About the Author: 

James Rogers is a patent attorney and former patent examiner with the United States Patent and Trademark Office. Previous to his work as a patent examiner, James was in house counsel for a diagnostic company and worked for several law firms in the intellectual property area. James received his law degree from Suffolk University Law School. He received an MS degree in biology with an emphasis on DNA recombinant technology from New York University and a PhD in molecular medicine from the University of South Florida College of Medicine with an emphasis on immunology. He is a member of the New York and Massachusetts bars.

Ypatent has additional information and links which supplements The Complete Patent Kit, Fourth Edition.

Companies:

Biotechne Simple Western (after preparing reagents they are loaded into a Simple Western plate which is placed with a cpaillary cartridhe into a Simple Western instrument. The Simple Western does protein analysis thorugh separation and immunoprobing in a capillary system.) 

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS): (see also capillary electrophoresis under Diagnostic Techniques)

Capillary electrophoresis-sodium dodecyl sulfate (CD-SDS) has emerged as a modern equilvalent of SDS-PAGE, offering superior reproductibility, sensitivity and throughput. (Wang WO 2019/152303)

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a modern equivalent of SDS-PAGE discussed below and offers superior reproducibility, sensitivty and throughput. During CD-SDS analysis of mAb products, minor peaks with shorter migraton times (LMW forms) than the intact antibody can be routinely observed. Unliked SDS-PAGE analysis, these LMW impurites cannot be extracted or subjected to further analyses. As a result, the idenitites of LMW impurites obersrved in CE-SDS methods are often proposed solely based on empirical knowledge. (Wang WL 2019/152303)

CE-SDS has emerged as a valuable alternative to conventional sodium dodecyl sulfate polycarylamide gele electrophoresis (SDS-PAGE) for the characterizaiton of automatic quantitation of biomolecules and particularly monoclonal antibodies. High separation efficiency, ease of operation and automation and reduced operating costs are the main reasons for this success. As a result, CE-SDS has found wide acceptance within the biopharmaceutical industry for the development, manufacturing and release of therapeutic mAbs. CE-SDS methods are in routine use for in process monitoring, purity assessment, detecing low level impurites and measuring size variant hterogeneity. (Schneider, “Tracking of antibody reduction fragment by capillary gel electrophoresis during the coupling to microparticles surface” J. Pharma. and Biomed. Analysis 53 (2010). 

CE-SDS is widely used for purity analysis of mAb therapeutics for release and stability to demonstrate product consistency and shelf life during the manurfacturing and life cycle of the product. The method separates molecule specific fragments and other impurities based on their electrophoretic mobility under denaturing conditions. Recently, US Pharmacopeia (USP) suggested that a universal mAb system suitability reference standard be adopted for CE-SDS and size exclusion chromatography. During sample prepration, antibodies are denatured in the presence of an ionic detergent which masks the native charge of the protein providing a net negative charge enabling electrophoretic mobility speration based almost exclusively on the hydrodynamic radius of the protein molecule(s). The disulfide bonds which are responsible for the tetrameric structure of mAbs are disrupted (reducing) or protected (non-reducing) during heat denaturation by reducing or alkylating reagents respectively. (Esterman “implementation of USP antibody standard for system suitability in capillary electrophoresis sodium dodecyl sulfate (CE-SDS) for rlease and stability methods” J. Pharmaceutical and Biomedical Analysis 128 (2016))

CE-SDS was used for seperation of denatured prtoein size variants under non-reduced or reduced conditions For non-reduced CE-SDS (nrCE-SDS) samples were denatured with sodium dodecyl suflate at 70C for 5 min in the present of iodoacetamide. for reduced CE-SDC (rCE-SDS), 2-mercaptoethanol (Bio-Rad) was added to the protein denaturation step to reduce the disulfide bonds. After denaturation, both non-reduced and reduced samples were injected int a bar fused silica capilary (Beckman Coulter) and separated based on hydrodynamic size resulting form an applied electric field in which migraiton of smaller sized proteins is inversiy related to overal size. (Katterle “How sable are new biologicals” Pharm, 80(11) 1557-1563 (2018). 

For Activatable Antibodies:

Carman (US 2019/0117789) discloses activatable antibodies that bind CD166. The activatable antibody (AA) includes an antibody that binds to CD166 including a heavy chain and light change, a masking moeity (MM) coupled to the Ab wherein the MM inhibits the binding of the AB to the CD166 when the AA is in an uncleaved state and a cleavable moeity (CM) coupled to the Ab, wehrein the CM is a substrate that functions for a protease. In some embdoiments an agent such as maytansinoid is conjugated to the AA. The anti-CD166 conjugated activatable antibody was activated with matriptase or MMP14 for 2 hours at 37C and mixed with intact conjugated activatable antibody. The mixture was then analysed by the The WES Capillary electrophoresis system using anti-human IgG (American Qualex Catalog #A110UK). Carman showed the ability to separate matriptase-activated or MMP14 activated conjugated activatable antibodies form intact conjugated activatable antibodies. 

Desnoyers (US Patent Application No: 16/632265) discloses a method of quantitating a level of activation of an activatable antibody which includes the steps of 1) loading at least one capillary with a stacking matrix and a separation matrix. the “stacking matrix” refers to a highly porous (relative to the separation matrix) material that functions to concentrate proteins present in the sample and “stack” them at the interface with the separation matrix so that the proteins start migration under electrophoresis conditions form the same physical starting point. Suitable stacking matrices include those used to prepare stacking gels for Western blotting such as acrylamide, SDS. Capillaries pre-loaded with stacking matrix and separation matrix are available commercially as Wes Separation Module. Electrophoresis causes the compounds in the sample to migrate through the separation gel at differential rates according to molecular size/weight. 2) the HMW and LMW compounds are immobilzied within each capilalry as by using UV light. 3) Each capilarry is immunoprobed with a first reagent such as an idiotypic antibody that is specific for the activatable antibody. 3) Detection of the first reagent can be accomplised for example with a second reagent that binds to the first reagent. Exemplary detectably labeled second r agents include HRP-conjugate anti-mouse secondary antibody. 

Vasiljeva (US Patent Application 2020/0377602) discloses polypeptides that include at least a first cleavable moiety (CM1) that is a substrate for at oast one matrix metalloprotease (MMP) and at leaset a second clevable moeity (CM2) that is a substrate for at least one serine protease to activatable antibodies which include these polypeptides. Vasiljeva disclosing measuring cleavage by capillary electrophoresis and analyzation with the Wes Western Blot protocol using goat anti-human IgG antibodies and anti-goat secondary anitobodies. The fraction of the cleaved activatable antibody was determined by quantifying the mobility of the higher mobility polypeptide corresponding to the cleaved antivatable antibody. 

Capillary electrophoresis-mass spectrometry (CE-MS):

CE-MS is a pwerful orthogonal technique capable of filling in gaps int he dientification, quantitation and isomeric resoluion of many small hydrophilic and charged metabolites. The metabolome is a large complex mixture of molecuels for which not one technique can optimally identify and measure it in it’s entirety. LC-MS, GC-MS and NMR have been widely used for metabolomics for teh past 20 years for a wide range of applications. CE-MS captures a unique metabolic chemical space beyond these standard methods pvoiding another window into metabolomics profiling. CE-MS is the most suited technique for analyzing posphorylated metabolites, amino acids or metabolites form teh TCA cycle and glycolytic pathways. A fused silica capillary contains surface charges of sianol groups present on the inner walls. The silanol groups on the capillary inner wall are ionized presenting an overall negative surface. Opposing ions in the electrolyte solution are attracted to the inner wall surface to achieve a balance of electric charges, resulting in the formation of a double layer with ionized silanol groups. Under these conditions, a potential diffference is created very close to the inner wall. The applicaiton of a voltage to both ends of the capillary attracts the positively charged ions of the diffuse double alyer to an anode. In contrast, the silanol groups cannot move due to the fixation on the wall surface and the entire electrolyte solution in the capillary is directed toward the anode with the migration of the positively charged ions, thereby generating a flow. The degree of mobility of any copound relative to tothers is due to variation in their ionic radius and size and charge of the electrolyte filing the capillary. A compound or metabilite with a larger ionic radius and smaller charge would ahve limited mobility compared to small, more polar species. Compounds with a small ionic radius and higher charge would have high obility. Hence controlling the electrical gradient across the capillary and pH of teh electrolyte solution are two of the most important parameters in controlling metabolite separation into the mass spectrometer. (Buko “Capillary electrophoresis mass spectrometry based metabolomics” J Applied Bioanalysis, 2017). 

