Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

Antibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual drug-linker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiple-level structural assessment protocols.     

Detection and identification of a serine to arginine sequence variant in a therapeutic monoclonal antibody

Sequence variants, also known as unintended amino acid substitutions in the protein primary structure, are one of the critical quality attributes needed to be monitored during process development of monoclonal antibodies (mAbs). Here we report on analytical methods for detection and identification of a sequence variant in an IgG1 mAb expressed in Chinese hamster ovary (CHO) cells. The presence of the sequence variant was detected by an imaged capillary isoelectric focusing (ICIEF) assay, showing a new basic species in mAb charge variant profile. The new basic variant was fractionated and enriched by ion-exchange chromatography, analyzed by reduced light and heavy chain mass determination, and characterized by HPLC-UV/MS/MS of tryptic and endoproteinase Lys-C peptide maps. A Serine to Arginine sequence variant was identified at the heavy chain 441 position (S441R), and confirmed by using synthetic peptides. The relative level of the S441R variant was estimated to be in the range of 0.3-0.6% for several mAb batches analyzed via extracted ion chromatogram (EIC). This work demonstrates the effectiveness of using integrated analytical methods to detect and identify protein heterogeneity and the importance of monitoring product quality during mAb bioprocess development.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies

Monoclonal antibodies are the fastest growing class of biologics in the pharmaceutical industry. The correlation between mAb glycosylation and aggregation has not been elucidated in detail, yet understanding the structure-stability relationship involving glycosylation is critical for developing successful drug formulations. We conducted studies of temperature-induced aggregation and compared the stability of both glycosylated and aglycosylated forms of a human IgG1. In parallel, we also performed molecular dynamics simulations of the glycosylated full antibody to gain an understanding of the polysaccharide surroundings at the molecular level. Aglycosylated mAbs are somewhat less stable and therefore aggregate more easily than the glycosylated form at the temperatures studied. Glycosylation seems to enhance solubility and stability of these therapeutics and thus might be important for long-term storage.     

Quantification of glycated IgG in CHO supernatants: A practical approach

Post-translational, nonenzymatic glycation of monoclonal antibodies (mAbs) in the presence of reducing sugars (in bioprocesses) is a widely known phenomenon, which affects protein heterogeneity and potentially has an impact on quality, safety, and efficacy of the end product. Quantification of individual glycation levels is compulsory for each mAb therapeutically applied in humans. We therefore propose an analytical method for monitoring glycation levels of mAb products during the bioprocess. This is a useful tool for process-design considerations, especially concerning glucose-feed strategies and temperature as major driving factors of protein glycation. In this study, boronate affinity chromatography (BAC) was optimized for determination of the glycation level of mAbs in supernatants. In fact, the complex matrix found in supernatants is an underlying obstacle to use BAC, but with a simple clean-up step, we found that the elution profile could be significantly improved so that qualitative and quantitative determination could be reached. Complementary analytical methods confirmed the performance quality, including the correctness and specificity of the results. For quantitative determination of mAb glycation in supernatants, we established a calibration procedure for the retained mAb peak, identified as glycated antibody monomers. For this approach, an available fully characterized mAb standard, Humira®, was successfully applied, and continuous monitoring of mAbs across three repetitive fed-batch processes was finally performed. With this practical, novel approach, an insight was obtained into glycation levels during bioprocessing, in conjunction with glucose levels and product titer over time, facilitating efficient process development and batch-consistency monitoring.     

Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies

ABSTRACT

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.     

Secondary mAb--vcMMAE conjugates are highly sensitive reporters of antibody internalization via the lysosome pathway

Monoclonal antibodies (mAb) selectively recognizing tumor surface antigens are an important and evolving approach to targeted cancer therapy. One application of therapeutic mAbs is drug targeting via mAb-drug conjugate (ADC) technology. Identification of mAbs capable of internalizing following antigen binding has been accomplished by tracking decline of surface-bound mAb or by internalization of a secondary mAb linked to a toxin. These methods may not be sufficiently sensitive for screening nor wholly predictive of the mAbs' capacity for a specific drug delivery. We have developed a highly selective and sensitive method to detect mAbs for cell internalization and drug delivery. This system uses secondary anti-human or anti-murine mAbs conjugated to the high-potency drug monomethyl auristatin E (MMAE) via a highly stable, enzymatically cleavable linker. Prior studies of this drug linker technology demonstrated internalization of a primary ADC leads to trafficking to lysosomes, drug release by lysosomal cathepsin B, and ensuing cell death. A secondary antibody--drug conjugate (2 degrees ADC) capable of binding primary mAbs bound to the surface of antigen-positive cells has comparable drug delivery capability. The system is sufficiently sensitive to detect internalizing mAbs in nonclonal hybridoma supernatants and is predictive of the activity of subsequently produced primary ADC. Because of their high extracellular stability, the noninternalized 2 degrees ADC are 100--1000-fold less toxic to cells over extended periods of time, permitting an assay in which components can be added without need for separate wash steps. This homogeneous screening system is amenable to medium-throughput screening applications and enables the early identification of mAbs capable of intracellular trafficking for drug delivery and release.     

