 1 ml SkillPak Bio prepacked columns (all from Tosoh Bioscience) containing the respective protein L resins. The Octave BIO system conssits of six pumps, a switching-valve block and a detector array. Each pump is designated for one buffer required in the process. Trhough the voalve block, up to eight columns can be addressed by different pumps in aprallel or connected to each other in series. The detector array provides precise control of up to four different process streams with regard to UV adsorption, conductivity, and pH. The method was used with Tosoh Bioscien’es ProComposer Method Wizard tool. When three of the columns are in the loading pahse, the remaining two go through the phases of wash and elute, then CIP and equilibration. Once the first column in the loading series is fully loaded, the column ports are switched into position against the flow stream. In MCC, product breakthrough is less of a risk druing laoding becasue of the secondary load columns. The columns were loaded to 85% of their previously determiend SBCs, resulting in loading masses of 45.3 mg/mL resin for the Tosoh resin. 

Mixed Mode Chromatography:

–HCIC:

Luo designed a swo step chromatographic separation method taht combined ABI-4FF and MMI-4FF resins for bioseparation. The ABI-4FF resin was first used to remove IgA at pH 4.5 and the flow through fraction from the frist step contained IgM and IgG. In order to capture IgM, the MMI-4FF resin was then used for purificaiton further at pH 4.5. After optimization, IgM purity of 65.2% and purificaiton factor of 28.3 were obtained. Heidebrechi also developed a scalable and cost efficient process that connected Capto MMC and MEP HyperCel to isolate bovine IgG from colostral whey.  (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

Optimization of Multi-Column Chromatography:

See also Tosoh Bioscience ProComposer Method Wizard tool  (converts batch method to MCC)

Gjoka (US 2017/0016864) disloses a method of determing an optimum operating binding capacity for a MCC where loading experiments are performed on a single column at different residence times and/or different flow rates to determine an optimum operating binding capacity for the MCC prcoess. Accurate prediction of the operating capacities enables estimation of many other multi-column process parameters such as productivity, cycle time, total number of columns and/or bufer ulitiziation. In one embodiment, the method includes (a) loading a target on a column at a first residence time and/or first flow rate, (b) loading the target on the column at a second residence time and/or flow rate wehre the first residence time and/or flow rate is different than the second residence time and/or flow rate, (c) generating breakthrough curves for the first reisdence time and/or first flwo rate and for the second residence time and/or second flow rate and (d) detemrining an optimum operating binding capacity fo the MCC process. Typically the second residence time is about double the first residence time and/or the second flow rate is about half the first flow rate. The inventors realized that if the columns are connected in series and loaded straight through to saturation (in the absence of valve switching to simulate countercurrent movement of the columns), the amount of target product bound by the first column immediately before breakthrough of the last column in the series is the ideal operating binding capacity. In other words, while one could masure the breakthrough curve by using a single column with a deterctor after it and the other breakthrough curve by  connecting two or mroe columns in series with a single detector at the end of the columns, the inventors found that the shape of the product brekathrough curve is almost completely dependent on the residence time applied whehn laoding a column. This enables generation of the two breakthrough curves with just a single column and single detector after the columns. To mimic the MMC one brekthrough curve is performed at a chosen residence time and to mimic two columns in series anouter breakthrough curve is perforemd at double the chosen residence time. The breakthrough curve at the chosen residne time is used to caclulate the amount bound to what would be the first column in series, where the area above the brekathrough curve is representative of the amount bound to the column. The break through at double the residence time is representative of the seocnd column in series and is necessary to inform how much product bould be laoed in MCC without incuring product low to the flow through. This brekathrough information is used to detemrine a limit as to how much proeduc is laoded and calculate a loading time or amount limit in the intial breathrough cruve. With these two breakthorugh curves it is possible to detemrine the amount bound ot the frist column before breathorugh of the seocnd column which is the oeprating binding capacity. This can be extned to three or more oclumns. For three columns, the seocnd breakthrough curve would be performed at three times the chosen residnece time and so on. 

Lin (US Patent Application No: 18/117,479, published as US 20230203092) discloses optimization for capturing proteins by multi-column continous chromatography (MCC) by performing a single time fo protein breakthrough experiment to obtain a protein breakthrough curve and integrating the breakthrough curve to obtain a single column loading capacity and establishing a linear relationship between the interconnected load time and the load residence time, solving for the optimal number of operation column for capaturing proteins and solving for the optimal load residence time for capturing proteins and solving for the maximum productivity of capturing proteins by MCC. In step 1, under the conditions of a set laoding protein concentration and arbitrary load residence time, a single time of protein breakthrough expeirment is performed to obtain a protein breakthroughcure. In step 2, under a set breathrough percentage (greater than or equal to 50%), integrating the breakthrough curce to obtain a singel column loading capacity of continuous chromatography and estalbishing a linear relationship between the interconnected load time and the load residence time through the single column loading cpacity. In step 3, using the linear relationship between the itnerconnected laod time and the load residence time to solve for the optimal number of operating columns for cpaturing proteins by MCC under the set laoding protien concetnraiton and protein breathrough perentage. 

Attachment of Protein A to a solid support, Generally

Methods of attaching protein ligands sucha s protein A and G to a solid support include ativating a media with a functional group (“activated group”) such as epoxide (epichlorohydran), cyanogen (cynogens bromide (CNB4), N,N-discuccinimidylacronate (DSC), aldehyde or an activated carboxylic acid (e.g., N-hydroxysuccinimide (NHS) esters), carbonyldiimidazole (CDI) activated esters). These activated groups can be attached directly ot the base matrix, as in the case of CNBr, or they can be part of a “oinker” or spacer molecule which is typically a liner chain of carbon, oxygen and nitrogen atoms, such as the ten membbered chain of carbon and oxygen found in the linker butane0diol diglycidyl ether (a common epxide coupling agent). The activated media is then equilibrated with the protein ligand under coupling cnoditions. For Protien A, typically 4-6 mg of progen (lgiand density) may be loaded per ml of media reusulting in an IgG maxim static capacity of 40 g/L. (Gian, US 7,833,723  and US 2007/0207500). 

Support-Arginine Linker – (amide bond) -ProteinA:  

Profy (US5,260,373) disclsoes covalently joining an immobilization support to an arginine containking linker and then joining the linker through an amide bond to Protein A.

Support- amino group –amide bond –linker –(carboxyl end) Protein A:  NH2 –R1 (protein A) – CO – NH –R2 (linker) — NH – Y (support)

Iwakura (US 2008/0051555) discloses a support having affinity for an antiboy that includes a peptide capable of binding to an antibody such as Protein A that is immobilzied at a carboxy end to an insoluble support haivng a primary amino group such as one contained in a lysine via an amide bond mediated by a linker sequence. 

An affinity separation matrix is often of organic nature and based on polymers that expose a hydrophilic surface to the aqueous media used, i.e., expose hydroxy (-OH) carboxy (-COOH) carboxamido (-CONH2), amino (-NH2), oligo or polyethylenoxy groups on their external and, if present also on internal surfaces). (Ander, US14/385336)

Cross-Linking

The support can be corsslinked, such as with hydroxyalkyl ether crosslinks. Crosslinker reagents producing such crosslinks can be e.g., epihalohydrins like epichlorohydrin diepoxides like butanediol diglycidyl ether, allylating reagents like ally halides or allyl glycidyl ether. Crosslinking is beneficial for the rigidity of the support and improves the chemical stability. Hydroxyalkyl ether corsslinks are alkli stable and do not cause significant nonspecific adsportion. (Ander, US14/385336)

Berg (US6,602,990) discloses a process for the production of a porous cross-linked polysaccharide gel by (a) preparing a solution of the polysacharide, (5) adding a bifunctional cross-linking agent have one active site and one inactive site (c) reacting hydroxylgroups of the polysaccharide with  the active site of the cross linking agent (3) forming a polysaccharide gel (e) activating the inactive site of the cross-linking agent (4) reacting the active site from (e) with hydroxylgroups of the polysaccharide gel.