Imaged capillary isoelectric focusing (iCIEF):

Charge profiels/isoelectric points (pI) of analytes can be determeind by imaged capillary isoelectric focusing (icIEF) analysis using an iCE3 system (ProteinSimple) equipped with a 5 cmx100 uM ID flurocarbon coated capillary Sampels are spearated according to theri pI and detected by using a whole column UV adsorption detector (280 nm) that avoids distrubing focused protein zones. Imaged capillary isoelectric focusing fo tthe analysis of therapeutic antibodies and antibody drug conjugates provides a fast separation and resolution of acidic, neutral and basis variants of an antibody. Katterle “How sable are new biologicals” Pharm, 80(11) 1557-1563 (2018).

Companies which assist in clinical trial development: Amarex

Center for Biologics Evaluation and Research (CBER)  (the center within the FDA which regulates biological products for human use under applicable federal laws).

Medicine and Healthcare Products Regulatory Agency (MHRA) (drug regulation in UK)

Chemical vs. Biological Products:

RNA:

While mRNA vaccines are considered as biological products, the classificaiton of chemical or biological products and the definition of gene therapy medicinal products may require updating or alignment to accommodate advancements in mRNA technology. Currently, plasmid DNA serves as the starting material for manufacturing mRNA va in vitro transcription method. Essentially, this involves using the plasmid. With advancements in cell free production technology, there may be a future where mRNA will be entirey synthetically produced. This raises the challenge of defining what constitutes a biological product.

Currently smaller RNAs such as siRNAs are typically considered chemical products due to their purely syntehtic nature. Barker, “Evolution of mRNA-based vaccines: a regulator’s view” Vaccine Insights 2024; 3(3), 97-105)

However, as mRNA becomes more syntehtically produced, utilizing cell free systems without biological components like plasmid, questions arise about whether they should still be classified as biological products. According to regulatory definitions, biological products typically involve substances produced or extracted form a biological source, necessitating specific testing and product controls. While mRNA has some similarities to shorter oligonucleotides like siRNAs, its great complexity and size warrant advanced characterization tools to ensure structural integrity, morphology and interactions with drug delivery systems. These are then more similar to biological products than to a well defined chemical product. Given that mRNA vacines fall under the category of immunogloical medicinal products, it may be appropriate for them to be classified as biological products. However, a mRNA production transitions away from plasmids toward cell free systems, updates to the definition may be necessary to accurately reflect the nature of these products. (Barker, “Evolution of mRNA-based vaccines: a regulator’s view” Vaccine Insights 2024; 3(3), 97-105)

Upstream manufacturing process of mRNA is maturing. cGMP quality plasmid, polymerases, and enzymes needed for in vitro synthesis of mRNA are available but can be costly. Poly(A) tails can be created by inclusion in the teplate or by use of enzyme. Capping options include high-efficiency, co-synthesis reagetns such as TriLink Biotechnology’s CleanCap and enzyme treatment with high efficiency. (Cytiva, “mRNA vaccines and therapeutics: current trends and perspectives” Fe 17, 2025.)

Accelerated Approval:

Accelerated Approval.   (FDA may approve drugs for serious or life-threatening diseases or conditions where there is an unmet medical need and the drug is shown to have an effect on a surrogate endpoint that is reasonably likely to predict a clinical benefit to patients (improving how patients feel or function, or whether they survive longer).

In 2012, Congrewss passed the Food and Drug Adminsitration Safety Innovations ACT (FDASIA). Section 901 of FDASIA amends the Federal Food, Drug, and Cosmetic Act to allow the FDA to base accelerated approval for drugs for serious conditions that fill an unmet medical need on whether the drug has an effect on a surrogate or an intermediate clinical endpoint. A surrogate endpoint used for accelerated approval is a marker -a laboratroy measurement, radiographic image, physical sign or other measure that is thought to predict clincial benefit, but is not itself a measure of clinical benefit. Likewise, an intermediate clinical endpoint is a measure of a therpaeutic effect that is considered reasonably likely to predict the clinical benefit of a drug, such as an effect on irreversible morbidity and mortality (IMM).

The FDA bases its decision on whether to accept the propsoed surrogate or intermedate clincial endpoint on the scientific support for that enpoint. Studies that demonstrate a drug’s effect on a surrogate or intermediate clincial endpoint msut be adequate and well control as required by the FD&*C Act.

Using surrogate or intermediate clincial endpoints can save valuable time in the drug approval process. Instead of haivng to wait to elarn if a drug actually extends survival for cnacer pateints, for exaple, the FDA may approve a drug based on evidence that the drug shrinks tumors, becasue tumor shrinkage is considered reasonably likely to predict a real clinical benefit.

Fast Track:

Fast track is a process designed to facilitate the development, and expedite the review of drugs to treat serious coditions and fill an unmet medical need. Determining wehther a condition is serious generally is based on wehther the drug will ahve an impact on such factors as survival, day-to-day functioning, or the likelihood that the codition, if left untreated, will progress form a less severe condition to a more serious one. AIDS, Alheimer’s, heart failrue and cancer are obvious examples of serious conditios.

Filling an unmet medical need is defined as providing a therapy where none exists or providing a therpay which may be potentially better than available therapy.

Fast track designation must be requrested by the drug company. A request for the designation may be made at the same time, or any time after, the applicaton submission for the investigation (study) of the drug under section 505(i) or section 351(a)(3) of the Public Health Service Act. FDA will review the request and make a deciion with 60 days based on whether the drug fills an unmet medical need to a serious condition. Once a drug recives Fast Track designation, early and frequent communication between the fDA and a drug company is encouraged throughout the entire drug development adn erview process.

Laws: 

FDA Development & Approval Process.

Priority Review.

Orphan Products Development

CFR Title 21:

21 CFR 211.65 (equipment construction)

21 CFR 600.11 (Physical establishment, equipment, animals and care)21 CFR 600.11 (Physical establishment, equipment, animals and care)

ICH Q7: Good Manufacturing Practice for Active Pharmaceutical Ingredients, US Fed. Reg. 66(186) 2000: 49-28-49029

FDA:  FDA blog

European Union good manufacturing practice

BIOSECURE act (proposed; US House of Representatives bill H.R. 7085; US Senate bill S. 3558) would restrict the US activity of 5 Chinese biotech companies on national security grounds. The act is expected to drive mroe business to CDMOs outside China).

Definitions:

ANDA: In order to being manufacturing a generic product, the manufacturer must submit an Abbreviated New Drug Application (ANDA) to the Orange Book before the patent on the branded product expires, certifying that the generic dose not infringe on the current patent. However, when submitting this ANDA, the eneric manufacturer must also certify for each unexpired patent in the ORange Book. Depending on the number of patents associated with a single product, this can create a substantial hurdle for generic entry, as it requires the gneeric manufacturer to go to the epxense of assessing each patent to determine infringement potential. (Campanelli, “Feeling evergreen: a case study of humira’s patent extension strategeis and retroactive assessment of seocnd-line patent validity”, 2022).

Biosimilars: Biosimilars cannot be approved by the FDA until the reference biologic has been on the market for at least 12 years.. This is different than in the small molecule drug space, where generics can enter a market after 5 years. As a result, biosimilars can only enter the market towards the end of the lifecycle of the reference biologic drug, which limits their overall potential earnings. (Campanelli, “Feeling evergreen: a case study of humira’s patent extension strategeis and retroactive assessment of seocnd-line patent validity”, 2022).

Section 351(K) of the Public Health Service Act (PHS) provides an abreviated licensure pathway for biological products shown to be biosimilar to, or interchangeable with, an FDA licensed reference product. As part of a demonstration of biosimilarity, a 351(k) applicaiton generally will contain data derived form a clinical study or studies sufficient to demonstrate safety, purity and potency in one or more appropriate conditions of use for which the reference product is licensed and for which the biosimilar or interchangeable biosimilar product applicant is seeking licensure. 351 (K)(2)(A)(i)(I)(cc) of the PHS Act. (Labeling for biosimilar and Interchangeable Biosimilar Products Guidance for Industry” September 2023).

FDA recommends that biosimilar and interchangeable biosimilar product labeling incorporate relevant data and information form the reference product labeling, with appropriate modificaitons.