The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures

To investigate the effect of dextran sulfate (DS), a widely used anti‐aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM‐0.025 and CS13‐1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13‐1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13‐1.00 cells, and showed the highest maximum mAb concentration. For SM‐0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N‐glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13‐1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3–1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113–1122, 2016     

Surfactants reduce aggregation of monoclonal antibodies in cell culture medium with improvement in performance of mammalian cell culture

Therapeutic monoclonal antibodies (mAbs) are biologics produced using mammalian cells and represent an important class of biotherapeutics. Aggregation in mAbs is a major challenge that can be mitigated by rigorous and reproducible upstream and downstream approaches. The impact of frequently used surfactants, like polysorbate 20, polysorbate 80, poloxamer 188, and 2-hydroxypropyl-beta-cyclodextrin, on aggregation of mAbs during cell culture was investigated in this study. Their impact on cell proliferation, viability, and mAb titer was also investigated. Polysorbate 20 and polysorbate 80 at the concentration of 0.01 g/L and poloxamer 188 at the concentration of 5 g/L were found to be effective in reducing aggregate formation in cell culture medium, without affecting the cell growth or viability. Furthermore, their presence in culture media resulted in increased cell proliferation as compared to the control group. Addition of these surfactants at the specified concentrations increased monomer production while decreasing high molecular weight species in the medium. After mAbs were separated, using protein “A” chromatography, flasks with surfactant exhibited improved antibody stability, when analyzed by DLS. Thus, while producing aggregation-prone mAbs via mammalian cell culture, these excipients may be employed as cell culture medium supplements to enhance the quality and yield of functional mAbs.     

Modulation of high mannose levels in N-linked glycosylation through cell culture process conditions to increase antibody-dependent cell-mediated cytotoxicity activity for an antibody biosimilar

The regulatory approval of a biosimilar product is contingent on the favorable comparability of its safety and efficacy to that of the innovator product. As such, it is important to match the critical quality attributes of the biosimilar product to that of the innovator product. The N-glycosylation profile of a monoclonal antibody (mAb) can influence effector function activities such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity. In this study, we describe efforts to modulate the high-mannose (HM) levels of a biosimilar mAb produced in a Chinese hamster ovary cell fed-batch process. Because the HM level of the mAb was observed to impact ADCC activity, it was desirable to match it to the innovator mAb's levels. Several cell culture process related factors known to modulate the HM content of N-glycosylation were investigated, including osmolality, ammonium chloride (NH₄Cl) addition, glutamine concentration, monensin addition, and the addition of alternate sugars and amino sugars to the feed medium. The process conditions evaluated varied in impact on HM levels, process performance and product quality. One condition, the addition of alternate sugars and amino sugars to feed medium, was identified as the preferred method for increasing HM levels with minimal disruptions to process performance or other product quality attributes. Interestingly, a secondary interaction between sugar and amino sugar supplemented feeds and osmolality was observed during process scale-up. These studies demonstrate sugar and amino sugar concentrations and osmolality are critical variables to evaluate to match HM content in biosimilar and their innovator mAbs.     

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation‐associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins

There are many factors that can influence the pharmacokinetics (PK) of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediated recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic antibodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation. Glycosylation of a mAb or Fc-fusion protein can have a significant impact on the PK of these molecules. mAb charge can be important and variants with pI values of 1–2 unit difference are likely to impact PK with lower pI values being favorable for a longer half-life. Most mAbs display target mediated drug disposition (TMDD), which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody (ADA) response and off-target binding, which require careful consideration during the discovery stage. mAbs are primarily absorbed through the lymphatics via convection and can be conveniently administered by the subcutaneous (sc) route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be reasonably estimated using cynomolgus monkey data and allometric scaling methods.     