Arginine Linkers:

Profy (EP0282308) describes an improved immobilized protein A resin which has high binding capacity for IgG1. The immobilization support material can be any support used in immunoassays such as filter paper, plastic beads, test tubes made from polyethylene, polystyrene, polypropylene or other suitable material. The linker consists of arginine coupled to the support directly or through a chemical chain of any lenght. The chemical chain can be another protein. The immunoglobulin binding prtoein is preferably protein A which can be joined to the linker through an amide bond.

Johansson 9US6,399,750) discloses a separation medium of the formula –B–X–Protein A-cys where B is a bridge which binds to the base matrix, X contains a heteroatom N or S (X is a thioether sulfur (-S-) and/or a secondary amine (-NH-) originating from Protein A-cys and Protein A-cys is recombinantly produced Protein A which contains cysteine in its amino acid sequence. The optimal molar ratio between the total IgG binding capacity and the amount of Protein A on the matrix may vary depending on the number of IgG bidning doamins that are present in the Protein A of the adsorbent.

PolyHistidine Linkers

Tamori (US 2013/0041135) discloses filler for affinity chromatography represented by the formula R-R2 where R represetns an amino acid sequence consisting of 4-300 amino acid residues containing a region consisting of 4-20 contiguous histidine residues and R2 represents an immunoglobulin binding sequence of 50-500 amino acids containing Z domain of Protein A. 

PolyProline Linkers

IChii (US 15/745,855, published as US 2018/0222939) discloses an affinity support which contains a ligand ofr the geenral formula R-R1 wherein R represents a linker that contains a polyproline bound to the solid support and R2 is an antibody bidning protein like Protein A. 

Fusion Proteins as Linkers

See also Carbon Filters

Companies:  Merk Group:  Merck KGaA   (subsidiaries: EMD Millipore Corporation)

Separation of Antibodies using Hydrophobic Chromatography Resins

Polystyrene or poly(ethyl)styrene or Vinylbenzene:

–cross-linked with Copolymers od divinylbenzene and ethyleneglycol methacrylate

Skudas (US 15/123110, published as US 2017/0073394) dicloses HCPs, antibody fragments and low MW substances can be separated from antibodies using a hydrophobic chromatography material made of cross-linked vinylbennzene, ethylstyrrene, poly(ethyl)styrene-divinylbenzene or poly(ethyl)styrene-divinylbenzene ethyleneglycol-dimethylacrylate). Preferably the resin is composed of polystyrene or poly(ethyl)styrene, which is cross-linked with copolymer of divinylbenzene and ethyleneglycol methacrylate in a ratio of 98:2 up to 10:90% by weight. The contacting is operated in flow through mode where the HCPs and fragments are absorbed to the hydrophobic chromatography material. 

Separation of Antibodies using Activated Carbon 

 Different Modes used

–Flow through mode: 

Bian (US 13/565463, published as US2013/0197200 and US 9096648; see also US 14/747029, published as US 2016/0016992; see also US 16/358,845, published as US 2019/0218250) discloses that carbonaceous material such as activated carbon can be incorporated into a chromatography column based antibody/ protein purification process in a flow through mode, resulting in reducing the burden of chromatography columns. The activated carbon can be used either upstream or downstream of a capture chromatography step to reduce the level of one or mroe impurities. In one embodiment, an antibody sample is contacted with an affinity media and the flow through contacted with a carbonaceous material such as activated carbon and the eluate from the activated carbon contacted with an anion exchange media. 

Ishihara (US 13/826195, published as US 2014/0046038) dicloses using activated carbon for the separation of proteins such as antibodies in a non-adsorption mode. Examples of the activated carbon include mineral based such as coal based and petroleum based activated carbon. Examples of plant based inlcude wood based activated carbon. The raw material of the activated carbon must be carbonacoues which includes materials such as sawdust, charcoal, ash, peat moss, peat or wood chip, coals such as lignite, brown coal or antracite, coal pitch, petroleum pitch, oil carbon, rayon, acrylonitril or phenol resin. 

Kozlov (US 14/891,724, published as US 2106/0090399) discloses a method of reducing antibody fragments from an antibody prepration by applying the same to activated carbon where the activated carbon binds the fragments and then removing the AC as by filtration or centrifugation. 

—-Protein A -AC (flow through) -AEX (flow through) -CEX

Kozlov (US 2018/0215786) also disclsoes a flow-through process for purifying a target molecule sucha s an antibody from a Protein A eluate which includes the steps fo contacting the eluate form the (a) Protein A column with activated carbon, (b) contacting a flow through from the AC with AEX and (c) contacting the flow through with CEX at a density of about 1-30 mM, wherein the eluate flow continously through steps (a)-(c). 

Singh (US 2013/0012689) discloses a continous profess for purifying a target molecule which includes flow through activated carbon followed by a flow through AEX and CEX media. 

Xenopoulos (US2015/0133636) discloses purification of an antibody/protein by from a Protein A eluate by contacting the eluate with activated carbon and then contacting the flow through with AEX, the flow through from this with CEX and obtaining the flow through sample comrpsing the antibody.

Xenopoulos (US 15/654876, published as US 2017/0320909) also discloses a method of purifying an antibody by precipitating contaminant(s) from a sample and then subjecting the clarified sample to a bind and elute chromatography step that incldues at least two separation units and then subjecting the eluate to a flow through purification step that includes two or more media, one of which is activated carbon and the other which are AEX or CEX. The process is advantageously practiced concurrently for at least a portion of two steps using surge tanks and/or static mixers. In a specific emobdiment the flow-through purificaiotn step is carried out as activated carbon followed by AEX and then CEX with an in-line static mixer and/or surge tank being used between AEX and CEX to change pH. 

Commercial Matrixs of Protien G

Protein-G Sepharose 4 Fast Flow: is manufactured by GE Healthcare (Hober, J. Chromatogr B, 2007, 848, pp. 40-47 and contains SpG immobilized as a ligand. 

Introduction

Protein G is a cell surface protein from Streptococcus: it contains multiple copies of two different small domains which can independenlty bind albumin and IgG. It is widely used as an affinity ligand for the purificaiton of IgG. (Palmer, J. Biotechnology 134 (2008) 222-230)

Compared to SPA, SPG displays a different binding sepctra for immunoglobulins from different species and subclasses thereof. The IgG binding domains of protein G are now widely used as an immunological tool for the affinity purification of monoclonal antibodies. Production of subfragments constructued by DNA technology have shown that an individaul C region is sufficient for full IgG binding. Nilsson (US6,740,734). 

Generally, protein G binds more IgG subclasses and with higher affinity than protein A. Protein G binds to human IgG of all four subclasses as well as to a number of mammalian monoclonal antibodies including those form mosue and rate, whereas protein A binds to neitehr human IgG3 nor to rat IgG. All IgGs that have been examined show a higher bnding affinity to protin G than to protin A. Protein G also interacts weakly with Fab, the antigen-bnding fragment of IgG but its binding affinity is only aobut 10% of the affinity for Fc. (Jones “Crystal structure of the C2 fragmetn of streptococcal protein G in complex with the Fc domain of human IgG” Structure, 1995, 3: 265-278). 

Protein G is a good choice for general purpose capture of antibodies at laboratory scale since it binds a broader range of IgG from eukaryotic species and binds more classes of IgG than protein A. Usually, protein G has greater affinity for IgG than protein A and exhibits minimal binding to albumin, resulting in cleaner preparations and greater yields. (GE Healthcare “Affinity Chromatography”, copyright 2014-2016) 

Unpredictability of IgG binding

Hober (Protein Engineering 15(1) pp. 835-842, 2002) discloses that while single mutations N7A and N36A in the C domain of SpG resulted in constructs with retained affinity to Fc, a significant decrease in affinity to IgG was observed when substituting Asn34.  see also Kossiakoff, (J of Immunological Methods, 415, pp. 24-30, 2014, citing Hober and stating the N7A/N36A double mutant led to a loss of Fab bnding affinity, presumably because of the critical contacts Asn36 provide in the Protein G-Fa complex.); see also US 2018/0044385)

Kossiakoff, (J of Immunological Methods, 415, pp. 24-30, 2014); see also US 2018/0044385) discloses that an 8 point mutations of the C2 domain of Protein G demonstrated a preference for the human IgG1 isotype as its affinity was decreased by a factor of 20-50 fold for IgG2, IgG3 and IgG4, suggesting subtile differences in domain orientation impact binding. 

