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Coronavirus in the crosshairs, Part 7: Diagnostic tests

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Post written by Simon L. Goodman, D.Phil.

With no foreseeable safe or effective therapy or vaccine against SARS-CoV-2, extensive population testing and quarantine are amongst the few scientifically rational means of protecting people and monitoring the pandemic.  To date (April 26, 2020) only 26.6  million tests for infection have been performed worldwide, with 3 million positives reported (1) by 129  of 195 countries.  Cases of infection were confirmed predominantly via nucleic acid amplification tests for viral RNA. Current antibody-based serological tests are qualitative, and poorly validated.  But there is little doubt that during epidemic and endemic infection, testing and obligate retesting will likely involve many 100s of millions of samples.

There are currently no validated US Food and Drug Administration (FDA)-approved diagnostic assays for SARS-CoV-2 or anti-SARS-CoV-2 antibodies in patient samples, but tests can receive  emergency use authorization (EUA) (2).  The lack of certification means that data from these tests is likely to be of lower quality than approved diagnostics, from the point of view of clinical specificity and selectivity.  However, in third-world countries, rapid point-of-care tests must become available as soon as possible.  To add to the difficulties that governments, the public, and healthcare providers face, there is an amoral escalating grey-market in dysfunctional serological home-test-kits (3).

Classes of virus tests

The coronavirus SARS-CoV-2 triggers a lethal and highly infectious disease, COVID-19, so rapid (minutes-to-hour) point-of-care tests are essential to reduce spread and establish the epidemiology of infection.  Classical laboratory culture methods, in any event necessitating rare Biosafety Level-3 facilities, are excluded. The level of anti-SARS-CoV-2 neutralizing antibodies needed for protective immunity, and the degree and duration of protective immunity developed, if any, remain unknown.  And there are no well-validated immunohistochemistry-capable anti-virus antibodies, so interstitial tissue distribution and loading remain unknown.

COVID-19 tests currently come in three classes:

1) Viral RNA amplification tests (Nucleic Acid Amplification Tests; NAAT);
2) Viral antigen tests, which are based on affinity detection methods (VADM); and
3) Serological tests, which detect any SARS-CoV-2-reactive antibodies.

Nasal, laryngeal or sputum swabs are currently accepted for tests 1) and 2), while pin-prick or venous blood is used for test 3). Swabbing is uncomfortable and exposes the sampler to virus, so a preprint suggesting detection in saliva of high viral titers by NAAT is of interest (4).

Tests 1) and 2) identify those who carry virus and may be infectious, even if asymptomatic.  Test 3) identifies people whose immune systems have responded to SARS-CoV-2 exposure, including those who may have active viremia. Only NAATs and a single VADM are quantitative at present.

A 4th class of test is currently unavailable: a commercial test that accurately detects neutralizing anti-SARS-CoV-2 antibodies, would be valuable because it could identify those likely to mount a strong response to a repeated infection.

Only a combination of such tests, including a serological test in particular, will identify the prevalence and exposure of populations to the virus.  However, as described below, current serological tests fall far below diagnostic standards (e.g., > 98% clinical selectivity; > 98% clinical specificity).  At present, only predominantly qualitative VADM and serological tests are available.  Infectivity is related to the amount of virus emitted by infected people, so routine quantitative tests might allow an accurate assessment of the progression of the pandemic.

Viral RNA detection

Automated commercial PCR and quantitative (Q-PCR) systems can rapidly detect and quantify (Q-PCR) SARS-CoV-2 RNA.  However, the high-throughput machinery and expertise needed to perform the test is generally found only in a sophisticated centralized laboratory environment.  Q-PCR laboratory-use only kits from 41 companies have received FDA EUAs, including those from:

Use of central laboratory facilities leads to slow readouts (1000-20000 samples per day). In the US, 21 laboratories have independently developed SARS-CoV-2 tests that are permitted for EUA usage as high-complexity molecular-based laboratory developed tests.

An exciting development is the use of isothermal amplification (IA) methods, which need less complex machinery and can be very fast (readout in < 15 mins). On March 27, 2020, the FDA issued an EUA for an IA system, ID NOW COVID-19 (Abbott laboratories), for rapid quantitative point-of-care use in SARS-CoV-2 detection.  The system has fast sample analysis time (15 min), but low throughput.