Evergreening (“patent thickening”): a legal strategy employed by any company with a proprietary patent by which they intend to extend the life of the original patent either through taking out new patents or by buying out a frustrating competitor. “Patent thicketing” invovles many patents being conferred at the same time, oten on overlapping aspects of the innovation. Each component of the drug is protected separately, for example the cotaing, the delivery system, the formulation, and the composition would all be protected under separate patetns. Onre reprot revealed that hte top 12 products on the market are protected by an average of 71 patents per drug. (Campanelli, “Feeling evergreen: a case study of humira’s patent extension strategeis and retroactive assessment of seocnd-line patent validity”, 2022).

Orange Book: The FDA maintains a list of approved drug products with therapeutic equivalence evlautions, known as the “Orange Book”. This keeps tract of all branded drugs with generic coutnerparts, as well as the use codes or cases for the branded products.

Orange Book Preface

The Orange Book

US Orphan Drug Act:

The US Orphan Drug Act was passed in 1983 to encourage research and development of therapies for rare diseases thorugh tax inventives, market exclusivity, and user fee exemptions. The act results in a marked increase in rare diease research, from fewer than 10 drugs before its passage to more than 300 drugs for rare dieases over the past 25 years.

Biosimilar Markets and Regulation

The pipeline of follow-on (biosimilar) products in developmetn includes nearly 800 biosimilars. See Biosimilarspipeline

BPCI Act:  

Biosimilars are defined as “similar” or highly similar” to the reference medicinal products following the EMA (European Medicines Agency ) adn the US FDA regulatory guildielines. In 2012, the EMA issued guidelines on the development of biosimilar monoclonal antibodies. In 2012 the FDA released draft guidance for teh development and regulatory review of biosimilars. In India, the biosimilars are termed as “similar biologics” in accordance with the guidelines issued by the Central Drugs Standard Control Organization (CDSCO).  (Bandyopadhyay “physicochemical and functional characterization of a biosimilar adalimumab ZRC-3197, Biosimilars, 2015: 5, pp. 1-18).

The BPCA Act was enacted to establish an abbreviated pathway for FDA licensure of biological products that are demonstrated to be biosimilar to or interchangeable with an FDA licensed reference product.

Guidance for Industry: Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a Reference Product, May 2014

Quality Considerations in Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product: Guidance for Industry

Contract Testing Organization (CTOs) and Contract Research Organization (CROs)

CTOS are specialized laboratories offering epxertise with characterization and preclinical testing. Examples include Charles River Laboratoreis and SGS. Others include BioAgilytix, Creative Proteomics, Eurofins Scientific, Intertek, Myoderm and SSCI.

CROs focus on managing clinical trials. Quintiles IMS and Parexel are examples. Others include Altasciences, IDDI, INC Research, inVentiv Health and Pharmaceutical Product Devleopment (PPD).

Good Manufacturing practice (CGMP):

World Health Organization (WHO)

21 CFR 211.65 states that equipement shall be constructed so that surfaces that contact components, in process materials, or drug products shall not be reactive, additive, or adsorptive so as to alter the safety, identity, strengh, quality, or purity of the drug product beyond the official or otehr established requriements.

US FDA CGMP for biologicals, 21 CFR 600.11 provies that all surfaces that come in contact with products shall be clean and free of surface solids, leacable contaminants, and other materials that will hasten the deterioration of the product or otherwise rend it less suitable for the intended use.

ICH Q7 Good Manufacturing Practice for Active Pharmacetuical Ingredients guidelines tates that equipment hsould be constructed so that surfaces that contact raw materials, intermediates, or APIs do not alter the quality of the itnermediates and APIs beyond official or other established specifications.

FDA Process:

Orange Book

A drug compound must pass three phases of huamn clinical trials in order to obtain FDA approval.

IND:

A threshold step is to gain the FDA’s permission to test the coumound in humans in the first place. After a drug developer has conducted preclinical studes (i.e., tested the compound in vitro and in animals, it submits an Investigational New Drug (IND) application to the FDA. An IND submission includes an investigator’s brochere, which disclsoes informaiton such as animal safety and preclinical efficacy data, clinical trail proposlas, and toxicology data. If the FDA approves the IND, then Phase I studies can commence.

Phase I:

Phase I studies involve administering the compound to a small group of healthy volunteers or advanced cancer patients with a vareity of tumor types. Phase I studies are conducted primarily to evaluate safety, to determine a safe dosgin range, and to idnetify any side effects.

Phase II:

Clinical trails do not focus on efficacy until Phase II, which typically involved adminsitering the compound to a specific patient population. The goals of a Phase II study include evaluating efficacy in specific patient populaiotns, determining dose tolerance and optimal dosage and identifying possible adverse effects and safety risks.

Phase III:

Phase III studies are large scale and are undertaken to evaluate clinical efficacy and safety in an expanded patient population. After completing Phase III studies, a developer submits a New Drug Applicaiton to the FDA for approval.

Simulated Moving Bed (SMB) chromatography:

Introduction:

A specific way of operating continous chromatography is called simulated moving bed (SMB) chromatography. In SMB all the chromatography columns are periodically and simultaneously moved in the direction opposite the sample flow. The movement of the columns is realized by appropriate redirections of inlet and outlet stream to/from the columns which requires a sophisticaed setup. Rose (WO 2017/140081)

The simulated moving bed (SMB) technology as been patetned about 40 years ago. it is a multi-column continous chromatographic binary separator involving a coutner-current between the liquid (eluent) and the separating medium (stationary pahse) packed in the columns. (Nicoud “Recent aspects in simulated moving bed” Aanlusis Magazien, 1998, 26, N 7). 

For large scale separations in actual productions continuous processes are much more economic than batch processes. The advantages of a continous process is for example high yield, less solvent consumption, less costly fractionation and analyses, better flexibility for the auntities to be purified. One way to realize a continuous chromatographic process is the so called simulated moving bed process (SMB). (Aumann, WO 2006/116886). 

In continous chromatography (SMB chromatography), several identical columns are connected in an arrangement that allows columns to be operated in series and/or in parallel, depending on the method requirements. Thus, all columns can be run in principle simultaneously, but slightly shifted in method steps. Compared to “conventional” chromatography, where a single chromatography cycle is based on consecutive steps of loading, washing, elution and regeneration, in continous chromatography based on multiple identical columns all these steps occur simulataentously but on different columns each. Continous chromatography operation results in a better utilization of chromatography resin, reduced prcoessing time and reduced buffer requriements. Rose (WO 2017/140081)

Aumann (US2011/0042310; US 2009/0050567; US8,496,836; US7,837,881) teaches a process for continuous purification of a mixture using columns that are grouped into at least four sections. After a switch time, the last column from the first section is moved to the first position of the second section, the last column of the second section is moved to the first position of the third seciton, the last column of the third section is moved to the first position of the fourth section and the last column of the fourth seciton is moved to become the first column of the first section. The column can be run in at least one batch mode step in which the outlet of one column is used to collect the desired intermediate product as well as in continous mode where the outlet of at least one column is fluidly connected with the inlet of at least one other column.

Bryntesson (WO2008/153472A1 and US2010/0176058) discloses a SMB method where a feed compriing a target compound is passed across a 1st adsorbent, the outflow passes to a 2nd adsorbent, the feed is redicted to the 2nd adsorbent, a wash liquid is passed across the 1st adorbent, this wash liquid outlfow is directed to a 3rd adsorbent and the second adsorbent outflow is directed to the 3rd adsorbent, the 1st adsorbent is regenerated, the feed is redicted to the 3rd adsorbent, wash liquid is passed across the 2nd adorbent, directed the wash liquid outflow to the 1st adsorbent, subsequently directly the outflow from the 3rd adsorbent to the 1st adsorbent, regenerating the 2nd adsorbent, redicteing the feed to the 1st adsorbent, washing the 3rd adsorbent and directly its outlfow to the 2nd adsorbent, directing the otuflow from the `st adsorbent to the 2nd adsorbent, regenerating the 3rd adsorbent and repeating all the steps.

Jeon (US 13/643146, published as US. 2013/0046080) discloses a chromatographic method for purifying an antibody where a sample is loaded on a 1st column, loading the outflow to a 2nd column, before washing the 1st column, stopping the loading of the sample and loading the sample on the 2nd column, loading the outflow from the 2nd column onto a third column and before washing of the 2nd column, stopping the loading of the sample on the 2nd column and loading the sample on the 3rd column and repeating the steps from the 1st to 3rd and 3rd to 1st columns.

Muller-Spath (US2014/0299547) teaches reducing the number of column to twofor the isolation of a product like an antibody comprising a first batch step, where said column are disconnected and a first column is loaded with feed and its outlet directed to waste and from a second column desired product is recovered and subsequently the second column is regnerated, a first interconnected step where the outlet of the first column is connect to the inlet of the second column, the first column is laoded beyond its DBC with feed and the outlet of the second column is directed to waste, a second batch step analogus to the first batch step but with exchanged column and a second interconnected step analogous to the first interconnected step but with exchanged column.