Structure of Protein G

Streptococcal protein G cinludes two or three domains that bind to the constant Fc region of most mammlain IgGs. Protein G is functionally related to staplylococcal protein A, with which it shares neither sequence nor structural homlogy. Sauer-Erikson (Structure, 1995, 3(3), 265-278). 

The beta-1, beta-2 and beta-3 domains of SpG carry out molecular recognition of both the Fc part of IgG and the Fab part of the homo sapiens IgG. The amino acid sequene of these domains differ by originating bacteria kinda and strains. Some subspecies lack beta-3 domain. (Koneka Corp, Japanese Patent Publication number 2009-195184)

B1 domain: is a 56 residue domain that foled into a four-stranded D-sheet and one a-helix. Despite its small size, it has two separate IgG binding sites on its surface, each interacting respectively with specific, independent istes on the Fab or Fc fragments of the antibody. Hellinga (US 6,663,862). 

C1,C2 C3 domains: Streptococcal protein G is a mltidomain protein with seaprate albumin binding and immunoglobulin binding regions. SPG exhibits a braod spectrum of binding to IgG subclasses, and bind to all four human subclasses of IgG as well as several animal IgG subclasses. The IgG binding region consists of three domains, C1, C2 and C3, exhibiting high sequence homology. The C2 domain is a small protein composed of only 55 residues. Hober (Protein Engineering 15(1) pp. 835-842, 2002)

The C2 domain of Protein G from Streptococcus is a multi-specific prtoein domain; it possess a high affinity for the Fc region of IgG but a much lower afifinity for the constant domain of the antibody fragment Fab. (Kossiakoff, J of Immunological Methods, 415, pp. 24-30, 2014); see also US 2018/0044385). 

there are only six differences in the amino acid sequences of the C1 and C3 domains of protien G. Nevertheless, the C3 domain binds IgG about seven times tighter than the C1 domain. (Jones “Crystal structure of the C2 fragmetn of streptococcal protein G in complex with the Fc domain of human IgG” Structure, 1995, 3: 265-278).

Where/What Protein G binds

It has been shown that binding to intact IgG as well as antibody fragments such as F(ab’)2 and Fc regions by protein G. Protein G also binds to immunoglobulins of most species including rat and goat and recognises most classes and subclasses. (Darcy , chapt 20 from Protein Chromatography: Methods and Protocols, Methods in Molecular Biology, vol. 681 (2011).

IgG binding:

Protein G recognizes a common site at the interface between CH2 and CH3 domains on the Fc part of human IgGA1, IgG2, IgG3 and IgG4 antibodies (Fcgamma) with high affinity. In addition, Protein G shows binding to the Fab portion of IgG antibodies through binding to the CH1 domain of IgG in combination with a CL domain of the kappa isotype. Protein G only binds to Fab from IgG1, IgG3 and IgG4 but not to Fab of IgG2. Binding affinity towards CH1 is significantly lower compared to its epitope on the Fc part.  (Hermans US13/982970). 

Protein G and protein A have developed different starties for binding to Fc. The protein G-Fc complex involves mainly charged and polar contacts, whereas protein A and Fc are held together through non-specific hydrophobic interactions and a few polar interactions. Erikson (Structure, 1995, 3(3), 265-278)

Protein G binds Fc at the hinge region that connects the CH2 and CH3 domains. There are three residues of the CH2 domain of Fc which are involved in the interfacial interacts: Ile253, Ser254 and Gln 311. In the CH3 domain, there are two areas which contribute to the interface: Glu380 and Glu382 and the residues His433-Gln438. All residues that interact with protein G are situated within loop regions of Fc except for Glu380, Glu382 and Gln438 which are exposed on one of the beta-hseets making up the CH3 domain. (Jones “Crystal structure of the C2 fragmetn of streptococcal protein G in complex with the Fc domain of human IgG” Structure, 1995, 3: 265-278).

Serum Albumin binding:

Streptococcal protein G (SpG) is capable of binding to both IgG and serum albumin. The structure of one of the three serum albumin binding domains has been deteremind, showing a three helix bundle domain, named ABD (ablumin binding domain) and is 46 amino acid residues in size. It has also been designated G148-Ga3. (Abrahmsen, WO/2009/016043)

Conditions used:

Proudfoot (Protein Expression and Purificaiton 8, 368-373 (1992) disclsoes purification of a Fab’ fragment by protein G sepharose. After the cell culture sueprnatant was applied to the column a peak of absorbance at 280 nm of weakly bound material was recoverd with a pH 7 wash (peak A). A further peak (peak B) could then be eluted from teh column with a low pH wash. Binding to teh protein G-Sepharose was efficient with no Fab’ ro F(ab’)2 deteced in the column flow through. 

Analogues of Protein G: Beta1/C2 domain mutants:

An SpG functional domain having an IgG binding ability is referred to as the beta domain or C domain. (Yoshida, US 16/585,895, published as US 2020/0031915)

The C2 domain of Protein G from Streptococcus is a multi-specific protein domain; it possesses a high affinity for the Fc region of the IgG, but a much lower affinity for the constant domain of the antibody fragment (Fab), which limits some of its applications. Accordingly, the Protein G interface has been engineered using phage display to create a Protein G variant with beta point mtuations to provide about 100 fold improved affinity over the parent domain for Fab fragments. A variant was also isolated having enhanced stability to basic conditions whcih is useful for regeneration. In some embodiments, the protin G Fab binding region is modified to have Tyr at position 16, Gln, His, or Tyr at position 452 or Tyr or Phe at position 40. Also disclsoed is pruificaiton of Fabs using the variants with a pH dependence of elution.  (See Kossiakoff (US 2018/0044385 and Baily, J of Immunoglobical Methods, 415 (2014) 24-30).

Deletions at N/C terminus:

The N-terminal of wild SpG-beta1 is Asp but it is Thr in wild type SpG-B1. The N-terminal is not contained in (Hober, Protein Engineering, 15(1), pp. 835-842, 2002) and the kind of amino acid at the N-temrinal makes no difference. (Yoshida, US 16/585,895, published as US 2020/0031915). 

Improved Alkali stability:

–Replacement of Asn residues: 

Protein G is a cell wall protein form group C and G streptococci. It is a type III Fc receptor which binds with high affinity to the Fc region of antibodies, in particular, IgG antibodies. It has been shown that Asn residues are the most susceptible at extreme alkaline pH and that replacement of all 3 Asn residues within the IgG binding domain of PrtG improves stability towards caustic alkali by about 8 fold (Palmer, J. Biotechnology 134 (2008) 222-230).

—-Substitution at Position 8:

Hober (Protein Engineering 15(1) pp. 835-842, 2002) disclsoes that the C2 domain contains three asparagine residues which were substituted for alanines since no alternative amino acids were found in the homologous sequences. Three single mutants were designated C2 N7A, C2 N34A and C2 N36A. 

Lindman (Biophysical J. 90, 2006, pp. 2911-2921) teaches a variant of protien G B1 domain having 3 mutations which include N8D. 

Palmer, (J. Biotechnology 134 (2008) 222-230) discloses that Asp8 lies within a beta-strand and that a N8T/N35A double mutant showed 17 fold more stability. 

Yoshida (US 16/585,895, published as US 2020/0031915) discloses an altered Protein G which can bind to an Fc region and a Fab region of an immunoglobulin and which has excellent chemical stability to an alkaline solution which includes for example the beta-1 region of SpG where the 8th position is substituted by Asp, Glu, His, Ile, Lys, Leu or Val. 

Mutants having higher binding activity for the Fab region (useful for purificaiton of Fab fragments):  

Since SpG has a weak binding affinity to a Fab region, the performance of a SpG affinity separation matrix product to maintain an antibody fragment which does not contain a Fc region and which contains a Fab region only is considered to be insufficient. Accordingly, efforts ahve been made to improve the binding affinity of SpG to a Fab region by introducing a mutation into SpG. (Murata and Yoshida, US 2017/03349457). 