On April 20, 2020, Baek et al. (5) reported a 15 minute, quantitative, one-tube, isothermal reverse transcription-loop-mediated isothermal amplification technique (Q-RT-LAMP) with a visual read out of phenol-red color change, possibly adaptable to microtiter plates.  This could prove valuable in those many COVID-19-plagued countries lacking ready access to Q-RT-PCR technology.

Although NAATs are state-of-the-art diagnostics for SARS-CoV-2 infection, it must be noted that “…a significant portion of patients who otherwise fit the diagnosis based on clinical and chest CT findings, including many hospitalized patients, have tested negative for viral RNA.” (italics added) (6).  This may include victims whose NAAT sinks below the limit of detection of Q-PCR after day 5 post infection (7).

Viral antigen detection

A number of companies provide point-of-care tests for viral antigen. These tests rely on SARS-CoV-2 antigen in patient samples being trapped  by antigen-targeting antibodies on a detectable mobile phase. For a few examples:

  • SD Biosensor’s lateral flow system has a well-documented limit of detection of ~2000 tissue culture infective dose / ml with 0/170 false positives specificity, but only 84% sensitivity over PCR (8) (i.e., resulting in 16% false negatives).
  • Coris BioConcept’s acute-phase screening kit uses immunochromatographic colloidal gold-based dip stick technology (analogous to capture ELISA). With a sensitivity of 5000 pfu /ml, the kit has a high positive predictive value (78-100%), but sensitivity over PCR of < 86%.  Both kits are reported to identify both SAR-CoV-2 and SAR-COV (vs other respiratory viruses and bacteria), and target conserved coronavirus nucleoprotein (9, 10).
  • Bioeasy uses time-resolved fluorescence analysis in a similar assay system, but the sensitivity and selectivity data are not reported (11).  Early phase infection was undetectable in a small sample set of urine and blood by VADM, while it could be identified in respiratory test probes (7).

The Foundation for Innovative New Diagnostics (FIND), a non-government organization, has identified some 10 (poorly validated) antigen tests marketed under the European CE label, and many tests of very dubious provenance are available on the internet.

Detecting anti-viral serological responses

Serological tests rely on detecting blood IgM and IgG antibodies, or mucus IgA and IgG, reacting with SAR-CoV-2.  The FDA has noted that owing to the urgency of the situation, they “… had not reviewed or authorized (or “approved”) … (these tests)… at least not initially, and … (they)… should not be used for diagnosing or excluding active SARS-CoV-2 infection.” (italics added).

To date (April 28, 2020), 7 serological tests from 6 companies have received EUA. These are based on:

Many more tests have been developed and marketed in Europe, including:

  • Immunochromatographic (Coris);
  • Lateral flow (Bioeasy; ElabScience; HighPlusTech; Senova; SD sensor; Premier/Hangzou); and
  • Classical plate-ELISA based for detection of anti-Covid-19 IgA and IgG (EuroImmun) (12).

FIND has identified over 60 CE labelled antibody tests (13).  Each provider uses immobilized recombinant SARS-CoV-2 proteins to capture antibodies from blood, which are in turn captured on, or recognized by, “anti-human-IgG” and or “anti-IgM” reagents.  As yet little detail of the validation of the reagents used in the tests has been published.  Whether any tests will ever become FDA-approved diagnostics is still an open question. The sensitivity of these tests is reportedly 1-2 logs below that of viral NAAT, and only the EuroImmun test is semi-quantitative.  For many rapid tests, the specificity and sensitivity has been challenged due to the low sample numbers, biased sampling and questionable reagents (14).

As yet only DiaSorin has claimed that their laboratory-based serological assay, detecting both S1 and S2 spike proteins of SARS-CoV-2, can preferentially detect neutralizing antibodies (15).   If their claim and methodology can be independently confirmed, the use of such an antibody combination in a rapid test kit may be a valuable step towards disease control.

Though many companies are now offering antibody tests, the World Health Organization has yet to recognize a single appropriate serological SARS-CoV-2 assay, none have been validated sufficiently for clinical approval by the FDA, and they remain as laboratory tests.  The quality of the antibodies used is currently unknown, and little data has been published on the conditions used to verify their specificity and selectivity.  As we saw in our recent webcast series, such a lack of validation can cause major problems when antibodies are used as a basis to make life and death decisions. In the case of SARS-CoV-2, properly validated tests are urgently needed.

Deciphering test results

There are 4 clear stages in the progression of SARS-CoV-2 infection, which should have distinct test profiles useful for making decisions on disease spread.