Rapid quantitation of monoclonal antibody N-glyco-occupancy and afucosylation using mass spectrometry

N-glyco-occupancy and afucoslyation level are two important quality attributes associated with N-glycosylation of therapeutic monoclonal antibodies (mAbs). We report here a fast mass spectrometry-based workflow for quantification of N-glycan site-occupancy and afucoslyation level of mAbs with improved throughput, precision, sensitivity and robustness. This method uses the deglycosylation after the first GlcNAc and inter-chain reduction of the mAbs, followed by liquid chromatography/mass spectrometry (LC-MS) analysis. The entire process can be completed within one hour, which provides a rapid quantitation of N-glyco-occupancy and afucosylation to support high-throughput cell line selection and process development for mAb biopharmaceuticals.     

Approaches to Quality Risk Management When Using Single-Use Systems in the Manufacture of Biologics

Biologics manufacturing technology has made great progress in the last decade. One of the most promising new technologies is the single-use system, which has improved the efficiency of biologics manufacturing processes. To ensure safety of biologics when employing such single-use systems in the manufacturing process, various issues need to be considered including possible extractables/leachables and particles arising from the components used in single-use systems. Japanese pharmaceutical manufacturers, together with single-use suppliers, members of the academia and regulatory authorities have discussed the risks of using single-use systems and established control strategies for the quality assurance of biologics. In this study, we describe approaches for quality risk management when employing single-use systems in the manufacturing of biologics. We consider the potential impact of impurities related to single-use components on drug safety and the potential impact of the single-use system on other critical quality attributes as well as the stable supply of biologics. We also suggest a risk-mitigating strategy combining multiple control methods which includes the selection of appropriate single-use components, their inspections upon receipt and before releasing for use and qualification of single-use systems. Communication between suppliers of single-use systems and the users, as well as change controls in the facilities both of suppliers and users, are also important in risk-mitigating strategies. Implementing these control strategies can mitigate the risks attributed to the use of single-use systems. This study will be useful in promoting the development of biologics as well as in ensuring their safety, quality and stable supply.KEY WORDS: biologics, manufacturing technology, quality risk management, regulatory science, single-use system     

Accelerating patient access to novel biologics using stable pool‐derived product for non‐clinical studies and single clone‐derived product for clinical studies

Cell cloning and subsequent process development activities are on the critical path directly impacting the timeline for advancement of next generation therapies to patients with unmet medical needs. The use of stable cell pools for early stage material generation and process development activities is an enabling technology to reduce timelines. To successfully use stable pools during development, it is important that bioprocess performance and requisite product quality attributes be comparable to those observed from clonally derived cell lines. To better understand the relationship between pool and clone derived cell lines, we compared data across recent first in human (FIH) programs at Amgen including both mAb and Fc‐fusion modalities. We compared expression and phenotypic stability, bioprocess performance, and product quality attributes between material derived from stable pools and clonally derived cells. Overall, our results indicated the feasibility of matching bioprocess performance and product quality attributes between stable pools and subsequently derived clones. These findings support the use of stable pools to accelerate the advancement of novel biologics to the clinic. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1476–1482, 2017     

Monoclonal antibodies: Longitudinal prescribing information analysis of hypersensitivity reactions

Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy’s life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3–4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use.     

Monoclonal antibodies: Longitudinal prescribing information analysis of hypersensitivity reactions

Monoclonal antibodies (mAbs) are known to cause hypersensitivity reactions (HSRs). The reactions pose a significant challenge to investigators, regulators, and health providers. Because HSRs cannot be predicted through the pharmacological basis of a therapy, clinical data are often relied upon to detect the reactions. Unfortunately, clinical studies are often unable to adequately characterize HSRs especially in therapies for orphan diseases. HSRs can go undetected until post-marketing safety surveillance when a large number of patients have been exposed to the therapy. The presented data demonstrates how hypersensitivity reaction warnings have changed over time in the prescribing information (PI), i.e., the drug package insert, through August 1, 2011 for 28 US-marketed mAbs. Tracking all PI revisions for each mAb over time revealed that hypersensitivity warning statements were expanded to include more severe manifestations. Over the course of a mAb therapy’s life cycle, the hypersensitivity warning is twice more likely to be upgraded than downgraded in priority. Approximately 85% of hypersensitivity-associated fatality warnings were added in PI revisions as a result of post-marketing experience. Over 60% (20/33) of revisions to hypersensitivity warnings occurred within 3–4 y of product approval. While HSRs are generally recognized and described in the initial PI of mAbs, fatal HSRs are most commonly observed in post-marketing surveillance. Results of this study suggest that initial product labeling information may not describe rare but clinically significant occurrences of severe or fatal HSRs, but subsequent label revisions include rare events observed during post-marketed product use.     