Thommes (US2004/0241878) teaches separating an antibody using a simulated moving bed (SMB) system where at certain points liquid streams may be introduced and at other points effluent streams may be withdrawn. Thommes (US 2007/0215534 and US Patent No: 8608960) also discloses a SMB system which incorporates a plurality of zones  such as an association zone, a wash zone, an elution zone. Suitable solid phases may include Protein A or G but can also include CEX. Use of continous chromatography in a continous counter-current mode where the columns are loaded to equilibrum capacity requires smaller column valumes compared to batch chromatgoraphy wehre the column are loaded to there DBC and requires using columns of 2-3 times the volume. In one embodiment, the feed is applied to 2 columns in parallel and makes a second pass through the 2 parallel columns. A carousel or similar apparatus may be used to move the columns relative to stationary valves and inlet and outlet streams. The columns moving to the left from the adsorption zone enter the adsorption wash zone. The effluent of the adsorption wash zone is fed back into the adsorption zone to minimize product loss. The adsorption was zone consists of 2 columns in series thus reducing the amount of buffer reuqired by about 40%. The product is leuted in an elution zone consisting of 2 columns in series. The eltuion zone is followed by an elution wash zone. 

Commercial SMB columns:

SMB systems have been designed by Novaept (Pompey, France), Tarpon (Worcester, Ma), Semba (Madison, Wi), Contichrom (Knauer, Germany) and GE Healthcare. (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

Cature SMB:

In captureSMB process, two identical columns are used to recover the target compound form the feed mixture. After the columns have been equilibrated, they are interconnected by connecting the outlet of the first column to the inlet of the second column. For a certain time, the feed solution is loaded onto the first column, The fact that there is a second column behind the first one allows for longer feeding compared to the batch case, because the eventual breakthrough is cought by the second column instead of being lost. After this time, the first column enters the recovery and regeneration step, which is equivalent to the batch process. During this step, the now diconnected second column is further loaded with feed. As soon as the first column is re-equilibrated and ready to be loaded again, the columns swap roles and ocntinue with the next interconnected step.  (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Periodic Counter-Current Chromatography systems (PCCS or MPCC):

In multi-colunn periodic counter-current chromatography (MPCC) the breakthrough protein form the first column is captured by the second column. The first column stops laoding when a set protein breakthrough percentage is reached and the loading switches tothe second column. The first column is then eluted and regenerated and multiple columns are alternated to acheive continuous chromatogrpahic separation, thereby improving productivity, resin capacity utilization and reducing buffer consumption and equipemnt size. Thus for a three column MPCC, columns 1 and 2 are connected in series for laoding and column 3 is eluted and subjected to recovery and regenreation R-R). When column 3 is completely eluted and regeneration, column 1 also reaches the set protein brekathrough percetnage. Columns 1 and 3 a re connected and unadsorbed protein in column 1 is washed to column 3. At the same time, feed is swtiched to column 2. After column 1 is cwashed, it is disconencted form column 3 and column 2 is connected to column 3 for continous loading, while column 1 is subjected to R-R. When column 1 is completely eluted and regeenrated column 2 also reaches the set protein breakthrough percetnage . Column 2 and 1 are connected in series and the unadsorbed rptoein in column 2 is washed to column 1. At the same time, the feed is switched to column 3. After column 2 is completely washed, it is disconnected form column 1 and column 3 is connected to column 1 for continous laoded bhile column 2 is subjected to the R-R. When column 2 is completely eluted and regenreated, column 3 also reaches the set protein brekathrough percetnage. Column 3 and 2 are connected in series and the unadsorbed rptoein in column 3 is washed to column 2. At the same time, the feed is switched to column 1. Lin (US Patent Application No: 18/117,479, published as US 20230203092)

In PCC, multiple columns are used to perform the same steps in parallel such that continuous feed is acheived and all other process steps are discrete in time. For PCC, a cycle is defined when each one of the columns has completed equilibraiton, load, wash, elution and regeneration steps in sequence. To proceed from one step to another, an automated column switching algorithm is employed. There are essentailly two decision points required for every column cycle –one at the start of protein brekathrough and the other at column saturation. These switch points in PCC are detemriend by a UV based control strategy developed by GE Healthcare that is based on a difference between the feed and column outlet V. In principle, column switching can be determined by an process analytical technology tool capable of in-line measurement of product concentration. (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

Konstantinov (US 2014/0255994, issued as US 9,650,412) discloses an integrated continous biomanufacturing process for producing a therapeutic protein such as an antibody which includes feeding a liguid culture medium into a first multi-column chromatogrpahy system (MCCS1) to capture the protein and then continously feeding the eluate which contains the protein into a second multi-column chromatgraphy ystem (MCCS2) to purify and polish the protein. The process can include the use of two or more mutli-column chromatography systems (MCCSs). A MCCS can include two or more chromatogrpahy columns or membranes or a combination of columns and membranes. The first and/or second MCCS can be a periodic counter current chromatogrpahy system (PCCS) which includes towo or more columns that are switched in order to allow for teh continous elution of the protein from the two or mroe columns. In PCCSs, multipe columns are used to run the same steps discretely and continously in a cyclic fasion. Since the columns are operated in series, the flow through and wash form one column is capture by another column. This unique feature allows for loading of the resin close to its static binding capacity instead of the DBC and is typical during batch mode chromatography. Once all the steps in the cycle are competed, the cycle is re-started. The system can include column switching based on teh UV absorbance difference betweenthe feed inlet and column outlet. During column laoding, the PCCC control system dtermiend the impurity baseline when the absorbance stabilizees. As the recombinant therapeutic prtoein breaks through, there is an increase in the outlet UV signal above the impurity baseline. At the point when deltaUV ahs reached a rpe-detemriend threshold (such as 3% breakthrough of the reocmbinatn therapetuic protein), the flow-through from column 1 is directed onto column 2 ineasted of to the wate. When column 1 is nearly saturated with recobmiannt threptuic protein adn the delta UV has reached a pre-detemriend vlue, the feed is switched to column 2. An important adantage of this deltaUV based column switching streegy is that it allows for uniform loading of the columns irrespective of the feed recobminant therapetuic prdcut concetnraiton and the oclumn capacity. In PCCCS, the resdience time (RT) of the protein on the column can be decreased without increasing the column size becasue the breakthrough form the frist column in the system can be captured on the second column in the system. 

3-column periodic counter-current chromatography (PCC) (3-C PCC):

The 3-column PCC process uses three identical columns. The first part of a switch consists of an interconnected wash step where the first, fully loaded column is washed and the wash is recylced into the third column, while the second column is loaded in a disconnected mode. Since the feed is applied to a disconnected column, this phase is denoted as athe batch pahse, with the duration tB and the feed flow rate BB. When the interconnected wash step is finished, the first and the third column are disconnected and the first column continues in the recvoery and regeneration procedure, with further wash steps, the elution, the CIP and the equilibraiton step. In the meantime, the two other columns are loaded in an interconnected manner using a flow rate QIc; therefore this part of the switch is called the interconnected phase, which last for the time tIC. After the interconnected phase is complete, all columns are moved one position upstream relative to the liquid phase flow, which denotes the beginning of a new switch. When three swithes have passed and the columns are back in the initial configuation, one cycle has passed. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Gadgil (US 16/340,822, published as US 2019/0263855) discloses a multi-column chromatography system such as a periodic counter current chromatography system (PCC) which includes carboxypeptidase B immobilized on sepharose.  The C-terminal lysine residues on H chain can be truncated by passing a harvest recovered form perfusion cell culture on a column which has CPB on sepharose. The CPB preferentially acts upon the basic amino acids, such as arginine and lysine and thus the resin can be used for removal of charged isoforms belowing to any class of antibodies. In one embodiment, a continuous process for reducing heterogeneity of an antibody includes a CPB-Sepharose column connect to a Protein A column. The flow through from the CPB-Sepharose column is directly loaded ontto the protein A column for capture step. The third and fourth columns can be selected from AEX, CEX, HIC and MM chromatography. In one embodiment, the multicolumns can be run either in series or in parallel. In one embodimentm the prcoess was carried out on AKTA pcc (three column periodic counter curent chromatography, 3C PCC) GE Healthcare, where one CPB-CNBR activated Sepharose 4B column (column 1) and two prtoein A columns (column 2) are connected parallely. The residence time maintined on column 1 is equivalent to 1.7 min and column 2 was about 4.4 min (as resin used is MabSelect SuRE LX). 