1) The newly infected carry replicating virus, detectable with NAAT.
2) The infected mount a delayed adaptive immune response producing anti-viral IgM 5-10 days after infection.
3) An IgG response begins some 14 days after infection.  This response may or may not produce neutralizing antibodies.
4) “Recovered” patients have a low viral titer, and so are NAAT-negative, and may or may not have detectable, and possibly neutralizing anti-SARS-CoV-2 IgG. 

Thus, people at stage 1 express only viral RNA, while those at stage 2 express viral RNA and anti-SARS-CoV-2 IgM and, later, IgA. At stage 3, the infected may not express viral RNA and may have SARS-CoV-2 IgG, while those at stage 4 may continue to express anti-SARS-CoV-2 IgG.  The duration, strength, and possible neutralizing nature of the primary and any secondary antibody response is currently unknown.

The COVID-19 pandemic has triggered a rapid effective scientific response.  Sadly, this has in general been obscured.  The demand for “tests” has driven a gold-rush – resulting in a morass of point-of-care lateral-flow kits. These frequently use unvalidated polyclonal antibodies. Given the volume of tests to be performed, this will drastically lower the quality of the resulting data.  The Antibody Society welcomes recent ongoing efforts to better define the performance of such kits (16), and we hope for more efforts along these lines in the coming weeks.

References

1.           Foundation for Innovative New Diagnostics. FIND Covid-19 tests. Geneva: FIND; 2020. Available from: https://www.finddx.org/covid-19/test-tracker/.
2.           US Food and Drug Administration. Policy for Diagnostic Tests for Coronavirus Disease-2019 during the Public Health Emergency Washington D.C.: FDA; 2020. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/policy-diagnostic-tests-coronavirus-disease-2019-during-public-health-emergency.
3.           Hahn SM, McMeekin JA. Coronavirus (COVID-19) Update: FDA Alerts Consumers About Unauthorized Fraudulent COVID-19 Test Kits. March 20, 2020. Available from: https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-alerts-consumers-about-unauthorized-fraudulent-covid-19-test-kits
4.           Wyllie AL, Fournier J, Casanovas-Massana A, Campbell M, Tokuyama M, Vijayakumar P, et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. medRxiv. 2020:2020.04.16.20067835.
5.           Baek YH, Um J, Antigua KJC, Park JH, Kim Y, Oh S, et al. Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2. Emerg Microbes Infect. 2020:1-31.
6.           Xiao SY, Wu Y, Liu H. Evolving status of the 2019 novel coronavirus infection: Proposal of conventional serologic assays for disease diagnosis and infection monitoring. J Med Virol. 2020;92(5):464-7. Available from: https://onlinelibrary.wiley.com/doi/full/10.1002/jmv.25702
7.           Woelfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Mueller MA, et al. Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster. Available from: medRxiv. 2020:2020.03.05.20030502.
8.           SD Biosensor. STANDARD Q COVID-19 Ag 2020. Available from: http://www.sdbiosensor.com/xe/product/7672.
9.           BioConcept C. COVID-19 Ag Respi-Strip [Product insert]. B – 5032 Gembloux, Belgium: Coris BioConcepts; 2020 [updated 8th April 2020]. Available from: https://www.corisbio.com/Products/Human-Field/Covid-19.php.
10.         Mousavizadeh L, Ghasemi S. Genotype and phenotype of COVID-19: Their roles in pathogenesis. J Microbiol Immunol Infect. 2020. Available from: https://doi.org/10.1016/j.jmii.2020.03.022
11.         Bioeasy. 2019-nCovIgG/IgM GICA rapid test kit 2020. Available from: http://en.bioeasy.com.tr/bioeasy-novel-coronavirus-2019-ncov-test-kits/.
12.         EuroImmun. SARS-CoV-2 test systems from EUROIMMUN [Supplier web site]. Lubeck: EuroImmun; 2020. Available from: https://www.coronavirus-diagnostics.com/.
13.         European Centre for Disease Prevention and Control.  An overview of the rapid test situation for COVID-19 diagnosis in the EU/EEA Stockholm: ECDC; 2020. Available at: https://www.ecdc.europa.eu/sites/default/files/documents/Overview-rapid-test-situation-for-COVID-19-diagnosis-EU-EEA.pdf
14.         Vogel G. First antibody surveys draw fire for quality, bias. Science. 2020;368(6489):350-1. Available at: https://science.sciencemag.org/content/368/6489/350.
15.         Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. 2020;181(2):281-92 e6. Available at: https://www.sciencedirect.com/science/article/pii/S0092867420302622.
16.         Whitman JD, Hyatt J, Mowery CT, Shy BS. Test performance evaluation of SARS-CoV-2 serological assays [Assay analysis Lateral flow tests]. UCSF Harvard consortium: University of California, San Francisco; 2020. Available from: https://covidtestingproject.org/.