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore “pandemic mAb” timelines need to be shortened. One acceleration tool is “deferred cloning” and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future “pandemic mAb.”     

Biotech Outsourcing Strategies cmc—Biologics stream: June 17, 2010, Copenhagen,Denmark

Now in its third year, the Biotech Outsourcing Strategies (BOS) meeting organized by Bio2Business took place at the Søhuset Conference Centre in Hørsholm, Copenhagen. The focus of this year''s event was the demanding and challenging area of chemistry, manufacturing and controls (CMC), and the meeting provided ample opportunity for lively discussion of the key issues surrounding this area. New for the 2010 conference, a biologics-focused lecture stream ran in parallel to the established small molecule stream. Both streams boasted a distinguished panel of keynote speakers who discussed all aspects of CMC from early stage scale-up through late stage clinical development. In addition to the keynote speakers, selected contract research organizations (CROs) gave short presentations on the solutions that they could provide to some of the challenges facing CMC. The meeting attracted more than 150 delegates from leading drug development companies and CRO service providers, and greatly facilitated the forging of new working relationships through pre-arranged one-to-one meetings. Moreover, exhibitions from event sponsors and considerable scheduled networking time over lunch and evening receptions further enhanced the highly productive and interactive nature of the meeting.Key words: outsourcing, chemistry manufacturing and controls, biopharmaceutical developments, contract manufacturingDr. Anne Bondgaard Tolstup (Symphogen) presented an overview of the key challenges for small companies involved in biopharmaceutical up-stream process (USP) development and good manufacturing practice (GMP). Founded in 2000, Symphogen focuses on cell line development and manufacturing of recombinant antibodies including polyclonal antibodies, a new class of therapeutics that Symphogen has brought into clinical development. Their therapeutic areas of interest are cancer, infectious diseases and immunoglobulin replacement therapy because there are many well-understood and novel targets for which new monoclonal antibody (mAb) products can be developed.Central to Dr. Tolstup''s presentation was a critical analysis of their experiences in contract manufacturing organization (CMO) outsourcing of three recombinant antibodies developed in-house. Symphogen''s first outsourcing experience came in 2004, which was fairly early in the life of the company, with the development of Sym001. Sym001 presented significant challenges for outsourcing because it was a new type of polyclonal antibody product comprising 25 recombinant antibodies. The challenge presented by the complexity of Sym001 was further complicated by the limited in-house experience in CMC development and a lack of equipment for USP development at Symphogen. For their USP development strategy, they wanted to employ a single batch manufacturing process that was novel at the time. Starting with 25 individual cell lines under GMP conditions, they expanded the cell lines, mixed them together and generated a polyclonal master cell bank. This was further expanded to give the polyclonal working cell bank that could then be used in USP manufacturing similar to those used for mAb production. To cope with the challenges faced with outsourcing such a novel production method, Symphogen formed an in-house CMC and regulatory team, sought advice from several experienced consultants and conducted extensive research into CMOs and regulatory agencies. After evaluation of several European CMOs, they formed a collaboration with Biovitrum (Stockholm, Sweden). The key criterion for this selection was a sense of strong commitment from the Biovitrum business development team to understand and accommodate customer demands during negotiation of technical work. Dr. Tolstup detailed the working infrastructure of the collaboration, stressing the importance of regular face-to-face meetings between the project teams, and the necessity for a joint steering team to mediate disagreements, thus keeping disputes out of the scientific groups. The collaboration was successful, and resulted in development of a consistent manufacturing process, and release and characterization tools to assess polyclonality. In 2007, Sym001 entered clinical trials and it is currently in Phase 2.In the case of Sym002, a smallpox drug comprising 26 antibodies that is being developed with funding from the United States (US) government, Dr. Tolstup discussed the difficulties that can be encountered when working with CMOs. She highlighted lack of face-to-face meetings, staff inexperience and inexperienced project management teams as less than ideal foundations for a mutually beneficial working relationship. Despite issues surrounding the collaboration, it resulted in two successful batch scale-ups of Sym002.The third CMO experience at Symphogen came in the manufacturing of Sym004, which comprises two chimeric IgG1 antibodies that target epidermal growth factor receptor domain III at uniquely positioned, non-overlapping epitopes. The manufacturing strategy for Sym004 included separate USP and down-stream process (DSP), preparation of drug substance for the two mAbs comprising Sym004, followed by formulation of the mAbs into one drug product by a 1:1 mix. They chose CMC biologics (Copenhagen, Denmark) to collaborate with because their technical equipment fit well with in-house technology at Symphogen. The scientific teams worked well together, leading to successful technology transfer and a