—-27Glu, K28, W43, Y45:

Hellinga (US 6,663,862 and WO 00/74728) discloses B1 Protein G mutants which exhibit binding activity for a Fab fragment of an IgG but substantially no binding activity for a Fc fragment of an IgG such as one having a mutation at the glutamate 27, lysine 28, tryptophan 43 and tyrosine 45. 

—-13T/S, 19V/L/I, 30V/L/I, 33F:

Yoshida (US 14/914439, published as US2016/0289306; see also Murata and Yoshida, US 2017/03349457) discloses a B1 domain of protein G with substitution of an amino acid residue at not less than the 13th, 19th, 30th and 33rd positions and having a higher binding force to the Fab region .  Specifically, a humanized monoclonal IgG was fragmented into a Fab fragment and a Fc fragment using pepain, and only the Fab fragment was seaprated and purified. 

(Koneka Corp, Japanese Patent Publication number 2009-195184) discloses variant protein G beta domains which bind more strongly to IgG-Fab fragment. 

Multimers of Protein G

Kossiakoff (Kossiakoff, J of Immunological Methods, 415, pp. 24-30, 2014); see also US 2018/0044385) discloses a fusion between to or mroe polypeptides of protein G variants. Multi-valency is a common feature of many biological systems that ahrness the simultaneous engagement of tethered ligands or multiple receptors. Biological processes use this as a means to increase the effective affinity of weak binding ligands as well as to qulitatively modify the activity of proteins through a multi-valent engagemnt and molecular crosslinking. Antibodies exploit multi-valency thorugh natrually occruing formats including the IgG (bivalent), IgA (tetravelnt) and IgM (decavalent). 

See also Plasma Protein Precipitation by Various Agents 

Nonionic, water-soluble polymers induce protein precipitaiton by excluding water from the solvation structure of a protein. PEG is the most widely studied and widely used polymer, but dextrans are also used for this purpose. Thylic acid, caroxymethl cellulose and polyethyleneimines precipitate proteins at much lower concetnration (usually e effect of polymers as precipitating agents is similar to partitioning in aqueous two-phase polymer systems. High PEG concentraitons are required to preciptiate low-molecular weight proteins, wehreas low concentraitons are required for high molecular weight proteins. Polyelectorlytes such as polyacrylic acid, caryleneimines preciptiate proteins at a much lower concentraiton than nonionic polymers. They act more like flocculants and adsrob to the prtoein. Thus, polyelectrolytes, unolke PEG, coprecipitate with the protein and can cause irreversible denaturation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)boxymethl cellulose and polyeth

In the mid 1960s Polson (Biochem. Biophys. Acta 82, 463-475 (1964) analyzed a variety of high molecular weight polymers for purifying proteins include polyethylne glycol (PEG), dextran and polyvinylpyrrolidone (PVP).

Antibodies can also be co-precipiated with negatively charged polymers. Selectivity parallels CEC Co-precipitation of non-IgG contaminants with positively charged polymers parallels the selectivity of AEC (Gagnon “Technology trends in antibody purificaiton, J of Chromatography A, 1221, 57-70 2012). See precipitation of antibodies with polyelectrolytes 

Precipitation with Non-ionic Organic polymers: 

Non-ionic organic polymers refers to a naturally occurring or synthetic hydrocarbon composed of linked repeating organic subunits that lack charged groups. It may be linear, cominatly linear with some branching, or dominantly branched. Examples include PEG, polypropylene glycol, polyvinylpyrrolidone (PVP). (Gagnon, US 14/766,131). 

1. Polyethylene glycol (PEG): 

Purification of proteins such as antibodies by precipitation with polyethylene glycol (PEG) has been described. It is typically performed as an aqueous phase technique, where PEG is dissolved in an aqueous protein preparation and causes the protein to precipitate from that solution. The size and concentration of PEG are known process variable, as is pH (Gagnon, US 14/766,131 published as US 2015/0376231). 

PEG is a nonionic polymer of ethylene glycol. It has been used for industrial prepariton of polyclonal antibodies for decades. In parallel with its polyclonal applicaitons, it is applied more frequently to m IgMs than to IgGs. (Gagnon, “Purificaiton tools for monoclonal antibodies” Validated Biosystems, Inc., 1996, pp. 1-269). 

Knevelman “High-Throughput screening techniques for rapid PEG-based precipitaiton of IgG4 mAb from clarified cell culture supernatant” pp. 697-705) discloses operating conditions of PEG precipitation of IgG4 as a function of mAb concentraiton, precipitatnt concentration and pH. 

Lain (“EPG Precipitaiton: A Powerful Tool for onoclonal antibody purificaiton” BioPharm, 2010, pp. 108) discloses optimization of PEG concentraiton and pH for precipitation of mAB. PEG was added as a 40% (w/w) stock soltuion to the desired final conetration and pH of the clarified media was adjusted to desired level with 2-M Tris in a 15 mL conical tube. The tube was then centrifuged and supernatant decanted. The pellet was redissolved in PBS. The final process resulted in a product yield of 90% and HCP reudction fo about 1 LRV. 

PEG in Combination with Protein-Precipitating salts (“kosmotropic salts”):

Common examples of “protein-precipitating salts” are ammonium sulfate, sodiu or potassium citrate, sodium or potassium pohsphate.  Such salts are commonly referred to as kosmotropic salts. Gagnon (US 14/766,131, published as US 2015/0376231) discloses precipitatin of an antibody with PEG in the presence of a non-protein precipitating salt

–PEG–Ammonium Sulfate:

Brooks (J. Immunoglogical Methods 155(I) 1992, pp. 129-132) discloses a emthod for the purificaiton of mouse monoclonal antibodies from hybridoma culture supernatants using polyethylene glycol 6000 (PEG 6000) and finally reprecipitating using an ammonium suiphate procedure. 

–PEG-Sodium Phosphate (antibody precipitated)

Gervais (WO 2009/016449) disclose purificaiton of antibodies using the steps of precipitation of the antibody using a PEG solution or sodium phosphate.

PEG in Combination with Non-Protein Precipitating salts (“Chatropic salts”):

“Non-protein-precipitating salts” refers to a salt that lacks the ability to mediate precipitation of a desired protein. Common examples include sodium or potassium chloride, sodium or potassium acetate, sodium or potassium thiocyantateor gaunidinium chloride. Gagnon (US 14/766,131, published as US 2015/0376231)

Gagnon (US 14/766,131, published as US 2015/0376231) discloses precipitatin of an antibody with PEG in the presence of non-protein precipitating salts such as sodium chloride, potassium chloride, sodium acetate, potassium acetate, sodium thiocyanate, potassium thiocyanate and guanidine chloride. 

–PEG + NACL:

Gagnon (US 14/766,131, published as US 2015/0376231) discloses precipitatin of an antibody with PEG in the presence of a non-protein precipitating salt such as NACL at a concentration of from about 0.2 – 2.0 M. 

PEG in Combination with Various Types of Chromatographies:

–PEG-AEX:

Tscheliessnig (J. Chromatography A, 1216 (2009) 7851-7864) discloses a two step purification strategy comprising PEG precipitation and AEX for a panel of IgM. 

–PEG-MF/TFF – CEX (bind and elute mode) –AEX –VF — VI –UF/DF:

Kuczewski (Biotechnolgy Journal 2011, 6, pp. 56-65) disclose expression of mAb in a cell line in an extreme density bioreactor process, clarification with enhanced cell settling, PEG precipitation, capturing the precipitate and washing using microfiltration, redissolving in an appropriate buffer at high concentration, CEX with a weak membrane in bind and elute mode, AEX membrane, viral inactivation. 

Coditions/Parameters for PEG Precipitation:

–PH:

Ramanam (US 2008/0214795) disclsoes isolating antibodies by precipitation using various precipitatns such as PEG. The pH of the solution comrising an antibody is adjusted to 0.5 pH unit of the pI of the antibody and PEG is added. 