James S. Huston – In Memoriam

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Post written by: Richard Begent, Ph.D., Emeritus Professor of Oncology, University College London

James S. Huston Jr, Ph.D., antibody engineer and founding President of The Antibody Society, died in Boston on March 25, 2020.

Jim Huston was a distinguished biophysicist and a pioneering antibody engineer; his creation of the single chain Fv (scFv) antibody was a seminal advance. These genetically encoded molecules could express the vast diversity of antibody repertoires, and could be used for specific target binding by themselves, incorporated into multifunctional molecules, attached to cell surfaces or applied in any number of formats relevant to biomedicine.

Antibodies with their multiple functions, including the capacity for specific binding to a range of targets, became practical pharmaceuticals with the advent of monoclonal antibodies as described by Köhler and Milstein in 1975. Genetic manipulation humanized the constant regions, making repeated administration feasible with widespread benefits for human health.

It was evident that the smallest target recognition moiety of antibodies, the variable region (Fv), if produced separately could be linked to many agents, conferring exquisite binding specificity. Since the VH and VL domains are separate in the native form of the Fv, they needed to be joined in a way that retained stability, the binding performance of the two components together, and appropriate flexibility.

Jim proposed doing this with one genetic construct that encoded a single-chain Fv (scFv) in which the VH and VL were joined by a flexible linker. The design issues were complex, but, consummate biophysicist that he was, he translated the requirements into a successful design for the linker. Working with colleagues at Creative Biomolecules, Massachusetts General Hospital and Harvard Medical School, an sFv reactive with digoxin was successfully produced and tested. The report (Huston et al 1988) of this work has been cited more than 2,300 times.

scFvs are readily expressed on the surface of filamentous bacteriophage and have often been the basis for naïve human antibody libraries with potential for rapid selection of desired binders from diverse libraries of many millions. This technology can be used for antibody discovery and humanization, and it has been the foundation of many successful commercial ventures. Jim’s own work included the demonstration of scFv fusion proteins and the first scFv intrabody therapy for the neurodegenerative condition, Huntington’s disease, an approach that is now being investigated in Parkinson’s disease.

The scFv format itself forms the targeting basis of T-cell recruiting agents, bispecific T-cell engagers (BiTEs), and chimeric antigen receptors (CARs), the antigen binding moiety of CAR-T cells. A number of products based on these formats are already licensed for clinical use, while several others are in development. This is an important beginning, but the potential for further applications is great because of the diversity of antibody repertoires and the robust nature of the sFv format.

Jim graduated in Chemistry from the University of Michigan and was awarded his Ph.D. for work on the Fd fragment of IgG and its domains, supervised by Professor Charles Tanford at Duke University. After postdoctoral research at Stanford and Harvard Medical Schools, he joined Creative Biomolecules in Boston in 1983 where he undertook the original work on scFvs. There followed numerous publications and patents relating to engineered antibodies and their applications.

Jim was one of the first people to see the long-term potential of antibody engineering and recognize how broad the applications could be. His lectures on this topic gave a unique experience in that one sometimes seemed to be discovering the meaning of his data simultaneously with him. Those who had the privilege of working with him benefited greatly not only from his generosity, enthusiasm, intellectual rigor and encouragement, but also from his ability to advise wisely or find the appropriate expert. He took the mission of advancing antibody engineering to an international level by serving as the scientific adviser to the Antibody Engineering & Therapeutics meetings for nearly 30 years. Over time, he brought the antibody engineering and therapeutics community together at meetings in San Diego and elsewhere. His insistence that scientific quality and education were the principal criteria for the program resulted in progressive growth and helped to cement a culture encompassing academia and industry. Building on this, he co-founded The Antibody Society in 2007 and was the Founding President and Chairman, remaining a Board Member until his death. He shared the gratification of many that, after a long gestation, antibody engineering is proving so beneficial to human health, with the promise of much more to come.