robust and scalable USP, and Phase 1 clinical studies are currently underway in the US. Dr. Tolstup concluded by summarizing the general learning points from CMO outsourcing at Symphogen by stating that good communication, flexibility and open-mindedness are key for successful CMO collaborations.Dr. Andreas Castan showcased the implementation of quality by design (QbD) in process development at Biovitrum. Although not an entirely new concept, QbD aims to design and build quality into product and manufacturing processes, instead of simply testing for quality after production. Regulatory agencies require monitoring and process robustness in order to reduce variability of manufacturing, and this is achieved at Biovitrum by the integration of process analytical technology (PAT) into QbD. PAT is a system for designing, analyzing and controlling manufacturing through measurements of critical quality and performance attributes, with the goal of ensuring final product quality. Dr. Castan stressed how incorporation of PAT in QbD can result in reduced variation and increased process understanding, and greatly facilitates technology transfer and scale-up. Linking performance of the manufacturing process to safety and efficacy of the product was discussed, with an emphasis on the need to define the critical quality attributes needed for assessment of the impact that the manufacturing process can have on product safety and efficacy. Dr. Castan indicated that quality attributes are usually assessed by conducting release tests used to assess a small subset of product characteristics, followed by more extensive characterization. By adopting a QbD approach the focus is much more on the process that allows broader assessment of the quality attributes, rather than on analyzing a small representative sample post-manufacture. Defining the relevant critical quality attributes of a product, however, can prove challenging, and Dr. Castan discussed the need for continual reassessment of quality attributes based upon structure-function relationships identified from biological characterization studies. Additionally, he pointed out the requirement for not just one, but a combination of different quality attributes.In a detailed case study, the process whereby Biovitrum defined the design space for one of their projects was explored. The USP comprised a mammalian cell culture fed-batch process, and the DSP consisted of three chromatographic steps, one virus reduction step and one ultrafiltration/diafiltration step, followed by an extensive analytical package to assess the critical quality attributes. Initial risk assessment starts with the manufacturing process and evaluates the operating parameters for each step. Each step is then rated and assigned a risk priority number and further scientific discussion determines which parameters are most likely to have an impact on the quality attributes, and therefore require further characterization. From the process risk assessment, Biovitrum then selected the best design for the project and established that a large number of analytical methods were required.At this point, Dr. Castan touched on experimental design, specifically the difference between running one factor at a time versus a sum of experiments design, which offers a more comprehensive and structured approach to exploration of design space. The characterization of the USP bioreactor step and the DSP capture step using the more structured design of experiments approach was discussed. From the failure mode and effects analysis, operational parameters were identified and augmented with axial points and repeats resulting in a total number of experiments required. The experimental results were then used to assess the reproducibility of the center points of one parameter, which then generated a reliable model necessary for plotting contour charts. With the design space established, it was then possible to identify the desirable product operating ranges.Some of the unique challenges and strategies for outsourcing biologics were evaluated by Dr. Torben Lund-Hansen (Lund-Hansen Consulting ApS). The talk began with an overview of how biologics differ from small molecules specifically with respect to their size, complex and often relatively undefined mechanism of activity, and requirement for post-translational modification. In addition, the source material for biologics can potentially lead to the transmission of adventitious agents that can be hard to identify and may not be easily measurable. As a result, there is a need for substantial in-process control and validation. Dr. Lund-Hansen highlighted how the bio-friendly nature of the manufacturing process aids the susceptibility of the process to microbial contamination, but that it is simply not possible to employ aseptic processing throughout, especially in the drug substance purification.In discussing the pre-clinical attributes of biologics, the difficulties with establishing the pharmacokinetics (PK) of biologics such as antibodies were raised by Dr. Lund-Hansen. While it is possible to measure the antibody in humans or animals for three to four weeks, the biological function may last for many more months, suggesting that PK may not be the most suitable method for assessing biologics. Furthermore, all biologics have an immunogenic potential that is obviously desirable in vaccines, but is a serious unwanted effect that can occur in single and chronic dosing of therapeutics. For example, in some cases up to 20–30% of patients may have an immune response to a protein drug, and this response may in turn cause a substantial decrease in the drug''s efficacy. As a consequence, regulatory agencies have issued very strict guidelines for measuring