PEG derviatives which can bind metal ions

Arnold discloses PEG derivatives that can bind metal ions such as Cu2+, Zn2+, Ni2+, Fe3+, Fe2+, Co2+ and Ca2+. The metallated derivatives can, in turn, be used in the extraction or precipitaiton of proteins that interact with these metals. For example, since copper and to some extent nicekl are known to interact with histidines, these metals are good hocies for separation of proteins that contain accessible histidines. If a metal chelate is used that carries a net positive harge, this chelate would be exxpected to interact more faorably with a protein whose surface metal binding sites are adjacent to regions of net negative charge. 

See also Virus inactivation (caprylic acid can be used as an agent to do this)

See also adjustment of pH with various agents under Agents used for fractionation See also AEX for the purification of immunoglobulin

In General

Chauntun (Archives of Biochemistry and Biophysics 89, 218-220 (196) discloses the precipiation of proteins from plama by caproic, htpylic, caprylic, pelargonic, capric and lauric acids. At low concentrsitons (0.04 M) these fatty acids form insoluble complexes, particulalry with alpah and beta globulins. At higher concentraiton (0.1M) albumin and all globulins are precipitated in appreciable amounts. 

Caprylate acid (also called octanoic acid) precipitation: 

The use of caprylic (octanoic) acid for the purificaiton of monoclonal or polyclonal antibodies such as antivenoms and human immunoglobulin is well established. The use of CA for the purificaiton of human immunoglobulin obtained from ethanol fractiona of plama also efficiently inactivates enveloped viruses. A CA final concentration of 3.5% is optimal to obtain immunoglobulins essentially free of albumin. It is proposed that CA binds to specific sites of the protein, thereby inducing partial unfolding of the protein, which exposes additional binding sites. The interfacial protein surface becomes hihgly hydrophobic and increases protein-protein attrraction, cusing association and precipitaiton of the macromolecular complexes. (Morales, Biotechnology and Applied Biochemistry, 59(1), 2012, pp. 50-54). 

Besides its role in IgG purification, caprylic acid in the non-ionized form also acts as an agent that can robustly inactive lipid-enveloped viruses within a few minutes when used at pH <6 and a concentration of >3.7 g/l. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. )

Chanutin (Archives of Biochemistry and Biophysics 89, 218-220 (1960) discloses precipitation of proteins from plasma by caproic, heptylic, caprylic, pelorgonic, capric and lauric acids. At low concentration (0.4 M) these fatty acids form insoluble complexes, particularly with alpha and beta globulins. At higher concentraiton (0.1M) albumin and all globulins are precipitated in appreciable amounts. It is also known that a considerable portion of the globulins of plasma is precipitated in the presence of caprylic acid at pH 4.2. 

Habeeb (Preparative Biochemistry, 14(1), 1-17 1984) discloses that carpylic acid can be used to precipitate nonimmuogloulin proteins from human plasma. In one embodiemnt, to 200 ml of plasma add 400 ml of 0.06 M acetate buffer pH 4, add caprylic acid, centrigue and filter if necessary to remove finely suspended particles and adjust H 6.2 with 1 N sodium hydroxide. The solution was then dialized against sodium acetate pH 5.7 and then mixed with 20 gm DEAE cellulose. 

Steinbuch (Archives of Biochemistry and Biophysic 134, 279-284 (1969) discloses that it is possible to botain IgG from plasma in good yeild with at least 90% purity using caprylic acid. The remaining impurities can be removed on DEAE-cellulose. In one embodiment, the pH of human plasma prtoein is adjusted to pH 4.8 with acetate buffer to form a precipitate and the supernatant solution is pH adjsuted to 4.7, centrifuged with the sueprnatant solution cotnaing pure IgG. 

Caprylic Acid -pH shift:

Lebing (WP 0893450) discloses a method for the purificaiton of Ig from human plasma which includes suspending the antibodies at pH 3.8-4.5 followed by addition of caprylic acid (or other source of caprylate) and a pH sift to pH 5-5.2 A precipitate of contaminating protins forms and is removed, while the majority of antibodies remain in solution. Sodium cpayrlate is again added to a final concentration of not less than about 15 mM and incubated udner conditions sufficient to reduce the titer of active virus (e.g., 1 hour at 25C). A precipitate (mainly caprylate) is removed and the clear solution is diluted with purified water to reduce ionic strengh. Anion exchange is then used. 

Source for Caprylic Acid

Parkkinen (WO2005/073252) disclsoes caprylic acid precipitation by adding caprylic acid as a free acid instead of adding it in the form of a salt, such as sodium caprylate as in US Patents Nos: 5,886,154 and 6,307,028. Caprylic acid slighly lowers the pH of the solution in contrast to sodium caprylate, which increases the pH. Accordingly, no pH shift to pH 5.0-5.2 takes place and virus inactivaiton is carried out at a lower pH. This is beneficial since at low pH the propotion of the non-ioniced form of carpylic acid is higher, and it is the non-ionized form of caprylic acid, which is effective in virus inaction. 

Caprylate acid -AEX

Alred (US 6,955,917 and US2003/0152966) discloses purification of antibodies form human plasma which involves suspension of the antibodies at pH 3.8 – 4.5 followed by additionl of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of contaminating proteins, lipids and caprylate forms is removed while the majority of the antibodies remain in solution. Sodium caparylate is again added, the precipitate removed and the clearl solution applied to AEX to obtain a high yeild of IgG. 

Buchacher (US7,553,938) discloses pararing antibody by adjusting pH of the starting solution to about 4.6-4.95, adding carpylate and/or heptanoate ions such that precipitate is formed and the antibodies are essentially present in the supernatant, incubating the supernatant under conditions of caprylate and/or heptanoate ion concentration, time, pH and termpature, optionally concentrating and DF, applying the filtered solution with at least one AEX such that contaminatns bind the resin while antiboides pass through and then virus inactivation. 

Lebing (US5,886,154) discloses that during human immunoglobulin preparation caprylic acid is generally recognized as an effective precipitating agent for most plasma proteins at pH 4.8, so long as parameters such as temperature and ionic strenght are optimized.  Lebing discloses suspending antibodies at pH 3.8 to 4.5 followed by addition of caprylic acid and a pH shift to pH 5.0-5.2. A prcipitate of contaminating proteins, lipids and caprylate forms and is removed while the majority of the antibodies remain in solution. Sodium caprylate is again added, the precipitate (mainly caprylate) is removed and the clear solution diluted with water to reduce ionic strenght. AEX is subsequently used to obtain IgG. ( See also caprylate in combination with other methods below and chromatography).   

Menyawi (US 14/900499, published as US 2016/0368970) discloses a process for the purificiton of IgG form plasma by providing an acidic solution comprising IgG  with a between 3.5-5.2, adjusting the pH to 5.2-6.2 while maintaining conductivity below 1.5 mScm using a multi-hydroxylated amino compound with or without carboxyl group , incubating for at least 15 mintues and removing any precipitate as by filtration, followed by ANEX, virus filtraiton and UF/DF. . 

Parkkinen (WO2005/073252) discloses preparing immunoglobulin by subjecting crude immunoglobulin solution to caprylic acid to remove protein aggregates and viruses, then IEX to purify the immunoglobulin, filtering to remove virus. 

Steinbuch and Audran (Arch. Biochem.Biophys. 134, 279-294, 1969) disclose a purificaiton for IgG with caprylate (i.e., octanoate, a C8 saturated fatty acid) as a precipitating agent. Non-immunoglublins were precipitated form human plasma after diltuion with an acetate buffer to reach a final pH of 4.8. After addition of caprylate under vigorous stirring an IgG enriched solution was obtained. Batch absorption of the supernatant on DEAE cellulose was used to clear additional impurities from the isolated fraction. 

Zhang discloses a method for preparing intravenous cytomegalovirus human immune globulin which uses caprylic acid precipitation and AEX for replacing the step of ethanol precipitation in the conventional cold ethanol method. 

—-Caprylic Acid – PEG –AEX — Virus remal

Parkkinen (WO2005/073252) discloses a process of subjecting a crude immunoglobulin solution to capylic acid treatment at a pH below 5 and then subjecting the sueprnatant solution to a protein precipitation such as  poleythylene glycol (PEG) to a concentraiton of 10-50 g/l or adsorbent such as fumed silica at a pH in excess of 5.0 to remove protein aggregates followed by AEX. 