4-column periodic counter-current chromatography (PCC) (4-C PCC):

The 4-column PCC process uses four identical columns that are loaded and eluted sequentially.  IIn the first part of the switch, the column in the first position, which has been washed in the previous switch, is eluted. Meanwhile, the second column undergoes the first part of the wash step, during which product from the liquid phase of the first column is directed to the cleaned and regenerated column in the last position. During this time, the column in the third position is disconnceted and loaded with feed. When the intercconencted part of the wash step is completed, the second column is disconnected and the recovery and regeneration continues. Product elution from the first column finishes during this phase, and CIP and regeneration follow. Meanwile, the other two column are interconnected and feed is applied to the third column. After this, one switch has passed and the columns are moved one position upstream. The 4-column PCC prcoess differs form the other processes in that there is more than one column in the recvoery and regeneration phase at any one time. If there is no constraint that the switching times in all positions must be the same, the recvoery and regeneraiton can be scheduled mroe efficiently in the 4-column PCC process. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Gdawat (“Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) discloses a 3 column PC cycle. At the begining of a cycle, the feed is laoded onto column 1 and the flow through goes to waste. (step 1) When the product start to breakthrough, the flow-through from column 1 is directed to column 2 to capture the unbound product. (step 2) Once column 1 is fully loaded, the feed is directly loaded onto column 2, while column 1 is washed onto column 3, eluted regenerated and requilibrated for the next cycle. (steps 3-4) Column 2 is now subjected to steps 3-6, which are identical to steps 1-4 for column 1. Finally, the same steps occur on column 3. 

–Commercial examples:

Gdawat (“Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) discloses a 4 column PCC system which is a custom modified AKTA system (GE Healthcare). The system was equipped with five UV minotors, three pmumps, multiple vales and a pH and conductivity meter and was pwoered by a Unicorn based custom control strategy. MabSelect SuRe, iminodiacetate adn hydrophobic interaction chromatgoraphy HIC) media were packed into three of four 1.6 cm x 6 cm, 16.6 cm x 10 cm, or 0.66 cm x 6 cm columns. 

Purification of Target Proteins using Continous Multi-column chromatography  each with the same ligand for each of the Target proteins

Bataille (US Patent application 15/125483, published as US 2017/0073396) disclsoes purification of three separate plasma proteins, immunoglobulin, albumin and fibrinogen, where muticolumn affinity chromatography comprising several chromatogrpahy columns having the same chromatography support are linked in series, the chromatography in series being used to purify the threee separate proteins. For example, a first mutlicolum affinity chromatography step where each of the columns includes an affinity ligand that binds to immunoglobulins is first conducted, the flow through fraction is then subjected to a second muticuloumn IEX or mixed mode or HIC multicolumn step where the fraction containing albumin is collected and the flow through is usbjected to a third multicolumn affinity chromatography series having an affinity ligand that binds to fibrinogen, at the conclusion of which the fration containing fibrinogen is collected. Each of the multicolumn chormatrpahy steps can for example be 3 columns in series and performed such that once loading and washing of the first column is completed, the first column is separated from the other columns and undergoes elution while the second column becomes the first column in the series and once the equilibration is completed, the first column is replaced in series in last position and the bycle of steps is repeated. 

Optimization of PCC:

Protein adsorption to a chromatogrpahy resin is dependent on the bidning conditions, size, biochemical proeprties of the target protein, the resin bead morphology (bead size, pore structure and phase ratio) and the functional ligand properties (type and density). Collectively, these factors influence the thermodynamic and mass transfer kinetics of protein-resin interations. The thermodynamics govern the equilibrium binding properties, such as static binding capacity and the binding isotherm, while mass transfer kinetics govern the rate of protein adsorption onto the resin. The quantitative aspect of mass trasnfer kinetics in protein binding is demonstrated by the shape and nature of the protein breakthrough curve. Protein breakthrough form a column occurs before all the protein accessible binding sites are unitized on the resin due to mass transfer resistance. Since columns are operated at a finite flow rate with a finite protein residence time, only a poriton of the static binding capacity can be utilzied. This capacity is the dynamic binding capacity at a particular residence time. Chromatographers have traditionally deisgned batch processes with relatively long target protein residence times to increase resin capacity utilization. However, this approach also resutls in low throughput and subsequent oversizing of the chromatogrpahic column to aceive the desired target throughput. In contrast to batch chromatogrpahy, target protein breakthrough is laoded onto a second column in PCC until the first column is almost fully saturated. This leads to full capacity utilization irrespective o most cmmonly used residence times. This can also be acheived in batch mode in convective systems (perfusion media with large proes and membrane chromatography) at short residence times wehre the mass transfer effects are negligible.  (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

(Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) isclsoes a mathematical framework for designing PCC for a given protein capture resin system. The scheduling required to perform continous captrue of a target protein using PCC is governed by the target prtoein breakthrough curve. For a given protien-capture resin system, the scheduling parameters can be detemriend from a mathematical transformation of the breakthrough curve. The optimal PCC operation can be defined using one of many potential bjective functions, including maximization of resin capacity, utlization, minimum recovery loss during was, resin lifetime usage or minimization of residence time. 

Countercurrent Tangential Chromatography:

Countercurrent trangential chromatography overcomes many of the limitations of conventional column chromatography by having the resin (in the form of a slurrry) flow through a series of static mixers and hollow fiber membrane modules. The microporous hollow fiber membranes retain the large resin particles while letting all disssolved species pass through the membrane and into the permeate. Thus, during the binding operation, the protein of interest stays bound to the resin while the impurites flow through the membrane and are removed as waste. The buffers used in the binding, washing, and elution steps flow countercurrent to the resin, enabling high resolution separation while reducing the amount of buffer needed for protein purification. For example, a concentrated slurry is pumped from slurry tank 1 into a first statis mixer (SM1) where it is mixed with recycled permeate form a second hollow fiber mrembrane module (HF2). The diluted slurry then passes into the frist hollow fiber module (HF1) where sufficient permeate is removed to return the slurry to its origianl concentration. This provides true countercurrent contacting, the slurry moves form stage 1 to stage 2 while the buffer (permeate) starts in stage 2 and moves to stage 1. For binding operations the concentrated slurry is then mixed with the protein mixture in the second statis mixer (SM2), pumped into HF2 and collected as a reconcentraed slurry in slurry tank 2. Permeate from the first module is sent to the permeate collection tank. For washing and elution, the concentrated slurry is mixed with an appropriate buffer in the second static mixer. (Zydner “Countercurrent tangential chromatography for large-scale protein purificaiton” Biotechnology and Bioengineering, 108(3), 2011), disclosing using a Macroprep 25Q strong anion exchange resin consisting of a methacryalte based modified with a quaternary amine functionality). 

The CCTX process utilizes the resin in the form of a slurry which flows thorugh a series of static mixers and hollow fiber membrane modules. The micro-porous hollow fiber membranes retain the large resin partciles while letting all dissolved species, including proteins and buffer components pass through the membrane an into the permeate. The buffers used in the binding, washing, elution, strippeing and equilbiraiton steps flow coutnercurrent to the resin slurry in a multi-stage confirguation, enabling hihg resolution separation while reducing the amoutn of ubffer need for protein purificaiton. (Shinkazh “Purificaiton of monoclonal antibodies form clarified cell culture fluid using Protein A capture continous coutnercurrent tangential chromatography” J Biotechnology. 213 (2015) 54-64). 

Shinkazh (US 2010/0193434; see also US 2017/0045483) discloses a module that consists of two or more interconnected tangential flow filters and static mixers. The chromatography resin flows thorugh this module in a single pass, while similar operations to a regular chromatographic process are performed on the resin (binding, washing, elution, regeneration and equilibration). The buffers for these operations are pumped into the module in a countercurrent direction to the flow of resin, and permeate solutiosn from latter stages are recyled back into previous stages. This creates concentration gradients in the permeate solution of the tangential flow filters in the countercurrent direction to resin flow, thus saving buffer volume and increasing process efficiency. The permeate solutions from binding, washing, equilibration and regeneraiton operations are put to waste. The permeate solution form the elution operation is the purified product stream which is collected in a separate product tank. In one embodiment, the module for CCTC includes a first input prot for receiving an input solutions, a first mixer for mixing the input solutions with a recyled solution from a second input port to produce a first mixer output, a stage 1 filter for concentrating the first mixer output to produce stage I retentate, wehrein stage I permeate exists the module from the stage 1 filter via a first output port, a second mixer for mixing the stage 1 retentate form teh stage I filter and an optional buffer solution from a second inpurt port and a stage 2 filter for concetnrating an output from the second mixer to produce stage 2 retnentate which exits the module form teh stage 2 filter via a second output port, wehrein stage 2 permeate exists the module form the stage 2 filter via a third output port. 