Jim’s many friends around the world will remember his love of life based on a deep Episcopalian faith, his pride and joy in his family, and the fortitude with which he bore illness over recent years.

The Antibody Society will honor Jim Huston and his many contributions to the field of antibody engineering at our next annual meeting.

Cyrus Chothia – In Memoriam

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Post contributed by: Prof. Andrew C.R. Martin, Professor of Bioinformatics and Computational Biology; SMB Graduate Tutor
Institute of Structural and Molecular Biology, University College London

Cyrus Chothia, FRS, (19 February 1942 – 26 November 2019), was an emeritus scientist at the Medical Research Council Laboratory of Molecular Biology (LMB) in Cambridge, UK where he was a fellow of Wolfson College. He studied at Durham University, followed by an MSc at Birkbeck College, University of London, a small college but famed for its involvement in the development of structural biology and x-ray crystallography, being the home of such luminaries as J.D. Bernal, Aaron Klug and Rosalind Franklin. This was followed by a PhD at University College London supervised by Peter Pauling, son of Linus Pauling. Cyrus was one of the founding fathers of structural bioinformatics and made a particular contribution in the antibody field. Amongst others, he worked with Nobel prize winner, Michael Levitt, Joel Janin, Alexey Murzin, Tim Hubbard and Anna Tramontano, but he is perhaps best known for his work with Arthur Lesk. His PhD students included a number of people who have gone on to make major contributions in bioinformatics, such as Alex Bateman, Steve Brenner, Mark Gerstein, Julian Gough, Sarah Teichmann, and Bissan Al-Lazikani.

Back in the early 1970s, Wu and Kabat had demonstrated the presence of hypervariable sequence regions in antibody variable domains that they suggested would form structural loops or complementarity-determining regions (CDRs) that come together in 3D to form the binding site. This was confirmed when Poljak solved the first antibody crystal structure, and it was assumed that the CDRs would also be extremely variable in structure. I first met Cyrus in the late 1980s when I was doing my DPhil in Oxford on modelling antibody combining sites with Anthony Rees. By that time, around eight structures of antibodies, or Bence Jones light chain dimers, were available and Cyrus, together with Arthur Lesk, compared these. They found that, with the exception of the third CDR of the heavy chain (CDR-H3), the structures of the remaining CDRs were remarkably conserved. Further they proposed that the presence of certain ‘key residues’ – either within the CDRs, or packing against them – would define the conformation [PMID: 3090684, 3681981, 2687698]. To be frank, Tony and I didn’t really believe it. After all, there were potentially billions of antibody sequences and they had looked at fewer than 10. Cyrus and Arthur came to visit us in Oxford, and I remember sitting in The Eagle and Child with them discussing these ideas. Cyrus was always modest and completely accepted that they may be wrong, but of course they turned out to be largely correct. As more structures became available, the rules evolved with the importance of other positions being recognized [PMID: 2118959], but the principle was completely upheld. When we published a paper on key residues in 1986, I spoke to him about one of the outliers that appeared not to follow the rules. His view (which was almost certainly correct!) was that the crystal structure was wrong. More recent analysis by ourselves and others has suggested that the rules aren’t always as precise as might once have been thought and the requirements for framework mutations outside the key residues in order to achieve good binding in antibody humanization supports the view that the precise conformation is influenced by other residues and the detail of the environment around the CDRs.

Cyrus introduced a definition of the ‘structural loops’ in antibodies. These are frequently referred to as the ‘Chothia CDRs’, a term that he did not like as, in his view, the CDRs were the sequence-variable regions defined by Wu and Kabat, while his definition related to regions that were structurally variable and he would not presume to redefine what had been done by Wu and Kabat. In fact, his definitions changed over his various papers as more structural information became available. He also introduced the Chothia numbering scheme for antibodies, which was based on Kabat numbering but corrected the insertion sites in CDR-L1 and CDR-H1 to be structurally correct. Unfortunately in 1989, they made an error such that the insertions in CDR-L1 were placed after residue L31 rather than L30. As another example of his humility and modesty, I happened to referee a paper of theirs in 1997 and recognized this error. He guessed that I was the referee, contacted me, and immediately accepted the correction.