immunogenicity, which should be of utmost consideration when developing biologics.Another challenging aspect of biologics development is that manufacturing facilities as well as the products must meet regulatory agencies'' specifications. Indeed, prior to obtaining a license the manufacturing facility must undergo inspection while being fully operational and manufacturing the complete product for which a biologics license is required. With this as context, Dr. Lund-Hansen discussed the challenges surrounding CMO selection for biologics. He advised choosing a strong and experienced CMO that can take the product all the way to market so that there is no need for technology transfers during the clinical development process. Furthermore, he stressed the need to work very closely with the CMO to understand the way the CMO works, and, crucially, not try to change their methods. The expectations from a customer point of view were discussed with respect to who is ultimately responsible for ensuring that CMC standards are met. Sometimes CMOs do not take responsibility for CMC and it can often be a sticking point in negotiations between customers and CMOs because of the large degree of risk involved.In assessing the interconnecting relationships between CMOs, sponsors and regulatory agencies, Dr. Lund-Hansen pointed out that quite often the sponsors are relatively inexperienced compared with regulatory agencies and CMOs that have worked with multiple sponsors. He called upon the CMO to educate without intimidating the customer, to be transparent in order to generate trust, and to communicate clearly and precisely the CMO strategy and cost. Additionally, the need to offer the customer a choice was raised with respect to the degree of flexibility within the working contract, and he suggested that a stretched time-line should result in a lower price to the customer.The presentation concluded with an evaluation of the huge amount of risk involved in the development of biotechnology products. Two types of risk were identified: the project risk, which should be carried by the customer and the technical risk, which should be shared between the customer and the CMO because the CMO should be in a better position to mitigate technical risk. Despite this, most CMOs prefer not to assume the technical risk, and this can lead to problems in contract negotiation.The next session featured short presentations from three CROs highlighting the technological solutions that they could provide to various CMC functions. Dr. Michael Becker summarized the technologies that Solvias offers in biopharmaceutical analysis. Solvias focuses on ensuring that the analytical technology that they develop for the customer meets deadlines and milestones to enable efficient product development. They provide analytical support from characterization of proteins through quality control methods to support stability studies, comparability studies and method transfer to CMO sites, focusing mainly on drug substance and drug development. Solvias adopted a fit for purpose approach that allows for innovation to meet customer demands, and they also seek to standardize wherever possible. The key technologies they offer are analysis by fragmented antibody capillary electrophoresis, peptide mapping and routine methods for amino acid analysis, and DNA sequencing.Eva Balslev Jørgensen described the biomanufacturing solutions offered by Novozymes BioPharma. In 2007, Novozymes BioPharma created BioBusiness as a division of Novozymes to strengthen the Novozymes brand position outside enzymes and offer value for customers interested in products with non-enzyme activities. They structure their services into three areas: manufacturing technologies, biomedical applications and manufacturing supplements. In manufacturing technologies, they offer a novel yeast expression system and an albumin infusion technology that can be used as a stabilizer for protein-based drugs. For biomedical applications, they developed the Recombumin excipient for vaccine formulation, albucult^®, which is a stabilizing agent and Hyacare^®, which is used for co-formulation in drug delivery. With respect to manufacturing supplements, they offer cell culture ingredients such as recombinant transferrin and recombinant human growth factors.The final CRO presentation was delivered by Dr. Roman Hlodan (Patheon) who discussed points to consider when developing biopharmaceuticals. Dr. Hlodan began with a look at factors affecting the development pathway by discussing business models, resources, drug delivery and expectations of investors. Regarding formulation choices, Dr. Hlodan suggested that perhaps refrigerated or ambient stored solutions offer the best option as they are cheap, easy to transport and carry the potential to be smoothly transferred to a more desirable delivery such as pre-filled syringe. If this type of formulation is not viable for the drug product, frozen solutions, which can minimize risk to product stability or lyophiles, which offer convenient transportation and storage, can be considered. To conclude his talk, Dr. Hlodan discussed some of the challenges that can arise when manufacturing, including drug substance availability, technology transfer and analytical considerations.The afternoon session began with discussion of approaches to biopharmaceutical formulation and development. Dr. Niels Johansen (ALK) noted that the company focuses on development of biopharmaceutical products for the diagnosis, treatment and prevention of allergies. Allergies are an immunological overreaction to allergens that exist in substances such as pollen, animal hair or food, and the prevalence of allergic diseases is increasing. Most treatments involve simple