Shinkazh (US 2020/0030717) disclsoes replacing the hollow fiber membranes of CCTC systems above by spiral micro-fluidic partcile sorter devices. These dvices seaprate resin form the dissolved species using Dean Vortices at the curved outer wall of the device channel to selectively attract partciles to one wall of the device. The devices accordingly do not reuqire a physical barreir such as a membrane for separating resin partciles from dissolved speies. 

Depression:

Bupropion is an antidepressant drug approved for depression. It has also been used off label for weight loss.  Bupropion is an antidepressant medication that works in the brain. It is approved for the treatment of major depressive disorder (MDD), seasonal affective disorder (SAD), and to help people quit smoking (smoking cessation).

Trintellix is a brand-name prescription antidepressant. It’s used in adults to treat a serious form of depression called major depressive disorder.

Selective Serotonin Reuptake Inhibitors:

Lexapro (escitalopram): Lexapro (escitalopram) is an antidepressant from the group of drugs called selective serotonin reuptake inhibitors (SSRIs) and is used to treat certain types of depression and anxiety. Lexapro works by balancing levels of serotonin in the brain and nerves. Lexapro (escitalopram) is good for treating depression and anxiety. It’s generally well-tolerated and has fewer drug interactions than other antidepressants. 

Escitalopram is a selective serotonin reuptake inhibitor while alprazolam is a benzodiazepine. Both can be used to treat anxiety, but escitalopram is also used to treat major depressive disorder while alprazolam is used to treat panic disorder.

Antihistamines:

Hydroxyzine: is an antihistamine that is also used to treat anziety. 

Benzodiazepines:

Benzodiazepines are central nervous system depressant medications that are often used for treating or managing anxiety disorders or insomnia. These medications have a sedative effect that can help people sleep better and feel less anxious. 

Xanax (Alprazolam): is a prescription drug that’s designed to address panic and anxiety disorders. This sedative is common and comes with warnings about addiction and abuse potential, as well as the risks of mixing it with other substances like alcohol.

Ativan (Lorazapam): is prescribed to reduce anxiety. Like many benzos, this medication works by slowing down brain activity and increasing relaxation. Because of its effects of sedation, there is a significant potential for drug abuse.

Valium (Diazapam): has a variety of approved uses, including treating anxiety, seizures, and alcohol withdrawal. Long-term Valium use can lead to dependence, tolerance, and dangerous withdrawal symptoms if you abruptly stop use.

See also Oxidation and Reduction in Biochemistry

Disulfide Bond Variants 

Disulfide reduced variants have one more disulfide bonding cystein(s) chemically reduced to the free thiol form. This variant can be monitored by HIC or by sizing methodology such as Capillary Electrophoresis with Sodium Dodecyl Sulfate (Harris, US2009/0202546).

Half-antibodies: Occasionally the disulfide bonds between the heavy chain polypeptides of antibodies are not formed, resulting in the formation of an antibody with no interchain disulfide bonds between two pairs of heavy and light chains. These antibodies have been called “half antibodies” (or “Hab”). Certain antibody classes and types are more susceptible to half antibody formation such as IgG4. In both natural and recombinant antibody production, a significant proportion of IgG4 antibodies at least as high as 35% are produced as half antibodies (Brower US 14/668820, published as US 2016/0108127).

Causes of Antibody Reduction in Cell Culture Production Processes:

Reducing enzymes: A very important structural feature of an antibody is the disulfide bonds that link its light and hiavy chains (inter-chain disulfides) together to form a quaternary complex. Under specific conditions, extensive reduction of the antibody’s inter-chain disulfides bonds are observed after harvest operations (centrifugation and filtration) and/or the first purificaiton step (i.e., protein A chromatography). On one such instance, it was estimated that as little as 10% of the antibody remained intact after the protein A step. This antibody reduction is not a unique issue to a specific antibody or a particular cell line. The reduction is reportedly caused by an active thioredoxin (Trx) system or other reducing enzymes with thioredoxin-like activity in the harvested cell culture fluid. The Trx system (i.e., Trx and TrxR) is one of the two intracellular enzymatic systems (the other is the glutaredoxin (Grx) system) that regualtes the cellular redox status and maintains a reducing environment in the cytosol. *Kao “Mechanism of antibody reduction in cell culture production processes” Biotechnology and Bioengineering” (2010). 

During purificaiton of IgG2, high level of fragment in the prcoess intermedaites was detected by HP-SEC. Fruther investigations traced the casue to be antibdoy reduction in the harvested cell culture fluid (HCCF). In addition, when the HCCF was redued, larger increase in aggregate content was observed druing low pH treatment. When purificaiton was initiated immediately after harvest, antibody reduction was not observed in the HCCF and aggregate formation during low pH treatment was minimized. This can be attributed to the shorter exposure time of the antibody to reducing enzymes in teh HCCF. Increasing cystine concentraitons also resulted in lower free thiol levels when the HCF was held. (Chun “Effects of antibody disulfide bond reduction on purificaiton process performance and final drug substance stability” Tiotechn Bioeng, 114(6) (2017). 

Methods to Decrease Reduction of Disulfide bonds

Reduction of the disulfide bonds that internally anchor disulfide bond-containing proteins can occur during manufactering. Reduction of the inter-chain disulfide bonds results in loss of functional antibodies and requires more coplex purification. During mAb process development, fragments and aggregates have to be removed to adequate levels due to their associated risks with increased immunogenicity and unknown effects on drug efficacy (Ahuja, US 2019/0112357)

Using Charged Depth Filter

Hoang (US 15/751,231, published as US 2018/0230180) discloses a method of reducing the perentage of reduced antigen-binding protein molecules using a charged depth filter.

Control of Disulfide bonds and Half-antibody formation using reduction/oxidation (redox) agents:

–-Addition of 2-MEA, reduced gluathione, oxidized glutathione, 2-mercaptoethanol, DTT, cystein, cystein, dithiobutylaine or sodium sulfite or Oxidizing metal ions (Cu2+, F32+, Fe3+, Zn2+)

—-During Cell Harvest

Brower (US 14/668820, published as US 2016/0108127) discloses a process to control the formation of disulfide bonds between polypeptides by contacting the polypeptides with a conditioned solution that includes predetermined solution parameters such as a redox reagent like 2-mercaptoethylamine (2-MEA), reduced glutahione, oxidized glutathione, 2-mercaptoethanol, dithiothreitol (DTT), cystine, dithiobutylamine or sodium sulfite. at a specific time point during the bioprocess. In some embodiments, the specific time point occurs during the step of viral inactivation, adjustment, chromatography, filtration, dilution, concentration or any bioprocess step that is cell free. In other embodimetns, the specific time point occurs during chromatogaphy.  In one embodiment, Brower discloses that the level of half antibody decreased in the presence of 1 mM 2-MEA but that higher levels (25 and 50 mM) results in increassed level of half-antibody and also generated H and L fragments.The level of half antibody also decreased within about 30 minutes in the presence of 2-MEA at 1-3 mM at pH 4.8. 

Chaderjian (“Effect of Copper Sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed” Biotechnol. Prog. 21: 550-3 (2005) discloses that cooper sulfate can at as an oxidizing agent, thereby facilitating disulfide bond formation when added to cell culture production medium. Purified antibody showed that either 50 or 100 uM copper sulfate additions reduce the level of free thiol by more than 10-fold. 

Dillon (WO 2006/047340) discloses a method of producing a IgG antibody by contacting the antibody that has been recombinantly produced by mammalian cells with a reduction/oxidation coupling reagent at a pH of about 5-11, optionally with a chaotropic agent. The method reduces the heterogeneity of the various antibody isotypes. For example, the method can be used to refold IgG4 molecules to decrease the present of IgG4 half molecuels. The redox coupling reagent may be any redox coupling reagents such a reduced glutatione and oxidized glutathione. 

Schotte (WO2010/125187) discloses a method for producing a domain antibody in a host other than E. coli, preferably yeast, by applying conditions that promote the formation of disulfide bridges in the domain antibody, specifically the addition of oxidizing agent, preferably oidizing metal ions such as Cu2+, Fe2+, Fe3+, Zn2+, or removing domain antibodies lacking at least one disulfide bridge or a combination of both. The inventor found that despite the high yield and funcitonality of domain antibodies produced in non-E coli hosts, there is a quantiatively significant fraction of product that represents a structural variant, specifically a product that lacks at least one disulfide bond. 