While I have focussed on his work on antibodies, he was widely known for his work in many areas of understanding protein structure. He was elected as a Fellow of the Royal Society (FRS) in 2000, for having “shown how the amino sequences of proteins determine their structure, function and evolution”. To name just a few of his contributions, he developed the SCOP classification of protein structure with Alexey Murzin [PMID: 7723011] and the SUPERFAMILY database with Julian Gough. He studied multi-domain proteins [PMID: 15093836], protein packing [PMID: 10388571] and was involved in functional annotation of more than 60,000 cDNAs from the mouse transcriptome [PMID: 12466851]. As well as his work on the conformation of the CDRs, he examined the packing of VH and VL domains in antibodies [PMID: 4093982] and examined the evolution of immunoglobulin domains in general [PMID: 7175935]. With Bissan Al-Lazikani, he published his final paper on canonical classes of antibody CDRs [PMID: 9367782], but then extended this into T-cell alpha-beta receptors [PMID: 10656805].

Cyrus made enormous contributions to our understanding of protein evolution in general as well as of the structure of antibodies. He will be hugely missed by the scientific community, and by me personally. His science and the many well-known scientists who did their PhDs with him or were influenced by him, are a huge and lasting legacy.

“Antibodies to Watch in 2020” at PEGS Europe

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Over the past decade, the ‘Antibodies to Watch’ article series has documented the results of the global biopharmaceutical industry’s efforts to bring innovative antibody therapeutics to patients in need. Dr. Janice Reichert, Executive Director of The Antibody Society, offered a preview of the 2020 version on Wednesday November 20, 2019 during the ‘Developing Successful Antibody Products’ session at PEGS Europe.

‘Antibodies to watch in 2020’ includes updates on recent and anticipated events relevant to antibody therapeutics in clinical development. Data for antibody therapeutics that were first approved in either the US or EU during 2019, as well as several products first approved in Russia or India, were provided. Antibody therapeutics undergoing regulatory review by the Food and Drug Administration or the European Medicines Agency as of November 2019 were also discussed. Brief summaries of antibody therapeutics in late-stage clinical study that may progress to regulatory review in late 2019 or 2020, based on public disclosures by the sponsoring companies, were included. In concluding, Dr. Reichert noted that the late-stage clinical pipeline is robust, and she anticipated that more antibody therapeutics will be in late-stage studies in 2020 than any year previously documented. Remarkably, compared to 2010, the number of antibody therapeutics currently in late-stage studies has nearly tripled (to 75 antibody therapeutics).

The ‘Antibodies to watch in 2020′ presentation can be downloaded here.

The Antibody Society was very pleased to see so many of our corporate sponsors in attendance at PEGS Europe!

Feeding drug development programs with sufficient antibody

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Author: Nick Hutchinson, Fujifilm Diosynth Biotechnologies

The demand for antibody and antibody-related therapeutics continues to increase. [1] The United States Food and Drug Administration has approved ~ 100 antibody therapeutics for a wide range of treatments. Nearly 600 antibody drugs are in clinical trials, [1] with ~75 of these in pivotal Phase 2 or Phase 3 studies.

Small or even virtual companies are developing many of these molecules. Technical teams working within these organizations must understand the activities needed to successfully commercialize the drugs. One critical activity is establishment of production strategies capable of supplying the material requirements of pre-clinical development, toxicology studies, clinical trials and then, if successful, market demand.

Patients cannot benefit from life-saving medicines if the drug’s launch is delayed due to lack of the material required for each phase of development. Furthermore, companies that miss clinical milestones suffer from delayed investments, thus reducing the opportunity to reach the clinic in a timely manner and capture market share, which lowers future revenues.

Many start-up biotech firms have a laser-like focus on the pre-clinical development of their antibody candidates, but sooner or later they must consider a manufacturing strategy that enables pre-clinical or clinical programs to stay on track.

Is manufacturability an obstacle to development?

One question drug developers should consider is the extent to which the manufacturability of the candidate is likely to be problematic and jeopardise material supply. Many of the standard, full-length antibodies have well-understood properties and are relatively easy to manufacture, allowing timely delivery to the clinic. However, there is an increasing number of modalities within this product class, [2] e.g., bispecifics, Fc-fusions and antigen-binding fragments, which may present additional production challenges. These can include challenges such as low expression from cell lines suitable for use in manufacturing, poor stability during purification processes or the need for non-standard analytical methods.