avoidance and symptomatic medicine, i.e., drugs that control symptoms but not the allergy. Allergy vaccinations, which involve injections of controlled doses of purified and standardized allergens extracted from natural allergen sources, are currently the only treatment that can change the course of the disease. Over time, the injections lead to desensitization of the immune system towards the allergen.Traditionally, allergy vaccinations are administered via subcutaneous injections over a period of three years and are used in the treatment of pollen, grass, dust, animal and bee/wasp venom allergies. In 2005 an alternative vaccination method, sublingual immunotherapy (SLIT), was developed. SLIT provides the potential for a more patient-friendly treatment and ALK strives to deliver an allergy vaccine that can provide fast drug release, solid dosage form for sublingual administration and good shelf life.In discussing drug product formulation, Dr. Johansen examined the requirements for compatibility between the complex mixture of proteins that make up the drug substance and the desired patient-friendly sublingual administration route. He also emphasized the importance of having a carefully designed manufacturing process because protein allergens are highly sensitive to heat and mechanical stress during processing. The question of carrying out in-house drug formulation and delivery development versus outsourcing to a CMO was then raised, and the conclusion was that the only viable option available to ALK as a small company was outsourcing to a CMO. Regarding CMO selection, Dr. Johansen emphasized examination of the reputation, available technology, and the size of the CMO with respect to it being able to take the product through development to market, when choosing a CMO. ALK formed a collaboration with Catalent, UK, the developer of Zydis^®, which is a freeze-dried oral solid dosage formulation technology that allows instant dissolution in the mouth and results in immediate release of the drug. Furthermore, the manufacturing processes of Zydis^® were ideal for formulation of proteins, which are generally stable in freeze-dried formulation. In 2006, ALK launched Grazax^®, which is a fast dissolving oral tablet developed using Zydis^® technology for the treatment of grass pollen-induced rhinitis and conjunctivitis.The strategies for creating outsourcing partnerships at Genmab were discussed by Dr Jesper Valbjørn. Genmab develops fully human antibodies for the treatment of cancers, particularly those for which there is an unmet medical need. Currently, the company has 9 Phase 3 studies of ofatumumab (Arzerra) and 2 Phase 3 studies of zalutumumab on-going, and 3 additional antibodies (daratumumab, RG4930, RG1512) in clinical development. In 2009 their first product, ofatumumab, was approved in the US for the treatment of chronic lymphocytic leukemia. Dr. Valbjørn began by examining the process for selecting a CMO. He noted that selection should be based upon the strategy and needs of the project, and he made clear distinctions between a strategic selection process, which is usually a long term, capacity-driven plan adopted by global companies and a tactical process, which is usually project-driven and includes a limited budget. Additionally, he emphasized that the need to plan for the lifetime of a project before seeking a CMO is key for the success of the project. In a more detailed breakdown of the selection process, Dr. Valbjørn described a method whereby a number of selection criteria are established, weighted and then used to screen potential CMOs.Contracting and project definition were touched upon briefly within the context of the overall process that starts with proposal requests, and includes technical and QA site visits with a review of findings and the forging of technical and quality agreements. Dr. Valbjørn stressed the importance of taking adequate time for this process because getting the agreement right from the start results in a win/win for both sides. Once the contract is in place, the next step is to effectively manage interactions with the CMO. At Genmab, this is done by setting well-defined expectations, goals and responsibilities and having alignment of teams across the company and the CMO. Dr. Valbjørn cited communication, including regular face-to-face meetings, as being important to the success of the project. Disputes are dealt with by a contract manger to keep disagreements out of the technical teams, and joint steering meetings occur every six to twelve months to evaluate performance and align expectations.With regard to technology transfer from the company to the CMO, Dr. Valbjørn stated that transparency with transfer development documentation is necessary in order to prevent problems arising at a later date. He also suggested running the process in the CMO''s development laboratory and training the CMO technicians in specialized analytical methods at your own site. The need to be data-driven and not to make assumptions about the knowledge of the CMO was also stressed. Dr. Valbjørn concluded the presentation with an analysis of the manufacturing process with respect to the engineering run and the GMP run. Engineering runs help to test the scale and performance of the process and minimize risk for the GMP run. Many CMOs are reluctant to carry out a GMP run without an initial engineering run; if the engineering run is eliminated, then there is a requirement to know the process and product well, and to communicate your priorities and risk profile/tolerance to the CMO upfront.Dr. Per Edebrink (RecipharmCobra Biologics) discussed finding the optimal characterization strategy for clinical study material. He began by giving a summary of critical