–—–Addition of Proxides to prevent antibody disulfide bond reduction:

During the manufacturing process, extensive reduction of antibodies has been observed after harvest operation or Protien A chromatography and multiple process parameters correlate to the extent of the reduction. For example, maintaining high levels of dissolved oxygen (DO) during harvest is vital to keep antibody molecuels intact. Mechanical shear forces, which cause cell lysis and cellular components to leak into harvest fluids also significantly contribues to the reduction. Other process parameters such as harvest hold time, media componetents (such as copper ions, cystein/cystine and pH) and termpature also have an effect on the extent of disulfide reduction. The reduciton of antibody products due to enzyme systems may be complete or partial Human IgG contain two H and 2 L chains that are held together by non-covalent interactions as well as inter chain disulfide bonds. There are a total of 12 intra chain disulfide bonds and one disulfide bond between L and H chains in all four IgG subclasses. The nubmer of inter-chain disulfide bonds between the two H chains is 2 for IgG1 and IgG4, 4 for IgG2 and 11 for IgG3. Additionally, the interchain disulfide bonds are more solvent acccessible than intra-chain bonds and the exposed cysteine residues are considered more reactive than non-exposed cysteine residues. Hydrogen proxide can oxidize reducing agents and prevent the reduction of disulfide bonds and prevent the generation of LMW species. There are many peroxide containing compounds, inorganic or oragnic that can be substituted for hydrogen peroxide. Two inorganic forms, sodium percabonate and sodium perborate, can also effectively suppress the reduction of disulfide bonds in a concentraiton dependent manner. (Du, “Using hydrogen peroxide to prevent antibody disulfide bond reduction during manufacturing process” MABS, 10(3), pp. 500-510 (2018). 

——-Addition of Peroxides + Red Light:

Color variation due to antibody process related impurities can be a major concern as a problem of lack of process control. One of the main sources of pink/red color in final drug substance has been identified as the vitamin B12-mAb complex.  Cyanocoblamin is the vitamin B12 form typically included in cell culture media as a cofactor required for DNA synthesis. Under cell culture conditions, however, cyanocobalamin is readily converted to hydroxo-cobalamin due to light exposure, with up to 80% converted after 10 d in culture. The red color intensity can be minimized by reducing the medium concentration of vitamin B12. Since disulfide reduction mediated mAb dissocaition can be prevented at the manufacturing scale( with putblished methods, the risk of red color can also be minimimized with these published methods. (Derfus “Red colored IgG4 casued by vitamin B12 form cell culture media combined with disulfide reduction at harvest” mAbs 2014).

The intensity of pink color depends on concentraitons of both free sulfhydryl groups on reduced mAb and hydroxocoalamin, the active form of vitamin B12. Both reactants are necessary to generate pink colr. Thus process control strategy can consider limiting either one or both factors. (Du, “Vitamin B12 association with mAbs: mechanism and potential mitigation strategeis” Biotechn. and Bioeng. 2018, 115: 900-909). 

Tan (US 16/612,175, published as US 20230103511) discloses preventing the reduction of antibodies by adding peroxides (e.g., hydrogen peroxide, sodium percarbonate or sodium perborate) to the harvest solution and inhibiting the conversion of vitamine B 12 (cyanocoalamin (CN-Cbl)) to hydroxocobalamin (HO-Cbl) by culturing cells under red light conditions during manufacture or cell harvest. Tan showed that the generation of pink colr during antibody manufacturing is dependent on concetnrations of both free sulfhydryl groups on reduced antibodies and hydroxocobalamin, the active form of vitamin B12. Both reactants are necessary and neither one alone is sufficient to generate pink color. The active form of vitamin B12 attaches to the free sulfhydryl group of the cystein located at H chain 134, LC 214, HC 321, 367 and 425. The five cysteins residues are distributed among both the Fab and the Fc regions. Cynocobalamin convertion to hydroxocobalamin is inhibited or reduced by replacing white light with red colored light (wavelenghts >600 nm) in the areas where the cell culture media is prepared and sotred and optionally in cell culture manufacturing and harvesting areas. 

–During Chromatography Process

Godavarti (US 7,825,223) discloses purifying an AB binding protein having an Fc region such as an anti-AB antibody by absorbing the antibody to an Fc binding agent such as Protein A, followed by a wash with a divalent cation such as copper, nickel, manganese, to remove impurities. 

Zou (WO 2006/060083) discloses subjecting column isolated preparations of polypetpides with a reduction/oxidation reagent such as cysteins and cystine, reduced and oxidized gluathione, dithiothreitol (DTT), 2-mercaptoethanol, hydrogen peroxide (oxidizer) and dithionitrobenzoate and a chatropic agent and isolating the refolded active protein produced from said contacting. In one embodiment, the redox reagent is cystein/cystine. The chaotropic agent can be urea, SDS or guanidine hydrochloride. In other embodimetns, refolding of the protein can be performed using other redox reagents such as copper. The refolding can occur on a column such as an affinity resin (e.g., Protein A) of a Fc domain containing polypeptide.

Isolation of domain antibodies with oxidizing agent(s)

Schotte (US13/266503 and US2012/0157664) discloses a method of producing a domain antibody in yeast by applying conditions such as addition of oxidizing agents, preferably oxidizing metal ions such as Cu2+, Fe2+, Fe3+ and Zn2+ that promote the formation of disulfide bridges in the domain antibodies and/or removing domain antibodies lacking at least one disulfide bride. The method results in the production of domain antibodies wherein the quality of the domain antibodies is improved with a reduced level of free thiol or its absence. 

Oxidation of Amino Acids:

Oxidation is a common degradation pathway that may occur during the life cycle of prtoeins and peptides. The resulting chemical modificaitons may affect in vitro stability and in vivo biologcial functions. In order to assess the potentail susceptibility of protein products to oxidation, different oxdiation stress conditions have been studies such as hydrogen peroxide (H2O2). , transition metal ions or UV exposure. Scuh treatment often result in a myriad of modificaitons on several amino acid residues, such as histidine, phenylalanine and tyrosine. However, methionine and tryptophan are the residues that are oxidized most cmmonly  and to the highest extent. (Folzer, “Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molcules” J of Pharmaceutical Sciences, 2015). 

Recombinant mAbs are constantly exposed to oxidizing environments, including dissolved oxygen, oxygen in teh air and free radicals such as those generated by reactions with metals and impurities form raw materials (e.g., peroxides). Forced oxidation is used to probe residues taht are susceptible to oxdiation and to udnerstand if there is a practical impact on this degradation pathway (e.g., oxidation of a residue in the CDR may affect binding and potency). The most commonly used approaches are incubation of recombinant mAbs with hydrogen peroxide or tert-butyl hydrogen peroxide for methionine residues. (Nowark “Forced degradation of recombinant monocloanl antibodies: A practical guide” mAbs (2017)). 

Oxidation of methionine: 

Although protein oxidation can occur at cysteine, tryptophan, lysine and other amino acids, methionine is often the most susceptible residue to oxidation. The most common product of methionine oxidation is methionine sulfoxide, which is more polar and less hydrophobic than methionine. Susceptible methionines are typically located on the surface of the protein and exposed to the solvent. Met oxidaiton can have adverse effects on proteins, including decreased stability and decreased biological activity. (Pan “Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn” Protein Science, 2008, 18, 424-433).

Folzer, “Selective oxidation of methionine and tryptophan residues in a therapeutic IgG1 molcules” J of Pharmaceutical Sciences, 2015) disclsoes that oxidation levels of Fc methionine was first quantified by prtoein A chromatogrpahy. the mAb species eluted at 23 min. The oxidized species formed upon inucation with oxidant eluted earlier (before 19.8 min) because of the lower affintiy of oxidized Fc methionine to protein A. The incubation of the mAb with 1% H2O2 for 24 hr at 5C as well as the incubation with 2% t-BHP for 120 h at 25C allowed for teh nearly complete oxidation of the methionine residues form teh Fc region. 

Anion exchange and hydrophilic interaction: 

Negatively charged mixed mode ligands below to the cation exchanger mixed mode (for example CaptoAdhere) (Melinda, US 20170058019)

Examples include Capto-QTM which is a strong anion exchange and is a trademark of GE Healthcare Bio-Sciences. 