One company I spoke to, for example, knew that they needed to increase the productivity of their cell cultures from below 0.5 g/L to greater than 3 g/L in order for the product to be commercially viable. Another company developing a monoclonal antibody explained that they needed a titer of ~ 10 g/L to ensure production efficiency was sufficiently high to allow them to be price competitive. A third company found that the isoelectric point of their Fc-fusion molecule was relatively low and they needed a tailored purification process for their product.

Companies developing standard IgG1, IgG2 or IgG4 products can leverage manufacturing platforms. [3] These allow production of different monoclonal antibodies with the required quality specifications and at high productivity with little process development. They offer a significant time- and cost-saving over the alternative, i.e, developing new processes for each new candidate. Companies with a pipeline of products may choose to invest in their own manufacturing platform, but, for many early-stage biotech companies it makes little sense to spend investors’ cash on production assets when there is considerable uncertainty around the likely success of a program. For this reason, many will outsource process development and manufacturing to a contract development and manufacturing organization (CDMO), many of whom will have their own established platform processes.

Early material supplies of antibody candidates

Cell line development scientists can generate stable, clonal cell banks derived from a production-ready host cell line in as little as 10 weeks following transfection. Cell cultures with transfectant pools can produce tens to hundreds of grams of material in as little as eight weeks following transfection. Scientists developing antibody therapeutics can use this antibody for their pre-clinical activities and initial formulation development experiments. In our experience, even at the pre-clinical stage, the drug development process can consume substantial amounts of material. Accurately determining material requirements at this stage will help ensure sufficient antibody is available.

Preclinical material supply might be met with bench-scale bioreactors, but we have worked on programs where the material requirements were sufficiently large that a 200-L mammalian cell culture run was needed, even though the cell line gave a high titer. This clearly demonstrated the utility of having a platform process because no additional process development on either the bioreactor conditions or the purification steps was needed. Expert developers of cell lines know that their host cell line will grow to high cell density under their platform conditions, and will select clones that combine high productivity with the desired product quality profile using high-throughput screening technologies.

Process development scientists operating platform processes typically allocate time, which would previously have been dedicated to manufacturing development, to the refinement of operating parameters and studies of manufacturing robustness that increase the likelihood of that full-scale production lots will be successfully released.

Supplying Toxicology and Early Clinical Material

Pilot-scale batches allow companies to predict large-scale manufacturing performance and refine scale-dependant process parameters. Companies often use material from the pilot-scale batch for toxicology work, stability studies and for generating reference standard, against which the first batch for clinical use can be released. It generally takes 6 – 8 months to reach this stage from the start of cell line development, yielding hundreds of grams of antibody, if not more.

For many companies, the demand for clinical-grade drug, manufactured to current Good Manufacturing Practices (GMP), can be met using bioreactors no larger in volume than 2000-L. The initial batch can be released within 12-14 months from the start of cell line development. Each batch can supply between 1 to 10 kilograms of antibody.

Modern, high-throughput manufacturing facilities provide enormous amounts of capacity such that with a robust, high-titer cell line no further scale-up may be required and firms can commercialize their product within the same facility they used for clinical lots. Others elect to scale-up still further to large-scale stainless steel manufacturing facilities, especially if the market demand is high and the overall process productivity is modest. More recently, firms are considering going to market with manufacturing processes that utilize smaller bioreactors operated in a continuous, perfusion mode. We believe such processes can yield over 15 kg of antibody from a 500-L bioreactor over a 4-week period. Deciding which approach to adopt is never easy because of uncertainties around factors such as dose requirements, overall market demand and competitive pressures. Experienced CDMOs will support customers through this decision-making process and will be able to provide invaluable advice.

In conclusion, many small biotech companies with new antibody drug assets can mitigate risks to drug development and commercialization timelines by thoroughly understanding the material supply requirements for preclinical, toxicology and clinical studies. Once they know this, they can determine how the need can be met by manufacturing organizations during process development and GMP production operations as part as an over-arching strategy for product commercialization.

[1] Kaplon H, Reichert JM. Antibodies to watch in 2019. MAbs. 2019;11(2):219-238. doi: 10.1080/19420862.2018.1556465.

[2] Scott M, Clark N. Next generation antibody therapeutics: Antibody fragments, dual-targeting strategies, and beyond… . European Pharmaceutical Review. 2009.

[3] Shukla AA, Wolfe LS, Mostafa SS, Norman C. Evolving trends in mAb production processes. Bioengineering & Translational Medicine. 2017;] 2(1): 58–69. doi: 10.1002/btm2.10061