Commercially available IMAC columns:

Profinity IMAC resins: The Profinity IMAC bead is a 60 um particle derivatized with iminodiacetic acid (IDA) which functions as the chelating ligand. The chemical structure of IDA allows hihgly selective binding of recombinant His-tagged prtoeins when charged with Ni2+ or other transition metals. As a result, target proteins can often be purified close to homegeneity in a single step (Protein Purifiaiton Profinity IMAC Resins, “Selective Profinity IMAC resins provide ultrahigh-purifty recombinabt His-Tagged Proteins” BioRad; see aslo US 6,423,666). 

ProPur Affinity spin columns: The Propur metal chelate Mini and midi kits are designed for simple, complete and rpaid purificaiton of His-tagged recombinant prtoein from bacterial cells, inset vectors, mammalian cells and yeast under native or denaturating condition. A highly comprised agarose resin is used in the Nunc spin column. The rapid Mini spin columns can be used to purify up to 24 protiens in a microcentrifuge simultaneously. The ProPur Midi spin columns can be used to purify up to 16 proteins in under 50 min using standard bench centrifuge. When a recombinant protein is expressed in E. coli, the protein elutes as insoluble aggregates called inclusion bodies. Denaturants sucy as urea completely unfold the target protein making the His tag much more accesible for interaction with the matrix. (Brownleader, “ProPur: His-tagged protein purification made easy’ Nature Methods, February 2007). 

Introduction:

In 1975, Porath introduced immboilized metal ion affinity chromatography (IMAC) for fractionating proteins. IMAC consists of derivatizing a resin with iminodiacetic acid (IDA) and chelating metal ions to the IDA derivativzed resin. The proteins are seaprated on the basis of their affinity for metal ions, which have been immobilized by chelation.

One of the most widely used methods for protein purificaiton is immobilized metal ion affintiy chromatogrpahy (IMAC) which allows rapid one-step purificaiton of fusion proteins. For such procedrues, proteins are engineered with affinity tags attached to the t’ or 3′ end of the target gene. Examples of such tags are hesahistidine and an 8-reisude peptide containing alternating histidine. The matrix is attached to chelating groups that immobilize transition metal ions such as Ni2+, Co2+, Cu2+ and Zn2+. Ni2+ is the most widely used metal ion. the simplicity of IMAC is very attractive as it lends itself to the bind-wash-elute mode of oepration if the appropriate buffer is selected. (Brownleader, “ProPur: His-tagged protein purification made easy’ Nature Methods, February 2007). 

Immobilized metal affinity chromatography (IMAC) is a type of affinity chromatography where proteins can be separated according to their affinity for metal ions that have been immobilized by chelation to an insoluble matrix. At pH values around neutal, the amino acids histidine, tryptophan and cystein form complexes with the chelated metal ions (e.g., Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+ and Fe2+). They can then be eluted by reducing the pH, increasing the mobile phase ionic strenght or adding ethylenediaminetetraacetic acid (0.05M) to the mobile phase. 

IMAC relies on the formation of weak corrdinate bonds between metal ions immobilized on a column and basic groups on proteins, mainly histidine residues. The absorbent can be formed by attaching to the matrix a suitable spacer arm plus a simple metal chelator, usually based on imino diaetate structures. These chelating ligands will bind tightly to metal ions, in particular to the divalent ions of the transition metals Fe, Co, Ni, Cu and Zn. (Vedadi WO03/025156).

IMAC is a versatile separation procedure that exploits differences in the affinities exhibited by many biopolymers for metals ions. The technique involves the chelation of a suitable metal ion onto a solid support matrix whose surface has previously been modified with a polydentate ligand. The resulting immobilized metal ion chelating complex then has the potential to coordinate with one or mroe elecggron donor groups residing on the surface of the interacting proteins. Separation selectivity is acheived on the basis of differences in the thermodynamic staiblities of the immobilized metal ion complexes with the vairous adsorbed proteins. Proteins whose adsorption complexes are the least stable will be eluted first, while proteins that form more stable omcplexes will be eluted later. The greater the diference in the equilibrium association constands (i.e., the larger the differences in the dissociation constants (KD) of the respective protein/immobilied metal ion coordination complexes, the higher the resolution obtained. (Hearn (US 2009/0143529). 

Hearn (US 2009/0143529) disloses a polyer substrate such as polysaccharides (e.g., agaroses, destrans, celluloses, etc) functionalized with at least one cyclic, metal ion coordinating ligand group which includes at least 3 nitrogen donoar atoms in the ring of the cyclic group, at lone of the nitrogen atoms having an optionaly substituted carboxy (lower alkyl) or optionally substituted phosphono (lower alkyl) group covalently attached thereto. It is advantageous that the functionality in a functionalized polymer substrate is covalently attached to the polymer substrate by means of a liner or spacer group. The IMA based systems sacheive a mixed modality of interaction with their target molecules that is based on a combination of coordination (electron donor/electron acceptor) and electrostatic (ion-exhcange) processes. The functionalized polmer substrates can be used to purify a desired protein of interest by virue of the presence of a metal ion such as divalent and trivalent metal ions such as Ca2+, Mg2+, Zon2+ and Fe3+ which iself is bound coordinatively to the cyclic ligand group in the functionality and which is in turn capable of binding coordinatively to donor atoms in the amino acid residues of a tag part of a fusion protein. 

His-tags:

The most important applicaiton of IMAC is purificaiton of recombinant proteins expressed in fusion with an epitope containing six or more histidine residues, the His tag. Due to the relatively high affinity and specificity of the His tag a single IMAC purificaiton step in most cases leads to a degree of purity of the target protein preparation taht is sufficient for many applicaitons. Block “Immobilized-metal affinity chromatography (IMAC): a review” Methods in Enzymology, 463 2009). The His-tag (also called 6xHis-tag) is one of the simplest and most widely used purificaiton tags, with six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or a resin for purificaiton. IMAC is the preferred choice as a first step during purificaiton of His-tagged proteins., although small batch reactions or spin columns with IMAC beads can be used for expression tests or small scale prepations. Metal ions are immobilized using linkages such as Ni(II)-nitrilotriacetic acid (Ni-NTA) or Co2+ carboxymethyl-asparatate on resinds and beads available from many commercial sources. Malhotra “Tagging for protein expression” chapter 16m Methods in Enzymology, volumen 463, 2009). 

The use of short histidine stretches or his tags, typically placed as an affinity tag at either the N-temrinus or C-terminus, enables the purification of the desired protein from the crude extract of the host cells in a single step. Different chromatographic supports and strategies are available of IMAC. The most widespread IMAC supports use either nitrilotriacetic acid (NTA) as a ligand for immobilizationg metals like nickel in affinity chromatogaphy (Ni-NTA) or different chelating Sepharose matrices. Although unviersally applicable, the use of his-tags and IMAC purificaiton is not recommended for proteins containing metals iones. Similarly, other aa like cysteine and nturally occurring histidine rich regions in host proteins may result in unwanted prtoein binding during IMAC purification. Arnau (Protein Expression and Purification 48 (2006) 1-13). 

cleavage:

The use of fusion tags for teh purificaiton of recombinant proteins is attractive for a number of reasons including effective capture of target proteins present in complex. In some circumstances, the removal of the tag is a crucial step partticularly in cases wehn the target protein is inteded for pharmaceutial applications in addition to cyrstallization and structural determination studies. Cleavage of fusion tags can be acheived by using either chemical or enzymatic methods. (Abdullah, Biotechnology and Bioengineerig, 92(4), 2005). 

Particular Types of Proteins Purified by IMAC

 IgG:

Histidine ligand affinity chromatography has been used to recover IgG. The antibodies were eluted from the adsorbents under very mild conditions, which ensures the structural integrity of the Abs (Vuayalakshmi “Antibody purification methods”, Applied Biochemistry & Biotechnology, 75, 1998.

Pentameric IgM including J chain and Dimeric IgA including J chain:

Brown & Simon (US Patent Application No: 14/476,559, published as US Patent 10385117; see also US Patent Application No: 16/401,322, published as US 2019/0256577) discloses purfication of pentamieric IgM including J chain as well as a dimeric IgA including J chain by adding secretory component which is tagged with an affinity or eipitope tage such as polyhistidine, psoing the mixture to a binding moeity immobilized on a solid phase resin such that the secretory IgA binds to the solid phage resin, washing and then eluting the secretory IgA therapeutic. 