quality attributes, looking at what processes can influence them and stressed the need to define them early in order to determine what characterization is required and aid design of the analytical test package. Although, it may not always be possible to know the critical quality attributes early in the pharmaceutical development, it is possible to predict what may be required from the International Conference on Harmonisation (ICH) guidelines for biopharmaceuticals. In particular, Dr. Edebrink highlighted Q6B, which outlines what structural characterization and physicochemical properties are needed. Regarding potential critical quality attributes of mAbs, Dr. Edebrink mentioned a number of different product variations, including Fc glycosylation, fragmentation in the hinge region, disulfide shuffling and lysine truncation. It is possible to identify some of these variations using intact mass protein analysis, and some example spectra were provided, including a reversed-phase high-performance liquid chromatography-ultraviolet (RP-HPLC-UV) mass spectrometry (MS) spectrum of a mAb showing the free light chain, hinge region fragments and the intact IgG.On the topic of glycoproteins, Dr. Edebrink discussed the effect that glycosylation may have on functional activity. Indeed, glycosylation can affect the biological properties of the glycoprotein, resulting in possible immunogenetic effects, and it can also reduce both the in vivo half-life and the shelf life of the glycoprotein. However, there are many ways to measure the extent of glycosylation of a glycoprotein and Dr. Edebrink showed how electrospray mass spectrometry, high performance anion exchange chromatography and matrix-assisted laser desorption/ionization MS can be used to assess glycoprotein glycosylation. It is also possible to test for sialylation, which can affect the half-life or activity of some biologics. LC-MS can be used to analyze the degree of sialylation and for peptide mapping. The final potential critical quality attribute (CQA) examined was deamidation, which can adversely affect activity. Deamidation is the degradation of asparagine and aspartate that proceeds via a succinamide intermediate. It is possible to test for deamidation using a number of different techniques and examples of using isoelectric focusing and peptide mapping coupled to LC-MS were shown. To conclude, Dr. Edebrink reiterated the need to identify potential CQAs as early as possible using the available knowledge, and assess the potential effect on the product. Furthermore, he highlighted peptide mapping by LC-MS as a powerful tool to provide detailed information on the presence and distribution of product variants.To conclude a highly informative and productive meeting, Dr. Jesper-Sonne Johansen (Novo Nordisk) presented a case study on world-class CMC development processes in mammalian, yeast and bacteria cell lines. In 2006, Novo Nordisk initiated a program to improve their CMC processes and set the ambitious goal of doubling the number of new projects in development within five years, which necessitated a doubling of capacity. They began by mapping all the CMC development processes, such as fermentation and analytical processes, and examined the number of people, time and resources allocated to each process, and assessed if there was room for improvement. They also examined how other companies carried out their CMC development and investigated opportunities for improvement and standardization of technologies going into the CMC process.The company then made two major modifications to their processes. First, they changed from continuous CMC development to a two-step development process in which the first step is to develop suitable processes for production of Phase 1 and 2 drug substance, focusing on delivering a high-quality product through a process that might not be optimized. The second step involves development of a robust manufacturing process and enhanced product knowledge. When implementing this new two-step development process, the decision was made to maintain resources present in Novo Nordisk and not to increase outsourcing.The second change that was implemented was the ‘five initiatives’ process. When examining project transfer from research to development and from development to production, they observed that a substantial amount of knowledge was lost. In order to combat this problem, they established technology transfer teams at each handover one year before the transition would occur to ensure efficient transitions. In addition, they set up technology groups that examined the technology used across research, development and production and, where possible, introduced standardized techniques.Regarding the qualitative outcome of these changes, for the two-step development process the result was a short and focused development time with clear goals and agreements on tasks. In the handover process, the transfer teams co-operated successfully and created knowledge across the chain from research to development to production. For the technology standardization, they developed standard guidelines for purifications and analytical methods. The same technology is now used in many projects, which has resulted in predictable outcomes of processes, and has greatly increased process knowledge. Remarkably, when Novo Nordisk reviewed the effect of implementing these approaches in standard development projects over two years, they found that overall resource use was half the previous level. Overall, the overhaul of CMC development at Novo Nordisk resulted in a two-fold increase in productivity between 2006 and 2008, and they were able to successfully double the number of development projects without increased use of resources.