Polyethyleneimine derivatized polymeric media:

Deorkar (US 2008/0203029) discloses useful mixed mode chromatographic media for bioseparations whcih media polymeric includes particles dervivatized with polyethyleneimine (PEI) which are further funcitonalized with appropriate reactants. Examples of polyyric anyhdride and akly chlorides. In one embodiment, an anion excahnged with mixed primary and secondary and tertiary amine sites is prepared by reaction polyethyleneimine with a polymeric support bearing epoxy or halo groups such as chloro, boromo, iodo groups. Such polymeric support can be any suitable synthetic polymer or natural polymer resin such as poly(meth)acrylate, cellulose.  Resin particle suitable for derviatizaiton with polyethyleneimine includes cellulose, agarose, eopoxidized or halogenated polystyrenes. Examples of suitable functionalizaiton reagents for reaction with the PEI surface derivatized polyemeric resin partciles include anyhdrides such as cyclic carboxylic anyhdrides such as glutaric and succinic anhydrides, unsatured carboxylic anhydrides such as maleic meta-bisulfite, alkyl chorides or anhydrides such as butryl chloride.  Examples of such commerically avaialble resins are Tosoh Biosciences Toyopeark AF-eopxy 650M apoxy activated Sepharose 6B. These materials can be reacted with one of the teminal amino group of polyethyleneimine of various molecular weights through the formation of chemically stable alpha-hydroxy amino groups. In one embodiment, the mixed mode media having weak anion, weak cation and strong cation sites were prepared by reacting the EEI derivtized polymer with an unsaturated carboxylic acid anyhdride followed by sulfunation.

Anion and hydrophobic: 

Polyamines with Hydrophobic Group:

Engstrand (US 2007/0259453) discloses a method of separating antibodies using a multi-modal separation matrix to adsorb undesired compounds. The multi-modal separation matrix includes first groups which are capable of interacting with negatively charged sites of the target compoudns and second groups, which are capable of at least one interaction othern than charge-charge itneraction with said target compounds. In one embodimetn, the first anion-exchanging groups are strong anion exchanges such as qin that the groups remain charged within a wide pH range. In another embodiment, the first groups are weak ion exchanges in that they are charged at certain pH values but may loose charge by a pH switch. The second groups may give electron acceptor-donor interactions (e.g., hydrogen-bonding, dipole-dipole)  and/or hydrophobic and/or hydrophilic interactions. The functional groups may be present on the same ligand or on different ligands. In one embodiment, the the ligands are coupled to the support via their frist groups, such as via amines resulting in quaternary amines. 

Johansson (US Patent No: 6,702,943) discloses a method for removal of a target substance by adsorption to a matrix carrying a pluality of lgidnas taht include anion-exchaning groups and a hydrophobic structure. 

Matsumoto, (US 2015/0344520; see also US Patent Applicaiton No: 16/349,617, published as US 2019/0345194) discloses a chromatography media obtained by adding polyamine to a base media containing porous particles and then modifying amino groups in the polyamine with a hydrophobic group has excellentsalt tolerance and excellent adsorption. In particular, the chromatography media contains a base media haivng porous particles and a polyamine bonded with the base media wherein 20-40% of amino groups in the polyamine is modified with a hydrophobic group. The polyamine is selected from the gorup of polyallylamine, polyvinylamine, chitosan, polysine, polyguanidine and polyornithine. 

Commerical Examples:

–Capto adhere: is a mixed mode resin, a combination of anion exchagne and hydrophobic interaction. It can be operated in flow through mode where the antibody does not bind but the contaminante does bind. It is commercially available from GE Healthcare Life Sciences (Shaban, US 14/809,211). 

Capto adhere is a strong anion exchanger with multimodal functionality that gives a different selectivity compared to traditional anion exhangers. The Capto adhere ligand, N-Benzyl-N-methyl ethanol amine, exhibits many functionalities for interaction. The most pronounced are ionic interaction, hydrogen bonding and hydrophobic interaction. Capto adhere is designed for post Protein A purification of mAbs. Removal of leached Protein A, aggregates, HCS, nucleic acids and viruses is performed in flow through mode. The medium is based on a rigid high flow agarose matrix that allows high flow velocities to be used. (GE Healthcare Life Science, Instructions 28-9064-05AC Affinity Chromatography Capto adhere). 

Commercial examples include Capto-Adhere which is a multimodal anion exchange and a trademark of GE Bio-Sciences. Capto Adhere is a mixed-mode chromatography support which exploits a combination of anion exchange and hydrophobic interaction functionalities (US 8,188,242). Its ligand, N-benzyl-methy ethanol amine, contains anion exchange, hydrophobic and hydrogen bonding interaction groups. It is a strong anion exchanger with a phenyl group for hydrophobic interactions and a hydroxyl group for hydrogen bonding. The Capto adhere ligand (N-Benzyl-N-methyl ethanolamine) exhibits multiple modes of protein-interactive chemistries, including ionic interaction, hydrogen bonding and hydrophobic interaction. The multimodal functionality of the resin confers it with an ability to remove antibody dimers and aggregates, leached prtoein A, host cell proteins (HCP), antibody/HCP complexes, process residuals and viruses. The resin is typically used in flow through mode in the context of production scale polishing step employing operational parameters designed to ahve the mAb pass directly through the column while the contaminants are adsorbed. Nti-Gyabaah US14/355014

Generic conditions for use of multimodal anion exchangers such as CAPTO adhere do not exist. Specific conditions must be developed for each protein. The recomended method development procedure consists of evaluting various combinations of pH and sodium chloride concentration in the hope of identifying conditions that preferentially favor retention of aggregates (US 2011/0166332).

Gagnon (US 2011/0166332 and WO2010030222) discloses a method of separating at least one intact non-aggregated protein (e.g., an IgG antibody) form a liquid preparation by contacting the preparation with a multimodal anion exchanger sucha s CAPTO-adhere in the presence of one or more species of protein excluded zwitterions such as glycine.Boschetti (WO2004/024318) discloses mixed mode ligands which comprises a cyclic group which can be a monocyclic group or a polycyclic group such as a aromatic group that is a cylic hydrocarbon containing only unsaturated carbon bonds to give an aromatic system. While any aromatic group can be used, suitable aromatic groups typically have one, two or three aromatic rings. Illustrative aromatic groups are phenyl and its substituted derivatives such as tolyl and xylyl. Bicycl

–MEP HyperCel is a commercially available example of a combination of anion exchange and hydrophobic interaction functionalities (US 8,188,242). The spearation  is based on its pH dependent ionizable dual mode hydrophobic ligand design. In the purificaiton of a monoclonal antibody, for example, IgG adsorbs to the MEP ligand mianly through hydrophobic interactions under physiological buffer conditions. Desorption is through electrostatic repulsion between positivley charged IgG molecules and ionizable pyridine ring of the ligand when the buffer pH is decreased to close to or below the ligand pKa of 4.8, which the ligand is positively charged. See also “HCIC” under Antibody purification schemes.

Other commercial examples include MEP-Hypercel and Capto Adhere. Ma (US14208043)

Anion exchange + hydrophobic interaction + hydrogen bonding and pi-pi bonding: 

ic aromatic group include fused individual rings and include napthyl. Polycyclic aromatic groups include anthracenyl and phenanthrenyl and groups such as acenanaphthylenyl that contain fused rings of different sizes. If an aromatic group is selected, it is preferred that the group be fused to a heterocylic (saturated to particaly saturated ring include at least one hetero atom such as N, S or O) or heteroaromatic group. Exemplary heterocyclic or heteroaromatic groups include thiazoline, thizolidone, imidazole, imidazoline, thiazole, traizoles, tetrzole, thiadiazole, imidazole, pyridine and morpholine. The ligand includes a linking group that optionally comprises a sulfur ion. The monocyclic or polycylic group is substituted with a sulfate, sulfonate, phospahte, or phosphaonate group. These groups are sufficiently acidic to exist as charged moieites with a large pH range such as from 2-12. The monocylic or polycyclic group is tethtered to the solid support by a linking group which includes a mercapto, ether, or amino containing moeity. The solid support operated via “mixed modes” of interaction. The monocyclic and polycyclic groups have a pK below 4 and thus are negatively charged within the pH ranges used. A biological substance such as an immunoglobuilin, is contacted with the substrate between pH 4-6, in which the range the biological substance bears a net postive or neutral charge.. When the pH is raied above about 8, the biological substance gains a net negative charge, thereby creating an electrostati repulsion between the negatively charged solid substrate and the negativey charged biological substance. Consequently, the biological substance is released and can be isolated.