Secretory Component:

Secrectory component (SC) is a protein that specifically binds to J-chain containing immunoglobulin. SC in its natural form is the extracelllar portion of the polymeric immunoglobulin receptor (pIgR) which usually gets associated during secretion with dimeric or polyemric IgA or pentameric IgM comprising a J chain. J chain containig IgA/IgM ibinds to the polyemric immunoglboulin receptor at the basolateral surface of eptithelial cells and is taken up into the cells by transcytosis. This receprtor complex then transits through the cellular compartments before being transported to the luminal surface of the peithelial cells. The transcytosed IgA/IgM-pIgR complex is then released through proteolysis, and part of the polymeric immunoglboulin receptor (pIgR) referred to as the natural secretory component, stays assocaited with the J chain contianing IgA/IgM, relasing secretory IgA/IgM. (Corthesy, WO 2013/132052). 

Corthesy, (WO 2013/132052) discloses adding a tag, such as a hexa-Histidine tag, which can aid in the purificaiton of the resulting prtoein. If such a tag is attached via a cleavalbe linker, the tag may be cleaved off prior to use in the invention. 

Nanobodies:

Beirnaert (Wo 2006/122786A2) discloses purificstion of His tagged anti-TNFalpha nanobodies using IMAC and elution using citric acid. The nanobodies were further purified on CEX. 

Rossi (WO2005/049649) teaches purification of IL-18 binding protein from a fluid by subjecting the fluid to immobilized metal ion affinity chromatography (IMAC), subjecting the eluate to HCIC, subjecting this eluate to CEX and subjecting the flow through to HIC and subjecting the eluate the eluate to reverse phase chromatography.

Particular Metal Binding – Chelating Agents

Iminodiacetic aicd

IMAC is based on the metal ion mediated interaction with protins. To acheive this interaction on solid phase, metal ions are adsorbed on a chelating resin and the resulting solid phase is used for protein adsorption. Chelation of metal ions occurs iva the intervention of selected ligands chemically attached on the matrix. The most popular chelating ligand is iminodiacetic acid. (Boschetti, Chapt 4, “Approaches to devise antiboy purificaiton processes by chromatography” Antibodies, volume 2, novel Technolgies and Thepaturic Use, 2004 

TREN chelated with copper:

The nobiological ligand TREN (Tris(2-aminoethyl)amine) is a quadridentate chelating ligand used in IMAC with four nitogen atoms, three of which are primary in nature adn the fourth one is tertiary. TREN chelated with copper and nickel ions has been employed in protein purificaiton. Due to its high amine residue content, TREN (without chelated metal ion) can serve as an anion exchanger. At a pH lower than 10, TREN is positvely charged and can adsorb negatively charged molecules, so this ligand could be an excellent candidate for the purificaiton of IgG from serum proteins. Bresolin (J of Chromatography B, 877 (2009) 17-23)

Boden (J Immunological Methods 181 (1995 225-232) discloses a rpaid, single step purificaiton of immunoglobulins from goat serum using immobilized metal ion affintiy chromatography (IMAC) on a high capacity gel, Novarose couple to tris(2-aminoethylamine (TREN) chelated with copper.

Bresolin (J of Chromatography B, 877 (2009) 17-23) discloses using Tris(2-aminoethyl)amine (TREN) as a chelating agent in IMAC for the purificaiton of IgG from human serum. 

Ribeiro (J Chromatography B, 861 (2008) 64-73) discloses purificaiton of IgG from human plasma using an affinity membrane complexed with Ni(II) prepared by coupling iminodiacetic acid (IDA) and Tris(2-ainoethyl)aine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. 

PEG chelated with a variety of metals

Arnold (US 5,283,339) discloses that certain PEG compounds are capable of chelating a variety of metals such as Zn2+, Ni2+, Fe3+, Fe2+, Co2+ and Ca2+ and can be used to precipitate proteins form solution. 

See also continous or SMB chromatography 

In General: 

Brown (WO2010/019148) discloses membrane chromatography run on either a standard chromatography system or a custom system like an AKTA exploer (GE Healthcare) equipped with pressure gauges, sensors, and pump plus pump controllers. Continous processing experiments were performed on the AKTA explore. The Q column flow through was pH adjusted in line and imediately loaded onto a Mustang S membrane. The Q column was packed with Q Sepharose Fast flow resin, the column outlet was attached to the inlet of a T connection and pH adjustment was accomplished in line by directly the B Pump to the opposite inlet of the T connection. The T connection provided adequate mixging and the pH adjsuted solution was directed to the inlet of the Mustang S membrane. Mustang S is a strong CEX and Mustang Q is a strong AEX. 

Coffman (US13/811178) teaches a method of protein purification using a column/resin that is arranged in tandem with the column of a second resin.

Vedadi (WO03/025156) discloses protein purification using one or more chromatography colun which are arranged so that teh flow through or eluate from one column loads directly onto another colun (e.g., the columns are arranged in tandem). In an exemplary embodiment, proteins are purified using a combination of an IEX, affinity and gel filtration column. In the embodiments, an automated process control system is included which includes various sensors, and pumps. 

Particular Schemes with In-Line Chromatography

AEX-Protein G Affinity:  

Qi (J. Biochem. Ciophys. Methods 49 (2001) 263-273) discloses injecting a diluted mouse serum onto a system whcih includes DEAE AEX and Protein G affinity columns coupled in series by a column switching technique. The advantage is that IgG and albumin could be separated and purified simultaneously. 

CEX-Mixed Mode:

Liu (US 2013/0079272) teaches loading a CEX and then a mixed mode material in continuous mode which is referred to as having the CEX and MM material eitehr direclty connected or other mechanisms which allows for continous flow.

Affinity Chromatography

–Protein A-AEX: 

Lundblad (WO2005/058452) discloses an automatic system in which a detector signal is monitored by a computer  comprising a plurality of chromatography column. Each column has an inlet end and an outlet end. In one bodmiment columns 1 and 2 are affinity columns each of which contains a ligand to which a protein of interest binds, column 3 is a desalting column and column 4 is an ion exchange column.

Shamashkin (Biotechnology and Bioengineering, 110(1) 2013) discloses a semi-continous downstream process where columsn and filters are linked and operated in tandem so as to eliminate the need for intermediate holding tanks. Shamashkin exemplifies a tandem process for the purificaiton of mAbs employing an Affinity Protein A capture step, followed by a flow through AEX step with the possibility of adding an in-line virus filtration step. All steps were linked sequentially and operated as one continous process using an AKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX was operated in a weak partitioning mode by a precise in-line titration of Protein A effluent. 

—-Protein A-AEX-CEX or CEX-AEX:

Duthe (US 14/889,397, published as US 2016/0083454) discloses a 3 step chromatography scheme of Protein A – CEX and AEX where each of the buffers used in the steps is Bis Tris. and the method is run in a closed system without buffer exchange or dilution.

Kulkarni (WO 2011/090719) discloses multiple chromatogrpahic steps wehre the low pH eluate from a protein A chromatogrpahy is further purified without the need of substantial pH adjustment. The eluate from the Protein A is loaded onto a CEX in Bind and elute mode without substantial adjustmnet of pH. A third purificaiton using AEX is performed in flow through mode without substantial adjustment of pH. 

Laursen (US7138120 and US2001/0051708; see also WO/1999/064462;  See also US 6,281,336) teaches a process for purifying IgG where AEX and CEX are preferably connected in series. Laursen teaches that the use of two serailly connected chromatography column makes the oerpation more practical because there is no need for an intermediary step of collecting the IgG containing fraction between the two IEX methods.

Mahajan (J Chromatography A, 1227 (2012) 154-162) discloses using Protein A and then loading the next column to capture the mAb. The continous operation is advantageous in that it can reduce both resin volumne and buffer consumption. However, the process is more complex than a batch process. The process utilizes a simulated moving bed concept where as the olumns are being used in series or parallel dpending on the process step. 

Soice (US2011/0065901 and WO2011/017514) discloses a method for purifying an Fc containing protein using the scheme of Affinity-CAEX-AEX which eliminates the need for a holding tank and/or a buffer exchange step. The purification process is referred to as a “connected process”. According to the prcoess, the sample is contacted with an affinity chromatography media, the Fc containing protein is eluted and contacted with CEX and eluted and then contacted with an AEX.

Williams (US2011/0073548) teaches a separation system comprising at least two sepration units such as a CEX and AEX unit which are connected in series outlet to inlet to form a line of separation units.

–Protein A – CEX:

Minakuchi (US15/022890, published as US 2016/0237113) discloses a method for purifying an antibody using a first carrier with an affinity ligand such as Protein A and a second carrier having a CEX group where the contacting occurs in a column having bothth carriers or where the columns are operated in-tandem and  where the affinit/Protein A chromatography and CEX are performed at one chromatography step such that and eluate is passed through the column where the pH of the eluate is 4.0 or less and the pKa of the CEX group is equal or greater than the pH of the eluate and the pKa of the CEX group is 4.0 or more. In one embodiment the CEX group is a carboxyl group. . 

Soice (WO2011/017514) discloses direct loading of an eluate from Protein A to CEX without the need for a holding tank. The chromatography column in effect becomes the “holding tank” storing the product while the buffer is switched to a buffer which works as an elution buffer for the loaded column and a loading buffer for the next column down stream. 

—-Protein A – low pH virus inactivation –HIC or CEX –UF/DF –AEX –NF

Ransohoff (US2013/0260419) teaches a continous process for the purification of antibody which includes clarification, Protein A, low pH viral inactivation, HIC, UF/DF, AEX, NF.

Ransohoff (WO 2012/078677) also teaches a continous process for the purificaiton of a mAB by conctating a product solution with an affinity ligand capable of complexing with the biological product such as protein A chromatography and continously transferring the purified product to a second unit operation comprising a low pH viral inactivation, CEX, UF/DF, AEX, NF. 

—-Protein A -AEX (flow through mode) – HIC (flow through mode)

Kremer (WO 2011/098526) discloses a method for purifying proteins such as antibodies using a single oeprational unit comprising both an AEX and HIC which are serially connected and which are operated in flow through mode. 

–Protein A-Multi-Mode (capto adhere)

Duthe (WO 2013/075849) disclsoes two chromatographic steps; one affinity and one multi-modal where all buffers can consist of Bis Tris in combination with NaCL, acetic acid and water. In one embodiment the affinity chromatography is Protein A. In one embodiment th eeluent obtained a the end of the first chromatographic step is passed over the second chromatography column with no treatment such as pH adjustment, buffer exchange or dilution.

—-Protein A — MM (capto adhere) — AEX

Duthe (US 14/889,397, published as US 2016/0083454) discloses a 3 step chromatography scheme of Protein A – MM (capto adhere) and AEX where each of the buffers used in the steps is Bis Tris. and the method is run in a closed system without buffer exchange or dilution. 

Mixed Mode – HA: Gagnon (US2009/0270596) discloses a method of purifying antibodies using mixed mode chromatography and HA where the first and second column tubes comprise inlets and outlets, wherein the outlet of the first column tube connects to the inlet of the second column tube such that the first column tube is in fluid communication with the second column tube. 

HIC-AEX: Urthaler (US2004/0002081) discloses a process for isolating a polynucleotide using a chromatographic secparation process characterized in that the process uses a combination of two steps selected from HIC and AEX and the two chromatographic steps may be carried out independently or, preferably in a single operation. To achieve a single operation, the device for the first step is directly linked to the chromatographic device for the second step by connecting the outlet of the first with the inlet of the second one. 

2 Unit Operation with Same or Similar Ligands (see also simulated moving bed chromatography)

Bryntessofn (WO 2008/153472) teach a simulated moving bed process wehrefor IgG adsorption to MabSelect affinity resin known as a 3C-PCC system where at least 3 substantially identically packed chromatography columns are operated in a semi-continous manner. Accordingy to the method, the absorbent is washed after binding of target compound and then the outlet of wash liquid from said adsorbent is subsequently passed onto another adsorbent for binding of target compound removed by said washing. Consequently, every time the feed is redicted to another adsorbent in the system, said adsorbent will have a small amount of target compound bound already. This allows for an efficient recovery of target compound. Chromatography resins may be Protein A based resins, IMAC resins, HIC resins, thiophilic resinds, IEX resins, multimodal resins. 

Mahajan (“improving affinity chromatography resin efficiency using semi-continous chromatography” J Chromatography A, 1227 (2012) 154-162 discloses operating Protein A affinity chromatography using several multiple smaller column in line. 3 Hi-Trap MabSelectu Sure columns were loaded individually at differen times until the 70% breakthrough point was achieves. The harvested cell culture fluid (HCCF) with unbound protein from the column was then laoded onoto the next column to capture the MAb, preventing any protein loss. 

Skudas (US 9,149,738) disclsoes a process of continous affinity chromatography by using at least three capture separation units have the same chromatography matrix such as Protein or IEX which are connected so that liquid can flow from one unit to the next and the last to the first, feeding the sample on a the first unit so that the first unit is at least part of the loading time in fluid communication with the next unit so that target molecules not bound to the frist unit can bind to the next unit and at the same time washing, eluting and/or reequilibreating one separation unit different from the separation unit that is being loaded and form the one in fluid communication, switching the feed to the enxt seapration unit and feeding the sample on the next unit so taht while the sample is being loaded on the next unit, this unit is at least part of the loading time in fluid communication with the unit after the next so taht target molecuels not bound can bind to the unit after the next and repeating the steps. In one embodim ent, the purified target molecuels that are lueted are further subjected to at least one flwo through purificaiton step.   See continous chromatography

Van Alstine (WO2011/035282) discloses the separation of complex mixtures of macromolecules such as proteins including polymer modified molecules using two or more columns in series whihc contain capture media with similar ligands such as IEX, IMAC, MM or Protein Affinity resin such that adsorption mobile phase flow through from the first column is used as adsorption mobile phase for the second or flow on column. For example, the macromolecules to be separated could be polymer modified proteins (e.g., PEGylated proteins). The two media used could have similar ligands and ligand density per unit area but different pore size distribution. The different proteins to be separated including modified proteins might differ in size such that the larger molcules are excluded from the first media  and thus flow through the first media. 

 Axial Flow Chromatography (AFC): Traditionally, chromatography systems use a cylindrically-shaped vessel. Buffers and samples containing products are pumped onto the top of the column and collected from the bottom. However, during processing, high pressure drops may occur, which sometimes prevent operation at high flow rates expecially for large pilot-scale or manufacturing-scale systems. (Demirci, 2012, 255-262, American J. of Biochemistry and Biotechnology).

Radial Flow Chromatography (RFC): was developed to reduce pressure drops in the system while maintaining high flow rates. In RFC, the column is cylindrical like AFC above, but the flow of the mobile phase passes from the outside of the cylindier through the resin bed to the inside of the cylinder. The packed bed is supported between two cylindrical fits and the gap between the frits represents the bed height or olumn lenght. The outer frit is the column inlet and thus the sample initially has a large area of stationary phase with witch to interact. The cross-sectional area of packing decreases progressively as the solute moves toward the center. The greatest advantage of RFC is oepration with less pressure drop under the same flow rates. (Demirci, 2012, 255-262, American J. of Biochemistry and Biotechnology).

Elimination of Intermediate Filtration Processes

Chen (WO2009/045897) teaches a protein purification system which includes one or more columns each with an adsorbent where a culture including a protein is flowed through the first column to provide a first eluate that is flowed the second conolumn without prior difiltration or ultra filtration of the first eluate. The process provides ebenefits for manufacture plant automation in that the process is capable of being operated on a high throughput and continous basis. The process is cpaable of handling high titer concentrations of about 5 g/L or even about 50 g/L.  One exemplified scheme is Pro-A-MEP (mercapto-ethylpyridine, which is a hydrophobic charge induction resin)-CHT (ceraminic hydroxyapatite)/AEX. For immobilization of the ligand an avidin/biotin chemsitry can be used.